@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} }