With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, Professor Bornhop at Vanderbilt University and Professor Flowers at Lehigh University are developing a unique sensing technique that greatly simplifies the measurement of molecular interactions. The method is termed backscattering interferometry (BSI). BSI is a simple and inexpensive way of studying chemical reactions, like those used to understand how a drug molecule works, to monitor environmental pollutes, or to diagnose disease from a blood test. BSI works by placing a sample into a microscale, glass or plastic, liquid-filled chamber that is illuminated by a laser like those used in a grocery store checkout. When the laser light is reflected back from the chamber it forms a set of alternating bright and dark spots (interference pattern) that are captured by a camera. These patterns are exquisitely sensitive to chemical or biochemical changes of the solution in the chamber. Most tools used to measure chemical interactions require the solution to be modified by tagging, labeling, or attachment to the chamber surface, yet in BSI none of these modifications are required to make the measurements. With support from this grant Professors Bornhop and Flowers are developing and validating the physical model for these "free-in-solution" measurements, by showing that the BSI signal originates from property changes in the solution, such as molecular shape (conformation) and the amount of water closely associated with the molecule (hydration). These properties have not previously been recognized or measured. The BSI model, termed the Free-solution Response Function (FreeSRF), is being expanded to a wide array of chemical and biochemical interactions (such as protein-to-protein, DNA-to-DNA interactions)to determine if FreeSRF can be used to predict chemical structural changes. Professor Bornhop is working with companies closely to commercialize a benchtop, compressed BSI that will allow more users to apply the developed approach to their own research. The students engaged in the project receive a broad range of scientific training, ranging from the construction and modification of the instrument, to the development and benchmarking of bioassays, to the development and refinement of the FreeSRF models. Professor Bornhop is also actively engaged in K-12 outreach through the Vanderbilt Student Volunteers for Science (VSVS) program and the Tennessee Governors' School program.

Back-scattering interferometry (BSI) is the refractive index (RI) sensor used in the studies performed under this NSF project. BSI is a free-solution, label-free technique with sensitivity that rivals fluorescence assays, is compatible with a wide range of analytes, can be used with complex matrices, and is fully quantitative. Professors Bornhop and Flowers are studying how intrinsic property changes report molecular binding events at unprecedented sensitivities (pM-fM) in complex milieu, even when the individual binding partners are undetectable by BSI. Having shown that interferometric, free-solution, label-free studies performed on BSI report conformation and hydration changes, two groups are working together to develop a model that provides a theoretical and experimental underpinning for the phenomenon being measured by BSI and other label-free methods. In addition, building on their theory that refines, or redefines, the basis for label-free free-solution RI measurements, the Free-Solution Response Function (FreeSRF), they are expanding investigations to a more diverse set chemical and biochemical interactions, including DNA and RNA hybridization, metal ion-chelator binding, and protein folding. It is anticipated that the research will impact on how scientists generally think about label-free sensing, going beyond BSI and SPR, leading to the development of new sensing/imaging systems and novel approaches to the analysis of ligand-protein interactions. A newly developed, highly compensated scattering interferometer (CSI), is being combined with FreeSRF to enable translation of the methodology to a broader scientific community. This bench-top CSI for mix-and-read free-solution assays enables a wide array of applications and serve as a foundation for an experiment-based curriculum that teaches students how understanding interaction determinations are critical in chemistry, biochemistry, molecular biology, and medical technology.

National Science Foundation (NSF)
Division of Chemistry (CHE)
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Lin He
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Vanderbilt University Medical Center
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