Anti-Brownian Trap Research Articles

Exploring Masses and Internal Mass Distributions of Single Carboxysomes in Free Solution Using Fluorescence and Interferometric Scattering in an Anti-Brownian Trap.

Carboxysomes are self-assembled bacterial microcompartments that facilitate carbon assimilation by colocalizing the enzymes of CO2 fixation within a protein shell. These microcompartments can be highly heterogeneous in their composition and filling, so measuring the mass and loading of an individual carboxysome would allow for better characterization of its assembly and function. To enable detailed and extended characterizations of single nanoparticles in solution, we recently demonstrated an improved interferometric scattering anti-Brownian electrokinetic (ISABEL) trap, which tracks the position of a single nanoparticle via its scattering of a near-infrared beam and applies feedback to counteract its Brownian motion. Importantly, the scattering signal can be related to the mass of nanoscale proteinaceous objects, whose refractive indices are well-characterized. We calibrate single-particle scattering cross-section measurements in the ISABEL trap and determine individual carboxysome masses in the 50-400 MDa range by analyzing their scattering cross sections with a core-shell model. We further investigate carboxysome loading by combining mass measurements with simultaneous fluorescence reporting from labeled internal components. This method may be extended to other biological objects, such as viruses or extracellular vesicles, and can be combined with orthogonal fluorescence reporters to achieve precise physical and chemical characterization of individual nanoscale biological objects.

Sensing the Phosphorylation State of Individual Peptides in Solution

Phosphorylation of biomolecules underlies a diverse array of signal transduction pathways and cellular decisions. However, single-molecule sensing of phosphorylation state remains challenging. We present a new detection scheme based on measuring the electric-field induced responses of single biomolecules in an anti-Brownian trap. Phosphorylation modifies the charge state of the biomolecule, which results in a shift in the molecule's response to external electric field (i.e. electrokinetic mobility). As a proof-of-principle demonstration, we monitored the phosphorylation of Kemptide by cAMP-dependent protein kinase A (PKA). For every molecule measured, we successfully resolved the phosphorylation state by its mobility. Moreover, by counting the number of unmodified and modified molecules, we tracked the progression of the phosphorylation reaction as a function of time. We envision the method to open up new possibilities in single-molecule observation of biological processes.

Tetherless, Precise and Extended Observation of Single-Molecule FRET in an Anti-Brownian Trap

A comprehensive understanding of biomolecules calls for the ability to observe single-molecule dynamics at the nanometer scale without constraints. Single-molecule Forster resonance energy transfer (smFRET) is a powerful tool for probing nanoscale dynamics, but existing modalities have limitations. Solution based confocal measurements are restricted by the short (~1ms) diffusion limited observation time. Surface immobilized measurements can extend the observation window, but at the expense of the molecule’s translational and rotational degrees of freedom. Moreover, there is always a concern that immobilization may perturb the biomolecule’s function. We overcome these limitations by combining smFRET optics with the capability to isolate individual molecules in solution using an Anti-Brownian ELectrokinetic (ABEL) trap. Our new platform, ABEL-FRET, enables photon-by-photon recording of smFRET trajectories over tens of seconds in solution, without tethering the molecule to a surface. We first demonstrate ABELFRET using short (~10bp) DNA rulers and achieve near shot-noise limited precision of ΔE~0.01 for 5,000 photons, which enables resolution of single base pair differences in a mixture of FRET-labeled dsDNA molecules. We also demonstrate the capability to make simultaneous measurements of donor fluorescence lifetime and smFRET.