Building a Nucleic Acid Nanostructure with DNA-Epitope Conjugates for a Versatile Approach to Electrochemical Protein Detection.

The recent surge of effort in nucleic-acid-based electrochemical (EC) sensors has been fruitful, yet there remains a need for more generalizable EC platforms for sensing multiple classes of clinically relevant targets. We recently reported a nucleic acid nanostructure for simple, economical, and more generalizable EC readout of a range of analytes, including small molecules, peptides, proteins, and antibodies. The nanostructure is built through on-electrode enzymatic ligation of three oligonucleotides for attachment, binding, and signaling. However, the generalizable detection of larger proteins remains a challenge. Here, we adapted the sensor to quantify larger proteins in a more generic manner through conjugating the protein's minimized antibody-binding epitope to the central DNA strand. This concept was verified using creatine kinase (CK-MM), a biomarker of muscle damage and several disorders for which rapid clinical sensing is important. DNA-epitope conjugates permitted a competitive immunoassay for the CK protein at the electrode via square-wave voltammetry (SWV). Sensing through a signal-off mechanism, the anti-CK antibody limit of detection (LOD) was 5 nM with a response time as low as 3 min. Antibody displacement by native protein analytes gave a signal-on response with the CK sensing range from the LOD of 14 nM up to 100 nM, overlapping with the normal (nonelevated) human clinical range (3-37 nM), and the sensor was validated in 98% human serum. While a need for improved DNA-epitope conjugate purification was identified, overall, this approach allows the quantification of a generic protein- or peptide-binding antibody and should facilitate future quantitative EC readouts of clinically relevant proteins that were previously inaccessible to EC techniques.

Ionic Strength and Hybridization Position near Gold Electrodes Can Significantly Improve Kinetics in DNA-Based Electrochemical Sensors.

A variety of electrochemical (EC) biosensors play critical roles in disease diagnostics. More recently, DNA-based EC sensors have been established as promising for detecting a wide range of analyte classes. Since most of these sensors rely on the high specificity of DNA hybridization for analyte binding or structural control, it is crucial to understand the kinetics of hybridization at the electrode surface. In this work, we have used methylene blue-labeled DNA strands to monitor the kinetics of DNA hybridization at the electrode surface with square-wave voltammetry. By varying the position of the double-stranded DNA segment relative to the electrode surface as well as the bulk solution's ionic strength (0.125-1.00 M), we observed significant interferences with DNA hybridization closer to the surface, with more substantial interference at lower ionic strength. As a demonstration of the effect, toehold-mediated strand displacement reactions were slowed and diminished close to the surface, while strategic placement of the DNA binding site improved reaction rates and yields. This work manifests that both the salt concentration and DNA hybridization site relative to the electrode are important factors to consider when designing DNA-based EC sensors that measure hybridization directly at the electrode surface.

Electrochemical Sensing of the Peptide Drug Exendin-4 Using a Versatile Nucleic Acid Nanostructure.

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.

Nonfaradaic Current Suppression in DNA-Based Electrochemical Assays with a Differential Potentiostat.

One of the key factors limiting sensitivity in many electrochemical assays is the nonfaradaic 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 nonfaradaic 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). Because 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.