The goal of this Phase II STTR project is to develop a real-time label-free biosensor that can analyze 96 samples at a time, compared to the 6 samples possible with current technologies. This platform will initially be demonstrated with G-protein-coupled receptors (GPCRs) and antibodies. What both applications have in common is the need for higher- throughput sensing, and demonstrating the integrated system for these will illustrate its versatility and potential contributions across the wide spectrum of biosensor applications. For the GPCR demonstration, our work with standard SPR instruments has shown that the choice of detergent(s) is critical for obtaining active solubilized receptor. However, standard low throughput SPR biosensors have two overwhelming drawbacks: (1) The analysis of 96 solubilization conditions requires more than two days and the receptor loses significant activity during this time, which makes it difficult to compare the results obtained at the beginning and end of the analysis and (2) the SPR instrument is limited to testing only one analysis buffer at a time, which means that the success of the entire assay depends on the initial choice of analysis buffer. In Phase I, we developed a 96-channel Continuous Flow Microspotter (CFM) printhead and demonstrated the ability to print GPCRs onto a sensor surface directly from crude media using up to 96 different analysis buffers. The GPCRs were also kept wetted and active throughout the printing process by the CFM's enclosed microchannel printing network. In the final experiment, we solubilized the GPCR CCR5 from whole cells using 192 different detergent conditions and spotted them onto an SPR sensor surface using our CFM printhead. We then tested the activity of the receptor and used the ligand binding results to determine that a certain combination of detergents best enhanced receptor activity. To run this analysis with a standard Biacore technology (e.g. T100) would have required four days of instrument time. In comparison, we were able to perform the analysis in less than 2 hours. In Phase II, we propose to integrate the 96-channel CFM from Phase I with a commercial SPR imager to enable automated interaction analysis in a highly parallel format. The following specific aims detail the combination of Wasatch's microfluidic technologies with the commercial IBIS SPR imager to produce a high-throughput label-free biosensor. 1. Refine the 96-channel CFM printhead design from Phase I to enable optimal performance when mounted on the IBIS SPR imager. 2. Mount the 96-channel CFM onto the IBIS SPR imager and optimize the fluidic parameters that affect platform sensitivity and uniformity. 3. Automate the CFM &SPR components within one seamless instrument: Automation of the flow cell positioning, sealing, fluid handling, valving, in-line degassing and temperature control. Integration of the CFM and SPR imager control software and data collection/analysis software. 4. Demonstrate use of the automated system with GPCRs and antibodies.

Public Health Relevance

Antibody analyses are one of the most common applications of biosensor technology and are typically straightforward. GPCRs are the hottest and most challenging system that biosensor users are tackling. Up to half of the drugs on the market today modulate some form of GPCR activity, and it is estimated that 25-50% of the total drug targets are in the GPCR families. GPCRs are the most studied of the major drug target classes, yet they are challenging to work with because they are normally membrane associated, present in low abundance, and unstable. By enabling the high throughput study of GPCRs, there is enormous potential for speeding drug development, treatments, and the associated health of patients with hundreds of different diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Small Business Technology Transfer (STTR) Grants - Phase II (R42)
Project #
5R42MH084372-03
Application #
8337293
Study Section
Special Emphasis Panel (ZRG1-IMST-G (10))
Program Officer
Delcarmen-Wiggins, Rebecca
Project Start
2008-08-01
Project End
2014-08-30
Budget Start
2012-09-01
Budget End
2014-08-30
Support Year
3
Fiscal Year
2012
Total Cost
$377,348
Indirect Cost
Name
Wasatch Microfluidics
Department
Type
DUNS #
171205177
City
Salt Lake City
State
UT
Country
United States
Zip Code
84123
Alonzo 3rd, Francis; Kozhaya, Lina; Rawlings, Stephen A et al. (2013) CCR5 is a receptor for Staphylococcus aureus leukotoxin ED. Nature 493:51-5