Single-molecule Binding Events Research Articles

(Invited) Combined Single-Molecule Raman and Conductance Spectroscopies

The nanoscale manipulation and characterization of individual molecules is necessary for understanding the intricacies of molecular structure which ultimately governs phenomena such as reaction mechanisms, catalysis, local effective temperatures, surface interactions, and charge transport phenomenon. Along these lines, the field of molecular-scale electronics has evolved tremendously in recent years: from the initial experiments claiming single-molecule conductance measurements to the development of robust and reproducible platforms that have begun providing a detailed understanding of the charge transport properties of these systems. It has now become possible to probe the chemical, structural, mechanical, and electrical properties of single-molecule devices to explore unique functional paradigms for applications. However, continued advances in in situ characterization of a molecular junction are needed to provide detailed information about the molecular configuration and its impact on the charge transport, reactions, and device behavior. Single-molecule conductance and Raman spectroscopies each provide unique perspectives into the behavior of molecular systems and reactions at the single-molecule level. In this talk we will discuss the development and implementation of system designed to simultaneously obtain conductance information and Raman spectra from a molecular junction to provide this information. This will include the development of a MEMS-based break junction system that allows facile integration with a Raman microscope to maximize the solid-angle and photon collection.This multi-dimensional information yields repeatable, self-consistent, verification of single-molecule resolution, and allows for detailed analysis of structural and configurational changes of the molecule in situ. We will discuss the correlation between single-molecule binding events and changes in Raman spectra (intensity, modes, etc.) and conductance to explore the possibility of obtaining single-molecule spectra from the molecule bound between two electrodes. We will further explore the utility of this system for in situ characterization of active single-molecule devices including electrically-active single-molecule switches and memory devices.

Single-molecule calorimeter and free energy landscape

The precise measurement of thermodynamic and kinetic properties for biomolecules provides the detailed information for a multitude of applications in biochemistry, biosensing, and health care. However, sensitivity in characterizing the thermodynamic binding affinity down to a single molecule, such as the Gibbs free energy ([Formula: see text]), enthalpy ([Formula: see text]), and entropy ([Formula: see text]), has not materialized. Here, we develop a nanoparticle-based technique to probe the energetic contributions of single-molecule binding events, which introduces a focused laser of optical tweezer to an optical path of plasmonic imaging to accumulate and monitor the transient local heating. This single-molecule calorimeter uncovers the complex nature of molecular interactions and binding characterizations, which can be employed to identify the thermodynamic equilibrium state and determine the energetic components and complete thermodynamic profile of the free energy landscape. This sensing platform promises a breakthrough in measuring thermal effect at the single-molecule level and provides a thorough description of biomolecular specific interactions.

Direct Measurement of Single-Molecule Ligand-Receptor Interactions.

Measuring the kinetics that govern ligand-receptor interactions is fundamental to our understanding of pharmacology. For ligand-gated ion channels, binding of an agonist triggers allosteric motions that open an integral ion-permeable pore. By mathematically modeling stochastic electrophysiological responses with high temporal resolution (ms), previous single channel studies have been able to infer the rate constants of ligands binding to these receptors. However, there are no reports of the direct measurement of the single-molecule binding events that are vital to how agonists exert their functional effects. For the first time, we report these direct measurements, the rate constants, and corresponding free energy changes, which describe the transitions between the different binding states. To achieve this, we use the super resolution technique: points accumulation for imaging in nanoscale topography (PAINT) to observe binding of ATP to orthosteric binding sites on the P2X1 receptor. Furthermore, an analysis of time-resolved single-molecule interactions is used to measure elementary rate constants and thermodynamic forces that drive the allosteric motions. These single-molecule measurements unequivocally establish the location of each binding states of the P2X1 receptor and the stochastic nature of the interaction with its native ligand. The analysis leads to the measurement of the forward and reverse rates from a weak ligand-binding state to a strong ligand binding state that is linked to allosteric motion and ion pore formation. These rates (kα = 1.41 sec-1 and kβ = 0.32 sec-1) were then used to determine the free energy associated with this critical mechanistic step (3.7 kJ/mol). Importantly, the described methods can be readily applied to all ligand-gated ion channels, and more broadly to the molecular interactions of other classes of membrane proteins.

Nanomolar ATP binding to single myosin cross-bridges in rigor: a molecular approach to studying myosin ATP kinetics using single human cardiomyocytes.

Our knowledge in the field of cardiac muscle and associated cardiomyopathies has been evolving incrementally over the past 60 years and all was possible due to the parallel progress in techniques and methods allowing to take a fresh glimpse at an old problem. Here, we describe an exciting tool used to examine the various states of the human cardiac myosin at the single molecule level. By imaging single Alexa647-ATP binding to permeabilised cardiomyocytes using total internal reflection fluorescence (TIRF) microscopy, we are able to acquire large populations of events in a short timeframe (~ 5000 sites in ~ 10 min) and measure each binding event with high spatio-temporal resolution. The applied algorithm decomposes the point pattern of single molecule binding events into individually distinct binding sites that enables us to recover kinetic parameters, such as bound or free time per site. This single molecule binding approach is a useful tool used to examine muscle contractility. Of particular importance is its application to probing the dynamic lifetimes and proportion of myosins in the super-relaxed state in human cardiomyopathies.

Detecting and Directing Single Molecule Binding Events on H-Si(100) with Application to Ultradense Data Storage.

Many diverse material systems are being explored to enable smaller, more capable and energy efficient devices. These bottom up approaches for atomic and molecular electronics, quantum computation, and data storage all rely on a well-developed understanding of materials at the atomic scale. Here, we report a versatile scanning tunneling microscope (STM) charge characterization technique, which reduces the influence of the typically perturbative STM tip field, to develop this understanding even further. Using this technique, we can now observe single molecule binding events to atomically defined reactive sites (fabricated on a hydrogen-terminated silicon surface) through electronic detection. We then developed a simplified error correction tool for automated hydrogen lithography, quickly directing molecular hydrogen binding events using these sites to precisely repassivate surface dangling bonds (without the use of a scanned probe). We additionally incorporated this molecular repassivation technique as the primary rewriting mechanism in ultradense atomic data storage designs (0.88 petabits per in2).

Beyond the TCR, Antigen Discrimination in T Cells Continues in the LAT:GRB2:SOS Protein Condensate

T cells activate in response to a stochastic series of single molecule binding events between T cell receptors (TCRs) and peptide ligands presented on major histocompatibility complexes (pMHCs) accumulated at a cell-cell interface. Discrimination between ligands with subtly different affinities ensures selective and robust immunological responses upon TCR triggering. Formation of protein condensates based on the scaffolding protein Linker for Activation of T cells (LAT) is a key intermediate step in TCR signaling. LAT is multi-phosphorylated on tyrosine residues by the kinase, ZAP70, which is activated by triggered TCRs. The phosphotyrosines recruit Grb2, which, via interactions with the multivalent GEF, SOS, forms an extended LAT:Grb2:SOS assembly that resembles a phase transition. We mapped single pMHC:TCR binding events in T cells expressing fluorescent protein-tagged LAT and show that LAT assemblies are discrete and have characteristic features that are independent of the originating binding event. Furthermore, LAT assemblies can become spatially uncoupled from the pMHC:TCR complex. These individual assemblies are capable of deforming the plasma membrane and creating local domains that may participate in protein segregation or control reaction kinetics. The probability of LAT assembly is a function of the pMHC affinity, suggesting a second layer of kinetic proofreading that exists beyond the triggered TCR. We recently introduced a molecular impulse-response assay which maps pMHC:TCR binding events to early T cell activation. Results here argue that LAT assemblies may confer a molecular memory that connects a series of spatiotemporally correlated binding events to cellular decision-making. By extending the impulse-response assay, we show that activating cells experience a characteristic, fast rate of LAT assembly. Taken together, we present new molecular dimensions to T cell activation that could provide actionable therapeutic insights.

Mapping the stochastic sequence of individual ligand-receptor binding events to cellular activation: T cells act on the rare events.

T cell receptor (TCR) binding to agonist peptide major histocompatibility complex (pMHC) triggers signaling events that initiate T cell responses. This system is remarkably sensitive, requiring only a few binding events to successfully activate a cellular response. On average, activating pMHC ligands exhibit mean dwell times of at least a few seconds when bound to the TCR. However, a T cell accumulates pMHC-TCR interactions as a stochastic series of discrete, single-molecule binding events whose individual dwell times are broadly distributed. With activation occurring in response to only a handful of such binding events, individual cells are unlikely to experience the average binding time. Here, we mapped the ensemble of pMHC-TCR binding events in space and time while simultaneously monitoring cellular activation. Our findings revealed that T cell activation hinges on rare, long-dwell time binding events that are an order of magnitude longer than the average agonist pMHC-TCR dwell time. Furthermore, we observed that short pMHC-TCR binding events that were spatially correlated and temporally sequential led to cellular activation. These observations indicate that T cell antigen discrimination likely occurs by sensing the tail end of the pMHC-TCR binding dwell time distribution rather than its average properties.

Spatially Resolved Sensitivity of Single-Particle Plasmon Sensors.

The high sensitivity of localized surface plasmon resonance sensors to the local refractive index allows for the detection of single-molecule binding events. Though binding events of single objects can be detected by their induced plasmon shift, the broad distribution of observed shifts remains poorly understood. Here, we perform a single-particle study wherein single nanospheres bind to a gold nanorod, and relate the observed plasmon shift to the binding location using correlative microscopy. To achieve this we combine atomic force microscopy to determine the binding location, and single-particle spectroscopy to determine the corresponding plasmon shift. As expected, we find a larger plasmon shift for nanospheres binding at the tip of a rod compared to its sides, in good agreement with numerical calculations. However, we also find a broad distribution of shifts even for spheres that were bound at a similar location to the nanorod. Our correlative approach allows us to disentangle effects of nanoparticle dimensions and binding location, and by comparison to numerical calculations we find that the biggest contributor to this observed spread is the dispersion in nanosphere diameter. These experiments provide insight into the spatial sensitivity and signal-heterogeneity of single-particle plasmon sensors and provides a framework for signal interpretation in sensing applications.

Fast, Background-Free DNA-PAINT Imaging Using FRET-Based Probes

DNA point accumulation in nanoscale topography (DNA-PAINT) enables super-resolution microscopy by harnessing the predictable, transient hybridization between short dye-labeled "imager" and complementary target-bound "docking" strands. DNA-PAINT microscopy allows sub-5 nm spatial resolution, spectrally unlimited multiplexing, and quantitative image analysis. However, these abilities come at the cost of nonfluorogenic imager strands, also emitting fluorescence when not bound to their docking strands. This has thus far prevented rapid image acquisition with DNA-PAINT, as the blinking rate of probes is limited by an upper-bound of imager strand concentrations, which in turn is dictated by the necessity to facilitate the detection of single-molecule binding events over the background of unbound, freely diffusing probes. To overcome this limitation and enable fast, background-free DNA-PAINT microscopy, we here introduce FRET-based imaging probes, alleviating the concentration-limit of imager strands and speeding up image acquisition by several orders of magnitude. We assay two approaches for FRET-based DNA-PAINT (or FRET-PAINT) using either fixed or transient acceptor dyes in combination with transiently binding donor-labeled DNA strands and achieve high-quality super-resolution imaging on DNA origami structures in a few tens of seconds. Finally, we also demonstrate the applicability of FRET-PAINT in a cellular environment by performing super-resolution imaging of microtubules in under 30 s. FRET-PAINT combines the advantages of conventional DNA-PAINT with fast image acquisition times, facilitating the potential study of dynamic processes.

Using Single Myosin and Small Myosin Ensembles to Examine Thin Filament Activation in a Laser Trap Assay

Thin filament activation in muscle requires both Ca++ binding to troponin and myosin strongly binding to actin. Despite extensive study, the relative importance of each of these processes to overall thin filament activation remains unclear. To determine the role of Ca++ we examined the effect of increasing Ca++ concentrations on the frequency of single molecule binding events in a single molecule laser trap assay using reconstituted thin filaments. The frequency of actomyosin binding events increased from 0.03 at low Ca++ (pCa7), to 0.30 at pCa6 to 0.44 events/sec at pCa5. Increasing the myosin concentration so that the regulated filament interacted with a mini-ensemble of myosin (∼6 heads) in the laser trap assay was performed to gain insight into the cooperative nature of actomyosin binding. Binding events were broadly distributed at the highest Ca++ (pCa5), generating forces from 0.2 to 6.75pN, but averaged 1.38±0.05pN. As Ca++ was decreased the high force events were lost, reducing the average force to 0.76pN at pCa6, 0.61 at pCa7 and 0.2pN at pCa9. In addition the total number of actomyosin interactions decreased from 3.7/sec at pCa5 to just 0.05/sec at pCa9. Finally, we measured filament velocity in a motility assay as a function of Ca++ and ATP to distinguish rigor activation vs. Ca++ activation in a large ensemble of myosin. At pCa9 filaments were activated at ATP levels <200 μM while at intermediate Ca++ levels (pCa6.5) filaments achieved only half of the maximum velocity observed at saturating Ca++ (pCa5). Preliminary modeling efforts suggest that these observations are consistent with a model where Ca++ regulates myosin's weak to strong binding transition but once bound strongly an actomyosin interaction activates the filament locally, facilitating neighboring molecules to bind.

Fixed-gap tunnel junction for reading DNA nucleotides.

Previous measurements of the electronic conductance of DNA nucleotides or amino acids have used tunnel junctions in which the gap is mechanically adjusted, such as scanning tunneling microscopes or mechanically controllable break junctions. Fixed-junction devices have, at best, detected the passage of whole DNA molecules without yielding chemical information. Here, we report on a layered tunnel junction in which the tunnel gap is defined by a dielectric layer, deposited by atomic layer deposition. Reactive ion etching is used to drill a hole through the layers so that the tunnel junction can be exposed to molecules in solution. When the metal electrodes are functionalized with recognition molecules that capture DNA nucleotides via hydrogen bonds, the identities of the individual nucleotides are revealed by characteristic features of the fluctuating tunnel current associated with single-molecule binding events.

Quantum dot-DNA origami binding: a single particle, 3D, real-time tracking study

The binding process of quantum dots and DNA origami was monitored using a 3D, real-time, single-particle tracking system. Single-molecule binding events were directly observed and precise measurements of the diffusion coefficient and second-order photon correlation function, g(2)(τ), were combined to distinguish free quantum dots from different conjugates of nQdot-origami.

Fabrication of nanoscale zero-mode waveguides using microlithography for single molecule sensing

We present a novel approach to the fabrication of zero-mode waveguides (ZMWs) using inexpensive processing techniques. Our method is capable of rapid fabrication of circular nanoapertures with diameters ranging from 70 nm to 2 μm, allowing us to perform a detailed characterization of the dependence of the fluorescence emission on the waveguide diameter. We also validated the use of the fabricated ZMWs by detecting single molecule binding events with a signal-to-noise ratio of ten.

Stochastic sensing of proteins with receptor-modified solid-state nanopores

Solid-state nanopores are capable of the label-free analysis of single molecules. It is possible to add biochemical selectivity by anchoring a molecular receptor inside the nanopore, but it is difficult to maintain single-molecule sensitivity in these modified nanopores. Here, we show that metallized silicon nitride nanopores chemically modified with nitrilotriacetic acid receptors can be used for the stochastic sensing of proteins. The reversible binding and unbinding of the proteins to the receptors is observed in real time, and the interaction parameters are statistically analysed from single-molecule binding events. To demonstrate the versatile nature of this approach, we detect His-tagged proteins and discriminate between the subclasses of rodent IgG antibodies.

DNA Nanoprobe for Intracellular Dynamics

We have developed a novel method to follow the dynamics of DNA interacting with the cellular environment in vivo using two-color correlation microscopy. A DNA probe is end-labeled with two quantum dots and transfected into an axenic strain of Dictyostelium discoideum. The motion of the quantum dots is observed with two-color fluorescence video microscopy. The computed time correlation functions of this two-particle motion reflect the fluctuations of the DNA probe as a result of its interactions with the cellular environment. Substantial differences between live cells and dead yet structurally intact cells point to a strong coupling of active, motor-driven fluctuations in the cell to the DNA probe. This suggests that the motion of native cellular DNA may similarly be driven by active processes instead of relying on purely thermal passive fluctuations. We also note that the difference between the autocorrelations of the center of mass motion and the relative motion of the two quantum dots is a sensitive measure for the effective length of the DNA probe on a length scale around one persistence length (∼ 50 nm). This paves the way for experiments with more complex DNA probes that can bind to intracellular proteins, and report single-molecule binding events through apparent length changes and consequently changes in this correlation measure.

Scanning force microscopy based amperometric biosensors

Cantilever-based biosensing has developed into an important research area especially for biomedical and clinical analysis. In particular, the possibility to scan miniaturized amperometric biosensors with high-fidelity distance control across biomedically relevant sample surfaces enables the determination of pertinent analytes such as, e.g., adenosine 5′-triphosphate (ATP). ATP is involved in a wide variety of important regulatory cellular mechanisms, and quantification of ATP has therefore been the focus of extensive research in recent years. Usually, scanning force microscopy-based biosensing relates to detection principles based on either mass-sensitive mechanical transduction, which can be detected as a frequency change of the cantilever, or a change in the force constant due to the absorption of molecules onto the sensor surface, as shown in Fig. 1a. Alternatively, a bimetallic cantilever can be used as a temperature sensing device for detecting calorimetric changes due to specific binding to an immobilized receptor, or due to absorption of molecules. Advanced microfabrication in combination with sophisticated surface functionalization schemes results in sensing interfaces tailored to specifically interact with analyte molecules, thus providing specificity and sensitivity of such miniaturized devices to the picomolar and even femtomolar concentration range [1]. Conventional cantilevertype biosensors do not provide a sharp tip, and are typically not applied for laterally resolved measurements in an imaging mode, which requires that the sensor is scanned across, e.g., a biological specimen at a controlled distance. Recently, the National Heart Lung and Blood Institute has published a report identifying demands for future developments related to diagnosis and therapy of cardiovascular, pulmonary, and hematologic diseases, which identifies in vivo nanosensors as a promising prospective application of nanotechnology for real-timemonitoring of biological signals in response to cardiac or inflammatory events [2]. Hence, besides measurements in complex background matrices, the analytical challenges encompass the ability of biosensors to provide information on structural changes along with chemical information on, e.g., regulatory processes at single cells or cell ensembles, ideally at a nanometer scale. Consequently, miniaturization of the transducer and sensing interface, along with positioning of such miniaturized sensors in close proximity to the sample surface providing laterally resolved quantitative information is a prerequisite. Alternatively to mass-sensitive cantilever-based sensing in bulk solution, atomic force microscopy (AFM) probes can be chemically functionalized, which enables detection and quantification of forces associated with single-molecule binding events at ambient conditions. Recently, this concept has evolved into a combined tool providing laterally resolved information along with force mapping, also known as topography and recognition imaging [3], which enables the visualization of single receptor binding sites at biological surfaces. The present article, however, focuses on the detection of chemical signaling events involving the secretion of transmitter molecules into the extracellular space, and associated efforts to detect such molecules and their concentration for enhancing fundamental understanding on disease-related cellular processes at a molecular level. Anal Bioanal Chem (2008) 390:239–243 DOI 10.1007/s00216-007-1670-8

Label-Free, Single-Molecule Detection with Optical Microcavities

Current single-molecule detection techniques require labeling the target molecule. We report a highly specific and sensitive optical sensor based on an ultrahigh quality (Q) factor (Q > 10(8)) whispering-gallery microcavity. The silica surface is functionalized to bind the target molecule; binding is detected by a resonant wavelength shift. Single-molecule detection is confirmed by observation of single-molecule binding events that shift the resonant frequency, as well as by the statistics for these shifts over many binding events. These shifts result from a thermo-optic mechanism. Additionally, label-free, single-molecule detection of interleukin-2 was demonstrated in serum. These experiments demonstrate a dynamic range of 10(12) in concentration, establishing the microcavity as a sensitive and versatile detector.

Detection of HSP60 on the membrane surface of stressed human endothelial cells by atomic force and confocal microscopy

The highly conserved and ubiquitous heat shock proteins (HSP) are essential for the cellular homeostasis and efficiently trigger cellular responses to stress conditions. Both microbial and human HSP act as dominant antigens in numerous infectious and autoimmune diseases such as atherosclerosis, inducing a strong immune-inflammatory response. In the present study, the surface localization of HSP60 on stressed and unstressed human umbilical venous endothelial cells (HUVECs) was investigated using sensitive high resolution microscopy methods and flow cytometry. Confocal laser scanning microscopy (CLSM) revealed an increase of HSP60 in the mitochondria and on the surface of heat-stressed living and fixed HUVECs compared to unstressed cells. Atomic force microscopy (AFM), which has developed as sensitive surface-probe technique in biology, confirmed the presence of HSP60 on the membrane of stressed cells at an even higher lateral resolution by detecting specific single molecule binding events between the monoclonal antibody AbII-13 tethered to AFM tips and HSP60 molecules on cells. The interaction force (force required to break a single AbII-13/HSP60 bond) was 59+/-2 pN, which correlated nicely to the 51+/-1 pN measured with isolated HSP60 attached to mica surfaces. Overall, we found clear evidence for the occurrence of HSP60 on the surface of stressed HUVECs in a very similar patchy distribution pattern in living and fixed cells. The relevance of our findings with respect to the role of HSP60 in atherogenesis is discussed.

In situ single-molecule detection of antibody-antigen binding by tapping-mode atomic force microscopy.

We performed in situ detection of specific and nonspecific binding during immunoreaction on surfaces at the same location before and after analyte was injected using tapping-mode atomic force microscopy (TM-AFM) in liquid and demonstrated the ability of TM-AFM to monitor the occurrence of single-molecule binding events and to distinguish nonspecific from specific binding by examining topographical change. Two antigen/antibody pairs were investigated: chorionic gonadotropin (hCG)/mouse monoclonal anti-hCG and goat IgG (anti-intact hCG)/ mouse monoclonal anti-goat IgG. Antibody (or antigen) molecules were covalently immobilized on uniform mixed self-assembled monolayers (SAMs) terminated with carboxylic acid and hydroxyl groups. Mixed SAMs allow the control of the density of immobilized antibody (or antigen) on surfaces to achieve the detection of individual antigens, antibodies, and antigen/antibody complexes. This in situ TM-AFM-based detection method allows the single-molecule detection of antigen/antibody binding under near-physiological environment and the distinction of nonspecific from specific binding. It could be extended into a microarray.

Detection of single-molecule interactions using correlated thermal diffusion.

Observation of discrete, single-molecule binding events allows one to bypass assumptions required to infer single-molecule properties from studies of ensembles of molecules. Optically trapped beads and glass microneedles have been applied to detect single-molecule binding events, but it remains difficult to identify signs of binding events given the large displacements induced by thermal forces. Here, we exploit thermal diffusion by using correlation between motion of optically trapped beads attached to both ends of a single actin filament to track binding events of individual myosin molecules. We use correlated diffusion to measure the stiffness of a single myosin molecule and estimate its thermal fluctuation in a poststroke state as comparable in amplitude to the measured stroke distance. The use of correlated diffusion to measure kinetics of single-molecule interactions and the stiffness of the interacting moieties should be applicable to any pair of interacting molecules, and not limited to biological motors.