3D Anti-Brownian ELectrokinetic (ABEL) trap for studies on nanoparticles and biomolecules

A three-dimensional ABEL trap with fast feedback cycle of 13.5 μs was developed for confocal microscopy studies on fluorescent nanoscale objects in aqueous solution. The trap contains a microfluidic cross-channel made by bonding two glass coverslips having 25 μm thick PDMS layers with tapered channels. The geometry was designed using COMSOL to enable four ±10 V potentials to drive the fluid via electroosmosis in any direction at a speed sufficient to counteract Brownian diffusion. Four laser beams are focused in the middle of the crossed channels to ~0.5 μm by a 1.2 NA objective. The foci partially overlap but are slightly offset in a tetrahedral pattern. When a molecule diffuses into the foci, fluorescence is excited by 304 MHz interleaved pulses in the four beams and collected onto two single-photon avalanche diodes to feed an FPGA circuit, which performs time-gated photon counting and applies an algorithm to adjust the voltages to trap the molecule. Results show trapping of 40 nm FluoSpheres—for a remarkable time of ~5 minutes—as well as 20 nm FluoSpheres and even single molecules of Streptavidin-Alexa 647. The 3D design avoids collisions of the trapped molecule with glass surfaces and sticking, which have limited previous biophysical studies using 1D and 2D ABEL traps.