A Force-Clamp Spectrometer and Software for Single Bond Cleavage Analysis
Lead Inventors: Julio M. Fernandez, Ph.D.Problem or Unmet Need:Chemical reactions involving bond cleavage can be detected using a variety of spectroscopic techniques. However, they are always detected in bulk or through coupled reactions, thus preventing examination of population heterogeneities or fine detail in reaction pathways.This technology comprises a stand-alone low-cost manufactured force-clamp spectrometer and its use in analysis of single-bond rupture using atomic force spectroscopy. Built on the principles of AFM, this stand-alone force-spectrometer has the addition of specialized feedback electronics enabling operation in constant force mode. This technology also includes customized software for automated data acquisition and analysis, developed specifically for use with the force spectrometer for various single-molecule studies. One aspect of the technology includes the use of the force spectrometer in an assay in which a polymer or another compound, acts as a substrate molecule with covalent bonds cross-linking its chain at known positions to create loops that open only upon cleavage of the bonds. In the assay, such a substrate is subject to a constant force in the force spectrometer, and its extension is measured over time. When a cross-linking bond is cleaved, the substrate extends by a known amount corresponding to the length of the loop. This creates a unique fingerprint of the specific reaction taking place. Precise control of the bond straining force allows for force spectroscopy of the reaction.
The force spectrometer device is small and cheap to manufacture The reaction fingerprint provides complete certainty of each event, therefore, control experiments are therefore not necessary Each individual bond cleavage is detected immediately, allowing for measurement of reaction kinetics Force spectroscopy enables the probing of chemical mechanisms that can elucidate aspects of a reaction, such as transition state geometry, not possible through any other method
Precise kinetic measurements of single bond cleavage reactions Study of disulfide bonds in protein mis/unfolding Study of mechanical resistance of chemical bonds such as those involved in protein-protein, antibody-protein and protein-living cell interaction High throughput screen for detecting efficient therapeutic agents Study of polymer elasticity A probe for studying structure and single-molecule biophysics of DNA and proteins Elucidation of mechanism details of enzyme function not available through any other method
This technology comprises a stand-alone low-cost manufactured force-clamp spectrometer and its use in analysis of single-bond rupture using atomic force spectroscopy. Built on the principles of AFM, this stand-alone force-spectrometer has the additio...
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