Microfluidic multifunctional probe array dielectrophoretic force spectroscopy with wide loading rates

The simultaneous investigation of a large number of events with different types of intermolecular interactions, from nonequilibrium high-force pulling assays to quasi-equilibrium unbinding events in the same environment, can be very important for fully understanding intermolecular bond-rupture mechanisms. Here, we describe a novel dielectrophoretic force spectroscopy technique that utilizes microsized beads as multifunctional probes for parallel measurement of intermolecular forces with an extremely wide range of force rate (10 ^-4 to 10 ⁴ pN/s) inside a microfluidic device. In our experiments, various forces, which broadly form the basis of all molecular interactions, were measured across a range of force loading rates by multifunctional probes of various diameters with a throughput of over 600 events per mm ², simultaneously and in the same environment. Furthermore, the individual bond-rupture forces, the parameters for the characterization of entire energy landscapes, and the effective stiffness of the force spectroscopy were determined on the basis of the measured results. This method of determining intermolecular forces could be very useful for the precise and simultaneous examination of various molecular interactions, as it can be easily and cost-effectively implemented within a microfluidic device for a range of applications including immunoassays, molecular mechanics, chemical and biological screening, and mechanobiology.