Diagnosis and treatment of medical conditions could be revolutionized by technology capable of rapid and
specific quantification of an arbitrary analyte in real time, over a wide concentration range. To quantify wide
ranging clinically relevant targets—small molecules, nucleic acids, or proteins—most method development has
drifted towards being target-focused and has lacked generalizability. Currently, the toolbox for potential point-of-
care (POC) analysis is a conglomerate of methods or specially targeted probes, and measurement of many
targets remains inaccessible to anything other than a large clinical laboratory. There is a pressing, unmet need
to develop a platform amenable to rapid, quantitative readout of multiple classes of clinically relevant targets.
Electrochemical (EC) sensors have attracted renewed interest for biomarker and drug quantification due to low
cost and adaptability to the POC. Still, current approaches (aptasensors, steric hindrance assays) are lacking in
generalizability or have complex, noncovalent structures that are not amenable to simple, drop-and-read
workflow. In this proposal, we describe our recent development of an innovative nucleic acid nanostructure that
exhibits unprecedented generalizability. Strong preliminary data shows this same nanostructure capable of
quantifying proteins and antibodies (streptavidin, anti-digoxigenin, anti-exendin-4), peptides (exendin-4), and
small molecules (biotin, digoxigenin, tacrolimus). The immunosuppressant drug, tacrolimus, can already be
quantified in its therapeutic range. Our objective in this funding period is not only to further develop this new and
promising technique, but also to develop a fully surface-confined version that allows true drop-and-read assay
workflow that is ideal for POC or real-time clinical measurements. In Aim 1, we will expand the utility of the DNA
nanostructure, and modification schemes will be adapted to the most efficient means of detecting proteins,
peptides, and small molecules. Nine targeted analytes are relevant to stress/heart disease, immunosuppression,
and diabetes monitoring. In Aim 2, we will use organic chemistry to make structural modifications to small
molecules or peptides appended to anchor-DNA to fine-tune antibody binding equilibria and improve competitive
assays for drop-and-read quantification. In Aim 3, we will develop a fully surface-confined sensor architecture
for drop-and-read workflow and real-time measurements. Antibody-DNA or Fab-fragment-DNA conjugates will
be used for tethering anchor molecules to the surface alongside DNA nanostructures. Finally, Aim 4 studies will
develop instrumentation for improved sensitivity and user experience with the assay. The rationale for this
research is to enable measurement of clinically relevant analytes previously inaccessible to EC, while providing
a generalizable framework for many other future analytes. The proposed work is significant as a first-of-its-kind
assay platform, which we expect to lead to an expanded list of future analytes, previously inaccessible to EC.
This proposal is thus innovative in both its technological approach and in its human health applications.
Preliminary evidence strongly supports feasibility, and the research team has a proven track-record of success.
