Abstract: We present an Anti-Brownian Electrokinetic trap (ABEL trap) capable of trapping individual fluorescently labeled protein molecules in aqueous buffer. The ABEL trap operates by tracking the Brownian motion of a single fluorescent particle in solution, and applying a time-dependent electric field designed to induce an electrokinetic drift that cancels the Brownian motion. The trapping strength of the ABEL trap is limited by the latency of the feedback loop. In previous versions of the trap, this latency was set by the finite frame rate of the camera used for video-tracking. In the present system, the motion of the particle is tracked entirely in hardware (without a camera or image-processing software) using a rapidly rotating laser focus and lock-in detection. The feedback latency is set by the finite rate of arrival of photons. We demonstrate trapping of individual molecules of the protein GroEL in buffer, and we show confinement of single fluorophores of the dye Cy3 in water.

© 2008 Optical Society of America

OCIS codes: (180.2520) Microscopy: Fluorescence microscopy; (180.5810) Microscopy:

Scanning microscopy;(300.6280) Spectroscopy : Spectroscopy, fluorescence and luminescence

 1. Introduction

The invention of traps for individual atoms and molecules in the gas phase led to new physical measurements (e.g. of the anomalous magnetic moment of the electron[fusion_builder_container hundred_percent=”yes” overflow=”visible”][fusion_builder_row][fusion_builder_column type=”1_1″ background_position=”left top” background_color=”” border_size=”” border_color=”” border_style=”solid” spacing=”yes” background_image=”” background_repeat=”no-repeat” padding=”” margin_top=”0px” margin_bottom=”0px” class=”” id=”” animation_type=”” animation_speed=”0.3″ animation_direction=”left” hide_on_mobile=”no” center_content=”no” min_height=”none”][1]), and new analytical techniques (e.g. ion cyclotron mass spectrometry[2]). Molecules in solution show much more complex behavior than molecules in the gas phase, but until now there have not existed devices capable of trapping single molecules in aqueous buffer. To address this issue we designed and built an Anti-Brownian Electrokinetic trap (ABEL trap) that grabs and holds single proteins in water at room temperature. We also used this device to temporarily confine single fluorophores of the dye Cy3 (molecular weight »500), although these dye molecules were not stably trapped for long times. These fluorophores have a mass smaller by a factor of 6£103 than that of the smallest objects previously trapped under comparable conditions.

In recent years there has been much interest in designing systems to track[3, 4, 5, 6, 7, 8, 9, 10] and trap[11, 12, 13, 14] small particles in solution. These systems all rely on observing the motion of a particle, and then canceling this motion either by translating the sample stage or by applying electrokinetic forces to the particle. The latency of the feedback loop determines the minimum size of particle that can be trapped and the minimum area to which a particle can be trapped. In the mechanical feedback systems, this latency is typically limited by the inertia of the feedback stage; in the electrokinetic systems the response of the particle to an applied field occurs faster than our 40 kHz measurement bandwidth, so the latency is limited by the bandwidth of the tracking system.

In the ABEL trap a small particle diffuses in a pancake-shaped fluid element defined by a nanofluidic cell. A fluorescence tracking system follows the two-dimensional Brownian motion of the particle, and a feedback system applies voltages that induce a combined electrophoretic and electroosmotic drift that cancels the Brownian motion. An earlier version of the ABEL trap used video tracking and computer-controlled feedback to achieve a latency of 4.5 ms, principally due to the 300 Hz maximum frame-rate of the camera. This latency set a lower bound of 20 nm on the diameter of objects that could be trapped in water (although smaller objects could be trapped by increasing the viscosity of the solution[14]). With this software based ABEL trap it was possible to observe the shape fluctuations of single DNA molecules in free solution[15, 16] and to control the motion of nanoparticles subject to arbitrary forcefields[17].

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