Electrochemical immuno-biosensor and method for detection of circulating protein biomarkers

An electrochemical immuno-biosensor-based method for detecting a target protein in a complex medium, the method comprising the steps of:

• selecting the target protein to be detected in the complex medium and providing a sample obtained from a subject, the target protein being an endogenous/circulating protein in the sample;
• selecting an antibody complementary to the selected target protein;
• preparing a recognition complex comprising the selected antibody, a fragment or a derivative thereof and at least one signaling DNA probe, each said at least one signaling DNA probe having a first end attached to the selected antibody, the fragment or the derivative thereof and a redox moiety covalently attached proximate to a second end of the at least one signaling DNA probe;
• mixing the sample of the complex medium with the recognition complex, such that, when present in the sample, the target protein binds with the antibody, the fragment or the derivative thereof of the recognition complex to form a signaling DNA-antibody-antigen complex, and when absent, the antibody, the fragment or the derivative thereof of the recognition complex remains free of the target protein;
• contacting the sample with an electrode comprising a high-curvature gold surface having a plurality of capturing DNA probes covalently attached to the gold surface at a first end, each said plurality of capturing DNA probes being longer than the at least one signaling DNA probe and comprising a sequence portion complementary toa sequence portion of the at least one signaling DNA probe, whereby the at least one signaling DNA probe of the recognition complex are capable of hybridizing with the plurality of capturing DNA probes to bring the redox moiety into proximity with the gold surface and generate an electrochemical current upon application of a voltage;
• an optimization step in which a surface density of the plurality of capturing DNA probes on the high-curvature gold surface and a dimension and/or curvature of the high-curvature gold surface are selected based on dose-response measurements obtained (i) with the at least one signaling DNA probe in the absence of the antibody, the fragment or the derivative thereof, (ii) with the antibody, the fragment or the derivative thereof present and the target protein absent, and (iii) with the antibody, the fragment or the derivative thereof present and the target protein present, thereby maximizing the current in the absence of the target protein and minimizing background attributable to steric hindrance of the antibody, the fragment or the derivative thereof, and achieving saturated surface hybridization at a minimum signaling DNA probe amount with a reduced effective concentration for target detection within a rapid assay time;
• and
• measuring the generated electrochemical current, wherein binding of the target protein to the antibody, the fragment or the derivative thereof of the recognition complex increases steric hindrance reducing hybridization of the at least one signaling DNA probe with the plurality of capturing DNA probes on the gold surface, thereby reducing the generated electrochemical current relative to the current obtained in the absence of the target protein.