Polymeric Enzyme-Gold Probes for Ultrasensitive Protein Blotting

Award Information
Agency: Department of Health and Human Services
Branch: N/A
Contract: 1R43GM084542-01
Agency Tracking Number: GM084542
Amount: $180,349.00
Phase: Phase I
Program: SBIR
Awards Year: 2008
Solicitation Year: 2008
Solicitation Topic Code: N/A
Solicitation Number: PHS2007-2
Small Business Information
DUNS: 784163446
HUBZone Owned: Y
Woman Owned: Y
Socially and Economically Disadvantaged: Y
Principal Investigator
 () -
Business Contact
Phone: (631) 205-9490
Email: hainfeld@nanoprobes.com
Research Institution
DESCRIPTION (provided by applicant): We propose to develop ultrasensitive detection reagents for protein blotting which will provide users with a means to detect as little as 0.001 to 0.0002 amol (10-21 to 2 x 10-22 mol) of a protein target in a two hour p rocedure. The high sensitivity will be provided in two ways. Firstly, the new probes will incorporate both enzymatic and gold nanoparticle labels in close proximity. When developed with a metallographic substrate, they generate a signal through two sensiti ve and mutually reinforcing processes: autometallographic development of the gold particle, and catalytic metal deposition by the enzyme, known as enzyme metallography. This combination will provide up to 100 times greater sensitivity than either method al one. Secondly, multiple copies of the enzyme-gold construct, together with multiple antibodies or other targeting agents, will be conjugated to a soluble polymer backbone, providing the probe with built-in signal amplification. The new conjugates will be u sed as a secondary detection reagents in Western blotting and other protein blotting methods. To evaluate their performance, the new probes will be compared head-to-head with chemiluminescent detection in experiments to quantitate the expression of _-1 and _-4 integrins and HER2 proteins, and to determine the relationship between integrin and HER2 protein expression in breast cancer. After evaluation in cultured cell lines, the reagents will be compared with chemiluminescence for the assessment of _-1 and _ -4 integrins and HER2 protein in two series of breast cancer cases, one constructed with HER2-overexpressing cases and one with EGFR-overexpressing cases. Comparison of the western blot results with immunohistochemical staining, in situ hybridization, and DNA microarray data will be used to assess the correlation between HER2 and integrin expression. The extent of direct binding interaction between _-4 integrins and HER2 protein will then be investigated using immunoprecipitation followed by western blottin g with the new reagents. This project will develop a new type of ultrasensitive detection reagent that will increase routine detection sensitivity within a short experimental time for protein blotting, and ultimately for many other methods such as in situ hybridization and nucleic acid blotting where detection sensitivity is critical. This will enable more precise quantitation of proteins from smaller samples by Western blotting, and more sensitive and precise visualization of gene copies. This will enable faster, more accurate diagnosis of cancer, infectious diseases and prion diseases, improve therapy selection, and provide new tools for the study of cancer at the molecular level.

* Information listed above is at the time of submission. *

Agency Micro-sites

SBA logo
Department of Agriculture logo
Department of Commerce logo
Department of Defense logo
Department of Education logo
Department of Energy logo
Department of Health and Human Services logo
Department of Homeland Security logo
Department of Transportation logo
Environmental Protection Agency logo
National Aeronautics and Space Administration logo
National Science Foundation logo
US Flag An Official Website of the United States Government