(Invited) Fast and Generalizable Electrochemical Sensing of Small Molecules, Peptides, and Proteins Using a Nucleic Acid Nanostructure with Analyte-DNA Conjugates (2022)

Sensors based on electrochemical (EC) readout offer low cost, miniaturization, and adaptability to the point-of-care (POC). Nonetheless, most EC sensors are specialized to a particular target, and there remains a need for a robust EC biosensor platform for the multitude of biomarkers that are not EC-active, do not undergo enzymatic conversion, or are not suited for potentiometry [1,2]. Impressively, aptamer-based EC sensors have been proven for sensing in living animals with temporal resolution as low as a few seconds [3], yet most method development has been target-focused, lacking generalizability [4]. Presently, the clinical EC toolbox is a conglomerate of targeted methods, and there is a pressing need to develop an EC platform amenable to rapid, generalizable, quantitative readout of multiple classes of clinically relevant targets. Direct EC sensing without added reagents or amplification steps should be ideal for this purpose. For example, the Kelley group recently developed a reagentless “molecular pendulum” EC sensor for a broad range of protein analytes [5]. Our group has been working to address this need and expand to more analyte classes for several years, and in 2019 we designed a versatile DNA-nanostructure architecture attached to gold electrode surfaces [6]. Initially, our sensors were validated with biotechnology controls, antibodies, and with a small molecule immunomodulatory drug in human serum. In this presentation, we discuss the expansion of the generalizability of our sensor platform, chiefly through custom synthesis of varied DNA-analyte bioconjugates to incorporate within the DNA-nanostructure. For peptide sensing, DNA-peptide conjugates were synthesized, purified, then ligated to the DNA-nanostructure. Sensors were validated for quantifying exendin-4 (4.2 kDa)—a human glucagon-like peptide-1 receptor agonist important in diabetes therapy—for the first time using direct EC methods, with an LOD of 6 nM [7]. Sensors for larger proteins were made using DNA-epitope conjugates. The antibody-binding epitope of creatine kinase MM (CK-MM) was conjugated into the nanostructure, allowing CK-MM sensing in the 10 to 100 nM range. Finally, DNA-steroid bioconjugates have been incorporated into the sensors. Sensing of testosterone throughout the clinically relevant range (for males) was accomplished from 1 to 50 nM (LOD of 0.9 nM), and cortisol could be easily detected in the 1 to 100 nM range, well below the 90 – 550 nM range in blood and nicely encompassing the 6 – 75 nM range in saliva. All of these sensors were functional in 98% human serum, and several detection ranges overlap with the clinical/therapeutic ranges, boding well for future applications in biosensing or therapeutic drug monitoring. Overall, this new DNA nanostructure platform provides a generalizable sensor with minimal workflow, direct-readout, and the capability to expand EC sensing to a wide variety of clinically important analytes. References: Turner, A. P., Chemical Society Reviews 2013, 42 (8), 3184-96. Wilson, G. S.; Johnson, M. A., Chem. Reviews 2008, 108 (7), 2462-81. Idili, A.; Gerson, J.; Kippin, T.; Plaxco, K. W., Anal. Chem. 2021, 93 , 4023-32. Labib, M.; Sargent, E. H.; Kelley, S. O., Chem. Reviews 2016, 116 (16), 9001-90. Das, J.; Gomis, S.; Chen, J. B.; Yousefi, H.; Ahmed, S.; Mahmud, A.; Zhou, W.; Sargent, E. H.; Kelley, S. O., Nature Chem. 2021, 13 (5), 428-434. Somasundaram, S.; Easley, C. J., J. Am. Chem. Soc. 2019, 141, 11721-11726. Khuda, N; Somasundaram, S.; Easley, C. J., ACS Sensors 2021, under revision.