Quantifying Intermolecular Interactions

Atomic force microscopes can generate a highly detailed topological map of a specimen by moving a stylus coupled to a cantilever over the sample surface, using a laser to measure deflection of the cantilever.1 Now, researchers at the University of California at Berkeley have adopted this principle to create a system capable of quantifying intermolecular interactions, with potential applications for both clinicians and high-throughput proteomics researchers.2 In a research article published in the September issue of Nature Biotechnology, Arun Majumdar, professor of mechanical engineering at Berkeley, and coauthors at Oak Ridge National Laboratory in Oak Ridge, Tenn., the University of Southern California in Los Angeles, and the Lawrence Berkeley National Laboratory at UC-Berkeley, describe a system to quantify prostate-specific antigen (PSA), a prostate cancer protein marker. The PSA test uses a cantilever 200 mm long and 0.5 mm wide, one side of which is coated with gold and conjugated to an antibody that recognizes PSA. Majumdar and his colleagues incubated the cantilever, which is a diving board-like device, with a simulated human serum that contained high concentrations of human plasminogen, human serum albumin, or bovine serum albumin, with varying concentrations of PSA. When the antibodies on the cantilever captured the PSA in the solution, the device bent a few nanometers in height. A laser beam aimed at the cantilever detected the deflection. Significantly, the researchers found that the amount of deflection was proportional to the PSA concentration, indicating that this assay can be used to determine whether, and at what concentration, PSA is present in a sample. Clinical laboratories generally test for PSA using ELISA, with a lower detection limit of 0.2 ng/ml. The scientists in this study detected PSA over the concentration range of 0.2 ng/ml to 60 mg/ml, which is as sensitive as ELISA. However, the authors note that they could actually achieve even higher sensitivity by changing the geometry (i.e., length) of the cantilever. Because the presence of high concentrations of "background" proteins didn't significantly affect the cantilever, the authors believe that the system will perform well when tested against actual human serum, says Majumdar. In February, Majumdar's group published an article to explain the science behind cantilever deflection.3 They demonstrated that the magnitude and direction of cantilever motion, in response to a binding event, depends on the interplay between two components of the Gibbs free energy of a reaction. The first component is conformational entropy, the result of changes in molecular configuration that occur during binding reactions such as DNA hybridization. The second component is intermolecular energy, such as electrostatic repulsive forces between adjacent molecules, which can be significantly affected by binding reactions. Majumdar notes that this cantilever approach offers a number of benefits over existing detection assays. First, the system requires no labeling or reporter molecules at all. In contrast, an ELISA requires using an antibody conjugated to a reporter enzyme, such as horseradish peroxidase. Second, the method is adaptable to a multiplexed format. For example, an array of cantilevers, each coated with a specific antibody, can test for multiple serum proteins. Such a test could improve both the speed and cost of cancer diagnostics. Third, the system can detect a wide variety of intermolecular interactions, including protein-protein, protein-DNA, DNA-DNA, and protein-ligand interactions. A group of scientists at IBM Research in Zurich, Switzerland, and Majumdar in his earlier publication, have already shown that cantilevers can readily detect single base pair mismatches in DNA duplexes.3-5 Finally, Majumdar notes that this technology could be used to develop a protein chip--the proteomic equivalent of a DNA microarray. Majumdar says that the potential commercial implications of this work have not escaped the authors' notice. Thomas Thundat, one of the coauthors of the Nature Biotechnology article, has patented the use of a single cantilever to detect an analyte. The group has filed for a second patent, covering arrays of cantilevers, which they have licensed to a start-up company, and they are in the process of applying for a third patent to cover an alternative multiplexing system. Jeffrey M. Perkel can be contacted at jperkel@the-scientist.com. References 1. C. Wright-Smith, C. Smith, "Atomic force microscopy," The Scientist, 15[2]:23, Jan. 22, 2001. 2. G. Wu et al., "Bioassay of prostate specific antigen (PSA) using microcantilevers," Nature Biotechnology, 19:856-60, Sept. 1, 2001. 3. G. Wu et al., "Origin of nanomechanical cantilever motion generated from biomolecular interactions," Proceedings of the National Academy of Sciences, 98:1560-4, Feb. 13, 2001. 4. J. Fritz et al., "Translating biomolecular recognition into nanomechanics," Science, 288:316-8, 2000. 5. K. Hansen et al., "Cantilever-based optical deflection assay for discrimination of DNA single nucleotide mismatches," Analytical Chemistry, 73:1567-71, 2001.

Quantifying Intermolecular Interactions

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