A Flexible Anti-Brownian Electrokinetic (ABEL) Trap for Single-Molecule Immobilization in Solution

Abstract

We demonstrate our ability to trap and probe individual fluorescent particles in solution using an improved anti-Brownian electrokinetic (ABEL) trap. Traditional single-molecule immobilization techniques include surface attachment and laser tweezers; the former technique often disrupts fragile biochemical systems, while the latter requires that molecules be conjugated to large beads. The ABEL trap circumvents these issues by tracking the motion of a particle via fluorescence, and applying electrokinetic feedback forces to cancel its Brownian motion.Our ABEL trap suppresses the Brownian motion of a fluorescent particle as follows. A laser beam is rapidly steered in a small scan pattern near the center of a microfluidic cell. An avalanche photodiode detects fluorescence photons from the molecule. A field-programmable gate array compares the precise arrival time of each photon with the known position of the laser, and generates a corresponding feedback voltage. The feedback voltages is amplified and applied to the trap. We use a broadband supercontinuum laser with an acousto-optic tunable filter to enable fluorescent tracking in any part of the visible spectrum, and we scan the laser using electro-optic deflectors that can function at up to 100 kHz. This combination of hardware enables precise spatial, temporal, and spectral control of our illumination and detection optics and can apply feedback at a latency of 2 μs, a better-than-tenfold improvement over previous trap designs. We hope that these improvements will enable us to trap single small-molecule fluorophores in solution.The flexibility of the ABEL trap makes it amenable for a wide variety of biophysical studies. Work is currently underway to apply the ABEL trap to study the dynamics of DNA in solution. In the future, we hope to apply the trap to study the kinetics of proteins such as proteorhodopsin.