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Separation and identification of different proteins is one of the most fundamental tasks in biochemistry that is typically achieved by electrophoresis and Western blot techniques. Yet, it is challenging to perform such an analysis with a small sample size. Using a principle analogous to these conventional approaches, we present a label-free, single-molecule technique to identify different proteins based on the difference in their size, charge, and antibody binding. We tether single protein molecules to a sensor surface with a flexible polymer and drive them into oscillation by applying an alternating electric field. By tracking the nanometer-scale oscillation of each protein molecule via high-resolution scattering microscopy, the size and charge of each protein molecule can be determined simultaneously. Changes induced by varying the buffer pH and antibody binding are also investigated, which allows us to further expand the separation ability and identify two different proteins in a mixture. We anticipate our technique will contribute to single protein analysis and biosensing.

Most label-free techniques rely on measuring refractive index or mass change on the sensor surface. Thus, it is challenging for them to measure small molecules or enzymatic processes that only induce a minor mass change on the analyte molecules. Here, we have developed a technique by combining Surface Plasmon Resonance sensing with an Oscillating Biomolecule Layer approach (SPR-OBL) to enhance the sensitivity of traditional SPR. In addition to the inherent mass sensitivity, SPR-OBL is also sensitive to the charge and conformational change of the analyte; hence it overcomes the mass limit and is able to detect small molecules. We show that the multimetric SPR-OBL measurement allows for sensing any changes regarding mass, charge, and conformation, which expands the detection capability of SPR.

Single-molecule detection can push beyond ensemble averages and reveal the statistical distributions of molecular properties. Measuring the binding kinetics of single proteins also represents one of the critical and challenging tasks in protein analysis. Here, we report total internal reflection-based evanescent scattering microscopy with label-free single-protein detection capability. Total internal reflection is employed to excite the evanescent field to enhance light-analyte interaction and reduce environmental noise. As a result, the system provides wide-field imaging capability and allows excitation and observation using one objective. In addition, this system quantifies protein binding kinetics by simultaneously counting the binding of individual molecules and recording their binding sites with nanometer precision, providing a digital method to measure binding kinetics with high spatiotemporal resolution. This approach does not employ specially designed microspheres or nanomaterials and may pave a way for label-free single-protein analysis in conventional microscopy.

Three-dimensional (3D) tracking of surface-tethered single particles reveals the dynamics of the molecular tether. However, most 3D tracking techniques lack precision, especially in the axial direction, for measuring the dynamics of biomolecules with a spatial scale of several nanometers. Here, we present a plasmonic imaging technique that can track the motion of ∼100 tethered particles in 3D simultaneously with sub-nanometer axial precision and single-digit nanometer lateral precision at millisecond time resolution. By tracking the 3D coordinates of a tethered particle with high spatial resolution, we are able to determine the dynamics of single short DNA and study its interaction with enzymes. We further show that the particle motion pattern can be used to identify specific and nonspecific interactions in immunoassays. We anticipate that our 3D tracking technique can contribute to the understanding of molecular dynamics and interactions at the single-molecule level.

Quantification of molecular interactions on a surface is typically achieved via label-free techniques such as surface plasmon resonance (SPR). The sensitivity of SPR originates from the characteristic that the SPR angle is sensitive to the surface refractive index change. Analogously, in another interfacial optical phenomenon, total internal reflection, the critical angle is also refractive index dependent. Therefore, surface refractive index change can also be quantified by measuring the reflectivity near the critical angle. Based on this concept, we develop a method called critical angle reflection (CAR) imaging to quantify molecular interactions on glass surface. CAR imaging can be performed on SPR imaging setups. Through a side-by-side comparison, we show that CAR is capable of most molecular interaction measurements that SPR performs, including proteins, nucleic acids and cell-based detections. In addition, we show that CAR can detect small molecule bindings and intracellular signals beyond SPR sensing range. CAR exhibits several distinct characteristics, including tunable sensitivity and dynamic range, deeper vertical sensing range, fluorescence compatibility, broader wavelength and polarization of light selection, and glass surface chemistry. We anticipate CAR can expand SPR′s capability in small molecule detection, whole cell-based detection, simultaneous fluorescence imaging, and broader conjugation chemistry., Here, the authors present a method for quantifying molecular interactions on a glass surface, based on measuring surface refractive index changes via the reflectivity near the critical angle. They demonstrate tunable sensitivity and dynamic range, deep vertical sensing range, also for intracellular signals.

Surface plasmon resonance microscopy (SPRM) is an excellent platform for in situ studying cell-substrate interactions. However, SPRM suffers from poor spatial resolution and small field of view. Herein, we demonstrate plasmonic scattering microscopy (PSM) by adding a dry objective on a popular prism-coupled surface plasmon resonance (SPR) system. PSM not only retains SPRM's high sensitivity and real-time analysis capability, but also provides ≈7 times higher spatial resolution and ≈70 times larger field of view than the typical SPRM, thus providing more details about membrane protein response to ligand binding on over 100 cells simultaneously. In addition, PSM allows quantifying the target movements in the axial direction with a high spatial resolution, thus allowing mapping adhesion spring constants for quantitatively describing the mechanical properties of the cell-substrate contacts. This work may offer a powerful and cost-effective strategy for upgrading current SPR products.

Measuring molecular binding is critical for understanding molecule-scale biological processes and screening drugs. Label free detection technologies, such as surface plasmon resonance (SPR), has been developed for analyzing the analytes in their natural forms. However, the specificity of these methods is solely relying on surface chemistry, and often have the nonspecific binding issues when working with samples in complex media. Herein, we show that single molecule based measurement can distinct specific and nonspecific binding processes by quantifying the mass and binding dynamics of individual bound analyte molecules, thus allowing the binding kinetic analysis in complex media such as serum. In addition, this single molecule imaging is realized in a commonly used Kretschmann prism coupled SPR system, thus providing a convenient solution to realize high resolution imaging on widely used prism coupled SPR systems.

Precise and sensitive detection of intracellular proteins and complexes is key to the understanding of signaling pathways and cell functions. Here, we present a label-free single-molecule pulldown (LFSMP) technique for the imaging of released cellular protein and protein complexes with single-molecule sensitivity and low sample consumption down to a few cells per mm

Measuring the binding kinetics of single proteins represents one of the most important and challenging tasks in protein analysis. Here we show that this is possible using a surface plasmon resonance (SPR) scattering technique. SPR is a popular label-free detection technology because of its extraordinary sensitivity, but it has never been used for imaging single proteins. We overcome this limitation by imaging scattering of surface plasmonic waves by proteins. This allows us to image single proteins, measure their sizes, and identify them based on their specific binding to antibodies. We further show that it is possible to quantify protein binding kinetics by counting the binding of individual molecules, providing a digital method to measure binding kinetics and analyze heterogeneity of protein behavior. We anticipate that this imaging method will become an important tool for single protein analysis, especially for low volume samples, such as single cells., Editor’s summary Plasmonic scattering microscopy (PSM) enables the imaging of single proteins on SPR instruments. The method enables measurement of protein size and binding kinetics and is fully compatible with simultaneous traditional SPR measurements.

Tethering a particle to a surface with a single molecule allows detection of the molecule and analysis of molecular conformations and interactions., Tethering a particle to a surface with a single molecule allows detection of the molecule and analysis of molecular conformations and interactions. Understanding the dynamics of the system is critical to all applications. Here we present a plasmonic imaging study of two important forces that govern the dynamics. One is entropic force arising from the conformational change of the molecular tether, and the other is solvent damping on the particle and the molecule. We measure the response of the particle by driving it into oscillation with an alternating electric field. By varying the field frequency, we study the dynamics on different time scales. We also vary the type of the tether molecule (DNA and polyethylene glycol), size of the particle, and viscosity of the solvent, and describe the observations with a model. The study allows us to derive a single parameter to predict the relative importance of the entropic and damping forces. The findings provide insights into single molecule studies using not only tethered particles, but also other approaches, including force spectroscopy using atomic force microscopy and nanopores.

Plasmonic scattering microscopy has advanced the evanescent detection approaches by offering wide-field single-molecule imaging capability. However, two limitations prevent the broader application of plasmonic single-molecule imaging. One is the heating effect accompanying the plasmonic enhancement, and the other is the complicated system structure resulting from the two-objective optical arrangement. Here, we report single-objective evanescent scattering microscopy. The evanescent field is created by total internal reflection instead of the surface plasmon resonance on the gold film. As a result, the sensing substrate without gold film produces little heat, and allows excitation and observation using one objective. In addition, this system enables quantification of protein binding kinetics by simultaneously counting the binding of individual molecules and recording their binding sites with nanometer precision, providing a digital method to measure binding kinetics with high spatiotemporal resolution. This work may pave a road for label-free single protein analysis in conventional microscopy.TeaserLabel-free single-molecule imaging on a total internal reflection fluorescence objective.

Charge sensitive optical detection (CSOD) technique is a label-free method for real-time measurement of molecular interactions. Traditional label-free optical detection techniques mostly measure the mass of a molecule, and they are less sensitive to small molecules. In contrast, CSOD detects the charge of a molecule, where the signal does not diminish with the size of the molecule, thus capable for studying small molecules. In addition, CSOD is compatible with the standard microplate platform, making it suitable for high-throughput screening of drug candidates. In CSOD, an optical fiber functionalized with the probe molecule is dipped into a well of a microplate where an alternate perpendicular electrical field is applied to the fiber, which drives the fiber into oscillation because of the presence of surface charge on the fiber. The binding of the target molecules changes the charge of the fiber, and thus the amplitude and phase of the oscillating fiber, which are precisely measured through tracking of the optical images of the fiber tip.

Charge sensitive optical detection (CSOD) technique is a label-free method for real-time measurement of molecular interactions. Traditional label-free optical detection techniques mostly measure the mass of a molecule, and they are less sensitive to small molecules. In contrast, CSOD detects the charge of a molecule, where the signal does not diminish with the size of the molecule, thus capable for studying small molecules. In addition, CSOD is compatible with the standard microplate platform, making it suitable for high-throughput screening of drug candidates. In CSOD, an optical fiber functionalized with the probe molecule is dipped into a well of a microplate where an alternate perpendicular electrical field is applied to the fiber, which drives the fiber into oscillation because of the presence of surface charge on the fiber. The binding of the target molecules changes the charge of the fiber, and thus the amplitude and phase of the oscillating fiber, which are precisely measured through tracking of the optical images of the fiber tip.

Measuring binding between molecules is critical for understanding basic biochemical processes, developing molecular diagnosis, and screening drugs. Here we study molecular binding at the single molecule level by attaching nanoparticles to the molecular binding pairs. We track the thermal fluctuations of the individual nanoparticles with sub-nanometer precision using a plasmonic scattering imaging technique and show that the fluctuations are controlled by the molecular binding pairs rather than by the nanoparticles. Analysis of the thermal fluctuations provides unique information on molecular binding, including binding energy profile, effective spring constant, and switching between single and multiple molecular binding events. The method provides new insights into molecular binding and also allows one to differentiate nonspecific binding from specific binding, which has been a difficult task in biosensors.

Three-dimensional (3D) tracking of surface-tethered single-particle reveals the dynamics of the molecular tether. However, most 3D tracking techniques lack precision, especially in axial direction, for measuring the dynamics of biomolecules with spatial scale of several nanometers. Here we present a plasmonic imaging technique that can track the motion of ∼100 tethered particles in 3D simultaneously with sub-nanometer axial precision at millisecond time resolution. By tracking the 3D coordinates of tethered particle with high spatial resolution, we are able to determine the dynamics of single short DNA and study its interaction with enzyme. We further show that the particle motion pattern can be used to identify specific and non-specific interactions in immunoassays. We anticipate that our 3D tracking technique can contribute to the understanding of molecular dynamics and interactions at the single-molecule level.

Label-free techniques for quantification of protein-protein interaction often requires protein samples separated from complex media using affinity purification tools such as magnetic nanoparticles. However, the separated proteins are attached to the nanoparticles and need additional preparation steps, including elution and immobilization to a sensor surface before measurement. To streamline this tedious process, we present a method that can directly quantify the protein binding kinetics on nanoparticles without elution and immobilization, by optically tracking the nanoparticle size change upon ligand binding. We measured antibody binding to nanoparticles with captured protein, which was pulled down from a different medium prior to the measurement. The source of noise for the method was also analyzed. We anticipate this method can simplify the workflow from protein separation to detection while providing sufficient binding kinetics and affinity information for protein studies.

Rapid and sensitive detection of biomarkers is the key to the diagnosis of acute diseases. One example is the detection of troponin in myocardial infarction. Here we report a gradient-based digital immunoassay method, which can achieve high-sensitivity cardiac troponin T (hs-cTnT) detection with only 1 microliter of plasma sample. We designed a multizone microfluidic channel functionalized with capture antibody specific to troponin. Taking advantage of limited sample volume, a troponin concentration gradient is created along the channel because of binding induced depletion. We quantified the concentration gradient by counting the detection antibody conjugated gold nanoparticles bound to different test zones with optical imaging. Differential counting between the zones removes most common noises and non-specific bindings. The total analytical time is about 30 minutes, and the limit of quantification is 6.2 ng/L. We examined 41 clinical plasma samples from 15 patients and the change in hs-cTnT concentration in serial samples showed good linear correlation with clinical results (R(2) = 0.98). Therefore, this simple and sensitive gradient-based digital immunoassay method is a promising technology for clinical hs-cTnT detection and could be adapted for detection of other biomarkers.

Most label-free detection technologies detect the masses of molecules, and their sensitivities thus decrease with molecular weight, making it challenging to detect small molecules. To address this need, we have developed a charge-sensitive optical detection (CSOD) technique, which detects the charge rather than the mass of a molecule with an optical fiber. However, the effective charge of a molecule decreases with the buffer ionic strength. For this reason, the previous CSOD works with diluted buffers, which could affect the measured molecular binding kinetics. Here, we show a technique capable of detecting molecular binding kinetics in normal ionic strength buffers. An H-shaped sample well was developed to increase the current density at the sensing area to compensate the signal loss due to ionic screening at normal ionic strength buffer, while keeping the current density low at the electrodes to minimize the electrode reaction. In addition, agarose gels were used to cover the electrodes to prevent electrode reaction generated bubbles from entering the sensing area. With this new design, we have measured the binding kinetics between G-protein-coupled receptors (GPCRs) and their small molecule ligands in normal buffer. We found that the affinities measured in normal buffer are stronger than those measured in diluted buffer, likely due to the stronger electrostatic repulsion force between the same charged ligands and receptors in the diluted buffer.

Charge is a fundamental property of a molecule, and precisely measuring it enables detection of the molecule and helps understand various chemical processes involving charge. Here we show a method to measure the charge of a single nanoparticle and binding of charged molecules to the nanoparticle using a conventional bright field optical microscope. The nanoparticle is tethered to an indium tin oxide surface with a polymer and driven into oscillation with an alternating electric field, which produces scattered light captured by a camera. The weak scattered light is separated from the intense bright field background using a Fourier transform filter, and the image contrast change provides the effective charge of the nanoparticle with precision of a few electron charges or less. This method allows us to detect DNA binding to the nanoparticles, demonstrating a simple method to detect and study molecules with a conventional optical microscope.

Membrane proteins play vital roles in cellular signaling processes and serve as the most popular drug targets. A key task in studying cellular functions and developing drugs is to measure the binding kinetics of ligands with the membrane proteins. However, this has been a long-standing challenge because one must perform the measurement in a membrane environment to maintain the conformations and functions of the membrane proteins. Here, we report a new method to measure ligand binding kinetics to membrane proteins using self-assembled virion oscillators. Virions of human herpesvirus were used to display human G-protein-coupled receptors (GPCRs) on their viral envelopes. Each virion was then attached to a gold-coated glass surface via a flexible polymer to form an oscillator and driven into oscillation with an alternating electric field. By tracking changes in the oscillation amplitude in real-time with subnanometer precision, the binding kinetics between ligands and GPCRs was measured. We anticipate that this new label-free detection technology can be readily applied to measure small or large ligand binding to any type of membrane proteins and thus contribute to the understanding of cellular functions and screening of drugs.

Measuring ligand-protein interactions is critical for unveiling molecular-scale biological processes in living systems and for screening drugs. Various detection technologies have been developed, but quantifying the binding kinetics of small molecules to the proteins remains challenging because the sensitivities of the mainstream technologies decrease with the size of the ligand. Here, we report a method to measure and quantify the binding kinetics of both large and small molecules with self-assembled nano-oscillators, each consisting of a nanoparticle tethered to a surface via long polymer molecules. By applying an oscillating electric field normal to the surface, the nanoparticle oscillates, and the oscillation amplitude is proportional to the number of charges on the nano-oscillator. Upon the binding of ligands onto the nano-oscillator, the oscillation amplitude will change. Using a plasmonic imaging approach, the oscillation amplitude is measured with subnanometer precision, allowing us to accurately quantify the binding kinetics of ligands, including small molecules, to their protein receptors. This work demonstrates the capability of nano-oscillators as an useful tool for measuring the binding kinetics of both large and small molecules.

Timely diagnosis of acute diseases improves treatment outcomes and saves lives, but it requires fast and precision quantification of biomarkers. Here we report a time-resolved digital immunoassay based on plasmonic imaging of binding of single nanoparticles to biomarkers captured on a sensor surface. The real-time and high contrast of plasmonic imaging lead to fast and precise counting of the individual biomarkers over a wide dynamic range. We demonstrated the detection principle, evaluated the performance of the method using procalcitonin (PCT) as an example, and achieved a limit of detection of ~ 3 pg/mL, dynamic range of 4-12500 pg/mL, for a total detection time of ~ 25 mins.

Human G protein-coupled receptors (GPCRs) respond to various ligands and stimuli. However, GPCRs rely on membrane for proper folding, making their biochemical properties difficult to study. By displaying GPCRs in viral envelopes, we fabricated a Virion Display (VirD) array containing 315 non-olfactory human GPCRs for functional characterization. Using this array, we found that 10 of 20 anti-GPCR mAbs were ultra-specific. We further demonstrated that those failed in the mAb assays could recognize their canonical ligands, suggesting proper folding. Next, using two peptide ligands on the VirD-GPCR array, we identified expected interactions and novel interactions. Finally, we screened the array with group B Streptococcus, a major cause of neonatal meningitis, and demonstrated that inhibition of a newly identified target, CysLTR1, reduced bacterial penetration both in vitro and in vivo. We believe that the VirD-GPCR array holds great potential for high-throughput screening for small molecule drugs, affinity reagents, and ligand deorphanization., G protein-coupled receptors (GPCRs) are important targets for drug discovery. Here, the authors develop a Virion Display array of 315 functional non-odorant GPCRs, providing a platform for high-throughput, unbiased screening for small molecule drugs, affinity reagents, and microbial interactions.

Luminol electrochemiluminescence (ECL) imaging was developed for the parallel measurement of active membrane cholesterol at single living cells, thus establishing a novel electrochemical detection technique for single cells with high analysis throughput and low detection limit. In our strategy, the luminescence generated from luminol and hydrogen peroxide upon the potential was recorded in one image so that hydrogen peroxide at the surface of multiple cells could be simultaneously analyzed. Compared with the classic microelectrode array for the parallel single-cell analysis, the plat electrode only was needed in our ECL imaging, avoiding the complexity of electrode fabrication. The optimized ECL imaging system showed that hydrogen peroxide as low as 10 μM was visible and the efflux of hydrogen peroxide from cells could be determined. Coupled with the reaction between active membrane cholesterol and cholesterol oxidase to generate hydrogen peroxide, active membrane cholesterol at cells on the electrode was analyzed at single-cell level. The luminescence intensity was correlated with the amount of active membrane cholesterol, validating our system for single-cell cholesterol analysis. The relative high standard deviation on the luminescence suggested high cellular heterogeneities on hydrogen peroxide efflux and active membrane cholesterol, which exhibited the significance of single-cell analysis. This success in ECL imaging for single-cell analysis opens a new field in the parallel measurement of surface molecules at single cells.

A luminol electrochemiluminescence assay was reported to analyze active cholesterol at the plasma membrane in single mammalian cells. The cellular membrane cholesterol was activated by the exposure of the cells to low ionic strength buffer or the inhibition of intracellular acyl-coA/cholesterol acyltransferase (ACAT). The active membrane cholesterol was reacted with cholesterol oxidase in the solution to generate a peak concentration of hydrogen peroxide on the electrode surface, which induced a measurable luminol electrochemiluminescence. Further treatment of the active cells with mevastatin decreased the active membrane cholesterol resulting in a drop in luminance. No change in the intracellular calcium was observed in the presence of luminol and voltage, which indicated that our analysis process might not interrupt the intracellular cholesterol trafficking. Single cell analysis was performed by placing a pinhole below the electrode so that only one cell was exposed to the photomultiplier tube (PMT). Twelve single cells were analyzed individually, and a large deviation on luminance ratio observed exhibited the cell heterogeneity on the active membrane cholesterol. The smaller deviation on ACAT/HMGCoA inhibited cells than ACAT inhibited cells suggested different inhibition efficiency for sandoz 58035 and mevastatin. The new information obtained from single cell analysis might provide a new insight on the study of intracellular cholesterol trafficking.

Nanodisc technology provides membrane proteins with a nativelike lipid bilayer and much-needed solubility and enables in vitro quantification of membrane protein binding with ligands. However, it has been a challenge to measure interaction between small-molecule ligands and nanodisc-encapsulated membrane proteins, because the responses of traditional mass-based detection methods scale with the mass of the ligands. We have developed a charge-sensitive optical detection (CSOD) method for label-free measurement of the binding kinetics of low molecular mass ligands with nanodisc-encapsulated membrane proteins. This microplate-compatible method is sensitive to the charge instead of the mass of a ligand and is able to measure both large and small molecules in a potentially high-throughput format. Using CSOD, we measured the binding kinetics between peptide and small-molecule ligands and a nanodisc-encapsulated potassium ion channel protein, KcsA-Kv1.3. Both association and dissociation rate constants for these ligands are obtained for the first time. The CSOD results were validated by the consistency of the values with reported binding affinities. In addition, we found that CSOD can tolerate up to 3.9% dimethyl sulfoxide (DMSO) and up to 10% serum, which shows its compatibility with realistic sample conditions.