Investigation of Redox Probe Location on Single-Stranded DNA Using Streptavidin-Based Electrochemical Biosensing Platform

Most electrochemical platforms utilize a thiolated capture DNA and 6-mercapto-1-hexanol (MCH) as a monolayer for performing electrochemical DNA biosensing (1–5). These platforms employ redox-labeled DNA, mostly using methylene blue (MB), to generate electrochemical signals following hybridization with capture DNA, resulting in the formation of double-stranded DNA (dsDNA) with MB positioned towards the electrode surface (1–5). While previous studies predominantly focus on using MB-labeled DNA to form dsDNA with thiolated capture DNA, this study explores the effect of MB positions at three different locations on biotinylated single-stranded DNA (DNA barcodes) within a non-thiolated electrochemical system that uses streptavidin-coated gold electrodes. We investigated biotinylated barcodes with MB labels positioned at three different locations: one near the 5’ end of the DNA, proximal to the electrode surface (MB1); one in the middle (MB2); and one near the 3’ distal end of the DNA (MB3). To analyze the performance of these three MB barcodes, we prepared streptavidin-coated nanostructured gold electrodes and measured current signals using square wave voltammetry (SWV) after the attachment of biotinylated MB barcodes with the streptavidin on the electrodes. We examined the time (ranging from 5 to 120 minutes) and temperature (room temperature (RT) and 37 ºC) kinetics for the barcodes, and we also determined the limit of detection (LOD) in buffer, covering concentrations from 0 to 1000 nM. Our barcode system was compared to the conventional thiol-DNA and MCH-backfilling-based electrochemical platform. Moreover, we explored the application of this platform as a signal-OFF system using complementary capture DNA. Our findings indicated that MBs located further from the electrode surface (MB2 and MB3) facilitated faster electron transfer than MB1, which was positioned closest to the electrode. This difference in electrochemical signal among the MB barcodes can be attributed to the flexibility of ssDNA, which makes methylene blue more accessible for electron transfer at the electrode surface in the cases of MB2 and MB3. During our examination of time and temperature kinetics, we observed that the current density signal from MB1 increased much more slowly than that from MB2 and MB3 at both temperatures. Notably, MB2 exhibited the fastest signal increase, showing significant response within 5 minutes at 37 ºC and 15 minutes at RT. The current density signal for MB2 and MB3 saturated after 30 minutes at 37 ºC, while the signal for MB1 continued to rise slowly. The reproducibility of the prepared system was confirmed when we tested another barcode sequence with similar MB positions. From the LOD analysis, we found that the lowest concentration was detectable for MB2 compared to the other barcodes. The streptavidin system (MB2 and MB3) demonstrated higher signaling and less variation than the capture DNA-based system, which required 4 µM of capture DNA to achieve the highest current density signal. When evaluating the signal-OFF assay, we observed a decrease in current density signals following the addition of complementary capture DNA to the MB-barcode immobilized streptavidin electrodes. MB1 exhibited the highest signal suppression (~80% suppression) among the other two barcodes (~60% suppression), likely due to its reduced flexibility after hybridization with capture DNA and the slower electron transfer through the streptavidin layer to the electrode surface. Overall, aside from the signal suppression study, the MB2 barcode showed better performance in terms of better LOD, faster electron transfer, higher current density signals than MB1 and MB3, and higher signal than a capture DNA-based system. Moreover, the results of this study will help as a guide for deciding the position of MB on the ssDNA in a signal-ON assay or on dsDNA in a signal-OFF assay and the use of a streptavidin-based electrochemical platform for biosensing applications. References: A. A. Lubin, B. Vander Stoep Hunt, R. J. White, K. W. Plaxco, Anal. Chem. 81, 2150–2158 (2009). R. Pandey et al., ACS Sensors. 7, 985–994 (2022). S. M. Traynor, G. A. Wang, R. Pandey, F. Li, L. Soleymani, Angew. Chemie. 132, 22806–22811 (2020). A. Victorious et al., Angew. Chemie Int. Ed. 61, e202204252 (2022). Z. Zhang, B. R. Adhikari, P. Sen, L. Soleymani, Y. Li, Adv. Agrochem. 2, 246–257 (2023).

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