@article{canoura_alkhamis_venzke_ly_xiao_2024, title={Developing Aptamer-Based Colorimetric Opioid Tests}, volume={3}, ISSN={["2691-3704"]}, DOI={10.1021/jacsau.3c00801}, abstractNote={Opioids collectively cause over 80,000 deaths in the United States annually. The ability to rapidly identify these compounds in seized drug samples on-site will be essential for curtailing trafficking and distribution. Chemical reagent-based tests are fast and simple but also notorious for giving false results due to poor specificity, whereas portable Raman spectrometers have excellent selectivity but often face interference challenges with impure drug samples. In this work, we develop on-site sensors for morphine and structurally related opioid compounds based on in vitro-selected oligonucleotide affinity reagents known as aptamers. We employ a parallel-and-serial selection strategy to isolate aptamers that recognize heroin, morphine, codeine, hydrocodone, and hydromorphone, along with a toggle-selection approach to isolate aptamers that bind oxycodone and oxymorphone. We then utilize a new high-throughput sequencing-based approach to examine aptamer growth patterns over the course of selection and a high-throughput exonuclease-based screening assay to identify optimal aptamer candidates. Finally, we use two high-performance aptamers with KD of ∼1 μM to develop colorimetric dye-displacement assays that can specifically detect opioids like heroin and oxycodone at concentrations as low as 0.5 μM with a linear range of 0–16 μM. Importantly, our assays can detect opioids in complex chemical matrices, including pharmaceutical tablets and drug mixtures; in contrast, the conventional Marquis test completely fails in this context. These aptamer-based colorimetric assays enable the naked-eye identification of specific opioids within seconds and will play an important role in combatting opioid abuse.}, journal={JACS AU}, author={Canoura, Juan and Alkhamis, Obtin and Venzke, Matthew and Ly, Phuong T. and Xiao, Yi}, year={2024}, month={Mar} }
 @article{yang_alkhamis_canoura_bryant_gong_barbu_taylor_nikic_banerjee_xiao_et al._2024, title={Exploring the Landscape of Aptamers: From Cross-Reactive to Selective to Specific, High-Affinity Receptors for Cocaine}, volume={2}, ISSN={["2691-3704"]}, DOI={10.1021/jacsau.3c00781}, abstractNote={We reported over 20 years ago MNS-4.1, the first DNA aptamer with a micromolar affinity for cocaine. MNS-4.1 is based on a structural motif that is very common in any random pool of oligonucleotides, and it is actually a nonspecific hydrophobic receptor with wide cross-reactivity with alkaloids and steroids. Despite such weaknesses preventing broad applications, this aptamer became widely used in proof-of-concept demonstrations of new formats of biosensors. We now report a series of progressively improved DNA aptamers recognizing cocaine, with the final optimized receptors having low nanomolar affinity and over a thousand-fold selectivity over the initial cross-reactants. In the process of optimization, we tested different methods to eliminate cross-reactivities and improve affinity, eventually achieving properties that are comparable to those of the reported monoclonal antibody candidates for the therapy of overdose. Multiple aptamers that we now report share structural motifs with the previously reported receptor for serotonin. Further mutagenesis studies revealed a palindromic, highly adaptable, broadly cross-reactive hydrophobic motif that could be rebuilt through mutagenesis, expansion of linker regions, and selections into receptors with exceptional affinities and varying specificities.}, journal={JACS AU}, author={Yang, Kyungae and Alkhamis, Obtin and Canoura, Juan and Bryant, Alexandra and Gong, Edward M. and Barbu, Mihaela and Taylor, Steven and Nikic, Dragan and Banerjee, Saswata and Xiao, Yi and et al.}, year={2024}, month={Feb} }
 @article{alkhamis_canoura_wu_emmons_wang_honeywell_plaxco_kippin_xiao_2024, title={High-Affinity Aptamers for <i>In Vitro</i> and <i>In Vivo</i> Cocaine Sensing}, volume={146}, ISSN={["1520-5126"]}, DOI={10.1021/jacs.3c11350}, abstractNote={The ability to quantify cocaine in biological fluids is crucial for both the diagnosis of intoxication and overdose in the clinic as well as investigation of the drug's pharmacological and toxicological effects in the laboratory. To this end, we have performed high-stringency in vitro selection to generate DNA aptamers that bind cocaine with nanomolar affinity and clinically relevant specificity, thus representing a dramatic improvement over the current-generation, micromolar-affinity, low-specificity cocaine aptamers. Using these novel aptamers, we then developed two sensors for cocaine detection. The first, an in vitro fluorescent sensor, successfully detects cocaine at clinically relevant levels in 50% human serum without responding significantly to other drugs of abuse, endogenous substances, or a diverse range of therapeutic agents. The second, an electrochemical aptamer-based sensor, supports the real-time, seconds-resolved measurement of cocaine concentrations in vivo in the circulation of live animals. We believe the aptamers and sensors developed here could prove valuable for both point-of-care and on-site clinical cocaine detection as well as fundamental studies of cocaine neuropharmacology.}, number={5}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Alkhamis, Obtin and Canoura, Juan and Wu, Yuyang and Emmons, Nicole A. and Wang, Yuting and Honeywell, Kevin M. and Plaxco, Kevin W. and Kippin, Tod E. and Xiao, Yi}, year={2024}, month={Jan}, pages={3230–3240} }
 @article{canoura_liu_alkhamis_xiao_2023, title={Aptamer-Based Fentanyl Detection in Biological Fluids}, volume={95}, ISSN={["1520-6882"]}, DOI={10.1021/acs.analchem.3c04104}, abstractNote={Fentanyl is a widely abused analgesic and anesthetic drug with a narrow therapeutic window that creates easy opportunities for overdose and death. Rapid, accurate, and sensitive fentanyl detection in biosamples is crucial for therapeutic drug monitoring and overdose diagnosis. Unfortunately, current methods are limited to either sophisticated laboratory-based tests or antibody-based immunoassays, which are prone to false results and are mainly used with urine samples. Here, we have utilized library-immobilized SELEX to isolate new aptamers─nucleic acid-based bioreceptors that are well-suited for biosensing─that can specifically bind fentanyl under physiological conditions. We isolated multiple aptamers with nanomolar affinity and excellent specificity against dozens of interferents and incorporated one of these into an electrochemical aptamer-based sensor that can rapidly detect fentanyl at clinically relevant concentrations in 50% diluted serum, urine, and saliva. Given the excellent performance of these sensors, we believe that they could serve as the basis for point-of-care devices for monitoring fentanyl during medical procedures and determining fentanyl overdose.}, number={49}, journal={ANALYTICAL CHEMISTRY}, author={Canoura, Juan and Liu, Yingzhu and Alkhamis, Obtin and Xiao, Yi}, year={2023}, month={Nov}, pages={18258–18267} }
 @article{alkhamis_canoura_willis_wang_perry_xiao_2023, title={Comparison of Aptamer Signaling Mechanisms Reveals Disparities in Sensor Response and Strategies to Eliminate False Signals}, volume={145}, ISSN={["1520-5126"]}, DOI={10.1021/jacs.3c03640}, abstractNote={Aptamers are nucleic acid-based affinity reagents that have been incorporated into a variety of molecular sensor formats. However, many aptamer sensors exhibit insufficient sensitivity and specificity for real-world applications, and although considerable effort has been dedicated to improving sensitivity, sensor specificity has remained largely neglected and understudied. In this work, we have developed a series of sensors using aptamers for the small-molecule drugs flunixin, fentanyl, and furanyl fentanyl and compare their performance─in particular, focusing on their specificity. Contrary to expectations, we observe that sensors using the same aptamer operating under the same physicochemical conditions produce divergent responses to interferents depending on their signal transduction mechanism. For instance, aptamer beacon sensors are susceptible to false-positives from interferents that weakly associate with DNA, while strand-displacement sensors suffer from false-negatives due to interferent-associated signal suppression when both the target and interferent are present. Biophysical analyses suggest that these effects arise from aptamer-interferent interactions that are either nonspecific or induce aptamer conformational changes that are distinct from those induced by true target-binding events. We also demonstrate strategies for improving the sensitivity and specificity of aptamer sensors with the development of a "hybrid beacon," wherein the incorporation of a complementary DNA competitor into an aptamer beacon selectively hinders interferent─but not target─binding and signaling, while simultaneously overcoming signal suppression by interferents. Our results highlight the need for systematic and thorough testing of aptamer sensor response and new aptamer selection methods that optimize specificity more effectively than traditional counter-SELEX.}, number={22}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Alkhamis, Obtin and Canoura, Juan and Willis, Connor and Wang, Linlin and Perry, Jacob and Xiao, Yi}, year={2023}, month={May}, pages={12407–12422} }
 @article{canoura_liu_perry_willis_xiao_2023, title={Suite of Aptamer-Based Sensors for the Detection of Fentanyl and Its Analogues}, volume={4}, ISSN={["2379-3694"]}, DOI={10.1021/acssensors.2c02463}, abstractNote={Fentanyl and its analogues are potent synthetic opioids that are commonly abused and are currently the number one cause of drug overdose death in the United States. The ability to detect fentanyl with simple, rapid, and low-cost tools is crucial for forensics, medical care, and public safety. Conventional on-site testing options for fentanyl detection─including chemical spot tests, lateral-flow immunoassays, and portable Raman spectrometers─each have their own unique flaws that limit their analytical utility. Here, we have developed a series of new aptamer-based assays and sensors that can detect fentanyl as well as several of its analogues in a reliable, accurate, rapid, and economic manner. These include colorimetric, fluorescent, and electrochemical sensors, which can detect and quantify minute quantities of fentanyl and many of its analogues with no response to other illicit drugs, cutting agents, or adulterants─even in interferent-ridden binary mixtures containing as little as 1% fentanyl. Given the high performance of these novel analytical tools, we foresee the potential for routine use by medical and law enforcement personnel as well as the general public to aid in rapid and accurate fentanyl identification.}, journal={ACS SENSORS}, author={Canoura, Juan and Liu, Yingzhu and Perry, Jacob and Willis, Connor and Xiao, Yi}, year={2023}, month={Apr} }
 @article{alkhamis_canoura_ly_xiao_2023, title={Using Exonucleases for Aptamer Characterization, Engineering, and Sensing}, volume={6}, ISSN={["1520-4898"]}, DOI={10.1021/acs.accounts.3c00113}, abstractNote={ConspectusAptamers are short, single-stranded nucleic acids that have been selected from random libraries to bind specific molecules with high affinity via an in vitro method termed systematic evolution of ligands by exponential enrichment (SELEX). They have been generated for diverse targets ranging from metal ions to small molecules to proteins and have demonstrated considerable promise as biorecognition elements in sensors for applications including medical diagnostics, environmental monitoring, food safety, and forensic analysis. While aptamer sensors have made great strides in terms of sensitivity, specificity, turnaround time, and ease of use, several challenges have hindered their broader adoption. These include inadequate sensitivity, bottlenecks in aptamer binding characterization, and the cost and labor associated with aptamer engineering. In this Account, we describe our successes in using nuclease enzymes to address these problems. While working with nucleases to enhance the sensitivity of split aptamer sensors via enzyme-assisted target recycling, we serendipitously discovered that the digestion of DNA aptamers by exonucleases is inhibited when an aptamer is bound to a ligand. This finding served as the foundation for the development of three novel aptamer-related methodologies in our laboratory. First, we used exonucleases to truncate nonessential nucleotides from aptamers to generate structure-switching aptamers in a single step, greatly simplifying the aptamer engineering process. Second, we used exonucleases to develop a label-free aptamer-based detection platform that can utilize aptamers directly obtained from in vitro selection to detect analytes with ultralow background and high sensitivity. Through this approach, we were able to detect analytes at nanomolar levels in biological samples, with the capacity for achieving multiplexed detection by using molecular beacons. Finally, we used exonucleases to develop a high throughput means of characterizing aptamer affinity and specificity for a variety of ligands. This approach has enabled more comprehensive analysis of aptamers by greatly increasing the number of aptamer candidates and aptamer-ligand pairs that can be tested in a single experiment. We have also demonstrated the success of this method as a means for identifying new mutant aptamers with augmented binding properties and for quantifying aptamer-target affinity. Our enzymatic technologies can greatly streamline the aptamer characterization and sensor development process, and with the adoption of robotics or liquid handling systems in the future, it should be possible to rapidly identify the most suitable aptamers for a particular application from hundreds to thousands of candidates.}, journal={ACCOUNTS OF CHEMICAL RESEARCH}, author={Alkhamis, Obtin and Canoura, Juan and Ly, Phuong T. and Xiao, Yi}, year={2023}, month={Jun} }
 @article{alkhamis_canoura_bukhryakov_tarifa_decaprio_xiao_2022, title={DNA Aptamer-Cyanine Complexes as Generic Colorimetric Small-Molecule Sensors}, volume={61}, ISSN={["1521-3773"]}, DOI={10.1002/anie.202112305}, abstractNote={Abstract}, number={3}, journal={ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, author={Alkhamis, Obtin and Canoura, Juan and Bukhryakov, Konstantin V and Tarifa, Anamary and DeCaprio, Anthony P. and Xiao, Yi}, year={2022}, month={Jan} }
 @article{canoura_alkhamis_liu_willis_xiao_2022, title={High-throughput quantitative binding analysis of DNA aptamers using exonucleases}, volume={12}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkac1210}, abstractNote={Abstract}, journal={NUCLEIC ACIDS RESEARCH}, author={Canoura, Juan and Alkhamis, Obtin and Liu, Yingzhu and Willis, Connor and Xiao, Yi}, year={2022}, month={Dec} }
 @article{jin_liu_alkhamis_canoura_bacon_xu_fu_xiao_2022, title={Near-Infrared Dye-Aptamer Assay for Small Molecule Detection in Complex Specimens}, volume={7}, ISSN={["1520-6882"]}, DOI={10.1021/acs.analchem.2c01095}, abstractNote={Aptamers are single-stranded oligonucleotides isolated in vitro that bind specific targets with high affinity and are commonly used as receptors in biosensors. Aptamer-based dye-displacement assays are a promising sensing platform because they are label-free, sensitive, simple, and rapid. However, these assays can exhibit impaired sensitivity in biospecimens, which contain numerous interferents that cause unwanted absorbance, scattering, and fluorescence in the UV-vis region. Here, this problem is overcome by utilizing near-infrared (NIR) signatures of the dye 3,3'-diethylthiadicarbocyanine iodide (Cy5). Cy5 initially complexes with aptamers as monomers and dimers; aptamer-target binding displaces the dye into solution, resulting in the formation of J-aggregates that provide a detectable NIR signal. The generality of our assay is demonstrated by detecting three different small-molecule analytes with their respective DNA aptamers at clinically relevant concentrations in serum and urine. These successful demonstrations show the utility of dye-aptamer NIR biosensors for high-throughput detection of analytes in clinical specimens.}, journal={ANALYTICAL CHEMISTRY}, author={Jin, Xin and Liu, Yingzhu and Alkhamis, Obtin and Canoura, Juan and Bacon, Adara and Xu, Ruyi and Fu, Fengfu and Xiao, Yi}, year={2022}, month={Jul} }
 @article{alkhamis_xiao_2022, title={Systematic Study of in Vitro Selection Stringency Reveals How To Enrich High-Affinity Aptamers}, volume={12}, ISSN={["1520-5126"]}, DOI={10.1021/jacs.2c09522}, abstractNote={Aptamers are oligonucleotide receptors with great potential for sensing and therapeutic applications. They are isolated from random libraries through an in vitro method termed systematic evolution of ligands by exponential enrichment (SELEX). Although SELEX-based methods have been widely employed over several decades, many aspects of the experimental process remain poorly understood in terms of how to adjust the selection conditions to obtain aptamers with the desired set of binding characteristics. As a result, SELEX is often performed with arbitrary parameters that tend to produce aptamers with insufficient affinity and/or specificity. Having a better understanding of these basic principles could increase the likelihood of obtaining high-quality aptamers. Here, we have systematically investigated how altering the selection stringency in terms of target concentration─which is essentially the root source of selection pressure for aptamer isolation─affects the outcome of SELEX. By performing four separate trials of SELEX for the same small-molecule target, we experimentally prove that the use of excessively high target concentrations promotes enrichment of low-affinity binders while also suppressing the enrichment of high-affinity aptamers. These findings should be broadly applicable across SELEX methods, given that they share the same core operating principle, and will be crucial for guiding selections to obtain high-quality aptamers in the future.}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Alkhamis, Obtin and Xiao, Yi}, year={2022}, month={Dec} }
 @article{yu_alkhamis_canoura_liu_xiao_2021, title={Advances and Challenges in Small‐Molecule DNA Aptamer Isolation, Characterization, and Sensor Development}, volume={60}, ISSN={1433-7851 1521-3773}, url={http://dx.doi.org/10.1002/anie.202008663}, DOI={10.1002/anie.202008663}, abstractNote={Abstract}, number={31}, journal={Angewandte Chemie International Edition}, publisher={Wiley}, author={Yu, Haixiang and Alkhamis, Obtin and Canoura, Juan and Liu, Yingzhu and Xiao, Yi}, year={2021}, month={Feb}, pages={16800–16923} }
 @article{liu_alkhamis_liu_yu_canoura_xiao_2021, title={Aptamer-Integrated Multianalyte-Detecting Paper Electrochemical Device}, volume={13}, ISSN={1944-8244 1944-8252}, url={http://dx.doi.org/10.1021/acsami.1c01822}, DOI={10.1021/acsami.1c01822}, abstractNote={On-site detection of multiple small-molecule analytes in complex sample matrixes would be highly valuable for diverse biosensing applications. Paper electrochemical devices (PEDs) offer an especially appealing sensing platform for such applications due to their low cost, portability, and ease of use. Using oligonucleotide-based aptamers as biorecognition elements, we here for the first time have developed a simple, inexpensive procedure for the fabrication of aptamer-modified multiplex PEDs (mPEDs), which can robustly and specifically detect multiple small molecules in complex samples. These devices are prepared via an ambient vacuum filtration technique using carbon and metal nanomaterials that yields precisely patterned sensing architecture featuring a silver pseudo-reference electrode, a gold counter electrode, and three gold working electrodes. The devices are user-friendly, and the fabrication procedure is highly reproducible. Each working electrode can be readily modified with different aptamers for sensitive and accurate detection of multiple small-molecule analytes in a single sample within seconds. We further demonstrate that the addition of a PDMS chamber allows us to achieve detection in microliter volumes of biological samples. We believe this approach should be highly generalizable, and given the rapid development of small-molecule aptamers, we envision that facile on-site multi-analyte detection of diverse targets in a drop of sample should be readily achievable in the near future.}, number={15}, journal={ACS Applied Materials & Interfaces}, publisher={American Chemical Society (ACS)}, author={Liu, Yingzhu and Alkhamis, Obtin and Liu, Xintong and Yu, Haixiang and Canoura, Juan and Xiao, Yi}, year={2021}, month={Apr}, pages={17330–17339} }
 @book{yu_xiao_2021, title={Aptamers that bind to natural and synthetic cannabinoids}, number={US10907163B1}, author={Yu, H.X. and Xiao, Y}, year={2021} }
 @inproceedings{canoura_liu_xiao_2021, title={Development of Aptamer-Based Sensors for Sensitive and Specific Detection of Fentanyl Opioids}, author={Canoura, J. and Liu, Y.Z. and Xiao, Y}, year={2021} }
 @article{liu_canoura_alkhamis_xiao_2021, title={Immobilization Strategies for Enhancing Sensitivity of Electrochemical Aptamer-Based Sensors}, volume={13}, ISSN={1944-8244 1944-8252}, url={http://dx.doi.org/10.1021/acsami.0c20707}, DOI={10.1021/acsami.0c20707}, abstractNote={Electrochemical aptamer-based (E-AB) sensors are a versatile sensing platform that can achieve rapid and robust target detection in complex matrices. However, the limited sensitivity of these sensors has impeded their translation from proof-of-concept to commercial products. Surface-bound aptamers must be sufficiently spaced to bind targets and subsequently fold for signal transduction. We hypothesized that electrodes fabricated using conventional methods result in sensing surfaces where only a fraction of aptamers are appropriately spaced to actively respond to the target. As an alternative, we presented a novel aptamer immobilization approach that favors sufficient spacing between aptamers at the microscale to achieve optimal target binding, folding, and signal transduction. We first demonstrated that immobilizing aptamers in their target-bound, folded state on gold electrode surfaces yields an aptamer monolayer that supports greater sensitivity and higher signal-to-noise ratio than traditionally prepared E-AB sensors. We also showed that performing aptamer immobilization under low ionic strength conditions rather than conventional high ionic strength buffer greatly improves E-AB sensor performance. We successfully tested our approach with three different small-molecule-binding aptamers, demonstrating its generalizability. On the basis of these results, we believe our electrode fabrication approach will accelerate development of high-performance sensors with the sensitivity required for real-world analytical applications.}, number={8}, journal={ACS Applied Materials & Interfaces}, publisher={American Chemical Society (ACS)}, author={Liu, Yingzhu and Canoura, Juan and Alkhamis, Obtin and Xiao, Yi}, year={2021}, month={Jan}, pages={9491–9499} }
 @article{yu_luo_alkhamis_canoura_yu_xiao_2021, title={Isolation of Natural DNA Aptamers for Challenging Small-Molecule Targets, Cannabinoids}, volume={93}, ISSN={0003-2700 1520-6882}, url={http://dx.doi.org/10.1021/acs.analchem.0c04592}, DOI={10.1021/acs.analchem.0c04592}, abstractNote={Aptamers are nucleic acid-based affinity reagents that are isolated via an in vitro process known as systematic evolution of ligands by exponential enrichment (SELEX). Despite their great potential for a wide range of analytical applications, there are relatively few high-quality small-molecule binding aptamers, especially for "challenging" targets that have low water solubility and/or limited moieties for aptamer recognition. The use of libraries containing chemically modified bases may improve the outcome of some SELEX experiments, but this approach is costly and yields inconsistent results. Here, we demonstrate that a thoughtfully designed SELEX procedure with natural DNA libraries can isolate aptamers with high affinity and specificity for challenging small molecules, including targets for which such selections have previously failed. We first isolate a DNA aptamer with nanomolar affinity and high specificity for (-)-trans-Δ9-tetrahydrocannabinol (THC), a target previously thought to be unsuitable for SELEX with natural DNA libraries. We subsequently isolate aptamers that exhibit high affinity and cross-reactivity to two other challenging targets, synthetic cannabinoids UR-144 and XLR-11, while maintaining excellent specificity against a wide range of non-target interferents. Our findings demonstrate that natural nucleic acid libraries can yield high-quality aptamers for small-molecule targets, and we outline a robust workflow for isolating other such aptamers in future selection efforts.}, number={6}, journal={Analytical Chemistry}, publisher={American Chemical Society (ACS)}, author={Yu, Haixiang and Luo, Yingping and Alkhamis, Obtin and Canoura, Juan and Yu, Boyang and Xiao, Yi}, year={2021}, month={Feb}, pages={3172–3180} }
 @book{yu_luo_xiao_2021, title={Materials and methods for colorimetric detection of small molecule targets}, number={US10948504B2}, author={Yu, H.X. and Luo, Y.P. and Xiao, Y}, year={2021} }
 @book{yang_yu_liu_xiao_2021, title={Method for isolating cross-reactive aptamer and use thereof}, number={US10907162B2}, author={Yang, W.J. and Yu, H.X. and Liu, Y.Z. and Xiao, Y}, year={2021} }
 @article{paudyal_wang_zhou_liu_cai_xiao_2021, title={Platinum-Nanoparticle-Modified Single-Walled Carbon Nanotube-Laden Paper Electrodes for Electrocatalytic Oxidation of Methanol}, volume={12}, ISSN={["2574-0970"]}, DOI={10.1021/acsanm.1c03186}, abstractNote={Platinum nanostructures have been used as electrocatalysts on various electrode subtrates for applications such as molecular sensing and fuel storage. Lightweight and flexible paper-based devices embedded with micro- or nanoscale metallic electrodes could prove highly useful for wearable devices and other portable applications. We here demonstrate that nanometer-thickness single-walled carbon nanotube (SWCNT)-laden paper prepared via ambient vacuum filtration offers an excellent conducting electrode substrate for the deposition of platinum through either electrodeposition or vacuum filtration. Characterization of the resulting paper electrodes reveals the formation of crystalline, interspersed, discrete spiky platinum nanoclusters (PtNCs) on the SWCNT film fabricated through electrodeposition, while those made via vacuum filtration feature interconnected spherical platinum nanoparticles (PtNPs). Using methanol as a benchmark molecule, we demonstrate that PtNP-SWCNT paper electrodes prepared via vacuum filtration have higher electrocatalytic efficiency compared to electrodes made via electrodeposition, and this is most likely due to the greater electroactive surface area of the PtNP-SWCNT paper electrode. This combination of the superior catalytic properties of platinum nanostructures with the flexible, thin, and conductive SWCNT paper substrate could prove highly valuable for the low-cost manufacturing of high-quality, disposable electrodes for a variety of applications.}, journal={ACS APPLIED NANO MATERIALS}, author={Paudyal, Janak and Wang, Po and Zhou, Fuyi and Liu, Yingzhu and Cai, Yong and Xiao, Yi}, year={2021}, month={Dec} }
 @article{canoura_yu_alkhamis_roncancio_farhana_xiao_2020, title={Accelerating Post-SELEX Aptamer Engineering Using Exonuclease Digestion}, volume={143}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/jacs.0c09559}, DOI={10.1021/jacs.0c09559}, abstractNote={The systematic evolution of ligands by exponential enrichment (SELEX) process enables the isolation of aptamers from random oligonucleotide libraries. However, it is generally difficult to identify the best aptamer from the resulting sequences, and the selected aptamers often exhibit suboptimal affinity and specificity. Post-SELEX aptamer engineering can improve aptamer performance, but current methods exhibit inherent bias and variable rates of success or require specialized instruments. Here, we describe a generalizable method that utilizes exonuclease III and exonuclease I to interrogate the binding properties of small-molecule-binding aptamers in a rapid, label-free assay. By analyzing an ochratoxin-binding DNA aptamer and six of its mutants, we determined that ligand binding alters the exonuclease digestion kinetics to an extent that closely correlates with the aptamer’s ligand affinity. We then utilized this assay to enhance the binding characteristics of a DNA aptamer which binds indiscriminately to ATP, ADP, AMP, and adenosine. We screened 13 mutants derived from this aptamer against all these analogues and identified two new high-affinity aptamers that solely bind to adenosine. We incorporated these two aptamers directly into an electrochemical aptamer-based sensor, which achieved a detection limit of 1 μM adenosine in 50% serum. We also confirmed the generality of our method to characterize target-binding affinities of protein-binding aptamers. We believe our approach is generalizable for DNA aptamers regardless of sequence, structure, and length and could be readily adapted into an automated format for high-throughput engineering of small-molecule-binding aptamers to acquire those with improved binding properties suitable for various applications.}, number={2}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Canoura, Juan and Yu, Haixiang and Alkhamis, Obtin and Roncancio, Daniel and Farhana, Rifat and Xiao, Yi}, year={2020}, month={Dec}, pages={805–816} }
 @inproceedings{liu_yu_alkhamis_moliver_xiao_2020, title={Achieving Near-ideal Binding Spectra using Subideal Bioreceptors}, author={Liu, Y.Z. and Yu, H.X. and Alkhamis, O. and Moliver, J. and Xiao, Y.}, year={2020} }
 @misc{xiao_2020, title={Beauty Through Simplicity – Problem-Driven Aptamer Science}, author={Xiao, Y.}, year={2020} }
 @article{yu_chen_liu_alkhamis_song_xiao_2020, title={Fabrication of Aptamer‐Modified Paper Electrochemical Devices for On‐Site Biosensing}, volume={60}, ISSN={1433-7851 1521-3773}, url={http://dx.doi.org/10.1002/anie.202008231}, DOI={10.1002/anie.202008231}, abstractNote={Abstract}, number={6}, journal={Angewandte Chemie International Edition}, publisher={Wiley}, author={Yu, Haixiang and Chen, Zhimin and Liu, Yingzhu and Alkhamis, Obtin and Song, Zhiping and Xiao, Yi}, year={2020}, month={Dec}, pages={2993–3000} }
 @inproceedings{xiao_2020, place={Chicago, Illinois, USA}, title={Isolation of Class-specific Aptamers and Development of Sensors for Instantaneous Detection of Synthetic Cathinones}, author={Xiao, Y.}, year={2020} }
 @article{alkhamis_yang_farhana_yu_xiao_2020, title={Label-free profiling of DNA aptamer-small molecule binding using T5 exonuclease}, volume={48}, ISSN={0305-1048 1362-4962}, url={http://dx.doi.org/10.1093/nar/gkaa849}, DOI={10.1093/nar/gkaa849}, abstractNote={Abstract}, number={20}, journal={Nucleic Acids Research}, publisher={Oxford University Press (OUP)}, author={Alkhamis, Obtin and Yang, Weijuan and Farhana, Rifat and Yu, Haixiang and Xiao, Yi}, year={2020}, month={Oct}, pages={e120–e120} }
 @book{xiao_luo_yu_2020, title={Materials and methods for colorimetric detection of small molecule targets}, number={US10725058B2}, author={Xiao, Y. and Luo, Y.P. and Yu, H.X.}, year={2020} }
 @book{xiao_yu_2020, title={Materials and methods for rapid and sensitive detection of small-molecule targets}, number={US10550395B2}, author={Xiao, Y. and Yu, H.X}, year={2020} }
 @book{xiao_yu_2020, title={Materials and methods for rapid and specific detection of synthetic cathinones}, number={US10683507B2}, author={Xiao, Y. and Yu, H.X}, year={2020} }
 @book{yang_yu_liu_xiao_2020, title={Method for isolating cross-reactive aptamer and use thereof}, number={US10655132B1}, author={Yang, W.J. and Yu, H.X. and Liu, Y.Z. and Xiao, Y}, year={2020} }
 @inproceedings{canoura_farhana_roncancio_xiao_2020, title={Screening of aptamer mutants for enhanced target specificity using a dual exonuclease digestion assay}, author={Canoura, J. and Farhana, R. and Roncancio, R. and Xiao, Y}, year={2020} }
 @article{liu_yu_alkhamis_moliver_xiao_2020, title={Tuning Biosensor Cross-Reactivity Using Aptamer Mixtures}, volume={92}, ISSN={0003-2700 1520-6882}, url={http://dx.doi.org/10.1021/acs.analchem.9b05339}, DOI={10.1021/acs.analchem.9b05339}, abstractNote={It is challenging to tune the response of biosensors to a set of ligands-for example, high cross-reactivity to a given target family while maintaining excellent specificity against interferents-due to the lack of suitable bioreceptors. We present a novel approach for controlling the cross-reactivity of biosensors by employing defined mixtures of aptamers with differing binding properties. As a demonstration, we develop assays for specific detection of a family of illicit designer drugs, the synthetic cathinones, with customized responses to each target ligand and interferent. We first use a colorimetric dye-displacement assay to show that the binding spectra of dual-aptamer mixtures can be tuned by altering the molar ratio of these bioreceptors. Optimized assays achieve broad detection of synthetic cathinones with minimal response towards interferents, and generally demonstrate better sensing performance than assays utilizing either aptamer alone. The generality of this strategy is demonstrated with a dual-aptamer electrochemical sensor. Our approach enables customization of biosensor responsiveness to an extent that has yet to be achieved through any previously-reported aptamer engineering techniques such as sequence mutation or truncation. Since multiple aptamers for the designated target family can routinely be identified via high-throughput sequencing, we believe our strategy offers a generally applicable method for generating near-ideal aptamer biosensors for various analytical applications, including medical diagnostics, environmental monitoring, and drug detection.}, number={7}, journal={Analytical Chemistry}, publisher={American Chemical Society (ACS)}, author={Liu, Yingzhu and Yu, Haixiang and Alkhamis, Obtin and Moliver, Jordan and Xiao, Yi}, year={2020}, month={Mar}, pages={5041–5047} }
 @inproceedings{yu_canoura_guntupalli_xiao_2019, title={A Cooperative-Binding Split Aptamer Assay for Rapid, Specific and Ultra-Sensitive Fluorescence Detection of Cocaine in Saliva}, author={Yu, H.X. and Canoura, J. and Guntupalli, B. and Xiao, Y}, year={2019} }
 @inproceedings{xiao_2019, title={Beauty Through Simplicity – Functionalized Aptamers for On-Site Small-Molecule Detection}, author={Xiao, Y.}, year={2019} }
 @inproceedings{liu_alkhamis_yu_xiao_2019, title={Development of Electrochemical Aptamer-Based Sensors for On-Site Synthetic Cathinone Detection}, author={Liu, Y.Z. and Alkhamis, O. and Yu, H.X. and Xiao, Y}, year={2019} }
 @book{xiao_guntupalli_paudyal_2019, title={Highly conductive porous paper-based metal thin films}, number={US 10290386US10290386B2}, author={Xiao, Y. and Guntupalli, B. and Paudyal, J}, year={2019} }
 @inproceedings{yu_yang_alkhamis_canoura_yang_xiao_2019, title={In Vitro Isolation of Small-Molecule-Binding Aptamers with Intrinsic Dye-Displacement Functionality}, author={Yu, H.X. and Yang, W.J. and Alkhamis, O. and Canoura, J. and Yang, K.-A. and Xiao, Y.}, year={2019} }
 @article{yang_yu_alkhamis_liu_canoura_fu_xiao_2019, title={In vitro isolation of class-specific oligonucleotide-based small-molecule receptors}, volume={47}, ISSN={0305-1048 1362-4962}, url={http://dx.doi.org/10.1093/nar/gkz224}, DOI={10.1093/nar/gkz224}, abstractNote={Abstract}, number={12}, journal={Nucleic Acids Research}, publisher={Oxford University Press (OUP)}, author={Yang, Weijuan and Yu, Haixiang and Alkhamis, Obtin and Liu, Yingzhu and Canoura, Juan and Fu, Fengfu and Xiao, Yi}, year={2019}, month={Mar}, pages={e71–e71} }
 @article{alkhamis_canoura_yu_liu_xiao_2019, title={Innovative engineering and sensing strategies for aptamer-based small-molecule detection}, volume={121}, ISSN={0165-9936}, url={http://dx.doi.org/10.1016/j.trac.2019.115699}, DOI={10.1016/j.trac.2019.115699}, abstractNote={Aptamers are nucleic acid-based affinity reagents that have gained widespread attention as biorecognition elements for the detection of targets such as ions, small molecules, and proteins. Over the past three decades, the field of aptamer-based sensing has grown considerably. However, the advancement of aptamer-based small-molecule detection has fallen short of the high demand for such sensors in applications such as diagnostics, environmental monitoring, and forensics. This is due to two challenges: the complexity of developing generalized sensing platforms and the poor sensitivities of assays targeting small molecules. This paper will review new approaches for the streamlined development of high-performance aptamer-based sensors for small-molecule detection. We here provide historical context, explore the current state-of-the art, and offer future directions—with emphasis placed on new aptamer engineering methods, the use of cooperative binding, and label-free approaches using fully-folded, high-affinity aptamers for small-molecule sensing.}, journal={TrAC Trends in Analytical Chemistry}, publisher={Elsevier BV}, author={Alkhamis, Obtin and Canoura, Juan and Yu, Haixiang and Liu, Yingzhu and Xiao, Yi}, year={2019}, month={Dec}, pages={115699} }
 @inproceedings{wang_yu_canoura_liu_alkhamis_fu_xiao_2019, title={Introducing Structure-Switching Functionality into Small-Molecule-Binding Aptamers via Nuclease-Directed Truncation}, author={Wang, Z.W. and Yu, H.X. and Canoura, J. and Liu, Y.Z. and Alkhamis, O. and Fu, F.F. and Xiao, Y}, year={2019} }
 @article{luo_yu_alkhamis_liu_lou_yu_xiao_2019, title={Label-Free, Visual Detection of Small Molecules Using Highly Target-Responsive Multimodule Split Aptamer Constructs}, volume={91}, ISSN={0003-2700 1520-6882}, url={http://dx.doi.org/10.1021/acs.analchem.9b00507}, DOI={10.1021/acs.analchem.9b00507}, abstractNote={Colorimetric aptamer-based sensors offer a simple means of on-site or point-of-care analyte detection. However, these sensors are largely incapable of achieving naked-eye detection, because of the poor performance of the target-recognition and signal-reporting elements employed. To address this problem, we report a generalizable strategy for engineering novel multimodule split DNA constructs termed "CBSAzymes" that utilize a cooperative binding split aptamer (CBSA) as a highly target-responsive bioreceptor and a new, highly active split DNAzyme as an efficient signal reporter. CBSAzymes consist of two fragments that remain separate in the absence of target, but effectively assemble in the presence of the target to form a complex that catalyzes the oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid, developing a dark green color within 5 min. Such assay enables rapid, sensitive, and visual detection of small molecules, which has not been achieved with any previously reported split-aptamer-DNAzyme conjugates. In an initial demonstration, we generate a cocaine-binding CBSAzyme that enables naked-eye detection of cocaine at concentrations as low as 10 μM. Notably, CBSAzyme engineering is straightforward and generalizable. We demonstrate this by developing a methylenedioxypyrovalerone (MDPV)-binding CBSAzyme for visual detection of MDPV and 10 other synthetic cathinones at low micromolar concentrations, even in biological samples. Given that CBSAzyme-based assays are simple, label-free, rapid, robust, and instrument-free, we believe that such assays should be readily applicable for on-site visual detection of various important small molecules such as illicit drugs, medical biomarkers, and toxins in various sample matrices.}, number={11}, journal={Analytical Chemistry}, publisher={American Chemical Society (ACS)}, author={Luo, Yingping and Yu, Haixiang and Alkhamis, Obtin and Liu, Yingzhu and Lou, Xinhui and Yu, Boyang and Xiao, Yi}, year={2019}, month={May}, pages={7199–7207} }
 @article{wu_belmonte_sykes_xiao_white_2019, title={Perspective on the Future Role of Aptamers in Analytical Chemistry}, volume={91}, ISSN={0003-2700 1520-6882}, url={http://dx.doi.org/10.1021/acs.analchem.9b03853}, DOI={10.1021/acs.analchem.9b03853}, abstractNote={It has been almost 30 years since the invention of SELEX (Systematic Evolution of Ligands by Exponential Enrichment) methodology and the description of the first aptamers. In retrospect over the past 30 years, advances in aptamer development and application have demonstrated that aptamers are potentially useful reagents that can be employed in diverse areas within analytical chemistry, biotechnology, biomedicine, and molecular biology. While often touted as artificial antibodies with an ability to be selected for any target, aptamer development, unfortunately, lags behind development of analytical methodologies that employ aptamers, hindering deeper integration into the application of analytical tool development. This perspective covers recent advances in SELEX methodology coupled with post-selection procedures all aimed at enhancing affinity and specificity of the selected aptamers - what we view as a critical barrier in the future role of aptamers in analytical chemistry. We discuss post-selection modifications that can be used for enhancing performance of the selected aptamers in analytical device by including understanding intermolecular interaction forces in the binding domain. While highlighting promising properties of aptamers that enable several analytical advances, we provide discussion on the challenges of penetration of aptamers in the analytical field.}, number={24}, journal={Analytical Chemistry}, publisher={American Chemical Society (ACS)}, author={Wu, Yao and Belmonte, Israel and Sykes, Kiana S. and Xiao, Yi and White, Ryan J.}, year={2019}, month={Nov}, pages={15335–15344} }
 @inproceedings{yang_yu_alkhamis_fu_xiao_2019, title={Rapid, naked-eye screening for synthetic cathinones based on cross-reactive aptamers}, author={Yang, W.J. and Yu, H.X. and Alkhamis, O. and Fu, F.F. and Xiao, Y}, year={2019} }
 @article{gao_zhao_huang_cheng_wang_han_xiao_lou_2019, title={Universal Design of Structure-Switching Aptamers with Signal Reporting Functionality}, volume={91}, ISSN={0003-2700 1520-6882}, url={http://dx.doi.org/10.1021/acs.analchem.9b03368}, DOI={10.1021/acs.analchem.9b03368}, abstractNote={Structure-switching aptamers (SSAs) offer a promising recognition element for sensor development. However, the generation of SSAs via in vitro aptamer selection technologies or post-selection engineering is challenging. Inspired by the two-domain structure of antibodies, we have devised a simple, universal strategy for engineering aptamers into SSAs with signal reporting functionality. These constructs consist of a 'constant' domain, comprising a split DNAzyme G-quadruplex (G4) region for signal transduction, and a 'variable' domain, comprising an aptamer sequence capable of specific target binding. In the absence of target, the G4-SSA construct folds into a parallel G4 structure with high peroxidase catalytic activity. Target binding disrupts the G4 structure, resulting in low enzymatic activity. We demonstrate that this change in DNAzyme activity enables sensitive and specific colorimetric detection of diverse targets including Hg2+, thrombin, sulfadimethoxine, cocaine, and 17β-estradiol. G4-SSAs can also achieve label-free fluorescence detection of various targets using a specific G4-binding dye. We demonstrate that diverse aptamers can be readily engineered into G4-SSA constructs independent of target class, binding affinity, aptamer length, or structure. This design strategy could broadly extend the power, accessibility and utility of numerous SSA-based biosensors.}, number={22}, journal={Analytical Chemistry}, publisher={American Chemical Society (ACS)}, author={Gao, Hualong and Zhao, Jiaoxing and Huang, Yang and Cheng, Xiao and Wang, Shuo and Han, Yu and Xiao, Yi and Lou, Xinhui}, year={2019}, month={Oct}, pages={14514–14521} }
 @inproceedings{xiao_2018, place={Orlando, USA}, title={Aptamer-based Assays for On-site Drug Detection}, author={Xiao, Y.}, year={2018} }
 @misc{xiao_2018, title={Beauty through simplicity – functionalized aptamers for on-site small-molecule detection}, author={Xiao, Y.}, year={2018} }
 @article{liang_canoura_yu_alkhamis_xiao_2018, title={Dithiothreitol-Regulated Coverage of Oligonucleotide-Modified Gold Nanoparticles To Achieve Optimized Biosensor Performance}, volume={10}, ISSN={1944-8244 1944-8252}, url={http://dx.doi.org/10.1021/acsami.7b16914}, DOI={10.1021/acsami.7b16914}, abstractNote={DNA-modified gold nanoparticles (AuNPs) are useful signal-reporters for detecting diverse molecules through various hybridization- and enzyme-based assays. However, their performance is heavily dependent on the probe DNA surface coverage, which can influence both target binding and enzymatic processing of the bound probes. Current methods used to adjust the surface coverage of DNA-modified AuNPs require the production of multiple batches of AuNPs under different conditions, which is costly and laborious. We here develop a single-step assay utilizing dithiothreitol (DTT) to fine-tune the surface coverage of DNA-modified AuNPs. DTT is superior to the commonly used surface diluent, mercaptohexanol, as it is less volatile, allowing for the rapid and reproducible controlling of surface coverage on AuNPs with only micromolar concentrations of DTT. Upon adsorption, DTT forms a dense monolayer on gold surfaces, which provides antifouling capabilities. Furthermore, surface-bound DTT adopts a cyclic conformation, which reorients DNA probes into an upright position and provides ample space to promote DNA hybridization, aptamer assembly, and nuclease digestion. We demonstrate the effects of surface coverage on AuNP-based sensors using DTT-regulated DNA-modified AuNPs. We then use these AuNPs to visually detect DNA and cocaine in colorimetric assays based on enzyme-mediated AuNP aggregation. We determine that DTT-regulated AuNPs with lower surface coverage achieve shorter reaction times and lower detection limits relative to those for assays using untreated AuNPs or DTT-regulated AuNPs with high surface coverage. Additionally, we demonstrate that our DTT-regulated AuNPs can perform cocaine detection in 50% urine without any significant matrix effects. We believe that DTT regulation of surface coverage can be broadly employed for optimizing DNA-modified AuNP performance for use in biosensors as well as drug delivery and therapeutic applications.}, number={4}, journal={ACS Applied Materials & Interfaces}, publisher={American Chemical Society (ACS)}, author={Liang, Pingping and Canoura, Juan and Yu, Haixiang and Alkhamis, Obtin and Xiao, Yi}, year={2018}, month={Jan}, pages={4233–4242} }
 @article{yu_yang_alkhamis_canoura_yang_xiao_2018, title={In vitro isolation of small-molecule-binding aptamers with intrinsic dye-displacement functionality}, volume={46}, ISSN={0305-1048 1362-4962}, url={http://dx.doi.org/10.1093/nar/gky026}, DOI={10.1093/nar/gky026}, abstractNote={Abstract Aptamer-based sensors offer a powerful tool for molecular detection, but the practical implementation of these biosensors is hindered by costly and laborious sequence engineering and chemical modification procedures. We report a simple strategy for directly isolating signal-reporting aptamers in vitro through systematic evolution of ligands by exponential enrichment (SELEX) that transduce binding events into a detectable change of absorbance via target-induced displacement of a small-molecule dye. We first demonstrate that diethylthiatricarbocyanine (Cy7) can stack into DNA three-way junctions (TWJs) in a sequence-independent fashion, greatly altering the dye's absorbance spectrum. We then design a TWJ-containing structured library and isolate an aptamer against 3,4-methylenedioxypyrovalerone (MDPV), a synthetic cathinone that is an emerging drug of abuse. This aptamer intrinsically binds Cy7 within its TWJ domain, but MDPV efficiently displaces the dye, resulting in a change in absorbance within seconds. This assay is label-free, and detects nanomolar concentrations of MDPV. It also recognizes other synthetic cathinones, offering the potential to detect newly-emerging designer drugs, but does not detect structurally-similar non-cathinone compounds or common cutting agents. Moreover, we demonstrate that the Cy7-displacement colorimetric assay is more sensitive than a conventional strand-displacement fluorescence assay. We believe our strategy offers an effective generalized approach for the development of sensitive dye-displacement colorimetric assays for other small-molecule targets.}, number={8}, journal={Nucleic Acids Research}, publisher={Oxford University Press (OUP)}, author={Yu, Haixiang and Yang, Weijuan and Alkhamis, Obtin and Canoura, Juan and Yang, Kyung-Ae and Xiao, Yi}, year={2018}, month={Jan}, pages={e43–e43} }
 @article{wang_yu_canoura_liu_alkhamis_fu_xiao_2018, title={Introducing structure-switching functionality into small-molecule-binding aptamers via nuclease-directed truncation}, volume={46}, ISSN={0305-1048 1362-4962}, url={http://dx.doi.org/10.1093/nar/gky305}, DOI={10.1093/nar/gky305}, abstractNote={Abstract We report a broadly applicable enzyme digestion strategy for introducing structure-switching functionality into small-molecule-binding aptamers. This procedure is based on our discovery that exonuclease III (Exo III) digestion of aptamers is greatly inhibited by target binding. As a demonstration, we perform Exo III digestion of a pre-folded three-way-junction (TWJ)-structured cocaine-binding aptamer and a stem–loop-structured ATP-binding aptamer. In the absence of target, Exo III catalyzes 3′-to-5′ digestion of both aptamers to form short, single-stranded products. Upon addition of target, Exo III digestion is halted four bases prior to the target-binding domain, forming a major target-bound aptamer digestion product. We demonstrated that target-binding is crucial for Exo III inhibition. We then determine that the resulting digestion products of both aptamers exhibit a target-induced structure-switching functionality that is absent in the parent aptamer, while still retaining high target-binding affinity. We confirm that these truncated aptamers have this functionality by using an exonuclease I-based digestion assay and further evaluate this characteristic in an electrochemical aptamer-based cocaine sensor and a fluorophore-quencher ATP assay. We believe our Exo III-digestion method should be applicable for the generation of structure-switching aptamers from other TWJ- or stem–loop-containing small-molecule-binding aptamers, greatly simplifying the generation of functionalized sensor elements for folding-based aptasensors.}, number={13}, journal={Nucleic Acids Research}, publisher={Oxford University Press (OUP)}, author={Wang, Zongwen and Yu, Haixiang and Canoura, Juan and Liu, Yingzhu and Alkhamis, Obtin and Fu, Fengfu and Xiao, Yi}, year={2018}, month={Apr}, pages={e81–e81} }
 @book{liang_yu_xiao_2018, title={Materials and methods for rapid visualization of NAD(P)H}, number={US10000790B2}, author={Liang, P.P. and Yu, H.X. and Xiao, Y}, year={2018} }
 @article{canoura_wang_yu_alkhamis_fu_xiao_2018, title={No Structure-Switching Required: A Generalizable Exonuclease-Mediated Aptamer-Based Assay for Small-Molecule Detection}, volume={140}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/jacs.8b04975}, DOI={10.1021/jacs.8b04975}, abstractNote={The binding of small molecules to double-stranded DNA can modulate its susceptibility to digestion by exonucleases. Here, we show that the digestion of aptamers by exonuclease III can likewise be inhibited upon binding of small-molecule targets and exploit this finding for the first time to achieve sensitive, label-free small-molecule detection. This approach does not require any sequence engineering and employs prefolded aptamers which have higher target-binding affinities than structure-switching aptamers widely used in current small-molecule detecting assays. We first use a dehydroisoandrosterone-3-sulfate-binding aptamer to show that target binding halts exonuclease III digestion four bases prior to the binding site. This leaves behind a double-stranded product that retains strong target affinity, whereas digestion of nontarget-bound aptamer produces a single-stranded product incapable of target binding. Exonuclease I efficiently eliminates these single-stranded products but is unable to digest the target-bound double-stranded product. The remaining products can be fluorescently quantified with SYBR Gold to determine target concentrations. We demonstrate that this dual-exonuclease-mediated approach can be broadly applied to other aptamers with differing secondary structures to achieve sensitive detection of various targets, even in biological matrices. Importantly, each aptamer digestion product has a unique sequence, enabling the creation of multiplex assays, and we successfully demonstrate simultaneous detection of cocaine and ATP in a single microliter volume sample in 25 min via sequence-specific molecular beacons. Due to the generality and simplicity of this assay, we believe that different DNA signal-reporting or amplification strategies can be adopted into our assay for target detection in diverse analytical contexts.}, number={31}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Canoura, Juan and Wang, Zongwen and Yu, Haixiang and Alkhamis, Obtin and Fu, Fengfu and Xiao, Yi}, year={2018}, month={Jul}, pages={9961–9971} }
 @inproceedings{yu_canoura_guntupalli_lou_xiao_2018, place={Orlando, USA}, title={Rapid and sensitive detection of small-molecule targets via enzyme-assisted target recycling based on cooperative binding split aptamers}, author={Yu, H.X. and Canoura, J. and Guntupalli, B. and Lou, X.H. and Xiao, Y}, year={2018} }
 @article{yu_canoura_guntupalli_alkhamis_xiao_2018, title={Sensitive Detection of Small-Molecule Targets Using Cooperative Binding Split Aptamers and Enzyme-Assisted Target Recycling}, volume={90}, ISSN={0003-2700 1520-6882}, url={http://dx.doi.org/10.1021/acs.analchem.7b03625}, DOI={10.1021/acs.analchem.7b03625}, abstractNote={Signal amplification via enzyme-assisted target recycling (EATR) offers a powerful means for improving the sensitivity of DNA detection assays, but it has proven challenging to employ EATR with aptamer-based assays for small-molecule detection due to insensitive target response of aptamers. Here, we describe a general approach for the development of rapid and sensitive EATR-amplified small-molecule sensors based on cooperative binding split aptamers (CBSAs). CBSAs contain two target-binding domains and exhibit enhanced target response compared with single-domain split aptamers. We introduced a duplexed C3 spacer abasic site between the two binding domains, enabling EATR signal amplification through exonuclease III's apurinic endonuclease activity. As a demonstration, we engineered a CBSA-based EATR-amplified fluorescence assay to detect dehydroisoandrosterone-3-sulfate. This assay achieved 100-fold enhanced target sensitivity relative to a non-EATR-based assay, with a detection limit of 1 μM in 50% urine. We further developed an instrument-free colorimetric assay employing EATR-mediated aggregation of CBSA-modified gold nanoparticles for the visual detection of low-micromolar concentrations of cocaine. On the basis of the generalizability of CBSA engineering and the robust performance of EATR in complex samples, we believe that such assays should prove valuable for detecting small-molecule targets in diverse fields.}, number={3}, journal={Analytical Chemistry}, publisher={American Chemical Society (ACS)}, author={Yu, Haixiang and Canoura, Juan and Guntupalli, Bhargav and Alkhamis, Obtin and Xiao, Yi}, year={2018}, month={Jan}, pages={1748–1758} }
 @inproceedings{wang_canoura_yu_xiao_2018, title={Utilizing nuclease screening of ligand-aptamer complexes to enhance specificity of an aptamer-based cocaine assay}, author={Wang, Z.W. and Canoura, J. and Yu, H.X. and Xiao, Y}, year={2018} }
 @article{yu_xu_liang_loh_guntupalli_roncancio_xiao_2017, title={A Broadly Applicable Assay for Rapidly and Accurately Quantifying DNA Surface Coverage on Diverse Particles}, volume={28}, ISSN={1043-1802 1520-4812}, url={http://dx.doi.org/10.1021/acs.bioconjchem.6b00660}, DOI={10.1021/acs.bioconjchem.6b00660}, abstractNote={DNA-modified particles are used extensively for applications in sensing, material science, and molecular biology. The performance of such DNA-modified particles is greatly dependent on the degree of surface coverage, but existing methods for quantitation can only be employed for certain particle compositions and/or conjugation chemistries. We have developed a simple and broadly applicable exonuclease III (Exo III) digestion assay based on the cleavage of phosphodiester bonds-a universal feature of DNA-modified particles-to accurately quantify DNA probe surface coverage on diverse, commonly used particles of different compositions, conjugation chemistries, and sizes. Our assay utilizes particle-conjugated, fluorophore-labeled probes that incorporate two abasic sites; these probes are hybridized to a complementary DNA (cDNA) strand, and quantitation is achieved via cleavage and digestion of surface-bound probe DNA via Exo III's apurinic endonucleolytic and exonucleolytic activities. The presence of the two abasic sites in the probe greatly speeds up the enzymatic reaction without altering the packing density of the probes on the particles. Probe digestion releases a signal-generating fluorophore and liberates the intact cDNA strand to start a new cycle of hybridization and digestion, until all fluorophore tags have been released. Since the molar ratio of fluorophore to immobilized DNA is 1:1, DNA surface coverage can be determined accurately based on the complete release of fluorophores. Our method delivers accurate, rapid, and reproducible quantitation of thiolated DNA on the surface of gold nanoparticles, and also performs equally well with other conjugation chemistries, substrates, and particle sizes, and thus offers a broadly useful assay for quantitation of DNA surface coverage.}, number={4}, journal={Bioconjugate Chemistry}, publisher={American Chemical Society (ACS)}, author={Yu, Haixiang and Xu, Xiaowen and Liang, Pingping and Loh, Kang Yong and Guntupalli, Bhargav and Roncancio, Daniel and Xiao, Yi}, year={2017}, month={Feb}, pages={933–943} }
 @article{yu_canoura_guntupalli_lou_xiao_2017, title={A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva}, volume={8}, ISSN={2041-6520 2041-6539}, url={http://dx.doi.org/10.1039/c6sc01833e}, DOI={10.1039/c6sc01833e}, abstractNote={A fluorescence assay based on a split aptamer featuring a cooperative-target-binding mechanism performs one-step, rapid detection of as low as 50 nM in 10% saliva without signal amplification.}, number={1}, journal={Chemical Science}, publisher={Royal Society of Chemistry (RSC)}, author={Yu, Haixiang and Canoura, Juan and Guntupalli, Bhargav and Lou, Xinhui and Xiao, Yi}, year={2017}, pages={131–141} }
 @inproceedings{yu_canoura_guntupalli_xiao_2017, title={A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva}, author={Yu, H.X. and Canoura, J. and Guntupalli, B. and Xiao, Y}, year={2017} }
 @inproceedings{roncancio_yu_xu_xiao_2017, title={A label-free aptamer-fluorophore assembly for highly sensitive and specific detection of cocaine}, author={Roncancio, D. and Yu, H.X. and Xu, X.W. and Xiao, Y}, year={2017} }
 @inproceedings{yu_canoura_guntupalli_xiao_2017, title={Cooperative-binding split aptamer assays for rapid, specific and ultra-sensitive detection of small-molecule targets in biofluid samples}, author={Yu, H.X. and Canoura, J. and Guntupalli, B. and Xiao, Y.}, year={2017} }
 @inproceedings{xiao_2017, title={Gold nanoparticle-based assays for rapid and sensitive colorimetric and electrochemical biosensing}, author={Xiao, Y.}, year={2017} }
 @misc{liang_guntupalli_yu_xiao_2017, title={Gold nanoparticle-based, paper devices for rapid and sensitive colorimetric and electrochemical biosensing}, author={Liang, P.P. and Guntupalli, B. and Yu, H.X. and Xiao, Y.}, year={2017} }
 @book{roncancio_yu_xu_xiao_2017, title={Materials and methods for rapid and specific detection of cocaine}, number={US9804178B2}, author={Roncancio, D. and Yu, H.X. and Xu, X.W. and Xiao, Y}, year={2017} }
 @inproceedings{yu_canoura_guntupalli_lou_xiao_2017, title={Rapid and sensitive detection of small-molecule targets via enzyme-assisted target recycling based on cooperative binding split aptamers}, author={Yu, H.X. and Canoura, J. and Guntupalli, B. and Lou, X.H. and Xiao, Y.}, year={2017} }
 @inproceedings{canoura_wang_yu_xiao_2017, title={Utilizing dual-enzyme selectivity for the identification of aptamer binding domains and the development of sensitive fluorescence detection of small-molecule targets}, author={Canoura, J. and Wang, Z.W. and Yu, H.X. and Xiao, Y.}, year={2017} }
 @inproceedings{canoura_wang_yu_xiao_2017, title={Utilizing nuclease screening of ligand-aptamer complexes to enhance specificity of an aptamer-based cocaine assay}, author={Canoura, J. and Wang, Z.W. and Yu, H.X. and Xiao, Y.}, year={2017} }
 @inproceedings{yu_canoura_guntupalli_xiao_2016, title={A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva}, author={Yu, H.X. and Canoura, J. and Guntupalli, B. and Xiao, Y.}, year={2016} }
 @inproceedings{yu_guntupalli_xiao_2016, title={A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva}, author={Yu, H.X. and Guntupalli, B. and Xiao, Y.}, year={2016} }
 @inproceedings{h.x._b._y_2016, title={A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva}, author={H.X., Yu and B., Guntupalli and Y, Xiao}, year={2016} }
 @inproceedings{roncancio_yu_xu_wu_liu_debord_lou_xiao_2016, title={A label-free aptamer-fluorophore assembly for highly sensitive and specific detection of cocaine}, author={Roncancio, D. and Yu, H.X. and Xu, X.W. and Wu, S. and Liu, R. and Debord, J. and Lou, X.H. and Xiao, Y.}, year={2016} }
 @inproceedings{guntupalli_liang_lee_yang_yu_canoura_he_li_weizmann_xiao_2016, title={Ambient filtration method to rapidly prepare highly conductive, paper-based porous gold films for electrochemical biosensing}, author={Guntupalli, B. and Liang, P.P. and Lee, J.H. and Yang, Y.H. and Yu, H.X. and Canoura, J. and He, J. and Li, W.Z. and Weizmann, Y. and Xiao, Y.}, year={2016} }
 @misc{xiao_2016, title={Bringing biosensors to the bedside}, author={Xiao, Y.}, year={2016} }
 @inproceedings{liang_yu_guntupalli_xiao_2016, title={Paper-based device for rapid visualization of NADH based on dissolution of gold nanoparticles}, author={Liang, P.P. and Yu, H.X. and Guntupalli, B. and Xiao, Y.}, year={2016} }
 @inproceedings{xiao_2016, title={Paper-based devices for rapid and sensitive colorimetric and electrochemical biosensing}, author={Xiao, Y.}, year={2016} }
 @article{xu_lou_wang_ding_yu_xiao_2016, title={Rapid, Surfactant-Free, and Quantitative Functionalization of Gold Nanoparticles with Thiolated DNA under Physiological pH and Its Application in Molecular Beacon-Based Biosensor}, volume={8}, ISSN={1944-8244 1944-8252}, url={http://dx.doi.org/10.1021/acsami.6b08350}, DOI={10.1021/acsami.6b08350}, abstractNote={The controlled attachment of thiolated DNA to gold nanoparticles (AuNPs) dictates many applications. This is typically achieved by either "aging-salting" processes or low-pH method, where either Na+ or H+ is used to minimize charge repulsion and facilitate attachment of thiolated DNA onto AuNPs. However, the "aging-salting" process takes a long time, and is prone to aggregation when used with larger AuNPs. Surfactants are needed to precoat and thereby enhance the stability of AuNPs. The low-pH method can disrupt the structural integrity of DNAs. We report here an oligoethylene glycol (OEG) spacer-assisted method that enables quantitative and instantaneous attachment at physiological pH without the need for surfactants. The method is based on our finding that an uncharged OEG spacer as short as six EG units can effectively shield against repulsion between AuNPs and DNAs, substantially enhancing both the adsorption kinetics and thermodynamics of thiolated DNAs. We applied this to thiolated DNAs of various lengths and thiol modification positions and to large AuNPs. Importantly, our method also allows for the direct immobilization of thiolated molecular beacons (MB), and avoids particle aggregation due to intermolecular hydrogen bonding. The prepared MB-AuNPs were successfully used for the fluorescent detection of target DNA at nanomolar concentrations. The OEG spacer appears to offer a highly effective parameter for tuning DNA adsorption kinetics and thermodynamics besides pH and salt, providing a novel means for highly controllable and versatile functionalization of AuNPs.}, number={40}, journal={ACS Applied Materials & Interfaces}, publisher={American Chemical Society (ACS)}, author={Xu, Qing and Lou, Xinhui and Wang, Lei and Ding, Xiaofan and Yu, Haixiang and Xiao, Yi}, year={2016}, month={Sep}, pages={27298–27304} }
 @inproceedings{canoura_wang_yu_ng_roncancio_xiao_2016, title={Utilizing nuclease screening of ligand-aptamer complexes to enhance specificity of an aptamer-based cocaine assay}, author={Canoura, J. and Wang, Z.W. and Yu, H.X. and Ng, B. and Roncancio, D. and Xiao, Y.}, year={2016} }
 @inproceedings{liang_yu_xiao_2015, title={A paper-based device for rapid visualization of NADH based on dissolution of gold nanoparticles}, author={Liang, P.P. and Yu, H.X. and Xiao, Y}, year={2015} }
 @article{guntupalli_liang_lee_yang_yu_canoura_he_li_weizmann_xiao_2015, title={Ambient Filtration Method To Rapidly Prepare Highly Conductive, Paper-Based Porous Gold Films for Electrochemical Biosensing}, volume={7}, ISSN={1944-8244 1944-8252}, url={http://dx.doi.org/10.1021/acsami.5b09612}, DOI={10.1021/acsami.5b09612}, abstractNote={Thin gold films offer intriguing material properties for potential applications including fuel cells, supercapacitors, and electronic and photonic devices. We describe here an ambient filtration method that provides a simple and novel way to generate rapidly porous and thin gold films without the need for sophisticated instruments, clean-room environments, and any postgrowth process or sintering steps. Using this approach, we can fabricate highly conductive gold films composed of gold nanoparticles layered atop a matrix of metallic single-walled carbon nanotubes on mixed cellulose ester filter paper within 20 min. These hybrid films (thickness ∼40 nm) exhibit fast electron transfer and excellent electrocatalytic properties that are similar to purchased gold films, but with a larger electroactive surface that lends itself to more sensitive analyte detection. We used the neurotransmitters dopamine and serotonin as benchmark analytes to demonstrate that our hybrid gold films can clearly discriminate the presence of both molecules in a mixture with resolution that greatly exceeds that of either purchased gold slides or electrodeposited gold films. Importantly, we postulate that this new approach could readily be generalized for the rapid fabrication of films from various other metals under ambient conditions, and could also be used as a prelude to transferring the resulting films onto glass or other flexible substrates.}, number={49}, journal={ACS Applied Materials & Interfaces}, publisher={American Chemical Society (ACS)}, author={Guntupalli, Bhargav and Liang, Pingping and Lee, Jung-Hoon and Yang, Yuehai and Yu, Haixiang and Canoura, Juan and He, Jin and Li, Wenzhi and Weizmann, Yossi and Xiao, Yi}, year={2015}, month={Dec}, pages={27049–27058} }
 @inproceedings{guntupalli_liang_yang_he_li_xiao_2015, title={Ambient filtration method to rapidly prepare highly conductive, paper-based porous gold films}, author={Guntupalli, B. and Liang, P.P. and Yang, Y.H. and He, J. and Li, W.Z. and Xiao, Y.}, year={2015} }
 @inproceedings{wang_yu_ng_roncancio_canoura_xiao_2015, title={Amplified colorimetric detection of cocaine in oral fluid based on exonuclease-assisted aptamer strand recycling}, author={Wang, Z.W. and Yu, H.X. and Ng, B. and Roncancio, D. and Canoura, J. and Xiao, Y.}, year={2015} }
 @misc{xiao_2015, title={Aptamer-based, exonuclease-amplified, paper device for point of collection screening of cocaine and methamphetamine in oral fluid}, author={Xiao, Y.}, year={2015} }
 @inproceedings{xiao_2015, title={Base-pair mismatch detection}, author={Xiao, Y.}, year={2015} }
 @misc{xiao_2015, title={Bringing biosensors to the bedside}, author={Xiao, Y.}, year={2015} }
 @article{zhao_liu_ding_zhao_yu_wang_xu_wang_lou_he_et al._2015, title={Nanoprobe-Enhanced, Split Aptamer-Based Electrochemical Sandwich Assay for Ultrasensitive Detection of Small Molecules}, volume={87}, ISSN={0003-2700 1520-6882}, url={http://dx.doi.org/10.1021/acs.analchem.5b01178}, DOI={10.1021/acs.analchem.5b01178}, abstractNote={It is quite challenging to improve the binding affinity of antismall molecule aptamers. We report that the binding affinity of anticocaine split aptamer pairs improved by up to 66-fold by gold nanoparticles (AuNP)-attached aptamers due to the substantially increased local concentration of aptamers and multiple and simultaneous ligand interactions. The significantly improved binding affinity enables the detection of small molecule targets with unprecedented sensitivity, as demonstrated in nanoprobe-enhanced split aptamer-based electrochemical sandwich assays (NE-SAESA). NE-SAESA replaces the traditional molecular reporter probe with AuNPs conjugated to multiple reporter probes. The increased binding affinity allowed us to use 1,000-fold lower reporter probe concentrations relative to those employed in SAESA. We show that the near-elimination of background in NE-SAESA effectively improves assay sensitivity by ∼1,000-100,000-fold for ATP and cocaine detection, relative to equivalent SAESA. With the ongoing development of new strategies for the selection of aptamers, we anticipate that our sensor platform should offer a generalizable approach for the high-sensitivity detection of diverse targets. More importantly, we believe that NE-SAESA represents a novel strategy to improve the binding affinity between a small molecule and its aptamer and potentially can be extended to other detection platforms.}, number={15}, journal={Analytical Chemistry}, publisher={American Chemical Society (ACS)}, author={Zhao, Tao and Liu, Ran and Ding, Xiaofan and Zhao, Juncai and Yu, Haixiang and Wang, Lei and Xu, Qing and Wang, Xuan and Lou, Xinhui and He, Miao and et al.}, year={2015}, month={Jul}, pages={7712–7719} }
 @article{liang_yu_guntupalli_xiao_2015, title={Paper-Based Device for Rapid Visualization of NADH Based on Dissolution of Gold Nanoparticles}, volume={7}, ISSN={1944-8244 1944-8252}, url={http://dx.doi.org/10.1021/acsami.5b04104}, DOI={10.1021/acsami.5b04104}, abstractNote={We describe a paper-based device that enables rapid and sensitive room-temperature detection of dihydronicotinamide adenine dinucleotide (NADH) via a colorimetric readout and demonstrate its value for monitoring NAD+-driven enzymatic reactions. Our system is based on NADH-mediated inhibition of gold nanoparticle (AuNPs) dissolution in a Au3+-cetyltrimethylammonium bromide (CTAB) solution. We fabricated a device consisting of a mixed cellulose ester paper featuring a wax-encircled, AuNP-coated film atop a cotton absorbent layer sandwiched between two plastic cover layers. In the absence of NADH, the Au3+-CTAB complex dissolves the AuNP layer completely, generating a white color in the test zone. In the presence of NADH, Au3+ is rapidly reduced to Au+, greatly decreasing the dissolution of AuNPs and yielding a red color that becomes stronger at increasing concentrations of NADH. This device exploits capillary force-assisted vertical diffusion, allowing us to apply a 25 μL sample to a surface-confined test zone to achieve a detection limit of 12.5 μM NADH. We used the enzyme glucose dehydrogenase as a model to demonstrate that our paper-based device can monitor NAD+-driven biochemical processes with and without selective dehydrogenase inhibitors by naked-eye observation within 4 min at room temperature in a small sample volume. We believe that our paper-based device could offer a valuable and low-cost analytical tool for monitoring NAD+-associated enzymatic reactions and screening for dehydrogenase inhibitors in a variety of testing contexts.}, number={27}, journal={ACS Applied Materials & Interfaces}, publisher={American Chemical Society (ACS)}, author={Liang, Pingping and Yu, Haixiang and Guntupalli, Bhargav and Xiao, Yi}, year={2015}, month={Jul}, pages={15023–15030} }
 @inproceedings{yu_wang_ng_canoura_xiao_2015, title={Simple, rapid and ultra-sensitive fluorescence detection of cocaine in oral fluid based on cooperative binding split aptamer}, author={Yu, H.X. and Wang, Z.W. and Ng, B. and Canoura, J. and Xiao, Y.}, year={2015} }
 @article{roncancio_yu_xu_wu_liu_debord_lou_xiao_2014, title={A Label-Free Aptamer-Fluorophore Assembly for Rapid and Specific Detection of Cocaine in Biofluids}, volume={86}, ISSN={0003-2700 1520-6882}, url={http://dx.doi.org/10.1021/ac503360n}, DOI={10.1021/ac503360n}, abstractNote={We report a rapid and specific aptamer-based method for one-step cocaine detection with minimal reagent requirements. The feasibility of aptamer-based detection has been demonstrated with sensors that operate via target-induced conformational change mechanisms, but these have generally exhibited limited target sensitivity. We have discovered that the cocaine-binding aptamer MNS-4.1 can also bind the fluorescent molecule 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND) and thereby quench its fluorescence. We subsequently introduced sequence changes into MNS-4.1 to engineer a new cocaine-binding aptamer (38-GC) that exhibits higher affinity to both ligands, with reduced background signal and increased signal gain. Using this aptamer, we have developed a new sensor platform that relies on the cocaine-mediated displacement of ATMND from 38-GC as a result of competitive binding. We demonstrate that our sensor can detect cocaine within seconds at concentrations as low as 200 nM, which is 50-fold lower than existing assays based on target-induced conformational change. More importantly, our assay achieves successful cocaine detection in body fluids, with a limit of detection of 10.4, 18.4, and 36 μM in undiluted saliva, urine, and serum samples, respectively.}, number={22}, journal={Analytical Chemistry}, publisher={American Chemical Society (ACS)}, author={Roncancio, Daniel and Yu, Haixiang and Xu, Xiaowen and Wu, Shuo and Liu, Ran and Debord, Joshua and Lou, Xinhui and Xiao, Yi}, year={2014}, month={Nov}, pages={11100–11106} }
 @inproceedings{roncancio_wu_yu_xu_liu_debord_lou_xiao_2014, title={A label-free aptamer-fluorophore assembly for highly sensitive and specific detection of cocaine}, author={Roncancio, D. and Wu, S. and Yu, H.X. and Xu, X.W. and Liu, R. and Debord, J. and Lou, X.H. and Xiao, Y.}, year={2014} }
 @article{wu_liang_yu_xu_liu_lou_xiao_2014, title={Amplified Single Base-Pair Mismatch Detection via Aggregation of Exonuclease-Sheared Gold Nanoparticles}, volume={86}, ISSN={0003-2700 1520-6882}, url={http://dx.doi.org/10.1021/ac4040373}, DOI={10.1021/ac4040373}, abstractNote={Single nucleotide polymorphism (SNP) detection is important for early diagnosis, clinical prognostics, and disease prevention, and a rapid and sensitive low-cost SNP detection assay would be valuable for resource-limited clinical settings. We present a simple platform that enables sensitive, naked-eye detection of SNPs with minimal reagent and equipment requirements at room temperature within 15 min. SNP detection is performed in a single tube with one set of DNA probe-modified gold nanoparticles (AuNPs), a single exonuclease (Exo III), and the target in question. Exo III’s apurinic endonucleolytic activity differentially processes hybrid duplexes between the AuNP-bound probe and DNA targets that are perfectly matched or contain a single-base mismatch. For perfectly matched targets, Exo III’s exonuclease activity facilitates a process of target recycling that rapidly shears DNA probes from the particles, generating an AuNP aggregation-induced color change, whereas no such change occurs for mismatched targets. This color change is easily observed with as little as 2 nM of target, 100-fold lower than the target concentration required for reliable naked eye observation with unmodified AuNPs in well-optimized reaction conditions. We further demonstrate that this system can effectively discriminate a range of different mismatches.}, number={7}, journal={Analytical Chemistry}, publisher={American Chemical Society (ACS)}, author={Wu, Shuo and Liang, Pingping and Yu, Haixiang and Xu, Xiaowen and Liu, Yuan and Lou, Xinhui and Xiao, Yi}, year={2014}, month={Mar}, pages={3461–3467} }
 @inproceedings{wu_liang_yu_xu_liu_lou_xiao_2014, title={Amplified single base-pair mismatch detection via the aggregation of exonuclease-sheared gold nanoparticles}, author={Wu, S. and Liang, P.P. and Yu, H.X. and Xu, X.W. and Liu, Y. and Lou, X.H. and Xiao, Y.}, year={2014} }
 @misc{xiao_2014, title={Bringing biosensors to the bedside}, author={Xiao, Y.}, year={2014} }
 @inproceedings{xu_guntupalli_yu_xiao_2014, title={Target recycling-based assay for rapid and accurate determination of DNA surface coverage on gold nanoparticles}, author={Xu, X.W. and Guntupalli, B. and Yu, H.X. and Xiao, Y.}, year={2014} }
 @inproceedings{guntupalli_liang_dai_yang_he_li_xiao_2013, title={Ambient filtration method to prepare paper-based electroactive nanoporous gold thin film for detection of dopamine and serotonin}, author={Guntupalli, B. and Liang, P.P. and Dai, J.Y. and Yang, Y.H. and He, J. and Li, W.Z. and Xiao, Y.}, year={2013} }
 @misc{xiao_2013, title={Bringing biosensors to the bedside}, author={Xiao, Y.}, year={2013} }
 @article{deborggraeve_dai_xiao_soh_2013, title={Controlling the function of DNA nanostructures with specific trigger sequences}, volume={49}, ISSN={1359-7345 1364-548X}, url={http://dx.doi.org/10.1039/c2cc36878a}, DOI={10.1039/c2cc36878a}, abstractNote={We report a hybridization-based switching mechanism with single-base specificity that can be readily integrated with functional DNA nanostructures. As an exemplar, we have developed a switchable DNAzyme (SDZ) that only becomes activated in the presence of a perfectly matched trigger sequence and operates effectively at room temperature.}, number={4}, journal={Chem. Commun.}, publisher={Royal Society of Chemistry (RSC)}, author={Deborggraeve, Stijn and Dai, Jian Yuan and Xiao, Yi and Soh, Hyongsok Tom}, year={2013}, pages={397–399} }
 @article{oh_plakos_xiao_eisenstein_soh_2013, title={In Vitro Selection of Shape-Changing DNA Nanostructures Capable of Binding-Induced Cargo Release}, volume={7}, ISSN={1936-0851 1936-086X}, url={http://dx.doi.org/10.1021/nn404079v}, DOI={10.1021/nn404079v}, abstractNote={Many biological systems employ allosteric regulatory mechanisms, which offer a powerful means of directly linking a specific binding event to a wide spectrum of molecular functionalities. There is considerable interest in generating synthetic allosteric regulators that can perform useful molecular functions for applications in diagnostics, imaging and targeted therapies, but generating such molecules through either rational design or directed evolution has proven exceptionally challenging. To address this need, we present an in vitro selection strategy for generating conformation-switching DNA nanostructures that selectively release a small-molecule payload in response to binding of a specific trigger molecule. As an exemplar, we have generated a DNA nanostructure that hybridizes with a separate 'cargo strand' containing an abasic site. This abasic site stably sequesters a fluorescent cargo molecule in an inactive state until the DNA nanostructure encounters an ATP trigger molecule. This ATP trigger causes the nanostructure to release the cargo strand, thereby liberating the fluorescent payload and generating a detectable fluorescent readout. Our DNA nanostructure is highly sensitive, with an EC50 of 30 μM, and highly specific, releasing its payload in response to ATP but not to other chemically similar nucleotide triphosphates. We believe that this selection approach could be generalized to generate synthetic nanostructures capable of selective and controlled release of other small-molecule cargos in response to a variety of triggers, for both research and clinical applications.}, number={11}, journal={ACS Nano}, publisher={American Chemical Society (ACS)}, author={Oh, Seung Soo and Plakos, Kory and Xiao, Yi and Eisenstein, Michael and Soh, H. Tom}, year={2013}, month={Nov}, pages={9675–9683} }
 @inproceedings{xiao_dai_paudyal_guntupalli_liang_pubillones_yang_li_wang_2013, title={Paper-based single-walled carbon nanotube thin film for catalytic detection of nicotinamide adenine dinucleotide in human blood}, author={Xiao, Y. and Dai, J.Y. and Paudyal, J. and Guntupalli, B. and Liang, P.P. and Pubillones, F. and Yang, Y.H. and Li, W.Z. and Wang, X.T}, year={2013} }
 @article{lou_zhao_liu_ma_xiao_2013, title={Self-Assembled DNA Monolayer Buffered Dynamic Ranges of Mercuric Electrochemical Sensor}, volume={85}, ISSN={0003-2700 1520-6882}, url={http://dx.doi.org/10.1021/ac401680c}, DOI={10.1021/ac401680c}, abstractNote={Sensors with wide dynamic ranges (DRs) are typically constructed by utilizing a set of ligands with varied affinities for the same target. We report here a novel buffer self-assembled monolayer (BSAM) strategy, to fabricate sensors with extraordinarily broad DRs using a single recognition ligand. We demonstrated the concept of BSAM by constructing the electrochemical mercuric sensors with different surface probe densities (SPD) on a gold electrode. These sensors are based on the coordination of Hg(2+) with a pair of thymine (T) formed between the two proximate poly(T) oligonucleotides on the electrode surface and Hg(2+) binding induced DNA strand displacement of ferrocene tagged poly(A). There are three types of T-Hg(2+)-T coordination: those formed between (a) two poly(T) strands where none are hybridized with poly(A) strands, thus contributing zero effect on releasing the signaling probe, (b) poly(A)/poly(T) hybridized and nonhybridized poly(T) strands, resulting in the release of a signaling probe from the surface; and (c) two poly(A)/poly(T) hybridized strands, causing the release of two signaling probes from the surface. The DRs from 10 pM to 0.1 mM at varied SPDs were observed, attributing to the tunable Hg(2+) storage capability of the poly(T) SAM formed on the surface due to the coordination mechanism of (a) and (b). The DR was able to be further extended to 1 mM by using the longer poly(T) strands. The ready-to-use sensor exhibited great selectivity against the common interferential metal ions. As demonstrated, the BSAM strategy is a facile way to fabricate sensors with tunable and wide DRs.}, number={15}, journal={Analytical Chemistry}, publisher={American Chemical Society (ACS)}, author={Lou, Xinhui and Zhao, Tao and Liu, Ran and Ma, Jie and Xiao, Yi}, year={2013}, month={Jul}, pages={7574–7580} }
 @inproceedings{xiao_2012, title={Biosensors, biotechnologies and biomaterials}, author={Xiao, Y.}, year={2012} }
 @article{feng_zhao_xiao_wu_ren_qu_2012, title={Electrochemical DNA three-way junction based sensor for distinguishing chiral metallo-supramolecular complexes}, volume={48}, ISSN={1359-7345 1364-548X}, url={http://dx.doi.org/10.1039/c2cc32496b}, DOI={10.1039/c2cc32496b}, abstractNote={Here we report a signal-on three-way junction based-electrochemical DNA sensor for distinguishing chiral metallo-supramolecular complexes with an enantioselective recognition ratio of about 3.5. This platform is easy to fabricate, simple to operate and readily regenerated.}, number={55}, journal={Chemical Communications}, publisher={Royal Society of Chemistry (RSC)}, author={Feng, Lingyan and Zhao, Chuanqi and Xiao, Yi and Wu, Li and Ren, Jinsong and Qu, Xiaogang}, year={2012}, pages={6900} }
 @article{ahmad_xiao_soh_2012, title={Selection is more intelligent than design: improving the affinity of a bivalent ligand through directed evolution}, volume={40}, ISSN={1362-4962 0305-1048}, url={http://dx.doi.org/10.1093/nar/gks899}, DOI={10.1093/nar/gks899}, abstractNote={Multivalent molecular interactions can be exploited to dramatically enhance the performance of an affinity reagent. The enhancement in affinity and specificity achieved with a multivalent construct depends critically on the effectiveness of the scaffold that joins the ligands, as this determines their positions and orientations with respect to the target molecule. Currently, no generalizable design rules exist for construction of an optimal multivalent ligand for targets with known structures, and the design challenge remains an insurmountable obstacle for the large number of proteins whose structures are not known. As an alternative to such design-based strategies, we report here a directed evolution-based method for generating optimal bivalent aptamers. To demonstrate this approach, we fused two thrombin aptamers with a randomized DNA sequence and used a microfluidic in vitro selection strategy to isolate scaffolds with exceptionally high affinities. Within five rounds of selection, we generated a bivalent aptamer that binds thrombin with an apparent dissociation constant (Kd) <10 pM, representing a ∼200-fold improvement in binding affinity over the monomeric aptamers and a ∼15-fold improvement over the best designed bivalent construct. The process described here can be used to produce high-affinity multivalent aptamers and could potentially be adapted to other classes of biomolecules.}, number={22}, journal={Nucleic Acids Research}, publisher={Oxford University Press (OUP)}, author={Ahmad, Kareem M. and Xiao, Yi and Soh, H. Tom}, year={2012}, month={Oct}, pages={11777–11783} }
 @misc{xiao_2011, title={Bringing biosensors to the bedside}, author={Xiao, Y.}, year={2011} }
 @article{ferguson_buchsbaum_wu_hsieh_xiao_sun_soh_2011, title={Genetic Analysis of H1N1 Influenza Virus from Throat Swab Samples in a Microfluidic System for Point-of-Care Diagnostics}, volume={133}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/ja203981w}, DOI={10.1021/ja203981w}, abstractNote={The ability to obtain sequence-specific genetic information about rare target organisms directly from complex biological samples at the point-of-care would transform many areas of biotechnology. Microfluidics technology offers compelling tools for integrating multiple biochemical processes in a single device, but despite significant progress, only limited examples have shown specific, genetic analysis of clinical samples within the context of a fully integrated, portable platform. Herein we present the Magnetic Integrated Microfluidic Electrochemical Detector (MIMED) that integrates sample preparation and electrochemical sensors in a monolithic disposable device to detect RNA-based virus directly from throat swab samples. By combining immunomagnetic target capture, concentration, and purification, reverse-transcriptase polymerase chain reaction (RT-PCR) and single-stranded DNA (ssDNA) generation in the sample preparation chamber, as well as sequence-specific electrochemical DNA detection in the electrochemical cell, we demonstrate the detection of influenza H1N1 in throat swab samples at loads as low as 10 TCID(50), 4 orders of magnitude below the clinical titer for this virus. Given the availability of affinity reagents for a broad range of pathogens, our system offers a general approach for multitarget diagnostics at the point-of-care.}, number={23}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Ferguson, B. Scott and Buchsbaum, Steven F. and Wu, Ting-Ting and Hsieh, Kuangwen and Xiao, Yi and Sun, Ren and Soh, H. Tom}, year={2011}, month={Jun}, pages={9129–9135} }
 @article{oh_ahmad_cho_kim_xiao_soh_2011, title={Improving Aptamer Selection Efficiency through Volume Dilution, Magnetic Concentration, and Continuous Washing in Microfluidic Channels}, volume={83}, ISSN={0003-2700 1520-6882}, url={http://dx.doi.org/10.1021/ac201269f}, DOI={10.1021/ac201269f}, abstractNote={The generation of nucleic acid aptamers with high affinity typically entails a time-consuming, iterative process of binding, separation, and amplification. It would therefore be beneficial to develop an efficient selection strategy that can generate these high-quality aptamers rapidly, economically, and reproducibly. Toward this goal, we have developed a method that efficiently generates DNA aptamers with slow off-rates. This methodology, called VDC-MSELEX, pairs the volume dilution challenge process with microfluidic separation for magnetic bead-assisted aptamer selection. This method offers improved aptamer selection efficiencies through the application of highly stringent selection conditions: it retrieves a small number (<10(6)) of magnetic beads suspended in a large volume (>50 mL) and concentrates them into a microfluidic chamber (8 μL) with minimal loss for continuous washing. We performed three rounds of the VDC-MSELEX using streptavidin (SA) as the target and obtained new DNA aptamer sequences with low nanomolar affinity that specifically bind to the SA proteins.}, number={17}, journal={Analytical Chemistry}, publisher={American Chemical Society (ACS)}, author={Oh, Seung Soo and Ahmad, Kareem M. and Cho, Minseon and Kim, Seon and Xiao, Yi and Soh, H. Tom}, year={2011}, month={Sep}, pages={6883–6889} }
 @article{olmsted_xiao_cho_csordas_sheehan_meiler_soh_bornhop_2011, title={Measurement of Aptamer–Protein Interactions with Back-Scattering Interferometry}, volume={83}, ISSN={0003-2700 1520-6882}, url={http://dx.doi.org/10.1021/ac202823m}, DOI={10.1021/ac202823m}, abstractNote={We report the quantitative measurement of aptamer-protein interactions using backscattering interferometry (BSI) and show that BSI can determine when distinct binding regions are accessed. As a model system, we utilized two DNA aptamers (Tasset and Bock) that bind to distinct sites of a target protein (human α-thrombin). This is the first time BSI has been used to study a multivalent system in free solution wherein more than one ligand binds to a single target. We measured aptamer equilibrum dissociation constants (K(d)) of 3.84 nM (Tasset-thrombin) and 5.96 nM (Bock-thrombin), in close agreement with the literature. Unexpectedly, we observed allosteric effects such that the binding of the first aptamer resulted in a significant change in the binding affinity of the second aptamer. For example, the K(d) of Bock aptamer binding to preformed Tasset-thrombin complexes was 7-fold lower (indicating higher affinity) compared to binding to thrombin alone. Preliminary modeling efforts suggest evidence for allosteric linkage between the two exosites.}, number={23}, journal={Analytical Chemistry}, publisher={American Chemical Society (ACS)}, author={Olmsted, Ian R. and Xiao, Yi and Cho, Minseon and Csordas, Andrew T. and Sheehan, Jonathan H. and Meiler, Jens and Soh, H. Tom and Bornhop, Darryl J.}, year={2011}, month={Dec}, pages={8867–8870} }
 @article{hsieh_white_ferguson_plaxco_xiao_soh_2011, title={Polarity-Switching Electrochemical Sensor for Specific Detection of Single-Nucleotide Mismatches}, volume={50}, ISSN={1433-7851}, url={http://dx.doi.org/10.1002/anie.201103482}, DOI={10.1002/anie.201103482}, abstractNote={Single-nucleotide polymorphisms (SNPs)—genetic variations that involve only a single DNA base-pair—can directly affect transcriptional regulations and protein functions. Thus, SNP genotyping serves as an important diagnostic for genetic diseases and drug responses. To date, methods of detecting such single-nucleotide mismatches can be broadly categorized into enzyme-aided and hybridizationbased approaches. The enzyme-aided approach typically involves a two-step, multi-component assay, in which a singlenucleotide-specific enzymatic reaction, such as primer extension, ligation, or cleavage, is coupled with a downstream detection of reaction products. As such, these methods are inherently complex, and the assay specificity is limited by both the activity of the enzyme and the sensitivity of the detection technique. In contrast, hybridization-based methods utilize DNA probes and various measurement techniques to report the hybridization difference between perfectly matched (PM) and single-nucleotide mismatched (SM) targets in a single-step. However, to resolve the small difference in thermodynamic stability between the two targets, these detection methods generally require complex probe designs and the careful control of hybridization conditions such as buffer composition, washing stringency, and melting temperature. In addition to these complex requirements, both enzymeaided and hybridization-based approaches are susceptible to false-positives because they can only measure the difference in the signal amplitude between PM and SM targets—and signal amplitude measurements are prone to fluctuation in target/probe concentrations, background contaminants, and other experimental perturbations (e.g., enzyme activity, washing stringency or temperature). Thus, for robust detection of single-nucleotide mismatches, there is a need for alternative sensor architectures that are less prone to errors from fluctuations in the signal amplitude. Toward this end, we present a single-step, room-temperature electrochemical sensor that detects single-nucleotide mismatches with a “polarity-switching” response. Our “bipolar” sensor reports a decreased output signal (signal-off) when hybridized with a PM target (Figure 1a, top right) but reports an opposite, increased signal (signal-on) when hybridized with a SM target (Figure 1a, bottom right). The output signal of the sensor is generated by the redox reporter methylene blue (MB), which is covalently attached to an electrodebound DNA probe. The polarity-switching response is achieved by tuning two key parameters—the structural flexibility of the probe and its interaction with the MB tag—that control the electron transfer between the MB tag and the electrode. In this work, we describe the design principles of the bipolar sensor and demonstrate its performance in discriminating SM and PM targets under various conditions. Furthermore, we elucidate the mechanism behind the polarity-switching behavior and quantify the relative contributions of the two parameters that govern the sensor output. The change in the output Faradaic current of our sensor is caused by alterations in the rate of electron transfer to the gold interrogating electrode, which is governed by the equilibrium probability of the DNA-boundMB tag approaching the electrode surface. In our sensor design, we exploited the structural flexibility of the DNA probe and the interaction between MB and DNA (e.g., intercalation and groove binding) to achieve mismatch detection through polarity switching. Regarding the probe flexibility parameter, the higher flexibility of single-stranded DNA (ssDNA) relative to rigid double-stranded DNA (dsDNA) increases the MB electron transfer rate and yields higher Faradaic currents. In parallel, the interaction between the MB tag and dsDNA decreases the electron transfer rate, thus reducing the Faradaic current. This decreased electron transfer is presumably due to the confinement of theMB tag within the DNA duplex, which lowers the probability of the MB approaching the electrode. Of note, this MB-dsDNA interaction depends on the DNA sequence, which therefore needs to be evaluated prior to sensor design. In the present case, we have experimentally determined that the interaction between MB and poly(thymine–adenosine) (T-A) duplexes effectively slows MB electron transfer rate com[*] Dr. Y. Xiao, Prof. H. T. Soh Materials Department, Department of Mechanical Engineering University of California, Santa Barbara Santa Barbara, CA 93106 (USA) E-mail: yixiao@physics.ucsb.edu tsoh@engr.ucsb.edu}, number={47}, journal={Angewandte Chemie International Edition}, publisher={Wiley}, author={Hsieh, Kuangwen and White, Ryan J. and Ferguson, Brian S. and Plaxco, Kevin W. and Xiao, Yi and Soh, H. Tom}, year={2011}, month={Oct}, pages={11176–11180} }
 @article{ahmad_oh_kim_mcclellen_xiao_soh_2011, title={Probing the Limits of Aptamer Affinity with a Microfluidic SELEX Platform}, volume={6}, ISSN={1932-6203}, url={http://dx.doi.org/10.1371/journal.pone.0027051}, DOI={10.1371/journal.pone.0027051}, abstractNote={Nucleic acid-based aptamers offer many potential advantages relative to antibodies and other protein-based affinity reagents, including facile chemical synthesis, reversible folding, improved thermal stability and lower cost. However, their selection requires significant time and resources and selections often fail to yield molecules with affinities sufficient for molecular diagnostics or therapeutics. Toward a selection technique that can efficiently and reproducibly generate high performance aptamers, we have developed a microfluidic selection process (M-SELEX) that can be used to obtain high affinity aptamers against diverse protein targets. Here, we isolated DNA aptamers against three protein targets with different isoelectric points (pI) using a common protocol. After only three rounds of selection, we discovered novel aptamer sequences that bind to platelet derived growth factor B (PDGF-BB; pI = 9.3) and thrombin (pI = 8.3) with respective dissociation constants (Kd) of 0.028 nM and 0.33 nM, which are both superior to previously reported aptamers against these targets. In parallel, we discovered a new aptamer that binds to apolipoprotein E3 (ApoE; pI = 5.3) with a Kd of 3.1 nM. Furthermore, we observe that the net protein charge may exert influence on the affinity of the selected aptamers. To further explore this relationship, we performed selections against PDGF-BB under different pH conditions using the same selection protocol, and report an inverse correlation between protein charge and aptamer Kd.}, number={11}, journal={PLoS ONE}, publisher={Public Library of Science (PLoS)}, author={Ahmad, Kareem M. and Oh, Seung Soo and Kim, Seon and McClellen, Forrest M. and Xiao, Yi and Soh, H. Tom}, editor={Antopolsky, MaximEditor}, year={2011}, month={Nov}, pages={e27051} }
 @article{wang_liu_teesalu_sugahara_kotamrajua_adams_ferguson_gong_oh_csordas_et al._2011, title={Selection of phage-displayed peptides on live adherent cells in microfluidic channels}, volume={108}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.1014753108}, DOI={10.1073/pnas.1014753108}, abstractNote={Affinity reagents that bind to specific molecular targets are an essential tool for both diagnostics and targeted therapeutics. There is a particular need for advanced technologies for the generation of reagents that specifically target cell-surface markers, because transmembrane proteins are notoriously difficult to express in recombinant form. We have previously shown that microfluidics offers many advantages for generating affinity reagents against purified protein targets, and we have now significantly extended this approach to achieve successful in vitro selection of T7 phage-displayed peptides that recognize markers expressed on live, adherent cells within a microfluidic channel. As a model, we have targeted neuropilin-1 (NRP-1), a membrane-bound receptor expressed at the surface of human prostate carcinoma cells that plays central roles in angiogenesis, cell migration, and invasion. We show that, compared to conventional biopanning methods, microfluidic selection enables more efficient discovery of peptides with higher affinity and specificity by providing controllable and reproducible means for applying stringent selection conditions against minimal amounts of target cells without loss. Using our microfluidic system, we isolate peptide sequences with superior binding affinity and specificity relative to the well known NRP-1-binding RPARPAR peptide. As such microfluidic systems can be used with a wide range of biocombinatorial libraries and tissue types, we believe that our method represents an effective approach toward efficient biomarker discovery from patient samples.}, number={17}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Wang, J. and Liu, Y. and Teesalu, T. and Sugahara, K. N. and Kotamrajua, V. R. and Adams, J. D. and Ferguson, B. S. and Gong, Q. and Oh, S. S. and Csordas, A. T. and et al.}, year={2011}, month={Apr}, pages={6909–6914} }
 @article{zuo_xia_patterson_soh_xiao_plaxco_2011, title={Two-Step, PCR-Free Telomerase Detection by Using Exonuclease III-Aided Target Recycling}, volume={12}, ISSN={1439-4227}, url={http://dx.doi.org/10.1002/cbic.201100592}, DOI={10.1002/cbic.201100592}, abstractNote={We report the sensitive detection of telomerase activity by using exonuclease III-aided target recycling to amplify the signal produced by a chimeric LNA-DNA molecular beacon. We demonstrate the specific detection of as few as 30 telomerase-positive breast cancer cells in a single-measurement fluorescence assay that avoids the problematic PCR and gel analysis of the current "gold-standard" assay.}, number={18}, journal={ChemBioChem}, publisher={Wiley}, author={Zuo, Xiaolei and Xia, Fan and Patterson, Adriana and Soh, H. Tom and Xiao, Yi and Plaxco, Kevin W.}, year={2011}, month={Nov}, pages={2745–2747} }
 @article{xia_white_zuo_patterson_xiao_kang_gong_plaxco_heeger_2010, title={An Electrochemical Supersandwich Assay for Sensitive and Selective DNA Detection in Complex Matrices}, volume={132}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/ja104998m}, DOI={10.1021/ja104998m}, abstractNote={In a traditional sandwich assay, a DNA target hybridizes to a single copy of the signal probe. Here we employ a modified signal probe containing a methylene blue (a redox moiety) label and a "sticky end." When a DNA target hybridizes this signal probe, the sticky end remains free to hybridize another target leading to the creation of a supersandwich structure containing multiple labels. This leads to large signal amplification upon monitoring by voltammetry.}, number={41}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Xia, Fan and White, Ryan J. and Zuo, Xiaolei and Patterson, Adriana and Xiao, Yi and Kang, Di and Gong, Xiong and Plaxco, Kevin W. and Heeger, Alan J.}, year={2010}, month={Oct}, pages={14346–14348} }
 @article{xia_zuo_yang_xiao_kang_vallee-belisle_gong_yuen_hsu_heeger_et al._2010, title={Colorimetric detection of DNA, small molecules, proteins, and ions using unmodified gold nanoparticles and conjugated polyelectrolytes}, volume={107}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.1005632107}, DOI={10.1073/pnas.1005632107}, abstractNote={We have demonstrated a novel sensing strategy employing single-stranded probe DNA, unmodified gold nanoparticles, and a positively charged, water-soluble conjugated polyelectrolyte to detect a broad range of targets including nucleic acid (DNA) sequences, proteins, small molecules, and inorganic ions. This nearly “universal” biosensor approach is based on the observation that, while the conjugated polyelectrolyte specifically inhibits the ability of single-stranded DNA to prevent the aggregation of gold-nanoparticles, no such inhibition is observed with double-stranded or otherwise “folded” DNA structures. Colorimetric assays employing this mechanism for the detection of hybridization are sensitive and convenient—picomolar concentrations of target DNA are readily detected with the naked eye, and the sensor works even when challenged with complex sample matrices such as blood serum. Likewise, by employing the binding-induced folding or association of aptamers we have generalized the approach to the specific and convenient detection of proteins, small molecules, and inorganic ions. Finally, this new biosensor approach is quite straightforward and can be completed in minutes without significant equipment or training overhead.}, number={24}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Xia, F. and Zuo, X. and Yang, R. and Xiao, Y. and Kang, D. and Vallee-Belisle, A. and Gong, X. and Yuen, J. D. and Hsu, B. B. Y. and Heeger, A. J. and et al.}, year={2010}, month={Jun}, pages={10837–10841} }
 @article{csordas_gerdon_adams_qian_oh_xiao_soh_2010, title={Detection of Proteins in Serum by Micromagnetic Aptamer PCR (MAP) Technology}, volume={49}, ISSN={1433-7851 1521-3773}, url={http://dx.doi.org/10.1002/anie.200904846}, DOI={10.1002/anie.200904846}, abstractNote={The effective diagnosis and prognosis of many diseases depends on the ability to quantitatively measure protein biomarkers from clinical samples at low concentrations. For example, fluctuations in serum concentrations of cytokines, such as platelet-derived growth factor-BB (PDGF-BB), can serve as indicators of tumor angiogenesis, whereas levels of virus-related proteins such as hemagglutinin can indicate progression of an infection. Accurate detection of diagnostic biomarkers in blood is often challenging because of its complex composition comprising thousands of proteins with concentrations ranging over 12 orders of magnitude. 5] Albumin, for example, constitutes approximately half of the total serum protein (30–50 mgmL ), while many important disease-related biomarkers exist at concentrations as low as 1 pg mL . 5] Enzyme-linked immunosorbent assay (ELISA) is a standard approach to detect protein biomarkers directly from blood. Unfortunately, this assay can suffer from a lengthy development period for specific antibodies, insufficient sensitivity, limited dynamic range, 8, 9] and long assay times involving multiple washing steps, thereby limiting its usefulness and making it impractical to implement at the point of care. Several groups have developed innovative approaches to improve the sensitivity and dynamic range of ELISAs by combining antibody-based molecular recognition with nucleic acid amplification-based detection, such as proximity ligation, immuno-PCR, and bio-barcode detection. However, because interferents in blood can inhibit many amplification reactions, there is a critical need for universal sample preparation systems that allow amplification-based detection of protein biomarkers from complex samples in a monolithic, disposable, and automated format. Herein, we report the micromagnetic aptamer PCR (MAP) detection system, which integrates high-gradient magnetic field sample preparation in a microfluidic device with aptamer-based real-time PCR readout, to achieve highly sensitive and quantitative detection of protein targets directly from complex samples. As a model, we demonstrate the capability to quantitatively detect the cancer biomarker PDGF-BB over a wide dynamic range (62 fm to 1 nm) in a complex background of serum with clearly discernable and reproducible PCR amplification signals. The detection assay starts with the incubation of a serum sample containing PDGF-BB target protein with magnetic beads coated with capture antibody and anti-PDGF PCR aptamers, which incorporate flanking PCR primer sequences (Figure 1A). As with ELISA, the use of dual-affinity reagents significantly increases the specificity of detection. After the incubation step, the sample was loaded into a micromagnetic separation (MMS) chip, in which magnetically labeled antibody–target–aptamer complexes were trapped by the high local magnetic field gradients generated by microfabricated ferromagnetic structures (MFSs) patterned within the microchannel. Meanwhile, nontarget serum proteins, unused reagents, and PCR contaminants were continuously washed out during separation (Figure 1B). After washing the trapped beads, the external magnetic field was removed, which demagnetized the MFSs thereby allowing magnetic target complexes to be eluted with phosphate-buffered saline containing 0.25 mm MgCl2 (PBSM; Figure 1C). The entire separation and purification process (trapping, washing, and bead release) required about 30 min. One microliter of collected eluent was directly subjected to real-time PCR analysis, which yielded a signal proportional to the concentration of target protein in the serum sample (Figure 1D). Note that real-time PCR calibration curves with and without magnetic beads verified that the presence of 1 10 antibodycoated magnetic beads per PCR reaction volume did not [*] S. S. Oh, Dr. Y. Xiao, Prof. H. T. Soh Materials Department, Department of Mechanical Engineering University of California, Santa Barbara Santa Barbara, CA 93106 (USA) E-mail: yixiao@physics.ucsb.edu tsoh@engr.ucsb.edu Dr. A. Csordas Institute for Collaborative Biotechnologies University of California, Santa Barbara (USA) Dr. A. E. Gerdon Department of Chemistry, Emmanuel College, Boston (USA)}, number={2}, journal={Angewandte Chemie International Edition}, publisher={Wiley}, author={Csordas, Andrew and Gerdon, Aren E. and Adams, Jonathan D. and Qian, Jiangrong and Oh, Seung Soo and Xiao, Yi and Soh, H. Tom}, year={2010}, month={Jan}, pages={355–358} }
 @article{xiao_dane_uzawa_csordas_qian_soh_daugherty_lagally_heeger_plaxco_2010, title={Detection of Telomerase Activity in High Concentration of Cell Lysates Using Primer-Modified Gold Nanoparticles}, volume={132}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/ja106513f}, DOI={10.1021/ja106513f}, abstractNote={Although the telomeric repeat amplification protocol (TRAP) has served as a powerful assay for detecting telomerase activity, its use has been significantly limited when performed directly in complex, interferant-laced samples. In this work, we report a modification of the TRAP assay that allows the detection of high-fidelity amplification of telomerase products directly from concentrated cell lysates. Briefly, we covalently attached 12 nm gold nanoparticles (AuNPs) to the telomere strand (TS) primer, which is used as a substrate for telomerase elongation. These TS-modified AuNPs significantly reduce polymerase chain reaction (PCR) artifacts (such as primer dimers) and improve the yield of amplified telomerase products relative to the traditional TRAP assay when amplification is performed in concentrated cell lysates. Specifically, because the TS-modified AuNPs eliminate most of the primer-dimer artifacts normally visible at the same position as the shortest amplified telomerase PCR product apparent on agarose gels, the AuNP-modified TRAP assay exhibits excellent sensitivity. Consequently, we observed a 10-fold increase in sensitivity for cancer cells diluted 1000-fold with somatic cells. It thus appears that the use of AuNP-modified primers significantly improves the sensitivity and specificity of the traditional TRAP assay and may be an effective method by which PCR can be performed directly in concentrated cell lysates.}, number={43}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Xiao, Yi and Dane, Karen Y. and Uzawa, Takanori and Csordas, Andrew and Qian, Jiangrong and Soh, H. Tom and Daugherty, Patrick S. and Lagally, Eric T. and Heeger, Alan J. and Plaxco, Kevin W.}, year={2010}, month={Nov}, pages={15299–15307} }
 @article{hsieh_xiao_tom soh_2010, title={Electrochemical DNA Detection via Exonuclease and Target-Catalyzed Transformation of Surface-Bound Probes}, volume={26}, ISSN={0743-7463 1520-5827}, url={http://dx.doi.org/10.1021/la100227s}, DOI={10.1021/la100227s}, abstractNote={We report a single-step, single-reagent, label-free, isothermal electrochemical DNA sensor based on the phenomenon of target recycling. The sensor exploits strand-specific exonuclease activity to achieve the selective enzymatic digestion of target/probe duplexes. This results in a permanent change in the probe structure that yields an increased faradaic current and liberates the intact target molecule to interact with additional detection probes to achieve further signal amplification. Using this architecture, we achieve an improved detection limit in comparison to hybridization-based sensors without amplification. We also demonstrate a 16-fold signal amplification factor at low target concentrations. Combined with the advantages of electrochemical detection and its ready integration with microelectronics, our approach may represent a promising path toward direct DNA detection at the point of care.}, number={12}, journal={Langmuir}, publisher={American Chemical Society (ACS)}, author={Hsieh, Kuangwen and Xiao, Yi and Tom Soh, H.}, year={2010}, month={Mar}, pages={10392–10396} }
 @article{oh_plakos_lou_xiao_soh_2010, title={In vitro selection of structure-switching, self-reporting aptamers}, volume={107}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.1009172107}, DOI={10.1073/pnas.1009172107}, abstractNote={
            We describe an innovative selection approach to generate self-reporting aptamers (SRAs) capable of converting target-binding events into fluorescence readout without requiring additional modification, optimization, or the use of DNA helper strands. These aptamers contain a DNAzyme moiety that is initially maintained in an inactive conformation. Upon binding to their target, the aptamers undergo a structural switch that activates the DNAzyme, such that the binding event can be reported through significantly enhanced fluorescence produced by a specific stacking interaction between the active-conformation DNAzyme and a small molecule dye,
            N
            -methylmesoporphyrin IX. We demonstrate a purely in vitro selection-based approach for obtaining SRAs that function in both buffer and complex mixtures such as blood serum; after 15 rounds of selection with a structured DNA library, we were able to isolate SRAs that possess low nanomolar affinity and strong specificity for thrombin. Given ongoing progress in the engineering and characterization of functional DNA/RNA molecules, strategies such as ours have the potential to enable rapid, efficient, and economical isolation of nucleic acid molecules with diverse functionalities.
          }, number={32}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Oh, S. S. and Plakos, K. and Lou, X. and Xiao, Y. and Soh, H. T.}, year={2010}, month={Jul}, pages={14053–14058} }
 @article{xia_zuo_yang_white_xiao_kang_gong_lubin_vallée-bélisle_yuen_et al._2010, title={Label-Free, Dual-Analyte Electrochemical Biosensors: A New Class of Molecular-Electronic Logic Gates}, volume={132}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/ja101379k}, DOI={10.1021/ja101379k}, abstractNote={An "XOR" gate built using label-free, dual-analyte electrochemical sensors and the activation of this logic gate via changing concentrations of cocaine and the relevant cDNA as inputs are described.}, number={25}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Xia, Fan and Zuo, Xiaolei and Yang, Renqiang and White, Ryan J. and Xiao, Yi and Kang, Di and Gong, Xiong and Lubin, Arica A. and Vallée-Bélisle, Alexis and Yuen, Jonathan D. and et al.}, year={2010}, month={Jun}, pages={8557–8559} }
 @article{xia_zuo_yang_xiao_kang_vallée-bélisle alexis_gong_heeger_plaxco_2010, title={On the Binding of Cationic, Water-Soluble Conjugated Polymers to DNA: Electrostatic and Hydrophobic Interactions}, volume={132}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/ja908890q}, DOI={10.1021/ja908890q}, abstractNote={Water-soluble, cationic conjugated polymer binds single-stranded DNA with higher affinity than it binds double-stranded or otherwise "folded" DNA. This stronger binding results from the greater hydrophobicity of single-stranded DNA. Upon reducing the strength of the hydrophobic interactions, the electrostatic attraction becomes the important interaction that regulates the binding between the water-soluble conjugated polymer and DNA. The different affinities between the cationic conjugated polymer and various forms of DNA (molecular beacons and its open state; single-stranded DNA and double-stranded DNA and single-stranded DNA and complex DNA folds) can be used to design a variety of biosensors.}, number={4}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Xia, Fan and Zuo, Xiaolei and Yang, Renqiang and Xiao, Yi and Kang, Di and Vallée-Bélisle Alexis and Gong, Xiong and Heeger, Alan J. and Plaxco, Kevin W.}, year={2010}, month={Feb}, pages={1252–1254} }
 @article{cho_xiao_nie_stewart_csordas_oh_thomson_soh_2010, title={Quantitative selection of DNA aptamers through microfluidic selection and high-throughput sequencing}, volume={107}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.1009331107}, DOI={10.1073/pnas.1009331107}, abstractNote={
            We describe the integration of microfluidic selection with high-throughput DNA sequencing technology for rapid and efficient discovery of nucleic acid aptamers. The Quantitative Selection of Aptamers through Sequencing method tracks the copy number and enrichment-fold of more than 10 million individual sequences through multiple selection rounds, enabling the identification of high-affinity aptamers without the need for the pool to fully converge to a small number of sequences. Importantly, this method allows the discrimination of sequences that arise from experimental biases rather than true high-affinity target binding. As a demonstration, we have identified aptamers that specifically bind to PDGF-BB protein with
            K
            
              d
            
             < 3 nM within 3 rounds. Furthermore, we show that the aptamers identified by Quantitative Selection of Aptamers through Sequencing have ∼3–8-fold higher affinity and ∼2–4-fold higher specificity relative to those discovered through conventional cloning methods. Given that many biocombinatorial libraries are encoded with nucleic acids, we extrapolate that our method may be extended to other types of libraries for a range of molecular functions.
          }, number={35}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Cho, M. and Xiao, Y. and Nie, J. and Stewart, R. and Csordas, A. T. and Oh, S. S. and Thomson, J. A. and Soh, H. T.}, year={2010}, month={Aug}, pages={15373–15378} }
 @article{zuo_xia_xiao_plaxco_2010, title={Sensitive and Selective Amplified Fluorescence DNA Detection Based on Exonuclease III-Aided Target Recycling}, volume={132}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/ja909551b}, DOI={10.1021/ja909551b}, abstractNote={A limitation of many traditional approaches to the detection of specific oligonucleotide sequences, such as molecular beacons, is that each target strand hybridizes with (and thus activates) only a single copy of the relevant probe sequence. This 1:1 hybridization ratio limits the gain of most approaches and thus their sensitivity. Here we demonstrate a nuclease-amplified DNA detection scheme in which exonuclease III is used to "recycle" target molecules, thus leading to greatly improved sensitivity relative to, for example, traditional molecular beacons without any significant restriction in the choice of target sequences. The exonuclease-amplified assay can detect target DNA at concentrations as low as 10 pM when performed at 37 degrees C, which represents a significant improvement over the equivalent molecular beacon alone. Moreover, at 4 degrees C we can obtain a detection limit as low as 20 aM, albeit at the cost of a 24 h incubation period. Finally, our assay can be easily interrogated with the naked eye and is thus amenable to deployment in the developing world, where fluorometric detection is more problematic.}, number={6}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Zuo, Xiaolei and Xia, Fan and Xiao, Yi and Plaxco, Kevin W.}, year={2010}, month={Jan}, pages={1816–1818} }
 @book{xiao_lubin_plaxco_2010, title={Signal-on architecture for electronic oligonucleotide-based detectors}, number={US7803542B2}, author={Xiao, Y. and Lubin, A.A. and Plaxco, K.W}, year={2010} }
 @article{xiao_lou_uzawa_plakos_plaxco_soh_2009, title={An Electrochemical Sensor for Single Nucleotide Polymorphism Detection in Serum Based on a Triple-Stem DNA Probe}, volume={131}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/ja905068s}, DOI={10.1021/ja905068s}, abstractNote={We report here an electrochemical approach that offers, for the first time, single-step, room-temperature single nucleotide polymorphism (SNP) detection directly in complex samples (such as blood serum) without the need for target modification, postwashing, or the addition of exogenous reagents. This sensor, which is sensitive, stable, and reusable, is comprised of a single, self-complementary, methylene blue-labeled DNA probe possessing a triple-stem structure. This probe takes advantage of the large thermodynamic changes in enthalpy and entropy that result from major conformational rearrangements that occur upon binding a perfectly matched target, resulting in a large-scale change in the faradaic current. As a result, the discrimination capabilities of this sensor greatly exceed those of earlier single- and double-stem electrochemical sensors and support rapid (minutes), single-step, reagentless, room-temperature detection of single nucleotide substitutions. To elucidate the theoretical basis of the sensor's selectivity, we present a comparative thermodynamic analysis among single-, double-, and triple-stem probes.}, number={42}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Xiao, Yi and Lou, Xinhui and Uzawa, Takanori and Plakos, Kory J. I. and Plaxco, Kevin W. and Soh, H. Tom}, year={2009}, month={Oct}, pages={15311–15316} }
 @article{swensen_xiao_ferguson_lubin_lai_heeger_plaxco_soh_2009, title={Continuous, Real-Time Monitoring of Cocaine in Undiluted Blood Serum via a Microfluidic, Electrochemical Aptamer-Based Sensor}, volume={131}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/ja806531z}, DOI={10.1021/ja806531z}, abstractNote={The development of a biosensor system capable of continuous, real-time measurement of small-molecule analytes directly in complex, unprocessed aqueous samples has been a significant challenge, and successful implementation has been achieved for only a limited number of targets. Toward a general solution to this problem, we report here the Microfluidic Electrochemical Aptamer-based Sensor (MECAS) chip wherein we integrate target-specific DNA aptamers that fold, and thus generate an electrochemical signal, in response to the analyte with a microfluidic detection system. As a model, we demonstrate the continuous, real-time (approximately 1 min time resolution) detection of the small-molecule drug cocaine at near physiological, low micromolar concentrations directly in undiluted, otherwise unmodified blood serum. We believe our approach of integrating folding-based electrochemical sensors with miniaturized detection systems may lay the groundwork for the real-time, point-of-care detection of a wide variety of molecular targets.}, number={12}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Swensen, James S. and Xiao, Yi and Ferguson, Brian S. and Lubin, Arica A. and Lai, Rebecca Y. and Heeger, Alan J. and Plaxco, Kevin W. and Soh, H. Tom.}, year={2009}, month={Mar}, pages={4262–4266} }
 @inbook{xiao_plaxco_2009, title={Electrochemical Approaches to Aptamer-Based Sensing}, ISBN={9780387737102 9780387737119}, url={http://dx.doi.org/10.1007/978-0-387-73711-9_7}, DOI={10.1007/978-0-387-73711-9_7}, booktitle={Functional Nucleic Acids for Analytical Applications}, publisher={Springer New York}, author={Xiao, Yi and Plaxco, Kevin W.}, year={2009}, pages={179–197} }
 @article{xiao_plakos_lou_white_qian_plaxco_soh_2009, title={Fluorescence Detection of Single-Nucleotide Polymorphisms with a Single, Self-Complementary, Triple-Stem DNA Probe}, volume={48}, ISSN={1433-7851 1521-3773}, url={http://dx.doi.org/10.1002/anie.200900369}, DOI={10.1002/anie.200900369}, abstractNote={Abstract}, number={24}, journal={Angewandte Chemie International Edition}, publisher={Wiley}, author={Xiao, Yi and Plakos, Kory J. I. and Lou, Xinhui and White, Ryan J. and Qian, Jiangrong and Plaxco, Kevin W. and Soh, H. Tom}, year={2009}, month={Jun}, pages={4354–4358} }
 @article{qian_lou_zhang_xiao_soh_2009, title={Generation of Highly Specific Aptamers via Micromagnetic Selection}, volume={81}, ISSN={0003-2700 1520-6882}, url={http://dx.doi.org/10.1021/ac900759k}, DOI={10.1021/ac900759k}, abstractNote={Aptamers are nucleic acid-based reagents that bind to target molecules with high affinity and specificity. However, methods for generating aptamers from random combinatorial libraries (e.g., systematic evolution of ligands by exponential enrichment (SELEX)) are often labor-intensive and time-consuming. Recent studies suggest that microfluidic SELEX (M-SELEX) technology can accelerate aptamer isolation by enabling highly stringent selection conditions through the use of very small amounts of target molecules. We present here an alternative M-SELEX method, which employs a disposable microfluidic chip to rapidly generate aptamers with high affinity and specificity. The micromagnetic separation (MMS) chip integrates microfabricated ferromagnetic structures to reproducibly generate large magnetic field gradients within its microchannel that efficiently trap magnetic bead-bound aptamers. Operation of the MMS device is facile and robust and demonstrates high recovery of the beads (99.5%), such that picomolar amounts of target molecule can be used. Importantly, the device demonstrates exceptional separation efficiency in removing weakly bound and unbound ssDNA to rapidly enrich target-specific aptamers. As a model, we demonstrate here the generation of DNA aptamers against streptavidin in three rounds of positive selection. We further enhanced the specificity of the selected aptamers via a round of negative selection in the same device against bovine serum albumin (BSA). The resulting aptamers displayed dissociation constants ranging from 25 to 65 nM for streptavidin and negligible affinity for BSA. Since a wide spectrum of molecular targets can be readily conjugated to magnetic beads, MMS-based SELEX provides a general platform for rapid generation of specific aptamers.}, number={13}, journal={Analytical Chemistry}, publisher={American Chemical Society (ACS)}, author={Qian, Jiangrong and Lou, Xinhui and Zhang, Yanting and Xiao, Yi and Soh, H. Tom}, year={2009}, month={May}, pages={5490–5495} }
 @article{zuo_xiao_plaxco_2009, title={High Specificity, Electrochemical Sandwich Assays Based on Single Aptamer Sequences and Suitable for the Direct Detection of Small-Molecule Targets in Blood and Other Complex Matrices}, volume={131}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/ja901315w}, DOI={10.1021/ja901315w}, abstractNote={We herein demonstrate a sandwich assay based on single aptamer sequences is suitable for the direct detection of small molecule targets in blood serum and other complex matrices. By splitting an aptamer into two pieces, we convert a single affinity reagent into a two-component system in which the presence of the target drives formation of a complex comprised of the target and the two halves of the aptamer. To demonstrate the utility of this approach we have used single anticocaine and anti-ATP aptamers to fabricate electrochemical sensors directed against the representative small molecules cocaine and ATP. Both targets are detected at low micromolar concentrations, in seconds, and in a convenient, general, readily reusable, electrochemical format. Moreover, both sensors are selective enough to deploy directly in blood, crude cellular lysates and other complex sample matrices.}, number={20}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Zuo, Xiaolei and Xiao, Yi and Plaxco, Kevin W.}, year={2009}, month={May}, pages={6944–6945} }
 @article{lou_xiao_wang_mao_zhao_2009, title={Label-Free Colorimetric Screening of Nuclease Activity and Substrates by Using Unmodified Gold Nanoparticles}, volume={10}, ISSN={1439-4227 1439-7633}, url={http://dx.doi.org/10.1002/cbic.200900355}, DOI={10.1002/cbic.200900355}, abstractNote={Nucleic acids are the molecules responsible for the conveyance of genetic information and have paramount importance in biological systems. Nucleases are specific enzymes capable of cleaving the phosphodiester bonds of nucleic acids between nucleotides either from the ends or internally. Nucleases play key roles in many biological processes such as DNA replication, repair, recombination, maturation, and degradation. These enzymes are also widely used in various biosensing techniques to achieve exceptional detection selectivity and sensitivity. For example, RNase H, which specifically digests the RNA strands in RNA–DNA heteroduplexes and does not affect single-stranded (ss) DNA, double-stranded (ds) DNA, ssRNA, and dsRNA, was utilized in a novel surface enzymatic amplification RNA microarray to enhance the sensitivity of surface plasmon resonance (SPR) for DNA oligonucleotides down to 1 fm. Nuclease activity varies greatly between substrates and many biosensing techniques are based on the high selectivity of these enzymes. Detection of nuclease activity includes viscometry, radioactive labeling, 12] gel electrophoresis, and fluorescence-based approaches. 14] A majority of these techniques are labor intensive and time consuming. Of the methods outlined above, only the fluorescence-based assay is suitable for high-throughput screening. However, this approach is very expensive due to the requirement of fluorophore-modified substrates. Therefore, it is desirable to screen nuclease activity and substrates with label-free substrates and without the need to use sophisticated instruments. Recently, Chad Mirkin’s group has used DNA-modified gold nanoparticles (AuNPs) as signal reporters to directly screen for endonuclease activity and inhibition by unaided visual inspection. In their method, endonucleases cleaved dsDNA in the absence of inhibitors and allowed the crosslinked AuNPs to separate into single AuNPs. This cleavage process was indicated by the instant color change from blue to red. In contrast, the crosslinked network of AuNPs remained aggregated and blue in the presence of inhibitors. Their method exploits the selectivity of nucleases for substrates and takes advantage of the unique optical properties of AuNPs, namely, the ability to obtain results without a detector. However, this method requires cumbersome preparation of modified AuNPs and the formation of crosslinked AuNPs, and thus is not suitable for the high-throughput screening of substrates. In addition, their method is limited to nucleases that have dsDNA molecules as their substrates, and thus is not suitable for ssDNA digest nucleases such as S1 nuclease. We describe here a simple, straightforward and general colorimetric method to measure nuclease activity and screen substrate using unmodified AuNPs (Scheme 1). By taking advant-}, number={12}, journal={ChemBioChem}, publisher={Wiley}, author={Lou, Xinhui and Xiao, Yi and Wang, Yi and Mao, Hongju and Zhao, Jianlong}, year={2009}, month={Aug}, pages={1973–1977} }
 @article{lou_qian_xiao_viel_gerdon_lagally_atzberger_tarasow_heeger_soh_2009, title={Micromagnetic selection of aptamers in microfluidic channels}, volume={106}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.0813135106}, DOI={10.1073/pnas.0813135106}, abstractNote={
            Aptamers are nucleic acid molecules that have been selected in vitro to bind to their molecular targets with high affinity and specificity. Typically, the systematic evolution of ligands by exponential enrichment (SELEX) process is used for the isolation of specific, high-affinity aptamers. SELEX, however, is an iterative process requiring multiple rounds of selection and amplification that demand significant time and labor. Here, we describe an aptamer discovery system that is rapid, highly efficient, automatable, and applicable to a wide range of targets, based on the integration of magnetic bead-based SELEX process with microfluidics technology. Our microfluidic SELEX (M-SELEX) method exploits a number of unique phenomena that occur at the microscale and implements a design that enables it to manipulate small numbers of beads precisely and isolate high-affinity aptamers rapidly. As a model to demonstrate the efficiency of the M-SELEX process, we describe here the isolation of DNA aptamers that tightly bind to the light chain of recombinant
            Botulinum
            neurotoxin type A (with low-nanomolar dissociation constant) after a single round of selection.
          }, number={9}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Lou, X. and Qian, J. and Xiao, Y. and Viel, L. and Gerdon, A. E. and Lagally, E. T. and Atzberger, P. and Tarasow, T. M. and Heeger, A. J. and Soh, H. T.}, year={2009}, month={Feb}, pages={2989–2994} }
 @article{xiao_uzawa_white_demartini_plaxco_2009, title={On the Signaling of Electrochemical Aptamer-Based Sensors: Collision- and Folding-Based Mechanisms}, volume={21}, ISSN={1040-0397 1521-4109}, url={http://dx.doi.org/10.1002/elan.200804564}, DOI={10.1002/elan.200804564}, abstractNote={Abstract}, number={11}, journal={Electroanalysis}, publisher={Wiley}, author={Xiao, Yi and Uzawa, Takanori and White, Ryan J. and DeMartini, Daniel and Plaxco, Kevin W.}, year={2009}, month={Jun}, pages={1267–1271} }
 @article{peng_wang_xiao_feng_zhao_ren_qu_2009, title={i-Motif Quadruplex DNA-Based Biosensor for Distinguishing Single- and Multiwalled Carbon Nanotubes}, volume={131}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/ja9051763}, DOI={10.1021/ja9051763}, abstractNote={The increasing worldwide demand for carbon nanotubes (CNTs) and increasing concern regarding how to safely develop and use CNTs are requiring a low-cost, simple, and highly sensitive CNT detection assay for toxicological evaluation and environmental monitoring. However, this goal is still far from being achieved. All the current CNT detection techniques are not applicable for automation and field analysis because they are dependent on highly expensive special instruments and complicated sample preparation. On the basis of the capability of single-walled carbon nanotubes (SWNTs) to specifically induce human telomeric i-motif formation, we design an electrochemical DNA (E-DNA) sensor that can distinguish single- and multiwalled carbon nanotubes both in buffer and in cell extracts. The E-DNA sensor can selectively detect SWNTs with a direct detection limit of 0.2 ppm and has been demonstrated in cancer cell extracts. To the best of our knowledge, this is the first demonstration of a biosensing technique that can distinguish different types of nanotubes. Our work will provide new insights into how to design a biosensor for detection of carbon nanotubes.}, number={38}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Peng, Yinghua and Wang, Xiaohui and Xiao, Yi and Feng, Lingyan and Zhao, Chao and Ren, Jinsong and Qu, Xiaogang}, year={2009}, month={Sep}, pages={13813–13818} }
 @article{white_phares_lubin_xiao_plaxco_2008, title={Optimization of Electrochemical Aptamer-Based Sensors via Optimization of Probe Packing Density and Surface Chemistry}, volume={24}, ISSN={0743-7463 1520-5827}, url={http://dx.doi.org/10.1021/la800801v}, DOI={10.1021/la800801v}, abstractNote={Electrochemical, aptamer-based (E-AB) sensors, which are comprised of an electrode modified with surface immobilized, redox-tagged DNA aptamers, have emerged as a promising new biosensor platform. In order to further improve this technology we have systematically studied the effects of probe (aptamer) packing density, the AC frequency used to interrogate the sensor, and the nature of the self-assembled monolayer (SAM) used to passivate the electrode on the performance of representative E-AB sensors directed against the small molecule cocaine and the protein thrombin. We find that, by controlling the concentration of aptamer employed during sensor fabrication, we can control the density of probe DNA molecules on the electrode surface over an order of magnitude range. Over this range, the gain of the cocaine sensor varies from 60% to 200%, with maximum gain observed near the lowest probe densities. In contrast, over a similar range, the signal change of the thrombin sensor varies from 16% to 42% and optimal signaling is observed at intermediate densities. Above cut-offs at low hertz frequencies, neither sensor displays any significant dependence on the frequency of the alternating potential employed in their interrogation. Finally, we find that E-AB signal gain is sensitive to the nature of the alkanethiol SAM employed to passivate the interrogating electrode; while thinner SAMs lead to higher absolute sensor currents, reducing the length of the SAM from 6-carbons to 2-carbons reduces the observed signal gain of our cocaine sensor 10-fold. We demonstrate that fabrication and operational parameters can be varied to achieve optimal sensor performance and that these can serve as a basic outline for future sensor fabrication.}, number={18}, journal={Langmuir}, publisher={American Chemical Society (ACS)}, author={White, Ryan J. and Phares, Noelle and Lubin, Arica A. and Xiao, Yi and Plaxco, Kevin W.}, year={2008}, month={Aug}, pages={10513–10518} }
 @article{cash_heeger_plaxco_xiao_2008, title={Optimization of a Reusable, DNA Pseudoknot-Based Electrochemical Sensor for Sequence-Specific DNA Detection in Blood Serum}, volume={81}, ISSN={0003-2700 1520-6882}, url={http://dx.doi.org/10.1021/ac802011d}, DOI={10.1021/ac802011d}, abstractNote={We describe in detail a new electrochemical DNA (E-DNA) sensing platform based on target-induced conformation changes in an electrode-bound DNA pseudoknot. The pseudoknot, a DNA structure containing two stem-loops in which the first stem's loop forms part of the second stem, is modified with a methylene blue redox tag at its 3' terminus and covalently attached to a gold electrode via the 5' terminus. In the absence of a target, the structure of the pseudoknot probe minimizes collisions between the redox tag and the electrode, thus reducing faradaic current. Target binding disrupts the pseudoknot structure, liberating a flexible, single-stranded element that can strike the electrode and efficiently transfer electrons. In this article we report further characterization and optimization of this new E-DNA architecture. We find that optimal signaling is obtained at an intermediate probe density ( approximately 1.8 x 10(13) molecules/cm(2) apparent density), which presumably represents a balance between steric and electrostatic blocking at high probe densities and increased background currents arising from transfer from the pseudoknot probe at lower densities. We also find that optimal 3' stem length, which appears to be 7 base pairs, represents a balance between pseudoknot structural stability and target affinity. Finally, a 3' loop comprised of poly(A) exhibits better mismatch discrimination than the equivalent poly(T) loop, but at the cost of decreased gain. Optimization over this parameter space significantly improves the signaling of the pseudoknot-based E-DNA architecture, leading to the ability to sensitively and specifically detect DNA targets even when challenged in complex, multicomponent samples such as blood serum.}, number={2}, journal={Analytical Chemistry}, publisher={American Chemical Society (ACS)}, author={Cash, Kevin J. and Heeger, Alan J. and Plaxco, Kevin W. and Xiao, Yi}, year={2008}, month={Dec}, pages={656–661} }
 @book{willner_xiao_pavlov_niazov_2007, title={Catalytic polynucleotide and its use for determination of analytes}, number={20070254282}, author={Willner, I. and Xiao, Y. and Pavlov, V. and Niazov, T.}, year={2007}, month={Nov} }
 @article{xiao_rowe_plaxco_2007, title={Electrochemical Detection of Parts-Per-Billion Lead via an Electrode-Bound DNAzyme Assembly}, volume={129}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/ja067278x}, DOI={10.1021/ja067278x}, abstractNote={An electrochemical lead sensor is based on lead-dependent changes in the conformational state of a surface-immobilized, methylene-blue modified DNAzyme assembly.}, number={2}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Xiao, Yi and Rowe, Aaron A. and Plaxco, Kevin W.}, year={2007}, month={Jan}, pages={262–263} }
 @article{xiao_qu_plaxco_heeger_2007, title={Label-Free Electrochemical Detection of DNA in Blood Serum via Target-Induced Resolution of an Electrode-Bound DNA Pseudoknot}, volume={129}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/ja074218y}, DOI={10.1021/ja074218y}, abstractNote={We have demonstrated a fully covalent, signal-on E-DNA architecture based on the target-induced resolution of a DNA pseudokont. In the absence of target, the electrode-bound DNA probe adopts a pseudoknot conformation that segregates an attached methylene blue (MB) from the electrode. Upon target binding, the pseudoknot is resolved, leading to the formation of a single-stranded DNA element that supports electron transfer from the methylene blue to the electrode.}, number={39}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Xiao, Yi and Qu, Xiaogang and Plaxco, Kevin W. and Heeger, Alan J.}, year={2007}, month={Sep}, pages={11896–11897} }
 @article{xiao_lai_plaxco_2007, title={Preparation of electrode-immobilized, redox-modified oligonucleotides for electrochemical DNA and aptamer-based sensing}, volume={2}, ISSN={1754-2189 1750-2799}, url={http://dx.doi.org/10.1038/nprot.2007.413}, DOI={10.1038/nprot.2007.413}, abstractNote={Recent years have seen the development of a number of reagentless, electrochemical sensors based on the target-induced folding or unfolding of electrode-bound oligonucleotides, with examples reported to date, including sensors for the detection of specific nucleic acids, proteins, small molecules and inorganic ions. These devices, which are often termed electrochemical DNA (E-DNA) and E-AB (electrochemical, aptamer-based) sensors, are comprised of an oligonucleotide probe modified with a redox reporter (in this protocol methylene blue) at one terminus and attached to a gold electrode via a thiol-gold bond at the other. Binding of an analyte to the oligonucleotide probe changes its structure and dynamics, which, in turn, influences the efficiency of electron transfer to the interrogating electrode. This class of sensors perform well even when challenged directly with blood serum, soil and other complex, multicomponent sample matrices. This protocol describes the fabrication of E-DNA and E-AB sensors. The protocol can be completed in 12 h.}, number={11}, journal={Nature Protocols}, publisher={Springer Science and Business Media LLC}, author={Xiao, Yi and Lai, Rebecca Y and Plaxco, Kevin W}, year={2007}, month={Nov}, pages={2875–2880} }
 @inbook{xiao_pavlov_niazov_dishon_kotler_willner_2006, place={Dordrecht}, series={NATO Security through Science Series (Series B: Physics and Biophysics - Programme for Security through Science (STS))}, title={Catalytic Beacons for the Detection of DNA and Telomerase Activity}, ISBN={1402033869}, url={http://dx.doi.org/10.1007/1-4020-3384-2_15}, DOI={10.1007/1-4020-3384-2_15}, abstractNote={Different new methods for the amplified and specific electronic or photonic detection of DNA will be addressed. Specific methods that will be described include: The amplified electrochemical detection of DNA by means of replication of the analyzed DNA on electrode supports, the incorporation of ferrocene redox-active groups into the replica, and the secondary activation of bioelectrocatalytic cascades. The use of rotating magnetic particles as sensing matrices for DNA using electrogenerated chemiluminescence as an optical readout signal. The magneto-mechanical detection of DNA on cantilever sensors exposed to an external magnetic field. In these systems, magnetic particles are attached to the target-replicated DNA associated with the lever, and the lever is mechanically deflected when subjected to an external magnet. The use of semiconductor quantum dots (QDs) for the photonic detection of DNA will be described by using nucleic acid-functionalized QDs that hybridize with the analyte DNA (CdSe-ZnS or CdTe quantum dots). The replication of the analyzed DNA while incorporating an appropriate dye allows, in the next step, to confirm the hybridization of the analyte DNA by fluorescence resonance energy transfer (FRET). The parallel detection of different DNA/RNA will be described by using QDs of different sizes and compositions. Catalytic nucleic acids that bind hemin and mimic peroxidase activity were employed for the amplified detection of DNA (or proteins). The DNAzyme leads to the generation of chemiluminescence in the presence of luminol/H2O2. Different DNAzyme-based catalytic schemes for the detection of DNA will be described, including the use of catalytic beacons and Au-nanoparticle/DNAzyme hybrid systems.}, booktitle={Defense against Bioterror}, publisher={Springer-Verlag}, author={Xiao, Y. and Pavlov, V. and Niazov, T. and Dishon, A. and Kotler, M. and Willner, I.}, editor={Morrison, D. and Milanovich, F. and Ivnitski, D. and Austin, T.R.Editors}, year={2006}, month={Jul}, pages={199–205}, collection={NATO Security through Science Series (Series B: Physics and Biophysics - Programme for Security through Science (STS))} }
 @book{willner_pavlov_zayatz_niazov_shlyahovsky_xiao_buron_2006, title={Novel sensors with nanoparticle probe}, number={WO2006008742}, author={Willner, I. and Pavlov, V. and Zayatz, M. and Niazov, T. and Shlyahovsky, B. and Xiao, Y. and Buron, R.}, year={2006} }
 @article{xiao_lubin_baker_plaxco_heeger_2006, title={Single-step electronic detection of femtomolar DNA by target-induced strand displacement in an electrode-bound duplex}, volume={103}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.0607693103}, DOI={10.1073/pnas.0607693103}, abstractNote={We report a signal-on, electronic DNA (E-DNA) sensor that is label-free and achieves a subpicomolar detection limit. The sensor, which is based on a target-induced strand displacement mechanism, is composed of a “capture probe” attached by its 5′ terminus to a gold electrode and a 5′ methylene blue-modified “signaling probe” that is complementary at both its 3′ and 5′ termini to the capture probe. In the absence of target, hybridization between the capture and signaling probes minimizes contact between the methylene blue and electrode surface, limiting the observed redox current. Target hybridization displaces the 5′ end of the signaling probe, generating a short, flexible single-stranded DNA element and producing up to a 7-fold increase in redox current. The observed signal gain is sufficient to achieve a demonstrated (not extrapolated) detection limit of 400 fM, which is among the best reported for single-step electronic DNA detection. Moreover, because sensor fabrication is straightforward, the approach appears to provide a ready alternative to the more cumbersome femtomolar electrochemical assays described to date.}, number={45}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Xiao, Y. and Lubin, A. A. and Baker, B. R. and Plaxco, K. W. and Heeger, A. J.}, year={2006}, month={Oct}, pages={16677–16680} }
 @article{xiao_piorek_plaxco_heeger_2005, title={A Reagentless Signal-On Architecture for Electronic, Aptamer-Based Sensors via Target-Induced Strand Displacement}, volume={127}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/ja056555h}, DOI={10.1021/ja056555h}, abstractNote={Thrombin binding stabilizes the alternative G-quadruplex conformation of the aptamer, liberating the methylene blue (MB)-tagged oligonucleotide to produce a flexible, single-stranded DNA element. This allows the MB tag to collide with the gold electrode surface, producing a readily detectable Faradaic current at thrombin concentrations as low as approximately 3 nM.}, number={51}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Xiao, Yi and Piorek, Brian D. and Plaxco, Kevin W. and Heeger, Alan J.}, year={2005}, month={Dec}, pages={17990–17991} }
 @article{xiao_pavlov_shlyahovsky_willner_2005, title={An OsII-Bisbipyridine-4-Picolinic Acid Complex Mediates the Biocatalytic Growth of Au Nanoparticles: Optical Detection of Glucose and Acetylcholine Esterase Inhibition}, volume={11}, ISSN={0947-6539 1521-3765}, url={http://dx.doi.org/10.1002/chem.200400988}, DOI={10.1002/chem.200400988}, abstractNote={Abstract}, number={9}, journal={Chemistry - A European Journal}, publisher={Wiley}, author={Xiao, Yi and Pavlov, Valeri and Shlyahovsky, Bella and Willner, Itamar}, year={2005}, month={Apr}, pages={2698–2704} }
 @article{pavlov_xiao_willner_2005, title={Inhibition of the Acetycholine Esterase-Stimulated Growth of Au Nanoparticles:  Nanotechnology-Based Sensing of Nerve Gases}, volume={5}, ISSN={1530-6984 1530-6992}, url={http://dx.doi.org/10.1021/nl050054c}, DOI={10.1021/nl050054c}, abstractNote={The acetylcholine esterase, AChE, mediated hydrolysis of acetylthiocholine (1) yields a reducing agent thiocholine (2) that stimulates the catalytic enlargement of Au NP seeds in the presence of AuCl(4)(-). The reductive enlargement of the Au NPs is controlled by the concentration of the substrate (1) and by the activity of the enzyme. The catalytic growth of the Au NPs is inhibited by 1,5-bis(4-allyldimethylammoniumphenyl)pentane-3-one dibromide (3) or by diethyl p-nitrophenyl phosphate (paraoxon; 4), thus enabling a colorimetric test for AChE inhibitors. The colorimetric assay was also developed on glass supports.}, number={4}, journal={Nano Letters}, publisher={American Chemical Society (ACS)}, author={Pavlov, Valeri and Xiao, Yi and Willner, Itamar}, year={2005}, month={Mar}, pages={649–653} }
 @article{xiao_lubin_heeger_plaxco_2005, title={Label-Free Electronic Detection of Thrombin in Blood Serum by Using an Aptamer-Based Sensor}, volume={44}, ISSN={1433-7851 1521-3773}, url={http://dx.doi.org/10.1002/anie.200500989}, DOI={10.1002/anie.200500989}, abstractNote={Blue, gold, and DNA: A methylene blue (MB) tagged, thrombin-binding DNA aptamer immobilized on a gold surface undergoes a large conformational change upon target binding (see schematic representation; eT: electron transfer). This folding produces a large, readily measurable change in redox current and allows the electrochemical detection of thrombin in blood serum.}, number={34}, journal={Angewandte Chemie International Edition}, publisher={Wiley}, author={Xiao, Yi and Lubin, Arica A. and Heeger, Alan J. and Plaxco, Kevin W.}, year={2005}, month={Aug}, pages={5456–5459} }
 @article{shlyahovsky_katz_xiao_pavlov_willner_2005, title={Optical and Electrochemical Detection of NADH and of NAD+-Dependent Biocatalyzed Processes by the Catalytic Deposition of Copper on Gold Nanoparticles}, volume={1}, ISSN={1613-6810 1613-6829}, url={http://dx.doi.org/10.1002/smll.200400057}, DOI={10.1002/smll.200400057}, abstractNote={Au/Cu core/shell nanoparticle assemblies on modified quartz or ITO slides are achieved through a biochemical reduction process, in which 1,4-dihydro-β-nicotinamide adenine dinucleotide (NADH) performs the Au-catalyzed reduction of Cu2+ ions (see scheme). Such assemblies are ideal for the optical or electrochemical detection of NADH and NAD+-dependent biocatalytic processes, such as the analysis of ethanol.}, number={2}, journal={Small}, publisher={Wiley}, author={Shlyahovsky, Bella and Katz, Eugenii and Xiao, Yi and Pavlov, Valeri and Willner, Itamar}, year={2005}, month={Feb}, pages={213–216} }
 @article{xiao_shlyahovsky_popov_pavlov_willner_2005, title={Shape and color of Au nanoparticles follow biocatalytic processes}, volume={21}, DOI={10.1021/la050308+}, abstractNote={The NAD(P)H-mediated growth of Au nanoparticles (NPs) in the presence of ascorbic acid, AuCl4-, and cetyltrimethylammonium bromide leads to the formation of shaped NP structures consisting of dipods, tripods, and tetrapods. The shaped particles exhibit a red-shifted plasmon absorbance at lambda = 680 nm, consistent with the existence of a longitudinal plasmon exciton. High-resolution transmission electron microscopy analysis of the tripod and tetrapod structures reveals directional growth along the <211> and <010> directions, respectively. The shaped Au NPs could be generated by a biocatalytic process using alcohol dehydrogenase, NAD+, and ethanol, and the resulting blue color provides a colorimetric test for ethanol.}, number={13}, journal={Langmuir}, author={Xiao, Y. and Shlyahovsky, B. and Popov, I. and Pavlov, V. and Willner, I}, year={2005}, pages={5659–5662} }
 @article{pavlov_xiao_gill_dishon_kotler_willner_2004, title={Amplified Chemiluminescence Surface Detection of DNA and Telomerase Activity Using Catalytic Nucleic Acid Labels}, volume={76}, ISSN={0003-2700 1520-6882}, url={http://dx.doi.org/10.1021/ac035219l}, DOI={10.1021/ac035219l}, abstractNote={A G-rich nucleic acid sequence binds hemin and yields a biocatalytic complex (DNAzyme) of peroxidase activity, namely, the biocatalyzed generation of chemiluminescence in the presence of H(2)O(2) and luminol. The DNAzyme is used as a label for the amplified detection of DNA, or for the analysis of telomerase activity in cancer cells, using chemiluminescence as an output signal. In one configuration, the analyzed DNA is hybridized with a primer nucleic acid that is associated with a Au surface, and the DNAzyme label is hybridized with the surface-confined analyte DNA. The DNA is analyzed with a detection limit of approximately 1 x 10(-)(9) M. In the second system, telomerase from HeLa cancer cells induces telomerization of a primer associated with a Au surface and the complementary DNAzyme units are hybridized with the telomere to yield the chemiluminescence. The detection limit of the system corresponds to 1000 HeLa cells in the analyzed sample.}, number={7}, journal={Analytical Chemistry}, publisher={American Chemical Society (ACS)}, author={Pavlov, Valeri and Xiao, Yi and Gill, Ron and Dishon, Arnon and Kotler, Moshe and Willner, Itamar}, year={2004}, month={Apr}, pages={2152–2156} }
 @article{pavlov_xiao_shlyahovsky_willner_2004, title={Aptamer-Functionalized Au Nanoparticles for the Amplified Optical Detection of Thrombin}, volume={126}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/ja046970u}, DOI={10.1021/ja046970u}, abstractNote={The catalytic enlargement of aptamer-functionalized Au nanoparticles amplifies the optical detection of aptamer-thrombin complexes in solution and on surfaces.}, number={38}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Pavlov, Valeri and Xiao, Yi and Shlyahovsky, Bella and Willner, Itamar}, year={2004}, month={Sep}, pages={11768–11769} }
 @article{xiao_pavlov_niazov_dishon_kotler_willner_2004, title={Catalytic Beacons for the Detection of DNA and Telomerase Activity}, volume={126}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/ja031875r}, DOI={10.1021/ja031875r}, abstractNote={DNA and telomerase activity are detected by a DNAzyme generated upon hybridization and opening of a functional catalytic beacon.}, number={24}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Xiao, Yi and Pavlov, Valeri and Niazov, Tamara and Dishon, Arnon and Kotler, Moshe and Willner, Itamar}, year={2004}, month={Jun}, pages={7430–7431} }
 @article{xiao_pavlov_levine_niazov_markovitch_willner_2004, title={Catalytic Growth of Au Nanoparticles by NAD(P)H Cofactors: Optical Sensors for NAD(P)+-Dependent Biocatalyzed Transformations}, volume={43}, ISSN={1433-7851 1521-3773}, url={http://dx.doi.org/10.1002/anie.200460608}, DOI={10.1002/anie.200460608}, abstractNote={Gold-plated gold: NAD(P)H cofactors mediate the catalytic growth of Au nanoparticles in the presence of AuCl4− and CTAB (cetyltrimethylammonium bromide; see picture). The process allows for the optical detection of the cofactors and the analysis of NAD(P)+-dependent biocatalyzed transformations in solutions and on surfaces.}, number={34}, journal={Angewandte Chemie International Edition}, publisher={Wiley}, author={Xiao, Yi and Pavlov, Valeri and Levine, Semion and Niazov, Tamara and Markovitch, Gil and Willner, Itamar}, year={2004}, month={Aug}, pages={4519–4522} }
 @article{niazov_pavlov_xiao_gill_willner_2004, title={DNAzyme-Functionalized Au Nanoparticles for the Amplified Detection of DNA or Telomerase Activity}, volume={4}, ISSN={1530-6984 1530-6992}, url={http://dx.doi.org/10.1021/nl0491428}, DOI={10.1021/nl0491428}, abstractNote={DNAzyme-functionalized Au−NPs act as catalytic labels for the amplified detection of DNA and telomerase activity on nucleic acid-functionalized gold surface. The DNAzyme stimulates, in the presence of hemin, H2O2, and luminol, the generation of chemiluminescence. For DNA analysis, a nucleic acid unit complementary to the analyzed DNA is tethered to the DNAzyme structure associated with the Au−NPs. For telomerase activity, a nucleic acid complementary to the telomer repeat units generated on the surface is tethered to the DNAzyme structure associated with the Au−NPs. The detection limit for the detection of DNA is 1 × 10-10 M. The method enables the detection of telomerase activity originating from 1000 HeLa cells.}, number={9}, journal={Nano Letters}, publisher={American Chemical Society (ACS)}, author={Niazov, Tamara and Pavlov, Valeri and Xiao, Yi and Gill, Ron and Willner, Itamar}, year={2004}, month={Jul}, pages={1683–1687} }
 @article{shi_xiao_willner_2004, title={Electrical contacting of glucose oxidase by DNA-templated polyaniline wires on surfaces}, volume={6}, ISSN={1388-2481}, url={http://dx.doi.org/10.1016/j.elecom.2004.08.004}, DOI={10.1016/j.elecom.2004.08.004}, abstractNote={Poly(aniline–aniline boronic acid) wires are generated on ds-DNA templates, and the resulting wires exhibit redox functions at neutral pH aqueous solutions. The association of flavin adenine dinucleotide (FAD) to the boronic acid ligand followed by the reconstitution of apo-glucose oxidase on the cofactor units yield an integrated enzyme-electrode where the biocatalyst reveals direct electrical contact with the electrode.}, number={10}, journal={Electrochemistry Communications}, publisher={Elsevier BV}, author={Shi, Lixin and Xiao, Yi and Willner, Itamar}, year={2004}, month={Oct}, pages={1057–1060} }
 @article{xiao_pavlov_gill_bourenko_willner_2004, title={Lighting Up Biochemiluminescence by the Surface Self-Assembly of DNA-Hemin Complexes}, volume={5}, ISSN={1439-4227}, url={http://dx.doi.org/10.1002/cbic.200300794}, DOI={10.1002/cbic.200300794}, abstractNote={The discovery of catalytic RNAs (ribozymes) has sparked scientific activities directed to the preparation of new biocatalysts and raised the suggestion that these biomolecules participated in the evolutionary process as preprotein catalysts. 2] Analogously, deoxyribozymes, catalytic DNAzymes, are not found in nature but extensive research efforts have demonstrated the successful synthesis of catalytic deoxyribozymes for many chemical transformations. 4] One interesting example of a catalytic DNA that reveals peroxidase-like activity includes a supramolecular complex between hemin and a single-stranded guanine-rich nucleic acid (aptamer). This complex was reported to catalyze the oxidation of 2,2 -azinobis(3-ethylbenzothiozoline)-6-sulfonic acid (ABTS) by H2O2, a common reaction used as an assay for peroxidase activity. It was suggested that the supramolecular docking of the guanine-quadruplex layers facilitates the intercalation of hemin into the complex and the formation of the biocatalytically active hemin center. Enzymes and, specifically, horseradish peroxidase (HRP) 9] are used as biocatalytic labels for the amplified detection of DNA-sensing events. The electrochemical amplified detection of DNA has been accomplished in the presence of different enzymes 8] and the chemiluminescent analysis of DNA in the presence of HRP has been reported. The integration of a DNA biocatalyst into DNA-detection schemes could provide a new method for the detection of nucleic acids that might reveal important advantages: 1) The catalytic DNA may substitute the protein-based biocatalysts, and thus eliminate nonspecific binding phenomena; 2) Tailoring of the DNA biocatalyst as part of the labeled nucleic acid might reduce the number of analytical steps for DNA detection. Here we report that two separated nucleic acids that include the segments A and B–constituting the single-stranded peroxidase deoxyribozyme, which forms a layered G-quadruplex structure (see Scheme 1)–self-assemble in the presence of hemin to form a biocatalyst for the generation of chemiluminescence in the presence of H2O2 and luminol. The effect of hybridization with the DNAzyme compounds on the resulting biochemiluminescence is discussed. We also demonstrate the self-assembly of biocatalytic, supramolecular hemin ±nucleic acid complexes on gold electrodes in monolayer configurations, and describe the biocatalytic and bioelectrocatalytic formation of chemiluminescence at the Acknowledgements}, number={3}, journal={ChemBioChem}, publisher={Wiley}, author={Xiao, Yi and Pavlov, Valeri and Gill, Ron and Bourenko, Tatyana and Willner, Itamar}, year={2004}, month={Feb}, pages={374–379} }
 @article{xiao_2003, title={"Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle}, volume={299}, ISSN={0036-8075 1095-9203}, url={http://dx.doi.org/10.1126/science.1080664}, DOI={10.1126/science.1080664}, abstractNote={The reconstitution of an apo-flavoenzyme, apo–glucose oxidase, on a 1.4-nanometer gold nanocrystal functionalized with the cofactor flavin adenine dinucleotide and integrated into a conductive film yields a bioelectrocatalytic system with exceptional electrical contact with the electrode support. The electron transfer turnover rate of the reconstituted bioelectrocatalyst is ∼5000 per second, compared with the rate at which molecular oxygen, the natural cosubstrate of the enzyme, accepts electrons (∼700 per second). The gold nanoparticle acts as an electron relay or “electrical nanoplug” for the alignment of the enzyme on the conductive support and for the electrical wiring of its redox-active center.}, number={5614}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Xiao, Y.}, year={2003}, month={Mar}, pages={1877–1881} }
 @article{xiao_kharitonov_patolsky_weizmann_willner_2003, title={Electrocatalytic intercalator-induced winding of double-stranded DNA with polyaniline}, ISSN={1359-7345 1364-548X}, url={http://dx.doi.org/10.1039/b301749d}, DOI={10.1039/b301749d}, abstractNote={The intercalation of doxorubicin into double-stranded DNA stimulates the electocatalyzed oxidation of aniline to polyaniline and its winding on the DNA template.}, number={13}, journal={Chemical Communications}, publisher={Royal Society of Chemistry (RSC)}, author={Xiao, Yi and Kharitonov, Andrei B. and Patolsky, Fernando and Weizmann, Yossi and Willner, Itamar}, year={2003}, pages={1540} }
 @article{ju_xiao_lu_chen_2002, title={Electrooxidative coupling of a toluidine blue O terminated self-assembled monolayer studied by electrochemistry and surface enhanced Raman spectroscopy}, volume={518}, ISSN={1572-6657}, url={http://dx.doi.org/10.1016/s0022-0728(01)00702-1}, DOI={10.1016/s0022-0728(01)00702-1}, abstractNote={A toluidine blue O (TBO) terminated self-assembled monolayer (SAM-I) was fabricated on a gold disk electrode by nucleophilic attack of the amino group in the reduced TBO on an immobilized carbonyl moiety of terephthaloyl chloride with cyclic scanning. The TBO units in the SAM-I were observed by surface enhanced Raman spectroscopy (SERS). The average TBO surface coverage of SAM-I was (2.5±0.5)×10−10 mol cm−2. Meanwhile, another TBO derived SAM (SAM-II) was prepared for comparison by activating the free carboxyl group of bound terephthaloyl chloride and then reacting it with the amino group of TBO. The average TBO surface coverage of SAM-II was only (6.8±0.1)×10−11 mol cm−2. Both SAMs possessed good electroactivity and showed identical properties. With an electrooxidative coupling of TBO units in SAM-I, a novel electroactive polymerized TBO monolayer (Film-I) was constructed, which displayed an improved stability and obvious electrochemical blocking effect. As a comparison, a poly(TBO) film (Film-II) was obtained by the electrochemical polymerization of TBO in solution on SAM-I for remedying the defectiveness of the knitted structure of Film-I. The SERS displayed more TBO units in Film-II than in Film-I. Film-II provided a much better barrier to the redox probe of K3[Fe(CN)6] due to the formation of the perfect structure.}, number={2}, journal={Journal of Electroanalytical Chemistry}, publisher={Elsevier BV}, author={Ju, Huangxian and Xiao, Yi and Lu, Xiaojun and Chen, Hongyuan}, year={2002}, month={Jan}, pages={123–130} }
 @article{hu_xiao_chen_2001, title={Anion adsorption on an Au colloid monolayer based cysteamine-modified gold electrode}, volume={17}, number={2}, journal={Chemical Research in Chinese Universities}, author={Hu, X.Y and Xiao, Y. and Chen, H.Y}, year={2001}, month={May}, pages={159–167} }
 @article{yi_huang-xian_hong-yuan_2000, title={Direct Electrochemistry of Horseradish Peroxidase Immobilized on a Colloid/Cysteamine-Modified Gold Electrode}, volume={278}, ISSN={0003-2697}, url={http://dx.doi.org/10.1006/abio.1999.4360}, DOI={10.1006/abio.1999.4360}, abstractNote={Direct electron transfer of immobilized horseradish peroxidase on gold colloid and its application as a biosensor were investigated by using electrochemical methods. The Au colloids were associated with a cysteamine monolayer on the gold electrode surface. A pair of redox peaks attributed to the direct redox reaction of horseradish peroxidase (HRP) were observed at the HRP/Au colloid/cysteamine-modified electrode in 0.1 M phosphate buffer (pH 7.0). The surface coverage of HRP immobilized on Au colloid was about 7.6 x 10(-10) mol/cm(2). The sensor displayed an excellent electrocatalytic response to the reduction of H(2)O(2) without the aid of an electron mediator. The calibration range of H(2)O(2) was 1. 4 microM to 9.2 mM with good linear relation from 1.4 microM to 2.8 mM. A detection limit of 0.58 microM was estimated at a signal-to-noise ratio of 3. The sensor showed good reproducibility for the determination of H(2)O(2). The variation coefficients were 3. 1 and 3.9% (n = 10) at 46 microM and 2.8 mM H(2)O(2), respectively. The response showed a Michaelis-Menten behavior at higher H(2)O(2) concentrations. The K(app)(M) value for the H(2)O(2) sensor was found to be 2.3 mM.}, number={1}, journal={Analytical Biochemistry}, publisher={Elsevier BV}, author={Yi, Xiao and Huang-Xian, Ju and Hong-Yuan, Chen}, year={2000}, month={Feb}, pages={22–28} }
 @article{xiao_ju_chen_1999, title={A reagentless hydrogen peroxide sensor based on incorporation of horseradish peroxidase in poly(thionine) film on a monolayer modified electrode}, volume={391}, ISSN={0003-2670}, url={http://dx.doi.org/10.1016/s0003-2670(99)00254-8}, DOI={10.1016/s0003-2670(99)00254-8}, abstractNote={A novel reagentless hydrogen peroxide sensor fabricated by the entrapment of horseradish peroxidase (HRP) into poly(thionine) film on a thionine self-assembled monolayer modified gold electrode is described. The direct electron transfer between the heme site of HRP and the electrode is mediated by thionine units incorporated in the polymer film. The sensor displays an excellent electrocatalytic response to the reduction of H2O2. The chronoamperometric current is proportional to the H2O2 concentration in the range of 6.2 μM to 9.4 mM with a correlation coefficient of 0.999. Moreover, such a modified electrode exhibits a good stability and reproducibility within the linear range of H2O2, the variation coefficients are 3.8% and 4.5% for 10 successive assays at a H2O2 concentration of 9.3 μM and 2.8 mM, respectively.}, number={3}, journal={Analytica Chimica Acta}, publisher={Elsevier BV}, author={Xiao, Y and Ju, H.-X and Chen, H.-Y}, year={1999}, month={Jun}, pages={299–306} }
 @article{hu_xiao_chen_1999, title={Adsorption characteristics of Fe(CN)63−/4− on Au colloids as monolayer films on cysteamine-modified gold electrode}, volume={466}, ISSN={1572-6657}, url={http://dx.doi.org/10.1016/s0022-0728(99)00113-8}, DOI={10.1016/s0022-0728(99)00113-8}, abstractNote={Self-assembled monolayers (SAMs) of chemisorbed cysteamine on gold electrode surfaces have been used as base interfaces for the deposition of the Au colloid. Different Au colloids, ranging in particles of diameter 2.6, 16, 42 and 51 nm, can be deposited on self-assembled cysteamine monolayers on a gold electrode. Fe(CN)64− adsorbed on Au colloid surfaces yields a pair of symmetric adsorption peaks with half-peak width (W1/2) of 90.2 mV and peak-to-peak separation (ΔEp) of 3 mV in 1.0 M KNO3 solution containing 1.00×10−3 M K3Fe(CN)6. The dependence of peak current on scan rate (ν) is linear. ΔEp of the adsorption wave increases while the Au colloid size decreases. The apparent specific capacitances of a bare gold electrode, the cysteamine-modified electrode and the Au colloid modified electrode measured by cyclic voltammetry at 500 mV are 57.8, 32.0 and 81.1 μF cm−2, respectively.}, number={1}, journal={Journal of Electroanalytical Chemistry}, publisher={Elsevier BV}, author={Hu, Xiao-Ya and Xiao, Yi and Chen, Hong-Yuan}, year={1999}, month={May}, pages={26–30} }
 @article{xiao_ju_chen_1999, title={Hydrogen peroxide sensor based on horseradish peroxidase-labeled Au colloids immobilized on gold electrode surface by cysteamine monolayer}, volume={391}, ISSN={0003-2670}, url={http://dx.doi.org/10.1016/s0003-2670(99)00196-8}, DOI={10.1016/s0003-2670(99)00196-8}, abstractNote={A novel strategy to construct a sensitive amperometric sensor of H2O2 was described. Horseradish peroxidase (HRP) was successfully immobilized on nanometer-sized Au colloids, which were supported by thiol-tailed groups of cysteamine monolayer. The thiol-tailed groups were formed through the covalent binding of glutaraldehyde on a cysteamine-modified gold electrode. With the aid of the catechol mediator in the solution, HRP-labeled Au colloids displayed excellent electrocatalytical response to the reduction of H2O2, which increased with decreasing size of Au colloids. The sensors responded to H2O2 in the concentration range of 0.39 μM–0.33 mM, and reached 95% of the steady-state current in less than 5 s with the detection limit of 0.15 μM. The response showed a Michaelis–Menten behavior at larger H2O2 concentrations. The KappM values for the sensors based on different sized HRP-labeled Au colloids (colloid A–D) were found to be 0.11, 0.094, 0.054 and 0.064 mM, respectively. The low KappM values demonstrated that HRP immobilized on Au colloids exhibited a high affinity to H2O2 with no loss of enzymatic activity.}, number={1}, journal={Analytica Chimica Acta}, publisher={Elsevier BV}, author={Xiao, Yi and Ju, Huang-Xian and Chen, Hong-Yuan}, year={1999}, month={May}, pages={73–82} }
 @article{ju_zhou_xiao_chen_1998, title={Amperometric Biosensor for Glucose Based on a Nanometer-Sized Microband Gold Electrode Coimmobilized with Glucose Oxidase and Poly(o-phenylenediamide)}, volume={10}, ISSN={1040-0397 1521-4109}, url={http://dx.doi.org/10.1002/(sici)1521-4109(199807)10:8<541::aid-elan541>3.0.co;2-j}, DOI={10.1002/(sici)1521-4109(199807)10:8<541::aid-elan541>3.0.co;2-j}, abstractNote={A nanometer-sized (88 mm) microband gold electrode for glucose detection is described. These noble metal ultramicrosensors are prepared by electrochemical copolymerization of o-phenylenediamide (PDA) and glucose oxidase at the nanometer-sized electrodes. Electropolymerization conditions of PDA at the microband surface are optimized. The current can rapidly (3s) reach a steady value upon the potential step from 0 to ˛0.6 V without stirring the solution. The permselectivity characteristics of polymer can eliminate the interference of ascorbic acid. The amperometric response to glucose shows a linear relation in the range of 0.5 mM to about 10 mM with the correlation coefficient of 0.9993. With further increasing of glucose concentration an obvious Michaelis-Menten’s response can be observed. The maximum steady current density is ca. 4.4 6 0.2 mA/cm 2 and the apparent Michaelis-Menten constant is 21.1 6 0.2 mM.}, number={8}, journal={Electroanalysis}, publisher={Wiley}, author={Ju, Huangxian and Zhou, Dongmei and Xiao, Yi and Chen, Hongyuan}, year={1998}, month={Jul}, pages={541–545} }