Developing a redox-mediated single NP detection for bioconjugate analysis.

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.

Grant Number: 5R21GM155760-02
NIH Institute/Center: NIH
Principal Investigator: Mario Alpuche-Aviles