Resistive Pulse Method Research Articles

Resistive pulse analysis of chiral amino acids utilizing metal-amino acid crystallization differences.

Here, we report a proof-of-concept resistive pulse method for analyzing chiral amino acids utilizing metal-amino acid crystallization differences. This method involves introducing an amino acid sample solution into a micropipette through a pressure-driven flow. The sample then mixes with a metal ion solution inside the pipette, forming metal-amino acid crystals. The crystal size depends on the enantiomeric excess (x) of chiral amino acid samples. Large x values lead to large crystals. The crystal size difference is then reflected in the resistive pulse size as they block the ionic transport in a micropipette to different extents. We used Cd-cystine crystallization as a model system and found approximately five times the mean current pulse size difference for racemic (x = 0) and L-only (x = +1) cystine samples. A similar result was observed for aspartate. Our discovery opens up new opportunities for micro/nanoscopic chiral amino acid analysis, which can potentially be used in single-cell analysis.

Nanopore electrochemical sensors for emerging hazardous pollutants detection

Nanopore electrochemical sensors have been developed to detect various emerging pollutants at the single-molecule level with high sensitivity and specificity due to their high resolution in past three decades, which is of great significance for emerging hazardous pollutants control. Electrochemical detection methods based on nanopore technology are primarily divided into two categories: resistive-pulse and rectification sensing. The main detection mechanism of the resistive-pulse method is based on the volume exclusion effect generated when the analyte enters through the nanopore, and it involves three primary detection strategies. The first approach involves nanopore technology for direct pollutant detection, where pollutant structure and concentration information is assessed based on nanopore-generated ionic current's blockade strength, duration, and frequency. The second strategy relies on weak intermolecular interactions when direct detection of pollutants is challenging. Molecular probes interact with target molecules to enhance detection sensitivity and achieve specific recognition. The third strategy is based on the biological/chemical reaction of pollutant molecules, through indirect detection method to achieve specific detection of target pollutant molecules. Additionally, relies on changes in surface charge polarity or nanopore spatial structure induced by the analyte molecules to achieve the detection of target molecules within the nanopore. This work demonstrates the research progress achieved in the application of nanopore electrochemical sensing technology to high-sensitivity detection of hazardous pollutants, utilizing the unique confined spatial characteristics of nanopores, combined with biorecognition techniques and principles of electrochemical reactions.

Solid-State Nanopore/Nanochannel Sensing of Single Entities

Solid-state nanopores/nanochannels, with their high stability, tunable geometry, and controllable surface chemistry, have recently become an important tool for constructing biosensors. Compared with traditional biosensors, biosensors constructed with solid-state nanopores/nanochannels exhibit significant advantages of high sensitivity, high specificity, and high spatiotemporal resolution in the detection single entities (such as single molecules, single particles, and single cells) due to their unique nanoconfined space-induced target enrichment effect. Generally, the solid-state nanopore/nanochannel modification method is the inner wall modification, and the detection principles are the resistive pulse method and the steady-state ion current method. During the detection process, solid-state nanopore/nanochannel is easily blocked by single entities, and interfering substances easily enter the solid-state nanopore/nanochannel to generate interference signals, resulting in inaccurate measurement results. In addition, the problem of low flux in the detection process of solid-state nanopore/nanochannel, these defects limit the application of solid-state nanopore/nanochannel. In this review, we introduce the preparation and functionalization of solid-state nanopore/nanochannel, the research progress in the field of single entities sensing, and the novel sensing strategies on solving the above problems in solid-state nanopore/nanochannel single-entity sensing. At the same time, the challenges and prospects of solid-state nanopore/nanochannel for single-entity electrochemical sensing are also discussed.

Application of Micropore Device for Accurate, Easy, and Rapid Discrimination of Saccharomyces pastorianus from Dekkera spp.

Traceability analysis, such as identification and discrimination of yeasts used for fermentation, is important for ensuring manufacturing efficiency and product safety during brewing. However, conventional methods based on morphological and physiological properties have disadvantages such as time consumption and low sensitivity. In this study, the resistive pulse method (RPM) was employed to discriminate between Saccharomyces pastorianus and Dekkera anomala and S. pastorianus and D. bruxellensis by measuring the ionic current response of cells flowing through a microsized pore. The height and shape of the pulse signal were used for the simultaneous measurement of the size, shape, and surface charge of individual cells. Accurate discrimination of S. pastorianus from Dekkera spp. was observed with a recall rate of 96.3 ± 0.8%. Furthermore, budding S. pastorianus was quantitatively detected by evaluating the shape of the waveform of the current ionic blockade. We showed a proof-of-concept demonstration of RPM for the detection of contamination of Dekkera spp. in S. pastorianus and for monitoring the fermentation of S. pastorianus through the quantitative detection of budding cells.

Effect of Electrolyte Concentration on Cell Sensing by Measuring Ionic Current Waveform through Micropores.

Immunostaining has been widely used in cancer prognosis for the quantitative detection of cancer cells present in the bloodstream. However, conventional detection methods based on the target membrane protein expression exhibit the risk of missing cancer cells owing to variable protein expressions. In this study, the resistive pulse method (RPM) was employed to discriminate between cultured cancer cells (NCI-H1650) and T lymphoblastoid leukemia cells (CCRF-CEM) by measuring the ionic current response of cells flowing through a micro-space. The height and shape of a pulse signal were used for the simultaneous measurement of size, deformability, and surface charge of individual cells. An accurate discrimination of cancer cells could not be obtained using 1.0 × phosphate-buffered saline (PBS) as an electrolyte solution to compare the size measurements by a microscopic observation. However, an accurate discrimination of cancer cells with a discrimination error rate of 4.5 ± 0.5% was achieved using 0.5 × PBS containing 2.77% glucose as the electrolyte solution. The potential application of RPM for the accurate discrimination of cancer cells from leukocytes was demonstrated through the measurement of the individual cell size, deformability, and surface charge in a solution with a low electrolyte concentration.

Solid-state nanopore hydrodynamics and transport.

The resistive pulse method based on measuring the ion current trace as a biomolecule passing through a nanopore has become an important tool in biotechnology for characterizing molecules. A detailed physical understanding of the translocation process is essential if one is to extract the relevant molecular properties from the current signal. In this Perspective, we review some recent progress in our understanding of hydrodynamic flow and transport through nanometer sized pores. We assume that the problems of interest can be addressed through the use of the continuum version of the equations of hydrodynamic and ion transport. Thus, our discussion is restricted to pores of diameter greater than about ten nanometers: such pores are usually synthetic. We address the fundamental nanopore hydrodynamics and ion transport mechanisms and review the wealth of observed phenomena due to these mechanisms. We also suggest future ionic circuits that can be synthesized from different ionic modules based on these phenomena and their applications.

Collision and Coalescence of Single Attoliter Oil Droplets on a Pipet Nanopore.

We describe the use of a quartz pipet nanopore to study the collision and coalescence of individual emulsion oil droplets and their subsequent nanopore translocation. Collision and coalescence of single toluene droplets at a nanopore orifice are driven primarily by electroosmosis and electrophoresis and lead to the fast growth of a trapped oil droplet. This results in a stepwise current response due to the coalesced oil droplet increasing its volume and its ability to partially block the nanopore's ionic current, allowing us to use the resistive-pulse method to resolve single droplet collisions. Further growth of the trapped oil droplet leads to a complete blockage of the nanopore and a nearly 100% current decay. The trapped oil droplet shows enormous mechanical stability at lower voltages and stays in its trapped status for hundreds of seconds. An increased voltage can be used to drive the trapped droplet into the pipet pore within several milliseconds. Simultaneous fluorescence imaging and amperometry were performed to examine droplet collision, coalescence, and translocation, further confirming the proposed mechanism of droplet-nanopore interaction. Moreover, we demonstrate the unique ability to perform fast voltammetric measurements on a nanopore-supported attoliter oil droplet and study its voltage-driven ion transfer processes.

Detection of protein aggregate morphology through single antifouling nanopore

In this report, we investigate the potential offered by nanopores for the detection of proteins aggregates. We focus our study on model proteins, such as ß-lactoglobulin, lysozyme and bovine serum albumin, for their different morphology (fibril or spherical) at the early stage of aggregation. In order to avoid the nanopore fouling a SiN nanopore was coated with PEG chain. The PEG polymers adopt a mushroom conformation which prevents the nonspecific adsorption of proteins aggregates. They also allow to extend the nanopore lifetime. Using resistive pulse method, we successfully detect all the protein aggregates. Our results reveal that the intensity of relative current blockade allows to discriminate the morphology of proteins aggregates.

Bioinspired Protein Channel-Based Scanning Ion Conductance Microscopy (Bio-SICM) for Simultaneous Conductance and Specific Molecular Imaging.

The utility of stochastic single-molecule detection using protein nanopores has found widespread application in bioanalytical sensing as a result of the inherent signal amplification of the resistive pulse method. Integration of protein nanopores with high-resolution scanning ion conductance microscopy (SICM) extends the utility of SICM by enabling selective chemical imaging of specific target molecules, while simultaneously providing topographical information about the net ion flux through a pore under a concentration gradient. In this study, we describe the development of a bioinspired scanning ion conductance microscopy (bio-SICM) approach that couples the imaging ability of SICM with the sensitivity and chemical selectivity of protein channels to perform simultaneous pore imaging and specific molecule mapping. To establish the framework of the bio-SICM platform, we utilize the well-studied protein channel α-hemolysin (αHL) to map the presence of β-cyclodextrin (βCD) at a substrate pore opening. We demonstrate concurrent pore and specific molecule imaging by raster scanning an αHL-based probe over a glass membrane containing a single 25-μm-diameter glass pore while recording the lateral positions of the probe and channel activity via ionic current. We use the average channel current to create a conductance image and the raw current–time traces to determine spatial localization of βCD. With further optimization, we believe that the bio-SICM platform will provide a powerful analytical methodology that is generalizable, and thus offers significant utility in a myriad of bioanalytical applications.

Characterization of liposomes and silica nanoparticles using resistive pulse method

The ability to precisely count inorganic and organic nanoparticles and to measure their size distribution plays a major role in various applications such as drug delivery, nanoparticles counting, and many others. Here we employ a simple resistive pulse method that allows translocations, counting, and measuring size and velocity distribution of silica nanoparticles and liposomes with diameters from 50nm to 250nm. This technique is based on the Coulter counter technique but has nanometer size pores. It was found that ionic current drops when nanoparticles enter the nanopore of a pulled micropipette, producing a clear translocation signal. Pulled borosilicate micropipettes with opening 50–350nm were used as the detecting instrument. This method provides a direct, fast and cost-effective way to characterize inorganic and organic nanoparticles in a solution.

Resistive-Pulse Detection of Multilamellar Liposomes

The resistive-pulse method was used to monitor the pressure-driven translocation of multilamellar liposomes with radii between 190 and 450 nm through a single conical nanopore embedded in a glass membrane. Liposomes (0% and 5% 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (sodium salt) in 1,2-dilauroyl-sn-glycero-3-phosphocholine or 0%, 5%, and 9% 1,2-dipalmitoyl-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine) were prepared by extrusion through a polycarbonate membrane. Liposome translocation through a glass nanopore was studied as a function of nanopore size and the temperature relative to the lipid bilayer transition temperature, T(c). All translocation events through pores larger than the liposome, regardless of temperature, show translocation times between 30 and 300 μs and current pulse heights between 0.2% and 15% from the open pore baseline. However, liposomes at temperatures below the T(c) were captured at the pore orifice when translocation was attempted through pores of smaller dimensions, but squeezed through the same pores when the temperature was raised above T(c). The results provide insights into the deformation and translocation of individual liposomes through a porous material.

Single-Nanoparticle Detection Using a Low-Aspect-Ratio Pore

We explored single-particle translocation through a low thickness-to-diameter aspect ratio Si(3)N(4) pore mimicking graphene nanopore structure by a resistive pulse method. Ionic conductance of 0.05 aspect ratio pores scales linearly with the diameter, indicating predominant contribution of the access resistance to the ion transport. We find that the access resistance changes little during particle translocation. Furthermore, we observe enhanced particle capture rates via the strong electric field extended outside the low-aspect-ratio pore mouth. We also demonstrate electrical discrimination of two different sized particles using the low-aspect-ratio pore sensor with the constant access resistance assumption. The present findings indicate the potential utility of nucleotide-sized graphene nanopores as an electrical sensing platform for single-base identification via transmembrane ionic current blockade detections.

Asymmetric track membranes: Relationship between nanopore geometry and ionic conductivity

The revealing of the “diodelike” properties of electrolyte-filled asymmetric nanopores in track membranes has given significant impetus to a detailed study of the properties of “track” nanocapillaries. Studying the behavior of electrolyte solutions in nanovolumes of a given geometry is very important for many applications, such as nanofluid technology, the resistive pulse method for detecting colloidal particles and molecules, modeling of biological membranes, etc. An attempt to find a quantitative relationship between the geometric shape of asymmetric nanopores and asymmetry in electrical conductivity has been made in this paper. The method of chemical etching in the presence of a surfactant was used for the formation of nanopores with different profiles. The pore structure was studied by electron microscopy. It has been found that the rectification ratio increases with the membrane thickness and depends strongly on the curvature of the pore profile in the selective layer. The maximum of the rectification has been observed in a 0.05–0.1M KCl. Simulation of the ionic conductivity of asymmetric nanopores by the Poisson-Nernst-Planck equation qualitatively explains the observed behavior. The effect of the asymmetry of electrical conductivity is well expressed even in cases when the pore radius in the selective layer is substantially greater than the Debye length. The modification of the pore surface by grafting of aminopropyltriethoxysilane results in the sign inversion of electric charge and a sharp change in the current-voltage characteristics of the membranes.

Resistive Pulse Analysis of Microgel Deformation During Nanopore Translocation.

Deformation of 570-nm radius poly(N-isopropylacrylamide-co-acrylic acid) microgels passing through individual 375- to 915-nm radius nanopores in glass has been investigated by the resistive-pulse method. Particle translocation through nanopores of dimensions smaller than the microgel yields electrical signatures reflecting the dynamics of microgel deformation. Translocation rates, and event duration and peak shape, are functions of the conductivities of microgel and electrolyte. Our results demonstrate that nanopore resistive-pulse methods provide new fundamental insights into microgel permeation through porous membranes.

Resistive-Pulse Detection of Short dsDNAs Using a Chemically Functionalized Conical Nanopore Sensor

To develop nanopore resistive-pulse sensors for the detection of short (50 base-pair [bp] and 100 bp) DNAs. Conically shaped nanopores were chemical etched into polyethylene terphthalate membranes. The as-etched membrane had anionic carboxylate sites on the pore walls. Neutral and hydrophilic ethanolamine functional groups were attached to these carboxylate sites using well-established EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) chemistry. The ethanolamine-functionalized pores were used to detect 50 and 100 bp DNAs via the resistive-pulse method. The resistive-pulse signature produced by the 50 bp DNA could be distinguished from that of the 100 bp DNA with these sensors. Attachment of ethanolamine to the carboxylate groups on the pore wall lowered the anionic charge density on the wall. This mitigated the problem of electrostatic rejection of the anionic DNAs from the pore and enabled the detection of these DNA analytes.

Direct detection of antibody-antigen binding using an on-chip artificial pore.

We demonstrate a rapid and highly sensitive all-electronic technique based on the resistive pulse method of particle sizing with a pore to detect the binding of unlabeled antibodies to the surface of latex colloids. Here, we use an on-chip pore to sense colloids derivatized with streptavidin and measure accurately their diameter increase on specific binding to several different types of antibodies. We show the sensitivity of this technique to the concentration of free antibody and that it can be used to perform immunoassays in both inhibition and sandwich configurations. Overall, our technique does not require labeling of the reactants and is performed rapidly by using very little solution, and the pore itself is fabricated quickly and inexpensively by using soft lithography. Finally, because this method relies only on the volume of bound ligand, it can be generally applied to detecting a wide range of ligand-receptor binding reactions.