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One of the key factors limiting sensitivity in many electrochemical assays is the non-faradaic or capacitive current. This is particularly true in modern assay systems based on DNA monolayers at gold electrode surfaces, which have shown great promise for bioanalysis in complex milieu such as whole blood or serum. While various changes in analytical parameters, redox reporter molecules, DNA structures, probe coverage, and electrode surface area have been shown useful, background reduction by hardware subtraction has not yet been explored for these assays. Here, we introduce new electrochemistry hardware that considerably suppresses non-faradaic currents through real-time analog subtraction during current-to-voltage conversion in the potentiostat. This differential potentiostat (DiffStat) configuration is shown to suppress or remove capacitance currents in chronoamperometry, cyclic voltammetry, and square-wave voltammetry measurements applied to nucleic acid hybridization assays at the electrode surface. The DiffStat makes larger electrodes and higher sensitivity settings accessible to the user, providing order-of-magnitude improvements in sensitivity, and it also significantly simplifies data processing to extract faradaic currents in square-wave voltammetry (SWV). Since two working electrodes are used for differential measurements, unique arrangements are introduced such as converting signal-OFF assays to signal-ON assays, or background drift correction in 50 % human serum. Overall, this new potentiostat design should be helpful not only in improving the sensitivity of most electrochemical assays, but it should also better support adaptation of assays to the point-of-care by circumventing complex data processing.

Electrochemical proximity assay (ECPA), was recently developed for direct protein quantitation as low as 158 fM. In this assay, the loop-like DNA structure holds methylene blue (MB-DNA) in close proximity with the gold surface when target is present, ensuring efficient electron transfer. However, for studying DNA binding dynamics on gold surfaces, investigations were carried by immobilizing SH-DNA onto SPR sensor surface to form self-assembled monolayer. DNA loop and MB-DNA oligonucleotides were alternately hybridized to the SH-DNA complementarily forming loop-like structure. Sensogram results showed increase in refractive angle (RA) when DNA alternately hybridized with complementary strands, however upon elimination of DNA loop or SH-DNA showed no discernible RA signal, suggesting loss in loop structure. We found that the SH-DNA binding on the gold surface was affected by free thiols moieties. Purification of SH-ssDNA led to signal enhancement in target binding in comparison to non-purified. We anticipate the feasibility of this study in improving the speed and sensitivity of ECPA-based biomarker recognition.

Rapid and specific quantitation of a variety of proteins over a wide concentration range is highly desirable for biosensing at the point-of-care, in clinical laboratories, and in research settings. Our recently developed electrochemical proximity assay (ECPA) is a target-flexible, DNA-directed, direct-readout protein quantitation method with detection limits in the low femtomolar range, making it particularly amenable to point-of-care detection. However, consistent quantitation in more complex matrices is required at the point-of-care, and improvements in measurement speed are needed for clinical and research settings. Here, we address these concerns with a reusable ECPA, where a gentle regeneration of the surface DNA monolayer (used to capture the proximity complex) is achieved enzymatically through a novel combination of molecular biology and electrochemistry. Strategically placed uracils in the DNA sequence trigger selective cleavage of the backbone, releasing the assembled proximity complex. This allows repeated protein quantitation by square-wave voltammetry (SWV)-as quickly as 3 min between runs. The process can be repeated up to 19 times on a single electrode without loss of assay sensitivity, and currents are shown to be highly repeatable with similar calibrations using seven different electrodes. The utility of reusable ECPA is demonstrated through two important applications in complex matrices: (1) direct, quantitative monitoring of hormone secretion in real time from as few as five murine pancreatic islets and (2) standard addition experiments in unspiked serum for direct quantitation of insulin at clinically relevant levels. Results from both applications distinguish ECPA as an exceptional tool in protein quantitation.

Electrochemical polymerization is a simple and direct technique often employed for immobilizing redox enzymes at an electrode surface. Besides these, it allows precise control over the amount, spatial distribution and orientation of the enzymes. Analytical expressions pertaining to the immobilization of enzyme by electrochemical polymerization on the electrode surface were obtained by Homotopy perturbation method (HPM). This expression further distinguishes the product of the enzyme reaction at the electrode surface from those occurring alongside the polymer employed for immobilization. These analytical results are compared with the available limiting case results and they are found to be in good agreement.