Although immunoassays are a critical part of bioanalytical chemistry, the rapid and selective analysis of biomolecules continues to be a complicated aspect of biomedical research. The long-term goal is to develop single-entity electrochemistry to improve the current methods, primarily based on enzyme-linked immunosorbent assay (ELISA) and related techniques. The PI aims to use electrochemical detection for simultaneous qualitative and quantitative analysis down to a single bioconjugate to allow rapid routine detection. The overall objective in this proposal is to use antibody/antigen interactions as proof of concept for single entity amperometry: measure the current vs. time at ultramicroelectrodes (UME, diam < 30 micrometers) that will respond to single antibody/antigen conjugates. The antibodies will be tagged to Au nanoparticles (NPs) and detected using electrochemical reactions. The central hypothesis is that we can measure currents from individual antibody/antigen conjugates by measuring the reaction rate of separate redox mediators. The rationale is that electrochemical detection of antigens should be possible under conditions relevant to biomedical analysis. We will achieve single biomolecule resolution by detecting individual bioconjugates interacting with an electrode surface. By isolating a single bioconjugate, we will demonstrate a detection limit of a single molecular antigen in the direct assay mode. The project will test the central hypothesis by pursuing the following specific aims: (1) developing a redox-mediated approach suitable for antibody-NP detection. (2) Antibody/Antigen detection in biological samples. We will study the selectivity of the technique and leveraging electron transfer (ET) through pinholes as a suitable mechanism to overcome biofouling. This aim also includes the effect of non-specific binding and agglomeration or aggregation. The distance between the electrode and the NP, attached to an antibody, is relatively large for tunneling, and the distance-dependence of ET will translate into a time-dependence of the experimental current that will be used to discriminate specific vs non-specific interactions. To design a redox-mediated scheme, we will use Marcus theory of ET to select the redox pairs. The detection schemes will be validated with assays used for ensemble measurements, and we will pursue the detection of single molecules using the novel redox-mediated single-entity electrochemical scheme. The applicant believes the proposed research is innovative because it focuses on single-entity electrochemistry that is expected to yield single molecule detection through antibody/NP conjugates. The proposed research is significant because it is expected to provide a novel way for the rapid and sensitive detection of antibody/antigen interactions, with the potential of enabling simultaneous qualitative and quantitative information with minimal supplies and electrochemical instrumentation.

The proposed research is relevant to public health because it focuses on developing a new and alternative strategy for sensing biomolecules through antigen-antibody interactions using electrochemical single nanoparticle detection. These measurements can potentially advance the detection of disease markers down to a single nanoparticle-bioconjugate, providing an efficient diagnosis before symptoms appear and guiding therapy and isolation decisions. Thus, the proposed research is relevant to the part of the NIH’s mission concerning reducing illness and disability.