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For an assay to be most effective in point-of-care clinical analysis, it needs to be economical, simple, generalizable, and free from tedious workflows. While electrochemistry-based DNA sensors reduce instrumental costs and eliminate complicated procedures, there remains a need to address probe costs and generalizability, as numerous probes with multiple conjugations are needed to quantify a wide range of biomarkers. In this work, we have opened a route to circumvent complicated multiconjugation schemes using enzyme-catalyzed probe construction directly on the surface of the electrode. With this, we have created a versatile DNA nanostructure probe and validated its effectiveness by quantification of proteins (streptavidin, anti-digoxigenin, anti-tacrolimus) and small molecules (biotin, digoxigenin, tacrolimus) using the same platform. Tacrolimus, a widely prescribed immunosuppressant drug for organ transplant patients, was directly quantified with electrochemistry for the first time, with the assay range matching the therapeutic index range. Finally, the stability and sensitivity of the probe was confirmed in a background of minimally diluted human serum.

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 bioanalytical sensors with oligonucleotide transducer molecules have been recently extended for quantifying a wide range of biomolecules, from small drugs to large proteins. Short DNA or RNA strands have gained attention recently due to the existence of circulating oligonucleotides in human blood, yet challenges remain for adequately sensing these targets at electrode surfaces. In this work, we have developed a quantitative electrochemical method which uses target-induced proximity of a single-branched DNA structure to drive hybridization at an electrode surface, with readout by square-wave voltammetry (SWV). Using custom instrumentation, we first show that precise control of temperature can provide both electrochemical signal amplification and background signal depreciation in SWV readout of small oligonucleotides. Next, we thoroughly compared 25 different combinations of binding energies by their signal-to-background ratios and differences. These data served as a guide to select the optimal parameters of binding energy, SWV frequency, and assay temperature. Finally, the influence of experimental workflow on the sensitivity and limit of detection (LOD) of the sensor is demonstrated. This study highlights the importance of precisely controlling temperature and SWV frequency in DNA-driven assays on electrode surfaces while also presenting a novel instrumental design for fine-tuning of such systems.

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.