Electrokinetic trapping at the one nanometer limit

Abstract

Anti-Brownian electrokinetic traps have been used to trap and study the free-solution dynamics of large protein complexes and long chains of DNA. Small molecules in solution have thus far proved too mobile to trap by any means. Here we explore the ultimate limits on trapping single molecules. We developed a feedback-based anti-Brownian electrokinetic trap in which classical thermal noise is compensated to the maximal extent allowed by quantum measurement noise. We trapped single fluorophores with a molecular weight of <1 kDa and a hydrodynamic radius of 6.7 Å for longer than one second, in aqueous buffer at room temperature. This achievement represents an 800-fold decrease in the mass of objects trapped in solution, and opens the possibility to trap and manipulate any soluble molecule that can be fluorescently labeled. To illustrate the use of this trap, we studied the binding of unlabeled RecA to fluorescently labeled single-stranded DNA. Binding of RecA induced changes in the DNA diffusion coefficient, electrophoretic mobility, and brightness, all of which were measured simultaneously and on a molecule-by-molecule basis. This device greatly extends the size range of molecules that can be studied by room temperature feedback trapping, and opens the door to further studies of the binding of unmodified proteins to DNA in free solution.A longstanding challenge in single-molecule spectroscopy has been to observe a small molecule in solution for an extended time, without surface tethering or other mechanical immobilization. Stable observation becomes more difficult as the particle decreases in size, because smaller objects diffuse more quickly, in accordance with the Stokes-Einstein relation. Gold nanoparticles as small as 18 nm in diameter, corresponding to a mass of 35 MDa, have been trapped using laser tweezers (1). Below this size laser tweezers fail because the trapping force is proportional to the volume of the trapped object. Real-time feedback provides an alternate strategy, and has been used to trap single atoms in vacuum (2). The anti-Brownian electrokinetic (ABEL) trap uses feedback to suppress Brownian motion in solution and can confine particles as small as the 800 kDa complex of the chaperonin GroEL (3-5). A 104 kDa protein, allophycocyanin, was recently studied in an ABEL trap in which the viscosity was increased with 50% glycerol to slow the Brownian motion (6). Past attempts to trap small-molecule fluorophores in aqueous solution resulted in transient confinement, but not stable trapping (3). Small-molecule fluorophores are the tiniest objects that one can conceive of trapping in aqueous solution. If a particular fluorophore can be trapped, then so too can any molecule to which it is attached.Laser tweezers and the ABEL trap both confine small objects in solution, but the two technologies enable different kinds of measurements. The optical forces of laser tweezers enable precise (subnanometer) localization of the trapped object, and permit application of precisely calibrated point forces for the pur...

Real-Time Calibration of a Feedback Trap

“…In the latter work, camera images are replaced by a continuously scanning laser and a single-pixel detector that detects individual photon counts. 3,27 Each individual count leads to an updated estimate of particle position, using a modified Kalman filter. The advantage of such a setup is vastly increased speed and a simplicity that comes from having instantaneous estimates of particle position (no camera exposure effects).…”

“…It is even possible to trap a single fluorescent dye molecule in water. 3,4 The feedback trap has had two major types of application. First, it has been used to probe the physical or chemical properties of the trapped object.…”

Twisting and subunit rotation in single FOF1-ATP synthase

“…New non-optical particle trapping devices such as the anti-Brownian electrokinetic trap [100][101][102][103][104] (ABEL trap) invented by Cohen and Moerner can be used to catch and actively push a single F O F 1 -ATP synthase to a target position for extended confocal FRET measurements [105]. Then, the different numbers of transported protons required to make one ATP molecule in the F O F 1 -ATP synthases of chloroplasts [3,106], bacteria [3] or mitochondria [107] (as calculated from ensemble measurements) can be compared to the step sizes of the rotary c-rings of the respective single F O motors.…”

Monitoring subunit rotation in single FRET-labeled FoF1-ATP synthase in an anti-Brownian electrokinetic trap

“…Thereby the arbitrary Brownian motion of the particle is compensated and the particle remains stationary. Several different ABELtrap versions have been realized [43][44][45][46][47] , with very fast feedback times in the microsecond time range [48][49][50][51][52] .…”