Electrokinetic Trap Research Articles

Measuring nanoparticle diffusion in an ABELtrap

Monitoring the Brownian motion of individual nanoscopic objects is key to investigate their transport properties and interactions with their close environment. Most techniques rely on transient diffusion through a detection volume or immobilisation, which restrict observation times or motility. We measure the diffusion coefficient and surface charge of individual nanoparticles and DNA molecules in an anti-Brownian electrokinetic trap (ABELtrap). This instrument is an active feedback trap confining the Brownian motion of a nanoparticle to the detection site by applying an electric field based on the particle’s current position. We simulate the Brownian motion of nanospheres in our sample geometry, including wall effects, due to partial confinement in the third dimension. The theoretically predicted values are in excellent agreement with our diffusion measurements in the ABELtrap. We also demonstrate the ABELtrap’s ability to measure varying sizes of DNA origami structures during denaturation.

Single-Molecule Measurements of Quenching and Photophysical Heterogeneity in Phycobiliproteins

Phycobilisomes, the membrane-associated light-harvesting antenna system of cyanobacteria, dynamically adjust to changing irradiation by modulating energy capture and transfer over a few seconds or longer, for example by genetic regulation, structural reorganization, or non-photochemical quenching by Orange Carotenoid Protein (OCP). Recent observation of excitation-dependent photodynamics in single intact surface-immobilized phycobilisomes, including “blinking” to dark or dim states, suggests that the phycobilisome itself may also possess intrinsic molecular mechanisms to regulate energy transfer. Here, we confine single phycobiliproteins in free solution to a small measurement region for several seconds at a time using an Anti-Brownian ELectrokinetic (ABEL) trap, and simultaneously monitor changes in multiple spectroscopic properties (brightness, fluorescence anisotropy, fluorescence lifetime, and emission spectrum). We are able to trap single intact phycobilisomes and isolated substructures thereof, including phycobilisomes quenched by OCP. We find that even the smallest constituent phycobilisome subunits exhibit surprisingly complex photodynamics. For example, in monomers of C-phycocyanin (C-PC), a phycobiliprotein that contains three covalently bound and chemically identical chromophores, we observe reversible switching among at least seven distinct photophysical states (Squires and Moerner, PNAS 2017). The spectroscopic properties of these states and the transitions among them allow us to identify each state as a unique combination of chromophores participating in energy transfer. A simple computational FRET model closely predicts the observed states, and additionally reveals that the primary acceptor chromophore in C-PC may sometimes act as a quencher. The complex photodynamics and quenching that we observe for individual subunits of the phycobilisome are likely one source of the dynamics observed for the full phycobilisome complex, and may provide an intrinsic mechanism for short-timescale modulation of energy transfer to the reaction center.

Revealing Conformational Variants of Solution-Phase Intrinsically Disordered Tau Protein at the Single-Molecule Level.

Intrinsically disordered proteins, such as tau protein, adopt a variety of conformations in solution, complicating solution-phase structural studies. We employed an anti-Brownian electrokinetic (ABEL) trap to prolong measurements of single tau proteins in solution. Once trapped, we recorded the fluorescence anisotropy to investigate the diversity of conformations sampled by the single molecules. A distribution of anisotropy values obtained from trapped tau protein is conspicuously bimodal while those obtained by trapping a globular protein or individual fluorophores are not. Time-resolved fluorescence anisotropy measurements were used to provide an explanation of the bimodal distribution as originating from a shift in the compaction of the two different families of conformations.

Revealing Conformational Variants of Solution‐Phase Intrinsically Disordered Tau Protein at the Single‐Molecule Level

AbstractIntrinsically disordered proteins, such as tau protein, adopt a variety of conformations in solution, complicating solution‐phase structural studies. We employed an anti‐Brownian electrokinetic (ABEL) trap to prolong measurements of single tau proteins in solution. Once trapped, we recorded the fluorescence anisotropy to investigate the diversity of conformations sampled by the single molecules. A distribution of anisotropy values obtained from trapped tau protein is conspicuously bimodal while those obtained by trapping a globular protein or individual fluorophores are not. Time‐resolved fluorescence anisotropy measurements were used to provide an explanation of the bimodal distribution as originating from a shift in the compaction of the two different families of conformations.

Direct single-molecule measurements of phycocyanobilin photophysics in monomeric C-phycocyanin

Phycobilisomes are highly organized pigment-protein antenna complexes found in the photosynthetic apparatus of cyanobacteria and rhodophyta that harvest solar energy and transport it to the reaction center. A detailed bottom-up model of pigment organization and energy transfer in phycobilisomes is essential to understanding photosynthesis in these organisms and informing rational design of artificial light-harvesting systems. In particular, heterogeneous photophysical behaviors of these proteins, which cannot be predicted de novo, may play an essential role in rapid light adaptation and photoprotection. Furthermore, the delicate architecture of these pigment-protein scaffolds sensitizes them to external perturbations, for example, surface attachment, which can be avoided by study in free solution or in vivo. Here, we present single-molecule characterization of C-phycocyanin (C-PC), a three-pigment biliprotein that self-assembles to form the midantenna rods of cyanobacterial phycobilisomes. Using the Anti-Brownian Electrokinetic (ABEL) trap to counteract Brownian motion of single particles in real time, we directly monitor the changing photophysical states of individual C-PC monomers from Spirulina platensis in free solution by simultaneous readout of their brightness, fluorescence anisotropy, fluorescence lifetime, and emission spectra. These include single-chromophore emission states for each of the three covalently bound phycocyanobilins, providing direct measurements of the spectra and photophysics of these chemically identical molecules in their native protein environment. We further show that a simple Förster resonant energy transfer (FRET) network model accurately predicts the observed photophysical states of C-PC and suggests highly variable quenching behavior of one of the chromophores, which should inform future studies of higher-order complexes.

Electrokinetic Size and Mobility Traps for On-site Therapeutic Drug Monitoring.

The extraction of target analytes from biological samples is a bottleneck in analysis. A microfluidic device featuring an electrokinetic size and mobility trap was formed by two nanojunctions of different pore size to extract and concentrate analytical targets from complex samples. The trap was seamlessly coupled with electrophoretic separation for quantitative analysis. The device was applied to the analysis of ampicillin levels in blood within 5 min and a linear response over the range of 2.5-20 μg mL(-1). This covers the recommended levels for treating sepsis, a critical condition with 30 to 50% mortality and unpredicted drug levels. The device provides a new opportunity for on-site therapeutic drug monitoring, which should enable quick and accurate dosing and may save lives in such critical conditions.

Electrokinetic Size and Mobility Traps for On‐site Therapeutic Drug Monitoring

AbstractThe extraction of target analytes from biological samples is a bottleneck in analysis. A microfluidic device featuring an electrokinetic size and mobility trap was formed by two nanojunctions of different pore size to extract and concentrate analytical targets from complex samples. The trap was seamlessly coupled with electrophoretic separation for quantitative analysis. The device was applied to the analysis of ampicillin levels in blood within 5 min and a linear response over the range of 2.5–20 μg mL−1. This covers the recommended levels for treating sepsis, a critical condition with 30 to 50 % mortality and unpredicted drug levels. The device provides a new opportunity for on‐site therapeutic drug monitoring, which should enable quick and accurate dosing and may save lives in such critical conditions.

Single-Molecule Identification of Quenched and Unquenched States of LHCII

In photosynthetic light harvesting, absorbed sunlight is converted to electron flow with near-unity quantum efficiency under low light conditions. Under high light conditions, plants avoid damage to their molecular machinery by activating a set of photoprotective mechanisms to harmlessly dissipate excess energy as heat. To investigate these mechanisms, we study the primary antenna complex in green plants, light-harvesting complex II (LHCII), at the single-complex level. We use a single-molecule technique, the Anti-Brownian Electrokinetic trap, which enables simultaneous measurements of fluorescence intensity, lifetime, and spectra in solution. With this approach, including the first measurements of fluorescence lifetime on single LHCII complexes, we access the intrinsic conformational dynamics. In addition to an unquenched state, we identify two partially quenched states of LHCII. Our results suggest that there are at least two distinct quenching sites with different molecular compositions, meaning multiple dissipative pathways in LHCII. Furthermore, one of the quenched conformations significantly increases in relative population under environmental conditions mimicking high light.

Single Molecule Studies of Tau Protein in the Abel Trap

Tau protein is an intrinsically disordered protein that stabilizes microtubules of neurons mainly in the central nervous system. Abnormalities in Tau are thought to be involved in Alzheimer's disease. The aggregation process of monomeric Tau into an oligomeric species and the intermediate conformational states that may facilitate aggregation are of particular interest. To investigate the disordered nature and aggregation of Tau protein, single, solution-phase Tau proteins and oligomers were isolated in a microfluidic trap, called the Anti-Brownian Electrokinetic (ABEL) Trap. The ABEL trap cancels Brownian motion of a molecule of interest via electrokinetic and/or electroosmotic flow, allowing prolonged examination of a single, fluorescently-labeled analyte in solution. Combining this technique with fluorescence anisotropy enabled studies into how monomer structure and aggregation changes with cellular conditions known to accelerate aggregation, including concentration effects, solution viscosity, heparin, and cellular crowding. Characteristics of disordered Tau were compared with those of a globular protein of similar size, microbial transglutaminase (MTG). The ultimate goal is to provide the missing link between non-aggregated, monomer Tau protein and the toxic species that result in devastating disease, offering insight into how this pathway may be upset.

Applications of Soil Moisture Sensor with Electrokinetic Ion Trap Mechanism

A sensor comprising a pair of stainless steel planar electrodes and a capacitance meter is proposed for the real-time monitoring of the moisture content in soil. As rain falls on the ground, the moisture content of the soil between the two electrodes increases. The resulting change in the dielectric constant of the sensing material (soil) produces a corresponding change in the capacitance signal, from which the moisture content can then be inversely derived. The measurement performance of the proposed sensor is enhanced by means of an ion trap mechanism comprising two graphite mesh electrodes positioned orthogonally to the measurement electrodes. A DC voltage is applied to the two electrodes such that the anions and cations in the water are trapped by the positive and negative electrodes, respectively, thereby minimizing their effects on the sensing operation. The experimental results show that the proposed sensor achieves a high degree of sensitivity (i.e., 1.27 μF/%) for gravimetric water contents ranging from 21 to 28%. Moreover, it is shown that the sensor has a repeatability of ±0.71% and ±0.55% for low and high gravimetric water contents, respectively.

Electron spin resonance of nitrogen-vacancy defects embedded in single nanodiamonds in an ABEL trap.

Room temperature optically detected magnetic resonance of a single quantum object with nanoscale position control is an outstanding challenge in many areas, particularly in the life sciences. We introduce a novel approach to control the nitrogen-vacancy (NV) centers hosted in a single fluorescent nanodiamond (FND) for which an anti-Brownian electrokinetic trap (ABEL) performs the position control and an integrated radiofrequency (RF) circuit provides enhanced magnetic flux density for ensemble spin-state control simultaneously. We demonstrate static magnetic field sensing in platforms compatible with ABEL trap. With the advances in the synthesis and functionalization of stable arbitrarily small FNDs, we foresee the use of our device for the trapping and manipulation of single molecular-sized FNDs in aqueous solution.

ChemInform Abstract: Single‐Molecule Spectroscopy of Photosynthetic Proteins in Solution: Exploration of Structure—Function Relationships

AbstractReview: 43 refs.

The regulatory switch of F1-ATPase studied by single-molecule FRET in the ABEL Trap.

F1-ATPase is the soluble portion of the membrane-embedded enzyme FoF1-ATP synthase that catalyzes the production of adenosine triphosphate in eukaryotic and eubacterial cells. In reverse, the F1 part can also hydrolyze ATP quickly at three catalytic binding sites. Therefore, catalysis of 'non-productive' ATP hydrolysis by F1 (or FoF1) must be minimized in the cell. In bacteria, the ε subunit is thought to control and block ATP hydrolysis by mechanically inserting its C-terminus into the rotary motor region of F1. We investigate this proposed mechanism by labeling F1 specifically with two fluorophores to monitor the C-terminus of the ε subunit by Förster resonance energy transfer. Single F1 molecules are trapped in solution by an Anti-Brownian electrokinetic trap which keeps the FRET-labeled F1 in place for extended observation times of several hundreds of milliseconds, limited by photobleaching. FRET changes in single F1 and FRET histograms for different biochemical conditions are compared to evaluate the proposed regulatory mechanism.

Sensing the Association States of Single Biomolecules by Motion Analysis in an Electrokinetic Trap

The different association states (i.e. bound/unbound, monomer/trimer, etc⋯) of a biomolecule are crucial to its function but yet difficult to differentiate at the single-molecule level. We describe a general approach to probe a single biomolecule's association states, and the time-dependent inter-conversion between these states in solution. Our approach is based on statistical analysis of molecular motions in an electrokinetic trap. This is possible because motion parameters (i.e. diffusion coefficient and electrokinetic mobility) are directly sensitive to intrinsic molecular properties of size and charge, which are modified as a molecule undergoes a change in association states. To accurately estimate single-molecule transport coefficients in solution, we employed photon-by-photon position tracking and developed an efficient algorithm based on Bayesian graphical models to analyze the raw tracking trajectories. As experimental demonstrations, we first used the method to resolve different populations in a multi-component mixture. Each measured single molecule, based on its size and charge characteristics reflected by transport properties, can be classified to a certain population. Differences as small as between the monomeric and trimeric forms of a fluorescent protein can be resolved. Second, to highlight the unique capability to probe time-dependent changes in molecular association states, we studied the hybridization kinetics of a 10-base ssDNA with its complementary partner. Digital, two-state, anticorrelated fluctuations in the measured diffusion coefficient and electrokinetic mobility were observed that directly sensed single-molecule binding and unbinding events in real time. We quantified the rates of the hybridization reaction by hidden Markov analysis of the time-dependent transport coefficients and investigated the destabilizing effect of a single-base mismatch.

Elucidation of the Photodynamics of Single Photosynthetic LH2 Complexes in Solution

Photosynthetic organisms function under low light by converting photoenergy to chemical energy with near-unity quantum efficiency and under high light by dissipating unused photoenergy to prevent formation of deleterious photoproducts. There is widespread interest in how this dual functionality is achieved, because this balance enables efficient light harvesting under the fluctuating intensities of sunlight at the earth's surface. One obstacle to characterizing the molecular mechanisms responsible for this balance is that the excited-state properties of photosynthetic proteins vary drastically between individual proteins (due to static heterogeneity) and even within a single protein over time (due to dynamic heterogeneity). In ensemble measurements, these excited-state properties appear as a static, average value. To overcome this averaging, we investigate light-harvesting complex 2 (LH2), the primary antenna in purple bacteria, at the single-molecule level. Using a novel technique, the Anti-Brownian ELectrokinetic (ABEL) trap, we study individual LH2 complexes in a solution-phase environment to eliminate perturbations due to immobilization schemes, which can alter the protein structure and function. Furthermore, we perform the first simultaneous measurements of fluorescence intensity, lifetime, and emission spectra from individual proteins. We identify three distinct functional conformations of LH2, two of which correspond to a quenched and an unquenched form, and observe transitions occurring between these forms on a timescale of seconds. Our results reveal that individual LH2 complexes undergo photoactivated switching to the quenched state, and thermally revert to the ground state. This is a previously unknown, reversible mechanism for dissipation of excitation energy activated by high light conditions, and may be one component by which photosynthetic organisms flourish under varying light intensities.

Single-molecule spectroscopy of photosynthetic proteins in solution: exploration of structure–function relationships

In photosynthetic light harvesting, absorbed photoenergy transfers through networks of pigment–protein complexes to a central location, known as the reaction center, for conversion to chemical energy. This process occurs with remarkable near-unity quantum efficiency. Pigment–protein complexes exhibit emergent properties very different from those of their component molecules, resulting from interactions among the pigments and between the pigments and the surrounding protein environment. Thus, the precise molecular mechanisms of the energy transport process are complex and coupled, obscuring their molecular origin. Furthermore, because these interactions are sensitive to the molecular structure, they vary from complex to complex and vary in time for each individual complex due to fluctuations of the protein's conformation. To explore the effect of complex-to-complex variation, we study individual photosynthetic proteins, one at a time. We use a solution-phase, single-molecule technique, known as the Anti-Brownian Electrokinetic (ABEL) trap, to elucidate the conformational dynamics of single photosynthetic pigment–protein complexes without introducing additional perturbations from immobilization strategies. After reviewing the principles of the ABEL trap, we demonstrate its application to the study of several photosynthetic pigment–protein complexes. We demonstrate that the ABEL trap approach can lead to an increased understanding of photosynthetic complexes by presenting three examples: (1) analysis of photodegradation pathways, (2) characterization of complex-to-complex heterogeneity, and (3) identification of distinct functional forms.

Single-Molecule Exploration of the Photodynamics of LHCII Complexes in Solution

Plants can safely dissipate excess excitation energy during light harvesting to prevent the formation of triplet chlorophyll, which can generate deleterious singlet oxygen. With this regulation, known as non-photochemical quenching (NPQ), efficient light harvesting and photosynthesis can be balanced under fluctuating sunlight intensity without damage to the photosynthetic machinery. NPQ has been extensively studied and key physiological regulatory factors have been identified. For example, it is known that the change in lumen pH or a pH gradient across thylakoid membrane can trigger an energy-dependent quenching (qE) pathway that activates on a timescale of seconds to minutes. However, understanding the molecular mechanism behind NPQ is still a challenge. One important question is whether, upon activation of qE, the number of quenched complexes increases or the degree of quenching in each complex changes. These two cases cannot be differentiated in ensemble-averaged measurements. Therefore, we use the Anti-Brownian ELectrokinetic (ABEL) trap to investigate single copies of light-harvesting complex II (LHCII), the primary antenna in higher plants. Different from other single-molecule techniques that utilize immobilization, perturbations due to surface attachment or encapsulation are avoided, and therefore the intrinsic dynamics and heterogeneity of individual complexes are revealed. We perform measurements of fluorescence intensity, excited-state lifetime, and emission spectra of single LHCII complexes in the ABEL trap. By analyzing the correlated changes in these properties over time, we observe that individual LHCII complexes are found in distinct forms with different extents of quenching. Comparing the results from different conditions known to correlate with qE activation will give a better understanding of NPQ at the molecular level.

Feedback-controlled electro-kinetic traps for single-molecule spectroscopy

A principal limitation of single-molecule spectroscopy in solution is the diffusion-limited residence time of a given molecule within the detection volume. A common solution to this problem is to immobilize molecules of interest on a passivated glass surface for extending the observation time to obtain reliable data statistics. However, surface tethering of molecules often introduces artifacts, particularly when studying the structural dynamics of biomolecules. To circumvent this limitation, we investigated alternative ways to extend single-molecule observation times in solution without surface immobilization. Among various possibilities, the so-called anti-Brownian electro-kinetic trap (or ABEL trap) seems best suited to achieve this goal. The essential part of that trap is a feedback-controlled electro-kinetic steering of a molecule’s position in reaction to its diffusive Brownian motion which is monitored by fluorescence, thus keeping the molecule within a sub-micron sized detection volume. Fluorescence trace recordings of over thousands of milliseconds duration on individual dye molecules within an ABEL trap have been reported. In this short review, we shall briefly discuss the principle and some results of ABEL trapping of individual molecules with possible extensions to future works.

Photo-Induced Conformational Flexibility in Single Solution-Phase Peridinin-Chlorophyll-Proteins

The peridinin-chlorophyll-protein (PCP) is an accessory light-harvesting complex found in red-tide dinoflagellates. PCP absorbs photons primarily in the blue-green spectral region via peridinin (Per) carotenoid pigments which then transfer excitations to chlorophyll (Chl) and ultimately downstream to photosystem II (PSII). Whereas the ultrafast dynamics of PCP are well-studied, much less is known about slower protein dynamics on time scales of milliseconds and seconds. Previous single-molecule studies of spectral emission and intensity have attached PCP to surfaces, but the native environment of PCP is in the lumen, meaning that a surface-attached environment could perturb its native conformations. To address this concern, we use the anti-Brownian electrokinetic (ABEL) trap to study single PCP monomers in solution for several seconds each. We measure, for the first time, simultaneous single-molecule intensity, lifetime, and spectral emission shifts for each trapped PCP monomer. The rate of reversible spectral redshifts depends linearly on irradiance over a factor of 30, indicating a light-induced mechanism which we attribute to a protein conformational change. Independent of these spectral shifts, our measurements of intensity and lifetime show reversible Chl quenching. In contrast to previous work, we show that this quenching cannot result from isolated photobleaching of Chl. These independent mechanisms arise from distinct conformational changes which maintain relatively stable fluorescence emission.

Single-molecule spectroscopy reveals photosynthetic LH2 complexes switch between emissive states

Photosynthetic organisms flourish under low light intensities by converting photoenergy to chemical energy with near unity quantum efficiency and under high light intensities by safely dissipating excess photoenergy and deleterious photoproducts. The molecular mechanisms balancing these two functions remain incompletely described. One critical barrier to characterizing the mechanisms responsible for these processes is that they occur within proteins whose excited-state properties vary drastically among individual proteins and even within a single protein over time. In ensemble measurements, these excited-state properties appear only as the average value. To overcome this averaging, we investigate the purple bacterial antenna protein light harvesting complex 2 (LH2) from Rhodopseudomonas acidophila at the single-protein level. We use a room-temperature, single-molecule technique, the anti-Brownian electrokinetic trap, to study LH2 in a solution-phase (nonperturbative) environment. By performing simultaneous measurements of fluorescence intensity, lifetime, and spectra of single LH2 complexes, we identify three distinct states and observe transitions occurring among them on a timescale of seconds. Our results reveal that LH2 complexes undergo photoactivated switching to a quenched state, likely by a conformational change, and thermally revert to the ground state. This is a previously unobserved, reversible quenching pathway, and is one mechanism through which photosynthetic organisms can adapt to changes in light intensities.