We are developing a new approach for performing massively parallel single-molecule force measurements using centrifugal force. This is accomplished with a new instrument that we call the Centrifuge Force Microscope.
SINGLE-MOLECULE CENTRIFUGATION: A NEW APPROACH FOR MASSIVELY-PARALLEL SINGLE-MOLECULE MANIPULATION
Precise manipulation of single molecules is leading to remarkable insights in physics, chemistry, biology and medicine. However, widespread adoption of single-molecule techniques is impeded by equipment cost and the laborious nature of making measurements one molecule at a time. We are developing a new approach to solve these issues: massively parallel single-molecule force measurements using centrifugal force. This approach is realized in a novel instrument that we call the Centrifuge Force Microscope (CFM), 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 have demonstrated 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 have verified 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.
Video of the rapidly rotating Centrifuge Force Microscope (CFM): A minaturize microscope mounted to a rotary stage enables micro-to-nanoscale motions to be observed within a dynamically controllable centrifugal force field. By tethering beads in the sample to the coverslip, thousands of single-molecule force experiments can be made in parallel, with forces ranging from femtoNewtons to microNewtons.
Antibody-antigen unbinding kinetics obtained from massively-parallel single-molecule measurements made with the Centrifuge Force Microscope (CFM): Force-dependent unbinding of digoxigenin and its antibody. Force clamps ranging from hundreds of femtoNewtons to several picoNewtons were applied using the CFM (filled triangles), as well as with the optical trap (open triangles). Each CFM data point was obtained from a single experiment lasting a few minutes, while optical trap data was collected serially over a period of many hours. Histograms of the rupture times were fit with a decaying exponential to obtain the off-rate at each force (inset).
Video showing the progression of a bond rupture experiment: Thousands of receptor-functionalized beads are brought into contact with a ligand-functionalized coverslip, then pulled away from the surface using a centrifugal force field. At the beginning of the movie, beads resting against the coverslip are in focus. Next, the objective is focused one-tether-length away from the coverslip. When the centrifugal force field is generated, unattached beads leave the surface immediately, whereas beads tethered by single-molecules are brought into focus and beads stuck to the surface remain blurry. The detachment of single-tethered beads over time can be used to determine the force-dependent off-rate.
MULTIPLEXED SINGLE-MOLECULE FORCE SPECTROSCOPY USING A CENTRIFUGE
Recently, to extend the capabilities of our approach, and to increase its accessibility, we have developed a new miniature version of the CFM that uses something that most biomedical researchers already have and use--the benchtop centrifuge. Our new design repurposes a benchtop centrifuge for high-throughput single-molecule experiments with high-resolution particle tracking, a large force range, temperature control and simple push-button operation. Furthermore, by incorporating DNA nanoswitches to enable repeated interrogation by force of single molecular pairs, we have demonstrated increased throughput, reliability and the ability to characterize population heterogeneity. We have performed spatiotemporally multiplexed experiments to collect 1,863 bond rupture statistics from 538 traceable molecular pairs in a single experiment, and have shown that 2 populations of DNA zippers can be distinguished using per-molecule statistics to reduce noise
Left: Benchtop CFM: The benchtop CFM device, consisting of the CFM unit itself (on the top), parts for transmitting the camera signal as well as a battery (on the right), fits into two standard buckets of a common laboratory centrifuge that are balanced by counterweights in the respective opposite buckets.
Right: Benchtop CFM: The picture on the top shows a DNA nanoswitch that forms a looped structure when a bond is formed between the attached reactive components (e.g. receptor-ligand pair shown in red and green); at one end it is attached to the sample stage and at the other to a bead (top). By applying centrifugal forces to the bead in the CFM device, the bond between the reactive components can be repeatedly ruptured, opening up the loop and increasing the length of the DNA tether (bottom), enabling highly reliable measurements of molecular interactions. In the CFM, many beads can be interrogated in parallel, enabling high-throughput single-molecule measurements (bottom left). In the video in the bottom right, the camera captures these rupture events in real time by registering the bead at a different spot.