Confirming Silent Translocation through Nanopores with Simultaneous Single-Molecule Fluorescence and Single-Channel Electrical Recordings.

Most of what is known concerning the luminal passage of materials through nanopores arises from electrical measurements. Whether nanopores are biological, solid-state, synthetic, hybrid, glass-capillary-based, or protein ion channels in cells and tissues, characteristic signatures embedded in the flow of ionic current are foundational to understanding functional behavior. In contrast, this work describes passage through a nanopore that occurs without producing an electrical signature. We refer to the phenomenon as "silent translocation." By definition, silent translocations are invisible to the standard tools of electrophysiology and fundamentally require a simultaneous ancillary measurement technique for positive identification. As a result, this phenomenon has been largely unexplored in the literature. Here, we report on a derivative of Cyanine 5 (sCy5a) that passes through the α-hemolysin (αHL) nanopore silently. Simultaneously acquired single-molecule fluorescence and single-channel electrical recordings from bilayers formed over a closed microcavity demonstrate that translocation does indeed take place, albeit infrequently. We report observations of silent translocation as a function of time, dye concentration, and nanopore population in the bilayer. Lastly, measurement of the translocation rate as a function of applied potential permits estimation of an effective energy barrier for transport through the pore as well as the effective charge on the dye, all in the absence of an information-containing electrical signature.

Mechanically Enhancing Planar Lipid Bilayers with a Minimal Actin Cortex.

All cells in all domains of life possess a cytoskeleton that provides mechanical resistance to deformation and general stability to the plasma membrane. Here, we utilize a two-dimensional scaffolding created by actin filaments to convey mechanical support upon relatively fragile planar bilayer membranes (black lipid membranes, BLMs). Robust biomembranes play a critical role in the development of protein nanopore sensor applications and might also prove helpful in ion-channel research. Our investigation utilizes a minimal actin cortex (MAC) that is formed by anchoring actin filaments to lipid membranes via a biotin-streptavidin-biotin bridge. We characterize the joined structure using various modes of optical microscopy, electrophysiology, and applied mechanical stress (including measurements of elastic modulus). Our findings show the resulting structure includes a thin supporting layer of actin. Electrical studies indicate that the integrity of the MAC-bilayer composite remains unchanged over the limits of our tests (i.e., hours to days). The actin filament structure can remain intact for months. Minimalistic layering of the actin support network produces an increase in the apparent elastic modulus of the MAC-derivatized bilayer by >100×, compared to unmodified BLMs. Furthermore, the resistance to applied stress improves with the number of actin layers, which can be cross-linked to arbitrary thicknesses, in principle. The weblike support structure retains the lateral fluidity of the BLM, maintains the high electrical resistance typical of traditional BLMs, enables relatively uninhibited molecular access to the lipid surface from bulk solution, and permits nanopore self-assembly and insertion in the bilayer. These interfacial features are highly desirable for ion-channel and nanopore sensing applications.

Design and Construction of a Multi-Tiered Minimal Actin Cortex for Structural Support in Lipid Bilayer Applications.

Artificial lipid bilayers have revolutionized biochemical and biophysical research by providing a versatile interface to study aspects of cell membranes and membrane-bound processes in a controlled environment. Artificial bilayers also play a central role in numerous biosensing applications, form the foundational interface for liposomal drug delivery, and provide a vital structure for the development of synthetic cells. But unlike the envelope in many living cells, artificial bilayers can be mechanically fragile. Here, we develop prototype scaffolds for artificial bilayers made from multiple chemically linked tiers of actin filaments that can be bonded to lipid headgroups. We call the interlinked and layered assembly a multiple minimal actin cortex (multi-MAC). Construction of multi-MACs has the potential to significantly increase the bilayer's resistance to applied stress while retaining many desirable physical and chemical properties that are characteristic of lipid bilayers. Furthermore, the linking chemistry of multi-MACs is generalizable and can be applied almost anywhere lipid bilayers are important. This work describes a filament-by-filament approach to multi-MAC assembly that produces distinct 2D and 3D architectures. The nature of the structure depends on a combination of the underlying chemical conditions. Using fluorescence imaging techniques in model planar bilayers, we explore how multi-MACs vary with electrostatic charge, assembly time, ionic strength, and type of chemical linker. We also assess how the presence of a multi-MAC alters the underlying lateral diffusion of lipids and investigate the ability of multi-MACs to withstand exposure to shear stress.

Temperature sculpting in yoctoliter volumes.

The ability to perturb large ensembles of molecules from equilibrium led to major advances in understanding reaction mechanisms in chemistry and biology. Here, we demonstrate the ability to control, measure, and make use of rapid temperature changes in fluid volumes that are commensurate with the size of single molecules. The method is based on attaching gold nanoparticles to a single nanometer-scale pore formed by a protein ion channel. Visible laser light incident on the nanoparticles causes a rapid and large increase of the adjacent solution temperature, which is estimated from the change in the nanopore ionic conductance. The temperature shift also affects the ability of individual molecules to enter into and interact with the nanopore. This technique could significantly improve sensor systems and force measurements based on single nanopores, thereby enabling a method for single molecule thermodynamics and kinetics.

The effects of diffusion on an exonuclease/nanopore-based DNA sequencing engine.

Over 15 years ago, the ability to electrically detect and characterize individual polynucleotides as they are driven through a single protein ion channel was suggested as a potential method for rapidly sequencing DNA, base-by-base, in a ticker tape-like fashion. More recently, a variation of this method was proposed in which a nanopore would instead detect single nucleotides cleaved sequentially by an exonuclease enzyme in close proximity to one pore entrance. We analyze the exonuclease/nanopore-based DNA sequencing engine using analytical theory and computer simulations that describe nucleotide transport. The available data and analytical results suggest that the proposed method will be limited to reading <80 bases, imposed, in part, by the short lifetime each nucleotide spends in the vicinity of the detection element within the pore and the ability to accurately discriminate between the four mononucleotides.

Information content in fluorescence correlation spectroscopy: binary mixtures and detection volume distortion.

When properly implemented, fluorescence correlation spectroscopy (FCS) reveals numerous static and dynamic properties of molecules in solution. However, complications arise whenever the measurement scenario is complex. Specific limitations occur when the detection region does not match the ideal Gaussian geometry ubiquitously assumed by FCS theory, or when properties of multiple fluorescent species are assessed simultaneously. A simple binary solution of diffusers, where both mole fraction and diffusion constants are sought, can face interpretive difficulty. In order to better understand the limits of FCS, this study systematically explores the relationship between detection-volume distortion, diffusion constants, species mole fraction, and fitting methodology in analyses that utilize a two-component autocorrelation model. FCS measurements from solution mixtures of dye-labeled protein and free dye are compared to simulations, which predict the performance of FCS under a variety of experimental circumstances. The results reveal a range of conditions necessary for performing accurate measurements and describe experimental scenarios that should be avoided. The findings also provide guidelines for obtaining meaningful measurements when grossly distorted detection volumes are utilized and generally assess the latent information contained in FCS datasets.

Accurate optical analysis of single-molecule entrapment in nanoscale vesicles.

We present a nondestructive method to accurately characterize low analyte concentrations (0-10 molecules) in nanometer-scale lipid vesicles. Our approach is based on the application of fluorescence fluctuation analysis (FFA) and multiangle laser light scattering (MALLS) in conjunction with asymmetric field flow fractionation (AFFF) to measure the entrapment efficiency (the ratio of the concentration of encapsulated dye to the initial bulk concentration) of an ensemble of liposomes with an average diameter less than 100 nm. Water-soluble sulforhodamine B (SRB) was loaded into the aqueous interior of nanoscale liposomes synthesized in a microfluidic device. A confocal microscope was used to detect a laser-induced fluorescence signal resulting from both encapsulated and unencapsulated SRB molecules. The first two cumulants of this signal along with the autocorrelation function (ACF) were used to quantify liposome entrapment efficiency. Our analysis moves beyond typical, nonphysical assumptions of equal liposome size and brightness. These advances are essential for characterizing liposomes in the single-molecule encapsulation regime. Our work has further analytical impact because it could increase the interrogation time of free-solution molecular analysis by an order of magnitude and form the basis for the development of liposome standard reference materials.

A distributed algorithm for multi-tau autocorrelation.

Network data-transfer times in distributed simulation environments can be reduced by performing data analysis at the remote source, if the analytical technique does not require the entire set of data at once. This novel multi-tau autocorrelation algorithm allows time-domain data records to be processed in discrete, distributed segments and combined at a later point in time. The new approach agrees with autocorrelation results performed by concatenating the discrete segments before correlation, but it operates with significantly shortened processing times. The multi-tau algorithm also benefits from reduced memory requirements since it does not require access to the entire data record at once, and from improved scalability since the multi-tau algorithm has order O(N), while fast Fourier transform autocorrelation algorithms have order O(N log N). This distributed algorithm has particular utility in simulations of fluorescence correlation spectroscopy or photon correlation spectroscopy.

Proximal Capture Dynamics for a Single Biological Nanopore Sensor.

Single nanopore sensors enable capture and analysis of molecules that are driven to the pore entry from bulk solution. However, the distance between an analyte and the nanopore opening limits the detection efficiency. A theoretical basis for predicting particle capture rate is important for designing modified nanopore sensors, especially for those with covalently tethered reaction sites. Using the finite element method, we develop a soft-walled electrostatic block (SWEB) model for the alpha-hemolysin channel that produces a vector map of drift-producing forces on particles diffusing near the pore entrance. The maps are then coupled to a single-particle diffusion simulation to probe capture statistics and to track the trajectories of individual particles on the µs to ms time scales. The investigation enables evaluation of the interplay among the electrophoretic, electroosmotic, and thermal driving forces as a function of applied potential. The findings demonstrate how the complex drift-producing forces compete with diffusion over the nanoscale dimensions of the pore. The results also demonstrate the spatial and temporal limitations associated with nanopore detection and offer a basic theoretical framework to guide both the placement and kinetics of reaction sites located on, or near, the nanopore cap.

Heterogeneous translational dynamics of rhodamine B in polyelectrolyte multilayer thin films observed by single molecule microscopy.

The lateral diffusion dynamics of rhodamine B (RB) in polyelectrolyte multilayer (PEM) thin films has been studied with single-molecule confocal fluorescence microscopy. The films were made with sodium poly(sodium 4-styrenesulfonate) (PSS) and poly(diallydimethlyammonium chloride) (PDDA). Analysis of the real-time emission intensity traces reveals three diverse components of translational motion: (1) fast diffusion of RB through the confocal detection volume; (2) reversible tracer adsorption processes; and (3) nanoconfined diffusion. These processes cover a wide range of time scales. Analysis via fluorescence correlation spectroscopy (FCS) involves multicomponent fitting of the autocorrelated emission data. The model includes a free Brownian diffusion parameter, D, and two rate constants of desorption, k(-1) and k(-2). For RB in a PSS/PDDA thin film made with 0.01 M NaCl in the polyelectrolyte buildup solutions, D = 1.7 x 10(-7) cm(2)/s, k(-1) = 30 s(-1), and k(-2) = 0.1 s(-1). FCS was also performed on RB/PEM samples made with NaCl concentrations of the buildup solutions ranging from 0.01 to 0.7 M. A weak dependence of D and k(-1) on NaCl concentration was observed while k(-2) increased linearly with [NaCl].

Numerical fluorescence correlation spectroscopy for the analysis of molecular dynamics under nonstandard conditions.

The suitability of mathematical models used to extract kinetic information from correlated data constitutes a significant issue in fluorescence correlation spectroscopy (FCS). Standard FCS equations are derived from a simple Gaussian approximation of the optical detection volume, but some investigations have suggested this traditional practice can lead to inaccurate and misleading conclusions under many experimental circumstances, particularly those encountered in one-photon confocal measurements. Furthermore, analytical models cannot be derived for all measurement scenarios. We describe a novel numerical approach to FCS that circumvents conventional analytical models, enabling meaningful analyses even under extraordinarily unusual measurement conditions. Numerical fluorescence correlation spectroscopy (NFCS) involves quantitatively matching experimental correlation curves with synthetic curves generated via diffusion simulation or direct calculation based on an experimentally determined 3D map of the detection volume. Model parameters are adjusted iteratively to minimize the residual differences between synthetic and experimental correlation curves. In order to reduce analysis time, we distribute calculations across a network of processors. As an example of this new approach, we demonstrate that synthetic autocorrelation curves correspond well with experimental data and that NFCS diffusion measurements of Rhodamine B remain constant, regardless of the distortion present in a confocal detection volume.

Membrane surface dynamics of DNA-threaded nanopores revealed by simultaneous single-molecule optical and ensemble electrical recording.

We describe a method for simultaneous single-molecule optical and electrical characterization of membrane-based sensors that contain ion-channel nanopores. The technique is used to study the specific and nonspecific interactions of streptavidin-capped DNA polymers with lipid bilayers composed of diphytanoyl phosphatidylcholine and diphytanoyl phosphatidylglycerol. Biotinylated DNA that is bound to fluorescently labeled streptavidin is electrophoretically driven into, or away from, the lumen of alpha hemolysin (alphaHL) ion channels by an external electric field. Confocal microscopy simultaneously captures single-molecule fluorescence dynamics from the membrane interface at different applied potentials. Fluorescence correlation analysis is used to determine the surface number density and diffusion constant of membrane-associated complexes. The dual optical and electrical approach can detect membrane-associated species at a surface coverage below 10(-5) monolayers of streptavidin, a sensitivity that surpasses most other in vitro surface analysis techniques. By comparing the change in transmembrane current to the number of fluorescent molecules leaving the bilayer when the electrical potential is reversed, we demonstrate the general utility of the approach within the context of nanopore-based sensing and discuss a mechanism by which DNA-streptavidin complexes can be nonspecifically retained at the membrane interface.