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.