The Anti-Brownian ELectrophoretic Trap (ABEL Trap): Fabrication and Software

The Anti-Brownian ELectrophoretic trap (ABEL trap) is a new device that allows a user to trap and manipulate fluorescent objects as small as 20 nm freely diffusing in solution. We describe in detail how to build an ABEL trap. CHALLENGES IN NANOMANIPULATION One of the outstanding challenges of nanotechnology is to develop a means to trap and manipulate individual molecules in solution. To study a biomolecule one would like to hold the molecule still, to turn it around, to pull on its ends, and to bring it into contact with other molecules—all in a highly controlled manner and in the molecule’s native environment. Such a level of control would also allow one to build custom molecular-assemblies designed for specific tasks. Laser tweezers have led to a revolution in the fields of nanomanipulation and biophysics. Singleand multiplebeam optical traps have been used to assemble structures from dielectric microparticles; to probe the intrinisic mechanical properties of DNA and RNA; and to probe the action of DNA-processive enzymes. Although less widespread, magnetic tweezers, and AC dielectrophoresis have also been used to trap and manipulate micron-scale objects. Unfortunately, all of these techniques fail for molecule-sized objects because the trapping force is proportional to the volume of the trapped object: to trap a 10 nm object requires a million times as much input power as to trap a 1-micron object. Biophysicists seeking to study individual molecules have a limited set of options for confining the molecule. One common practice is to immobilize the molecule on a surface. Unfortunately surface chemistry is notoriously finicky, and there is a persistent doubt whether the tied-down molecule acts the same as its free-solution comrades. Another technique is to immobilize the molecule in the pores of a gel. However, if the pores are small enough to confine the molecule, then it is difficult to get the molecule into the pores in the first place. Finally, in fluorescence correlation spectroscopy (FCS), molecules in free-solution are observed as they diffuse through a tightly focused laser beam. While these molecules may be in their native environment, the short residence times of FCS (typically a few milliseconds), limit the technique to the study of fast processes. Many biological processes occur on the timescale of seconds to hours. As a concrete example of the problem facing single-molecule researchers, nobody has found a way to observe the catalytic cycle of the chaperonin GroEL, which takes 7 – 15 seconds, without disturbing this cycle. The Paul trap and Dehmelt’s improvements on the Penning trap allowed scientists to observe individual gas-phase ions for long times. These traps have been used in basic physics to measure the magnetic moment of the electron, and are also widely used as an analytical tool in massspectrometry. To-date, no equivalent tool has been developed for studying single-molecules in solution. Feedback control is widely used to stabilize the motion of stochastic systems, where the stochasticity may arise from quantum, thermal, or manufacturing fluctuations. In particular, feedback may be used to cancel the Brownian motion of a single nanoscale object in solution, over some finite bandwidth. In contrast to passive trapping schemes, feedback trapping has the advantage that the applied potential need not have any local minima. Recent proposals have discussed using feedback to track the Brownian motion of individual fluorescent molecules in solution. For nanoscale objects, electrophoretic forces are far stronger than either magnetic or optical forces and are thus most amenable to inclusion in a feedback system. THE ANTI-BROWNIAN ELECTROPHORETIC TRAP (ABEL TRAP) Over the past year and a half, we have developed an Anti-Brownian ELectrophoretic trap (ABEL trap): a device that uses quasi-DC electric fields and digital feedback to manipulate individual nanoscale objects in solution at ambient temperature. We have trapped individual 20 nm polystyrene nanospheres, 100 nm lipid vesicles, ~700 nm (radius of gyration) molecules of λ-DNA, and particles of tobacco mosaic virus. Unlike other trapping schemes, the trapping performance of the ABEL trap scales favorably for small particles. The ABEL trap works by monitoring the Brownian motion of the particle (via fluorescence microscopy), and then applying a feedback voltage to the solution so that the electrophoretic drift exactly cancels the Brownian motion. The drift can be due either to the direct action of the field on the object’s charge, or to an electroosmotic flow which creates a drag on the object. The incarnation described here relies on electroosmotic flow, although we have used both effects to trap objects. The ABEL trap works on any object that can be imaged optically and that can be dispersed in water. The ABEL trap is non-invasive, is gentle enough to handle biological molecules, and can trap objects far smaller than can be trapped with laser tweezers. In this article we describe in detail how to build an ABEL trap and the software that controls the ABEL trap.