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The recent surge of effort in nucleic acid-based electrochemical (EC) sensors has been fruitful, and some have even shown real-time quantification of drugs in the blood of living animals. Yet there remains a need for more generalizable EC platforms for the detection of multiple classes of clinically relevant targets. Our group has recently reported a nucleic acid nanostructure that permits simple, economical, and generalizable EC readout of a wide range of analytes (small molecules, peptides, large proteins, or antibodies). The DNA nanostructure is built through on-electrode enzymatic ligation of three oligonucleotides for attachment, binding, and signaling. However, the signaling mechanism predominantly relies on tethered diffusion of methylene blue at the electrode surface, limiting the detection of larger proteins that have no readily available small molecule binding partners. In this study, we adapted the nanostructure sensor to quantify larger proteins in a more generic manner, through conjugating the proteins minimized antibody-binding epitope to the central DNA strand of the nanostructure (DNA-peptide conjugate). This concept was verified using creatine kinase (CK-MM), an important biomarker of muscle damage, myocardial infarction, overexertion/rhabdomyolysis, or neuromuscular disorders where clinical outcomes could be improved with rapid sensing. DNA-epitope conjugates permitted a competitive immunoassay protocol at the electrode surface for quantifying CK protein. Square-wave voltammetry (SWV) signal suppression was proportional to the amount of surface-bound antibody with a limit of detection (LOD) of 5 nM and a response time as low as 3 minutes, and displacement of antibody by native CK-MM protein analyte could also be assayed. CK was quantified from the LOD of 14 nM up to 100 nM, overlapping well with the normal (non-elevated) human clinical range of 3 37 nM, and the sensor response was validated in 98% human serum. While a need for improved DNA-epitope conjugate purification was identified, overall this approach not only allows the detection of a generic protein- or peptide-binding antibody, but it also should facilitate future quantitative EC readout of various clinically relevant protein analytes that were previously inaccessible to EC techniques.

Although endogenous peptides and peptide-based therapeutics are both highly relevant to human health, there are few approaches for sensitive biosensing of this class of molecules with minimized workflow. In this work, we have further expanded on the generalizability of our recently developed DNA nanostructure architecture by applying it to electrochemical (EC) peptide quantification. While DNA-small molecule conjugates were used in a prior work to make sensors for small molecule and protein analytes, here DNA-peptide conjugates were incorporated into the nanostructure at the electrode surfaces, and antibody displacement permitted rapid peptide sensing. Interestingly, multivalent DNA-peptide conjugates were found to be detrimental to the assay readout, yet these effects could be minimized by solution-phase bioconjugation. The final biosensor was validated for quantifying exendin-4 (4.2 kDa)─a human glucagon-like peptide-1 receptor agonist important in diabetes therapy─for the first time using EC methods with minimal workflow. The sensor was functional in 98% human serum, and the low nanomolar assay range lies between the injected dose concentration and the therapeutic range, boding well for future applications in therapeutic drug monitoring.

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

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.

Septal correction is the commonest surgery by rhinologists worldwide. We aimed at studying the comfort level of the patient with standard postoperative nasal packing with Merocel and placing quilting sutures in septum leaving the nose unpacked in the postoperative period. We conducted the study in the tertiary care centre enrolling 82 patients in a quasi-randomised method of odd and even numbers placing them in the nasal packing group and the quilting group respectively. We used analogue scoring method for subjective assessment of comfort level in the postoperative period and the surgeon objectively assessed the patient on follow up. The results were tabulated and analysed. Postoperative pain, headache and sleep disturbance was significantly more in the nasal packing group. We found that the crusting is commonly seen in patients in the nasal packing group. Quilting the nasal septum and leaving the nasal cavity unpacked increases the comfort level of the patient in the postoperative period. The resultant pain, headache and sleep disturbance caused by nasal packing can be significantly avoided by using quilting the septum without nasal packing. We also observed that by avoiding nasal packing postoperatively, the patients were more comfortable and compliant with the treatment regimen and follow-up.

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.

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.