@article{mohammad_talton_hetzler_gongireddy_wei_2023, title={Unidirectional trans-cleaving behavior of CRISPR-Cas12a unlocks for an ultrasensitive assay using hybrid DNA reporters containing a 3' toehold}, volume={8}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkad715}, abstractNote={CRISPR-Cas12a can induce nonspecific trans-cleavage of dsDNA substrate, including long and stable λ DNA. However, the mechanism behind this is still largely undetermined. In this study, we observed that while trans-activated Cas12a didn't cleave blunt-end dsDNA within a short reaction time, it could degrade dsDNA reporters with a short overhang. More interestingly, we discovered that the location of the overhang also affected the susceptibility of dsDNA substrate to trans-activated Cas12a. Cas12a trans-cleaved 3' overhang dsDNA substrates at least 3 times faster than 5' overhang substrates. We attributed this unique preference of overhang location to the directional trans-cleavage behavior of Cas12a, which may be governed by RuvC and Nuc domains. Utilizing this new finding, we designed a new hybrid DNA reporter as nonoptical substrate for the CRISPR-Cas12a detection platform, which sensitively detected ssDNA targets at sub picomolar level. This study not only unfolded new insight into the trans-cleavage behavior of Cas12a but also demonstrated a sensitive CRISPR-Cas12a assay by using a hybrid dsDNA reporter molecule.}, journal={NUCLEIC ACIDS RESEARCH}, author={Mohammad, Noor and Talton, Logan and Hetzler, Zach and Gongireddy, Megha and Wei, Qingshan}, year={2023}, month={Aug} }
 @article{hetzler_wang_krafft_jamalzadegan_overton_kudenov_ligler_wei_2022, title={Flexible sensor patch for continuous carbon dioxide monitoring}, volume={10}, ISSN={["2296-2646"]}, DOI={10.3389/fchem.2022.983523}, abstractNote={Monitoring and measurement of carbon dioxide (CO2) is critical for many fields. The gold standard CO2 sensor, the Severinghaus electrode, has remained unchanged for decades. In recent years, many other CO2 sensor formats, such as detection based upon pH-sensitive dyes, have been demonstrated, opening the door for relatively simple optical detection schemes. However, a majority of these optochemical sensors require complex sensor preparation steps and are difficult to control and repeatably execute. Here, we report a facile CO2 sensor generation method that suffers from none of the typical fabrication issues. The method described here utilizes polydimethylsiloxane (PDMS) as the flexible sensor matrix and 1-hydroxypyrene-3,6,8-trisulfonate (HPTS), a pH-sensitive dye, as the sensing material. HPTS, a base (NaOH), and glycerol are loaded as dense droplets into a thin PDMS layer which is subsequently cured around the droplet. The fabrication process does not require prior knowledge in chemistry or device fabrication and can be completed as quickly as PDMS cures (∼2 h). We demonstrate the application of this thin-patch sensor for in-line CO2 quantification in cell culture media. To this end, we optimized the sensing composition and quantified CO2 in the range of 0-20 kPa. A standard curve was generated with high fidelity (R2 = 0.998) along with an analytical resolution of 0.5 kPa (3.7 mm Hg). Additionally, the sensor is fully autoclavable for applications requiring sterility and has a long working lifetime. This flexible, simple-to-manufacture sensor has a myriad of potential applications and represents a new, straightforward means for optical carbon dioxide measurement.}, journal={FRONTIERS IN CHEMISTRY}, author={Hetzler, Zach and Wang, Yan and Krafft, Danny and Jamalzadegan, Sina and Overton, Laurie and Kudenov, Michael W. and Ligler, Frances S. and Wei, Qingshan}, year={2022}, month={Sep} }