Electrokinetic Trap Research Articles

Label-Free Anti-Brownian Trapping of Single Nanoparticles in Solution.

Today, biomolecular nanoparticles are prevalent as diagnostic tools and molecular delivery carriers, and it is particularly useful to examine individuals within a sample population to quantify the variations between objects and directly observe the molecular dynamics involving these objects. Using interferometric scattering as a highly sensitive label-free detection scheme, we recently developed the interferometric scattering anti-Brownian electrokinetic (ISABEL) trap to hold a single nanoparticle in solution for extended optical observation. In this perspective, we describe how we implemented this trap, how it extends the capabilities of previous ABEL traps, and how we have begun to study individual carboxysomes, a fascinating biological carbon fixation nanocompartment. By monitoring single nanocompartments for seconds to minutes in the ISABEL trap using simultaneous interferometric scattering and fluorescence spectroscopy, we have demonstrated single-compartment mass measurements, cargo-loading trends, and redox sensing inside individual particles. These experiments benefit from rich multiplexed correlative measurements utilizing both scattering and fluorescence with many exciting future capabilities within reach.

Quantifying Microsecond Solution-Phase Conformational Dynamics of a DNA Hairpin at the Single-Molecule Level.

Quantifying the rapid conformational dynamics of biological systems is fundamental to understanding the mechanism. However, biomolecules are complex, often containing static and dynamic heterogeneity, thus motivating the use of single-molecule methods, particularly those that can operate in solution. In this study, we measure microsecond conformational dynamics of solution-phase DNA hairpins at the single-molecule level using an anti-Brownian electrokinetic (ABEL) trap. Different conformational states were distinguished by their fluorescence lifetimes, and kinetic parameters describing transitions between these states were determined using two-dimensional fluorescence lifetime correlation (2DFLCS) analysis. Rather than combining fluorescence signals from the entire data set ensemble, long observation times of individual molecules allowed ABEL-2DFLCS to be performed on each molecule independently, yielding the underlying distribution of the system's kinetic parameters. ABEL-2DFLCS on the DNA hairpins resolved an underlying heterogeneity of fluorescence lifetimes and provided signatures of two-state exponential dynamics with rapid (<millisecond) transition times between states without observation of the substantially stretched exponential kinetics that had been observed in previous measurements on diffusing molecules. Numerical simulations were performed to validate the accuracy of this technique and the effects the underlying heterogeneity has on the analysis. Finally, ABEL-2DFLCS was performed on a mixture of hairpins and used to resolve their kinetic data.

Single-molecule fluorescence multiplexing by multi-parameter spectroscopic detection of nanostructured FRET labels

Multiplexed, real-time fluorescence detection at the single-molecule level can reveal the stoichiometry, dynamics and interactions of multiple molecular species in mixtures and other complex samples. However, fluorescence-based sensing is typically limited to the detection of just 3–4 colours at a time due to low signal-to-noise ratio, high spectral overlap and the need to maintain the chemical compatibility of dyes. Here we engineered a palette of several dozen composite fluorescent labels, called FRETfluors, for multiplexed spectroscopic measurements at the single-molecule level. FRETfluors are compact nanostructures constructed from three chemical components (DNA, Cy3 and Cy5) with tunable spectroscopic properties due to variations in geometry, fluorophore attachment chemistry and DNA sequence. We demonstrate FRETfluor labelling and detection for low-concentration (<100 fM) mixtures of mRNA, dsDNA and proteins using an anti-Brownian electrokinetic trap. In addition to identifying the unique spectroscopic signature of each FRETfluor, this trap differentiates FRETfluors attached to a target from unbound FRETfluors, enabling wash-free sensing. Although usually considered an undesirable complication of fluorescence, here the inherent sensitivity of fluorophores to the local physicochemical environment provides a new design axis complementary to changing the FRET efficiency. As a result, the number of distinguishable FRETfluor labels can be combinatorically increased while chemical compatibility is maintained, expanding prospects for spectroscopic multiplexing at the single-molecule level using a minimal set of chemical building blocks.

Precise electrokinetic position and three-dimensional orientation control of a nanowire bioprobe in solution.

Owing to Brownian-motion effects, the precise manipulation of individual micro- and nanoparticles in solution is challenging. Therefore, scanning-probe-based techniques, such as atomic force microscopy, attach particles to cantilevers to enable their use as nanoprobes. Here we demonstrate a versatile electrokinetic trap that simultaneously controls the two-dimensional position with a precision of 20 nm and 0.5° in the three-dimensional orientation of an untethered nanowire, as small as 300 nm in length, under an optical microscope. The method permits the active transport of nanowires with a speed-dependent accuracy reaching 90 nm at 2.7 μm s-1. It also allows for their synchronous three-dimensional alignment and rotation during translocation along complex trajectories. We use the electrokinetic trap to accurately move a nanoprobe and stably position it on the surface of a single bacterial cell for sensing secreted metabolites for extended periods. The precision-controlled manipulation underpins developing nanorobotic tools for assembly, micromanipulation and biological measurements with subcellular resolution.

Mechanism of ADP-Inhibited ATP Hydrolysis in Single Proton-Pumping FoF1-ATP Synthase Trapped in Solution.

FoF1-ATP synthases in mitochondria, in chloroplasts, and in most bacteria are proton-driven membrane enzymes that supply the cells with ATP made from ADP and phosphate. Different control mechanisms exist to monitor and prevent the enzymes' reverse chemical reaction of fast wasteful ATP hydrolysis, including mechanical or redox-based blockade of catalysis and ADP inhibition. In general, product inhibition is expected to slow down the mean catalytic turnover. Biochemical assays are ensemble measurements and cannot discriminate between a mechanism affecting all enzymes equally or individually. For example, all enzymes could work more slowly at a decreasing substrate/product ratio, or an increasing number of individual enzymes could be completely blocked. Here, we examined the effect of increasing amounts of ADP on ATP hydrolysis of single Escherichia coli FoF1-ATP synthases in liposomes. We observed the individual catalytic turnover of the enzymes one after another by monitoring the internal subunit rotation using single-molecule Förster resonance energy transfer (smFRET). Observation times of single FRET-labeled FoF1-ATP synthases in solution were extended up to several seconds using a confocal anti-Brownian electrokinetic trap (ABEL trap). By counting active versus inhibited enzymes, we revealed that ADP inhibition did not decrease the catalytic turnover of all FoF1-ATP synthases equally. Instead, increasing ADP in the ADP/ATP mixture reduced the number of remaining active enzymes that operated at similar catalytic rates for varying substrate/product ratios.

Ratiometric sensing of redox environments inside individual carboxysomes trapped in solution.

The rapid diffusion of biological nanoparticles in solution impedes our ability to continuously monitor individual particles and measure their physical and chemical properties. To overcome this, we previously developed the interferometric scattering anti-Brownian electrokinetic (ISABEL) trap, which uses scattering to localize a particle and applies electrokinetic forces to counteract Brownian motion, thus enabling extended observation. Here we present an improved ISABEL trap that incorporates a near-infrared scatter illumination beam and rapidly interleaves 405- and 488-nm fluorescence excitation beams, and apply this trap to the study of carboxysomes. Carboxysomes are bacterial microcompartments that self-assemble to sequester the enzyme rubisco and provide a favorable chemical environment for the fixation of carbon dioxide. With the ISABEL trap, we monitored the internal redox environment of individual carboxysomes labeled with the ratiometric redox reporter roGFP2. Carboxysomes were observed to vary widely in both redox-dependent fluorescence and scattering contrast (reporting on mass). Furthermore, we used redox sensing to explore the chemical kinetics within intact carboxysomes, where bulk measurements may contain unwanted contributions from aggregates or interfering fluorescent proteins. Overall, we demonstrate the ISABEL trap's ability to sensitively monitor chemical information inside single nanoscale biological objects, enabling new experiments on these systems.

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.

Ratiometric Sensing of Redox Environments Inside Individual Carboxysomes Trapped in Solution.

Diffusion of biological nanoparticles in solution impedes our ability to continuously monitor individual particles and measure their physical and chemical properties. To overcome this, we previously developed the interferometric scattering anti-Brownian electrokinetic (ISABEL) trap, which uses scattering to localize a particle and applies electrokinetic forces that counteract Brownian motion, thus enabling extended observation. Here we present an improved ISABEL trap that incorporates a near-infrared scatter illumination beam and rapidly interleaves 405 and 488 nm fluorescence excitation reporter beams. With the ISABEL trap, we monitored the internal redox environment of individual carboxysomes labeled with the ratiometric redox reporter roGFP2. Carboxysomes widely vary in scattering contrast (reporting on size) and redox-dependent ratiometric fluorescence. Furthermore, we used redox sensing to explore the chemical kinetics within intact carboxysomes, where bulk measurements may contain unwanted contributions from aggregates or interfering fluorescent proteins. Overall, we demonstrate the ISABEL trap’s ability to sensitively monitor nanoscale biological objects, enabling new experiments on these systems.

A bottom-up perspective on photodynamics and photoprotection in light-harvesting complexes using anti-Brownian trapping.

Single-molecule fluorescence spectroscopy allows direct, real-time observation of dynamic photophysical changes in light harvesting complexes. The Anti-Brownian ELectrokinetic (ABEL) trap is one such single-molecule method with useful advantages. This approach is particularly well-suited to make detailed spectroscopic measurements of pigment-protein complexes in a solution phase because it enables extended-duration single-molecule observation by counteracting Brownian motion. This Perspective summarizes recent contributions by the authors and others that have utilized the unique capabilities of the ABEL trap to advance our understanding of phycobiliproteins and the phycobilisome complex, the primary light-harvesting apparatus of cyanobacteria. Monitoring the rich spectroscopic data from these measurements, which include brightness, fluorescence lifetime, polarization, and emission spectra, among other measurable parameters, has provided direct characterization of pigments and energy transfer pathways in the phycobilisome, spanning scales from single pigments and monomeric phycobiliproteins to higher order oligomers and protein-protein interactions of the phycobilisome complex. Importantly, new photophysical states and photodynamics were observed to modulate the flow of energy through the phycobilisome and suggest a previously unknown complexity in phycobilisome light harvesting and energy transport with a possible link to photoadaptive or photoprotective functions in cyanobacteria. Beyond deepening our collective understanding of natural light-harvesting systems, these and future discoveries may serve as inspiration for engineering improved artificial light-harvesting technologies.

Characterizing physical properties of single carboxysomes in the Interferometric Scattering Anti-Brownian ELectrokinetic trap

Anti-Brownian traps enable extended study of single particles in free solution by closed-loop feedback forces that directly counteract Brownian motion monitored by fluorescence. The longer observation times of individual particles allow for detailed characterization of photophysical properties, dynamics, and infrequent events. We have recently invented the Interferometric Scattering Anti-Brownian ELectrokinetic (ISABEL) trap, which uses scattered light in place of fluorescence to detect the particle position for feedback.

Classification of DNA-FRET based single-molecule spectroscopic labels in anti-Brownian electrokinetic (ABEL) trap

Fluorescent tags are usually distinguished by their color, making fluorescence multiplexing at the single-molecule level challenging since only a few tags can be distinguished due to the overlap in their emission spectra and the low signal-to-noise ratios inherent in single-molecule measurements. We have engineered a series of spectroscopically distinguishable single-molecule labels whose emission is characterized by unique combinations of photophysical parameters including brightness, lifetime, and emission spectra.

Probing DNA-protein interactions using single-molecule diffusivity contrast

Single-molecule fluorescence investigations of protein-nucleic acid interactions require robust means to identify the binding state of individual substrate molecules in real time. Here, we show that diffusivity contrast, widely used in fluorescence correlation spectroscopy at the ensemble level and in single-particle tracking on individual (but slowly diffusing) species, can be used as a general readout to determine the binding state of single DNA molecules with unlabeled proteins in solution. We first describe the technical basis of drift-free single-molecule diffusivity measurements in an anti-Brownian electrokinetic trap. We then cross-validate our method with protein-induced fluorescence enhancement, a popular technique to detect protein binding on nucleic acid substrates with single-molecule sensitivity. We extend an existing hydrodynamic modeling framework to link measured diffusivity to particular DNA-protein structures and obtain good agreement between the measured and predicted diffusivity values. Finally, we show that combining diffusivity contrast with protein-induced fluorescence enhancement allows simultaneous mapping of binding stoichiometry and location on individual DNA-protein complexes, potentially enhancing single-molecule views of relevant biophysical processes.

Fast ATP-Dependent Subunit Rotation in Reconstituted FoF1-ATP Synthase Trapped in Solution.

FoF1-ATP synthases are ubiquitous membrane-bound, rotary motor enzymes that can catalyze ATP synthesis and hydrolysis. Their enzyme kinetics are controlled by internal subunit rotation, by substrate and product concentrations, and by mechanical inhibitory mechanisms but also by the electrochemical potential of protons across the membrane. Single-molecule Förster resonance energy transfer (smFRET) has been used to detect subunit rotation within FoF1-ATP synthases embedded in freely diffusing liposomes. We now report that kinetic monitoring of functional rotation can be prolonged from milliseconds to seconds by utilizing an anti-Brownian electrokinetic trap (ABEL trap). These extended observation times allowed us to observe fluctuating rates of functional rotation for individual FoF1-liposomes in solution. Broad distributions of ATP-dependent catalytic rates were revealed. The buildup of an electrochemical potential of protons was confirmed to limit the maximum rate of ATP hydrolysis. In the presence of ionophores or uncouplers, the fastest subunit rotation speeds measured in single reconstituted FoF1-ATP synthases were 180 full rounds per second. This was much faster than measured by biochemical ensemble averaging, but not as fast as the maximum rotational speed reported previously for isolated single F1 complexes uncoupled from the membrane-embedded Fo complex. Further application of ABEL trap measurements should help resolve the mechanistic causes of such fluctuating rates of subunit rotation.

A Novel Microfluidic Device for Blood Plasma Filtration.

The use of whole blood and some biological specimens, such as urine, saliva, and seminal fluid are limited in clinical laboratory analysis due to the interference of proteins with other small molecules in the matrix and blood cells with optical detection methods. Previously, we developed a microfluidic device featuring an electrokinetic size and mobility trap (SMT) for on-chip extract, concentrate, and separate small molecules from a biological sample like whole blood. The device was used to on-chip filtrate the whole blood from the blood cells and plasma proteins and then on-chip extract and separate the aminoglycoside antibiotic drugs within 3 min. Herein, a novel microfluidic device featuring a nano-junction similar to those reported in the previous work formed by dielectric breakdown was developed for on-chip filtration and out-chip collection of blood plasma with a high extraction yield of 62% within less than 5 min. The filtered plasma was analyzed using our previous device to show the ability of this new device to remove blood cells and plasma proteins. The filtration device shows a high yield of plasma allowing it to detect a low concentration of analytes from the whole blood.

Single-molecule FRET dynamics of molecular motors in an ABEL trap.

Single-molecule Förster resonance energy transfer (smFRET) of molecular motors provides transformative insights into their dynamics and conformational changes both at high temporal and spatial resolution simultaneously. However, a key challenge of such FRET investigations is to observe a molecule in action for long enough without restricting its natural function. The Anti-Brownian ELectrokinetic Trap (ABEL trap) sets out to combine smFRET with molecular confinement to enable observation times of up to several seconds while removing any requirement of tethered surface attachment of the molecule in question. In addition, the ABEL trap's inherent ability to selectively capture FRET active molecules accelerates the data acquisition process. In this work we exemplify the capabilities of the ABEL trap in performing extended timescale smFRET measurements on the molecular motor Rep, which is crucial for removing protein blocks ahead of the advancing DNA replication machinery and for restarting stalled DNA replication. We are able to monitor single Rep molecules up to 6seconds with sub-millisecond time resolution capturing multiple conformational switching events during the observation time. Here we provide a step-by-step guide for the rational design, construction and implementation of the ABEL trap for smFRET detection of Rep in vitro. We include details of how to model the electric potential at the trap site and use Hidden Markov analysis of the smFRET trajectories.

Revisiting Subunit Rotation in FOF1-ATP Synthase by Single-molecule FRET in an ABELtrap

Since 20 years we apply intensity-based and time-resolved single-molecule FRET measurements to study subunit rotation and regulatory conformational changes of individual FoF1-ATP synthases in liposomes, either driven by ATP hydrolysis or during ATP synthesis. However, observation times of freely diffusing proteoliposomes in a confocal microscope are limited by Brownian motion. In addition, arbitrary trajectories through the confocal detection volume cause large intensity fluctuations in smFRET traces resulting in varying precision for the smFRET distance measurement within the data for single enzyme at work. To counteract diffusive motion actively in real time, we have built a fast anti-Brownian electrokinetic trap (ABELtrap, invented by A. E. Cohen and W. E. Moerner) with a laser focus pattern generated by electro-optical beam deflectors and controlled by a programmable FPGA chip. We recorded surprisingly fast subunit rotation of FoF1-ATP synthases at different ATP concentrations, and analyzed Michaelis-Menten kinetics as extracted from single-molecule FRET traces of individual membrane enzymes hold in solution by the ABELtrap. Substeps of the rotor and ADP inhibition were identified by extended single-enzyme observation in the ABELtrap.

Interferometric Scattering Enables Fluorescence-Free Electrokinetic Trapping of Single Nanoparticles in Free Solution.

Anti-Brownian traps confine single particles in free solution by closed-loop feedback forces that directly counteract Brownian motion. Extended-duration measurements on trapped objects allow detailed characterization of photophysical and transport properties as well as observation of infrequent or rare dynamics. However, this approach has been generally limited to particles that can be tracked by fluorescence emission. Here we present the Interferometric Scattering Anti-Brownian ELectrokinetic (ISABEL) trap, which uses interferometric scattering rather than fluorescence to monitor particle position. By decoupling the ability to track (and therefore trap) a particle from collection of its spectroscopic data, the ISABEL trap enables confinement and extended study of single particles that do not fluoresce, only weakly fluoresce, or exhibit intermittent fluorescence or photobleaching. This new technique significantly expands the range of nanoscale objects that may be investigated at the single-particle level in free solution.

Single-molecule trapping and spectroscopy reveals photophysical heterogeneity of phycobilisomes quenched by Orange Carotenoid Protein

The Orange Carotenoid Protein (OCP) is a cytosolic photosensor that is responsible for non-photochemical quenching (NPQ) of the light-harvesting process in most cyanobacteria. Upon photoactivation by blue-green light, OCP binds to the phycobilisome antenna complex, providing an excitonic trap to thermally dissipate excess energy. At present, both the binding site and NPQ mechanism of OCP are unknown. Using an Anti-Brownian ELectrokinetic (ABEL) trap, we isolate single phycobilisomes in free solution, both in the presence and absence of activated OCP, to directly determine the photophysics and heterogeneity of OCP-quenched phycobilisomes. Surprisingly, we observe two distinct OCP-quenched states, with lifetimes 0.09 ns (6% of unquenched brightness) and 0.21 ns (11% brightness). Photon-by-photon Monte Carlo simulations of exciton transfer through the phycobilisome suggest that the observed quenched states are kinetically consistent with either two or one bound OCPs, respectively, underscoring an additional mechanism for excitation control in this key photosynthetic unit.

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.

Integrated Microfluidic Devices Fabricated in Poly (Methyl Methacrylate) (PMMA) for On-site Therapeutic Drug Monitoring of Aminoglycosides in Whole Blood.

On-site therapeutic drug monitoring (TDM) is important for providing a quick and accurate dosing to patients in order to improve efficacy and minimize toxicity. Aminoglycosides such as amikacin, gentamicin, and tobramycin are important antibiotics that have been commonly used to treat infections of chronic bacterial infections in the urinary tract, lung, and heart. However, these aminoglycosides can lead to vestibular and auditory dysfunction. Therefore, TDM of aminoglycosides is important due to their ototoxicity and nephrotoxicity. Here, we have developed a hot embossed poly (methyl methacrylate) (PMMA) microfluidic device featuring an electrokinetic size and mobility trap (SMT) to purify, concentrate, and separate the aminoglycoside antibiotic drugs amikacin, gentamicin, and tobramycin. These drugs were separated successfully from whole blood within 3 min, with 30-fold lower detection limits compared to a standard pinched injection. The limit of detections (LOD) were 3.75 µg/mL for gentamicin, 8.53 µg/mL for amikacin, and 6.00 µg/mL for tobramycin. These are sufficient to cover the therapeutic range for treating sepsis of 6–10 μg/mL gentamicin and tobramycin and 12–20 μg/mL of amikacin. The device is simple and could be mass produced via embossing or injection molding approaches.