Polarity‐Switching Electrochemical Sensor for Specific Detection of Single‐Nucleotide Mismatches

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