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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