One of the important applications for biosensors is to help chemists and molecular biologists sort through huge “libraries” of chemical compounds that potentially have important pharmaceutical properties. For example, pharmaceutical researchers work to understand the protein-protein interactions that take place within our cells that lead to a particular disease, and they and wish to find chemical molecules that effectively stop a critical interaction from occurring, thus preventing the disease. Because the interaction of small chemical molecules with proteins is currently not completely predictable, researchers often test hundreds of thousands of different chemicals. It is important to perform these tests quickly, so the ability to perform them in a “high-throughput” fashion will reduce the amount of time it takes to test an entire library.
We are collaborating with research groups in the University of Illinois Chemistry department and the Beckman Institute to use photonic crystal biosensors for high-throughput screening for applications in Parkinson’s disease and cancer.
A label-free method has been developed to observe the biological activity of human breast cancer cells using photonic crystal (PC) biosensors incorporated within 96-well microplates. This method is used to study cell attachment, proliferation, and detachment induced by the exposure of cells to potential drug compounds. Figure 1 shows detection of breast cancer cell attachment and proliferation over a 24-hour time course. The biosensors and associated imaging instrument enable quantitative measurements and visualization of cell populations attached to the sensor surface with single cell resolution. This assay does not require the use of proprietary reagents or stains that typically induce the death of the cells under study. As such, repeated measurement of the same cells can be made without removing them from their culture environment, which allows for the direct determination of proliferation and apoptosis rates. Furthermore, the assay is simpler and more rapid than alternative cell proliferation assays and can be used for high-throughput screening applications. Using this method, the effect of 61 different plant extracts on breast cancer cells has been studied, in which some extracts were shown to reduce cell proliferation while others enhanced the rate of proliferation. The results are applicable to a wide range of cell types and compound libraries, and an assay for human pancreatic cancer cells is currently under development.
This research is being conducted in collaboration Dr. Kenneth Watkin in the University of Illinois Department of Speech and Hearing Science. The plant extracts were kindly provided by Dr. R. Chowdhury of the University of Dhaka, Bangladesh. We are thankful for support from the National Science Foundation (NSF), SRU Biosystems, and the United States Agency for International Development (USAID).
Small molecule screening for Parkinson’s disease
Protein-DNA interactions are essential for fundamental cellular processes such as transcription, DNA damage repair, and apoptosis. As such, small molecule disruptors of these interactions could be powerful tools for investigation of these biological processes, and such compounds would have great potential as therapeutics. Unfortunately, there are few methods available for the rapid identification of compounds that disrupt protein-DNA interactions. We have demonstrated that photonic crystal (PC) technology can be utilized to detect protein-DNA interactions and can be used in a high-throughput screening mode to identify compounds that prevent protein-DNA binding. The PC technology is used to detect binding between protein-DNA interactions that are DNA sequence-dependent (the bacterial toxin-antitoxin system MazEF), and those that are DNA sequence-independent (the human Apoptosis Inducing Factor [AIF]). The PC technology was further utilized in a screen for inhibitors of the AIF-DNA interaction, and through this screen, aurin tricarboxylic acid (ATA) was identified as the first in vitro inhibitor of AIF. Figure 1 shows the elevated percent inhibition (~80%) shown by ATA in comparison to 1000 other small molecules. The generality and simplicity of the photonic crystal method should enable this technology to find broad utility for identification of compounds that inhibit protein-DNA binding.
The research is being conducted in collaboration with Dr. Paul Hergenrother in the University of Illinois departments of Chemistry and Biochemistry.
- “A Label-Free High Throughput Optical Technique for Detecting Small Molecule Interactions,” B. Lin, J. Pepper, P. Li, H. Pien, and B.T. Cunningham, Biosensors and Bioelectronics, Vol. 17, No. 9, 827-834, September 2002.
- “A New Method for Label-Free Imaging of Biomolecular Interactions,” P. Li, B. Lin, J. Gerstenmaier, and B.T. Cunningham, Sensors and Actuators B, Vol. 99, 6-13, 2004.
- “Label-Free Assays on the BIND System,” B.T. Cunningham, P. Li, S. Schulz, B. Lin, C. Baird, J. Gerstenmaier, C. Genick, F. Wang, E. Fine, and L. Laing, Journal of Biomolecular Screening, Vol 9, 481-490, 2004.
- “A Label-Free Biosensor-Based Cell Attachment Assay for Characterization of Cell Surface Molecules,” B. Lin, P. Li, and B.T. Cunningham, Sensors and Actuators B, Vol 114, No. 2, 559-564, 2006.
- “Label-free Detection of Biomolecular Interactions: Applications in Proteomics and Drug Discovery,” B.T. Cunningham and L.L. Laing, Expert Reviews in Proteomics, Vol 3, No. 3, 271-281, 2006.
- “A label-free photonic crystal biosensor imaging method for detection of cancer cell cytotoxicity and proliferation,” L. Chan, S. Gosangari, K. Watkin, and B.T. Cunningham, Apoptosis, Vol. 12, No, 6, 1061-1068, 2007.
- “Label-free imaging of cancer cells using photonic crystal biosensors and application to cytotoxicity screening of a natural compound library,” L.L. Chan, S. Gosangari, K.L. Watkin, and B.T. Cunningham, Sensors and Actuators B, Vol. 132, 418-425, 2008.
- “Advantages and application of label-free detection assays in drug screening,” B.T. Cunningham and L.G. Laing, Expert Opinions in Drug Discovery, Vol. 3, No. 8, 891-901, 2008.
- “General Method for Discovering Inhibitors of Protein-DNA Interactions using Photonic Crystal Biosensors,” L.L. Chan, M.F. Pineda, J. Heeres, P. Hergenrother, and B T. Cunningham, ACS Chemical Biology, Vol. 3, No. 7, 437-448, 2008.
- “A method for identifying small molecule aggregators using photonic crystal biosensor microplates,” L.L. Chan, E.A. Lidstone, K.E. Finch, J.T. Heeres, P.J. Hergenrother, and B.T. Cunningham, Journal of the Association for Laboratory Automation (JALA), Vol. 14, No. 6, 348-359, 2009.
- “Identifying modulators of protein-protein interactions using photonic crystal biosensors,” J.T. Heeres, S.-H. Kim, B.J. Leslie, E.A. Lidstone, B.T. Cunningham, and P.J. Hergenrother, Journal of the American Chemical Society, Accepted, November 2009.
- “Photonic crystal integrated microfluidic chip for determination of kinetic reaction rate constants,” C.J. Choi, I.D. Block, B. Bole, D. Dralle, B.T. Cunningham, IEEE Sensors Journal, Vol. 9, No. 12, 1697-1704, 2009.
- “Detection of growth factor binding to gelatin and heparin using a photonic crystal biosensor,” L.L. Chan, A. Morgan, A. Sendemir-Urkmez, R. Jamison, and B.T. Cunningham, Materials Science and Engineering C, Vol. 30, 686-690, 2010.
- “Cytotoxicity screening of bangladeshi medicinal plant extracts on pancreatic cancer cells,” S. George, S.V. Bhalerao, E.A. Lidstone, I.S. Ahmad, A. Abbasi, B.T. Cunningham, K.L. Watkin, BMC Complementary and Alternative Medicine, Vol. 10, No. 52, 2010.
- “Cytotoxicity Effects of Amoora rohituka and chittagonga on Breast and Pancreatic Cancer Cells,” L.L. Chan, S. George, I. Ahmad, S.L. Gosangari, A. Abbasi, B.T. Cunningham, and K.L. Watkin, Evidence-Based Complementary and Alternative Medicine, 2011, Article ID 860605.
- “Label-free imaging of cell attachment with photonic crystal enhanced microscopy,” E.A. Lidstone, V. Chaudhery, A. Kohl, V. Chan, T.W. Jensen, L.B. Schook, R. Bashir, and B.T. Cunningham, Analyst, Vol. 136, No. 18, 3608-3615, 2011. DOI: 10.1039/C1AN15171A. (featured on inside cover).