Massively parallel single-molecule manipulation using centrifugal force.
Precise manipulation of single molecules has already led to remarkable insights in physics, chemistry, biology, and medicine. However, widespread adoption of single-molecule techniques has been impeded by equipment cost and the laborious nature of making measurements one molecule at a time. We have solved these issues by developing an approach that enables massively parallel single-molecule force measurements using centrifugal force. This approach is realized in an instrument that we call the centrifuge force microscope in which objects in an orbiting sample are subjected to a calibration-free, macroscopically uniform force-field while their micro-to-nanoscopic motions are observed. We demonstrate high-throughput single-molecule force spectroscopy with this technique by performing thousands of rupture experiments in parallel, characterizing force-dependent unbinding kinetics of an antibody-antigen pair in minutes rather than days. Additionally, we verify the force accuracy of the instrument by measuring the well-established DNA overstretching transition at 66 +/- 3 pN. With significant benefits in efficiency, cost, simplicity, and versatility, single-molecule centrifugation has the potential to expand single-molecule experimentation to a wider range of researchers and experimental systems.
Halvorsen
Ken
K
The Rowland Institute at Harvard, Harvard University, Cambridge, Massachusetts, USA.
Wong
Wesley P
WP
D000906:Antibodies / Q000737:chemistry; D002498:Centrifugation / Q000191:economics / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000737:chemistry; D004076:Digoxigenin / Q000737:chemistry; D004867:Equipment Design; D007700:Kinetics; D008853:Microscopy / Q000191:economics / Q000295:instrumentation / Q000379:methods*; D009690:Nucleic Acid Conformation; D052898:Optical Tweezers; D013057:Spectrum Analysis / Q000191:economics / Q000295:instrumentation / Q000379:methods*
D000906:Antibodies; D004247:DNA; D004076:Digoxigenin