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Electrochemical sensors are well studied for detecting DNA, RNA, small molecules, inorganic ions, etc. Using the latest DNA-based sensors, a change in the location of a redox reporter attached to an electrode-bound nucleic acid gives rise to faradaic current changes, which can be monitored by square wave voltammetry (SWV) and used for sensing of various analytes. However, the nonfaradaic current generated along with the faradaic current has often been viewed as a limiting factor in sensor performance and either subtracted or ignored. Nonfaradaic current can be suppressed somewhat by surface blocking and reduced electrode surface area, and our group recently introduced a differential potentiostat (DiffStat) that provided efficient, in-hardware subtraction. On the other hand, removal of nonfaradaic current may lead to loss of important information embedded within. Moreover, a change in temperature can affect both faradaic and nonfaradaic currents by altering the movement of DNA strands and other ions present in the solution. Usually, faradaic current is proportional to the temperature. However, temperature-dependent changes in nonfaradaic current have not been well studied in DNA monolayer-based sensors. In this study, the changes in both faradaic and nonfaradaic currents were monitored in an electrochemical DNA hybridization assay. A thiolated DNA (thio-DNA) monolayer was assembled onto gold electrodes, and current was measured at different square wave frequencies (1 to 1000 Hz). The DNA analyte and the methylene blue tagged DNA (MB-DNA) were hybridized with the thio-DNA separately to compare the signals. Using an in-house built Peltier controller, the effects of temperature were monitored during operation of an analyte-induced proximity assay with SWV readout. In-house written MATLAB code was developed to automatically analyze each set of raw SWV data. Within this code, a polynomial baseline was calculated in the vicinity of the redox potential of MB-DNA (around -200 mV) to allow faradaic and nonfaradaic currents to be separated, post assay. Two-dimensional heatmaps were generated to study the variation of these currents with both temperature and square wave frequency. The faradaic current increased up to a certain temperature and then decreased due to dissociation (or melting) of the DNA from the surface. The nonfaradaic current, on the other hand, increased with temperature in a viscosity-dependent manner and also increased with the square wave frequencies as expected from a capacitive, single exponential time decay. Current changes exhibited by the DNA sensors upon analyte binding and temperature fluctuation suggested that both faradaic and nonfaradaic signals could be used for multidimensional analysis of sensor responses in the future. Figure 1

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.

Thiol-terminated single-stranded DNA (SH ss-DNA) monolayer assembly on gold as a probe is a significant model for various biosensors. In electrochemical proximity assay (ECPA), SH ss-DNA with complementary DNA loop moves a methylene blue (MB)-conjugated oligonucleotide (MB ss-DNA) close to a gold surface, which forms a DNA loop like structure, for transferring electron when target protein is present. After treatment of urea in DI water, the MB ss-DNA and DNA loop dissociate from the DNA loop like structure for reusability. Surface plasmon resonance (SPR) is a powerful optical technique for studying dynamic matrix of affinity biosensors. In this study, we first confirmed DNA complementarities in flow mode with surface plasmon resonance detection. SPR results showed discriminative refractive angle change (DRA) in each DNA hybridization. However, the loss in DNA loop structure (SH-ssDNA or DNA loop) showed inconspicuous sensogram. Furthermore, we studied the effects of free thiols, which is a result of cleavage of disulfide precursors by reductant, in SH ss-DNA monolayer assembly on gold surface. Comparison of DRA in pure SH-ss DNA and non-purified SH-DNA in several concentrations showed free thiols can compete with SH-ss DNA on gold surface. DNA loop and MB ss-DNA added to two solutions is used for double confirmation of the recognition event. Mixture of different ratios of SH-ss DNA and free thiols were used to further study the competition between SH-ss DNA and free thiols. Moreover, we investigated reusable DNA Loop formation capable of several uses in simple matrix. Urea dissolved in DI water was utilized to dissociate DNA loop and MB ss-DNA from DNA loop-like structure. Optimization of concentration and pH of urea were performed. Study dynamics of this reusable DNA loop structure can improve sensitivity and feasibility of ECPA-based marker.

Surface plasmon resonance (SPR) is a well-established optical technique for studying DNA-DNA binding (hybridization) in the fields of molecular biology and biomedical sensing. With the emergence of high performance protein-to-DNA switches as electrochemical biosensors, the importance of understanding DNA binding dynamics at gold surfaces has significantly increased. One such method, the electrochemical proximity assay (ECPA), has been shown capable of direct protein quantitation as low as 20 fM. We report here the SPR study of a DNA loop model of ECPA that mimics probe/target binding with a DNA loop. In the assay, the loop-like structure holds methylene blue (MB) in close proximity with the gold surface when target is present, ensuring efficient electron transfer. Investigations on hybridization dynamics were carried out by immobilizing thiolated capture DNA onto the gold surface of the SPR sensor chip to form self-assembled monolayer. DNA loop oligonucleotides and MB-DNA sequences were alternately hybridized to the complementary strands to form a loop-like structure. Sensogram results showed increase in refractive angle (RA) in real time (flow-mode) analysis of binding sequence of the DNA complementarities. In order to verify this binding recognition event, one of the counterpart DNA probe sequences (DNA loop or SH-DNA) were eliminated which showed no discernable RA signal, suggesting loss in loop structure. Further investigations on analysis of time required for thiolated DNA to self-assemble onto the gold surface and optimization of concentrations under stopped-flow (batch mode) conditions were carried out. With this proof-of-concept dynamics of the DNA loop model system, we anticipate that this study can significantly improve the speed and sensitivity of ECPA-based biomarker recognition.