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.
Many important biomarkers of disease or human health status must still be sent away for measurement in clinical laboratories, including biomarkers for which the physician desires rapid readout such as heart disease markers, immunosuppressant drug monitoring, or diabetes markers. While electrochemical biosensors have been very useful for a few select markers (e.g. glucose), most biomarkers remain inaccessible. This project seeks to develop a DNA-based nanostructure that can be attached to electrode surfaces and ultimately permit simple, rapid measurement of multiple biomarkers that were previously not measurable with electrochemistry.
