Building a nucleic acid nanostructure with DNA-epitope conjugates for a versatile electrochemical protein detection platform

Abstract

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.

An antibody-based molecular switch for continuous small-molecule biosensing

“…We believe that our antibody-switch design represents a valuable addition to the rapidly growing toolbox of protein-based biosensors. For example, Easley and coworkers have made use of competitive binding of solution-phase antibodies to enable small-molecule sensing using antigen-modified DNA structures on electrochemical sensors (35)(36)(37). However, because these sensors rely on the introduction of free antibody into solution to achieve competitive binding, they are unsuitable for continuous sensing.…”

An Electrochemical Proximity Assay (ECPA) for Antibody Detection Incorporating Flexible Spacers for Improved Performance

“…30 Major advantages of electrochemical biosensors are easy-tooperate instrumentation, cost-effective sensing elements, and availability of various efficient and rapid transduction methods [31][32][33] ; and the high sensitivity, good specificity, and compatibility with miniaturization have made electrochemical systems indispensable for point-of-care diagnostics. [34][35][36] Different types of electrochemical sensors such as electrochemical aptamer-based sensors (E-AB), 34,[37][38][39][40] E-DNA scaffolds, 36,[41][42][43] a molecular pendulum, 25 and a bowtie-shaped DNA nanostructure for immunosensing, [44][45][46] are promising for point-of-care measurements 47 . Previously our group has reported the electrochemical proximity assay (ECPA) capable of detecting proteins as low as the fM range.…”