An Electrochemical Proximity Assay (ECPA) Based Sensor for Antibody Detection

Abstract

Biomarker detection is vitally important for disease diagnosis. Critical biomarkers which are present in ultra-low concentrations in body fluids have been detected with immunoassays such as ELISA and improved versions such as Alpha-LISA and digital ELISA. Although these techniques have fM-pM range detection limits, the requirement of special instruments, reagents, and labor-intensive workflows have directed researchers to develop simpler assay techniques. The ideal alternative assay will be cost effective, with simple mix-and-read workflows, yet will not compromise its specificity and sensitivity. Analyte-driven hybridization of short complementary DNA strands have gained substantial attention for these reasons. Molecular pincer assays and proximity ligation assays (PLA) have successfully demonstrated sensitivities comparable to ELISA, using antibody-DNA conjugates. These techniques rely on fluorescence detection, which is sensitive and specific, but is challenging in terms of miniaturization and point-of-care (POC) use. Electrochemical (EC) detection, on the contrary, can be easily miniaturized and employs simple instrumentation with high sensitivity. We have previously introduced the electrochemical proximity assay (ECPA), where insulin and thrombin have been detected at fM or pM concentrations, respectively. ECPA leverages target driven hybridization of a methylene blue (redox reporter) tagged DNA to a thiolated DNA attached to a gold surface. In this work, we have developed a novel assay for antibody detection, leveraging the ECPA concept. Here, simultaneous binding of recognition elements to paratopes of the antibody results in hybridization of methylene blue (MB) tagged DNA to thiolated-DNA (see schematic), and generated current is proportional to the antibody concentration. Using free-solution, thermoflourimetric analysis (TFA) of similar strands, we showed that probe flexibility is very important for sensor response, where ssDNA with polyethylene glycol (PEG) linkers improved sensitivities significantly. Using information from TFA, we used the same modification and demonstrated that analyte-dependent EC current was increased after ssDNA attachments to the electrode were substituted with more flexible PEG linkers (see data). This modification was introduced to both the MB-DNA and thiolated-DNA, allowing direct antibody sensing of 45 nM. With optimization in the near future, this new ECPA-based antibody sensor should be useful for clinical monitoring of the immune response. Figure 1