Resistive Pulse Sensing Research Articles

Complementary Nanoparticle Characterization by Resistive-Pulse Sensing, Electron Microscopy, and Charge Detection Mass Spectrometry.

Nanotechnology has provided novel modalities for the delivery of therapeutic and diagnostic agents. In particular, nanoparticles (NPs) can be engineered at a low cost for drug loading and delivery. For example, silica NPs have proven useful as a controlled release platform for anti-inflammatory drugs. Despite the wide-ranging potential applications for NPs, robust characterization across all size ranges remains elusive. Electron microscopy (EM) is the conventional tool for measuring NP diameters. However, imitations in throughput and the inability to provide comprehensive information on physical properties, such as mass and density, without underlying assumptions, hinder a complete analysis. In addition, assessing sample heterogeneity, aggregation, or coalescence in solution by traditional EM analysis is not possible. Resistive-pulse sensing (RPS) provides a high throughput, solution-phase method for characterizing particle heterogeneity based on volume. Complementing these methods, charge detection mass spectrometry (CD-MS), a single particle technique, provides accurate mass information for heterogeneous samples including NPs. By combining EM, RPS and CD-MS, accurate volume, mass, and densities were obtained for silica NPs of various sizes. The results show that the density for 20 nm silica NPs is close to the density of fused silica (2.2 g/cm3). Larger silica NPs were found to have densities that were either smaller or larger, while also falling outside the range of densities usually found for silica colloids and NPs (1.9-2.3 g/cm3). Lower densities are attributed to pores (i.e., porous particles). For one sample, the mass distribution showed two components attributed to two populations of particles in the sample with different densities. The synergistic combination of EM, RPS, and CD-MS measurements outlined here for NP samples, allows much more extensive information to be obtained than from any of the techniques alone.

Integrated In-Plane Nanofluidic Devices for Resistive-Pulse Sensing.

Single-particle (or digital) measurements enhance sensitivity (10- to 100-fold improvement) and uncover heterogeneity within a population (one event in 100 to 10,000). Many biological systems are significantly influenced by rare or infrequent events, and determining what species is present, in what quantity, and the role of that species is critically important to unraveling many questions. To develop these measurement systems, resistive-pulse sensing is used as a label-free, single-particle detection technique and can be combined with a range of functional elements, e.g., mixers, reactors, filters, separators, and pores. Virtually, any two-dimensional layout of the micro- and nanofluidic conduits can be envisioned, designed, and fabricated in the plane of the device. Multiple nanopores in series lead to higher-precision measurements of particle size, shape, and charge, and reactions coupled directly with the particle-size measurements improve temporal response. Moreover, other detection techniques, e.g., fluorescence, are highly compatible with the in-plane format. These integrated in-plane nanofluidic devices expand the toolbox of what is possible with single-particle measurements.

Fabrication of low aspect ratio solid-state pores from sub-micron to microscales utilizing crossing blades

This article introduces a novel method for fabricating low aspect ratio pores in various sizes, from sub-micron to microscales. This technique utilizes the intersection of two collided blades as a soft lithography mold. The theoretical details of the fabrication method, including the pore’s shape and geometrical parameters, were inspected, modeled, and discussed. Multiple mechanical and electrochemical measurements were performed to characterize the fabricated pores. Pore fabrication results showed that a wide range of pore sizes, from 350 nanometers to more than 30 micrometers, can be constructed by employing this method. Electrochemical impedance spectroscopy (EIS) implied that the blades-intrusion pores have the same electrochemical behavior as the ones on 2D materials. Meanwhile, the durability of the fabricated pores against the pressure of two bars ensures great mechanical stability of these pores. Fabricated pores with sizes of 2.3 µm and 20 µm were successfully employed as sensing gates in resistive pulse sensing (RPS) for detecting polystyrene beads of 600 nm, 1 µm, and 2 µm in diameter and downsized yeast cells, respectively. A distinct scatter of pulses of different particle sizes in the pulse amplitude-width plane verified the blades-intrusion pore’s capability as a sensing gate.

Assessment of Tunable Resistive Pulse Sensing (TRPS) Technology for Particle Size Distribution in Vaccine Formulations - A Comparative Study with Dynamic Light Scattering.

A comparative assessment was performed to evaluate the potential of particle sizing by an ensemble based conventional dynamic light scattering (DLS) technique and an emerging technology based on tunable resistive pulse sensing (TRPS) using particle by particle approach by evaluating three different types of vaccine formulations representing three case studies and showing the limitation of each technique, instrument variability, sensitivity, and the resolution in mixed population. Three types of in-house vaccine formulations- a protein antigen, an outer membrane vesicle and viral particles were simultaneously evaluated by TRPS based Exoid and two DLS instruments-Zetatrac and Zetasizer for particle size distribution, aggregates, and resolution of polydisperse species. The data from first case study show the risk of possible size overestimation and size averaging in polydisperse samples in DLS measurements which can be addressed by the TRPS analysis. It also shows how TRPS may be utilized only to large size antigens due to its limited size range. The second case study highlights the difference in the sensitivities of two DLS instruments working on the same principle. The third case study show that how TRPS can better resolve the large aggregate species compare to DLS in polydisperse samples. This analysis shows that TRPS can be used as an orthogonal technique in addition to conventional DLS based methods for more precise and in-depth characterization. Both techniques are efficient in size characterization and produce comparable results, however the choice will depend on the type of formulation and size range to be evaluated.

Critical parameters to standardize the size and concentration determination of nanomaterials by nanoparticle tracking analysis

The size and concentration are critical for the diagnostic and therapeutic applications of nanomaterials but the accurate measurement remains challenging. Nanoparticle tracking analysis (NTA) is widely used for size and concentration determination. However, highly repeatable standard operating procedures (SOPs) are absent. We adopted the “search-evaluate-test” strategy to standardize the measurement by searching the critical parameters. The particles per frame are linearly proportional to the sample concentration and the measured results are more accurate and repeatable when the concentration is 108-109 particles/ml. The optimal detection threshold is around 5. The optimal camera level is such that it allows clear observation of particles without diffractive rings and overexposure. The optimal speed is ≤ 50 in AU and ∼ 10 μl/min in flow rate. We then evaluated the protocol using polydisperse polystyrene particles and we found that NTA could discriminate particles in bimodal mixtures with high size resolution but the performance on multimodal mixtures is not as good as that of resistive pulse sensing (RPS). We further analyzed the polystyrene particles, SiO2 particles, and biological samples by NTA following the SOPs. The size and concentration measured by NTA differentially varies to those determined by RPS and transmission electron microscopy.

Bridging Ionic Current Rectification and Resistive-Pulse Sensing for Reliable Wide-Linearity Detection.

As two mainstream ionic detection techniques, ionic current rectification (ICR) suffers from large fluctuations in trace level detection, while resistive-pulse sensing (RPS) encounters easy clogs in high-concentration detection. By rationally matching the nanopore size with the DNA tetrahedron (TDN), this work bridges the two techniques to achieve reliable detection with wide linearity. As a representative analyte, miRNA-10b could specifically combine with and release TDN from the interior wall, which thus induced the simultaneous generation of distinct ICR and RPS signals. The ICR signals could be attributed to the balance between the effective orifice and surface charge density of the inner wall, while the RPS signals were induced by the complex of miRNA-10b and TDN passing through the nanopore. Such an operation contributed to a wide detection range of 1 fM-1 nM with a good linearity. The feasibility of this method is also validated in single-cell and real plasma detection.

Simultaneous Multipass Resistive-Pulse Sensing and Fluorescence Imaging of Liposomes.

Simultaneous multipass resistive-pulse sensing and fluorescence imaging have been used to correlate the size and fluorescence intensity of individual E. coli lipid liposomes composed of E. coli polar lipid extracts labeled with membrane-bound 3,3-dioctadecyloxacarbocyanine (DiO) fluorescent molecules. Here, a nanopipet serves as a waveguide to direct excitation light to the resistive-pulse sensing zone at the end of the nanopipet tip. Individual DiO-labeled liposomes (>50 nm radius) were multipassed back and forth through the orifices of glass nanopipets' 110-150 nm radius via potential switching to obtain subnanometer sizing precision, while recording the fluorescence intensity of the membrane-bound DiO molecules. Fluorescence was measured as a function of liposome radius and found to be approximately proportional to the total membrane surface area. The observed relationship between liposome size and fluorescence intensity suggests that multivesicle liposomes emit greater fluorescence compared to unilamellar liposomes, consistent with all lipid membranes of the multivesicle liposomes containing DiO. Fluorescent and nonfluorescent liposomes are readily distinguished from each other in the same solution using simultaneous multipass resistive-pulse sensing and fluorescence imaging. A fluorescence "dead zone" of ∼1 μm thickness just outside of the nanopipet orifice was observed during resistive-pulse sensing, resulting in "on/off" fluorescent behavior during liposome multipassing.

Nanofluidic resistive pulse sensing for characterization of extracellular vesicles.

This paper describes the development, design and characterization of a resistive pulse sensing (RPS) system for the analysis of size distributions of extracellular vesicles (EVs). The system is based on microfluidic chips fabricated using soft-lithography and operated in pressure-driven mode. This fabrication approach provided reproducible pore dimensions and the best performing chip design enabled, without calibration, sizing of both 252 nm and 460 nm test particles within 8% of theoretically calculated values, based on the size specifications provided by suppliers. The number concentration measurement had higher variations and without calibration provided estimates within an order of magnitude, for sample concentrations across 4 orders of magnitude. The RPS chips could also measure successfully EVs and other biological nanoparticles in purified samples from cell culture media and human serum. A compact, fast and inexpensive RPS system based on this design could be an attractive alternative to current gold-standard techniques for routine characterization of EV samples.

Detection and identification of single ribonucleotide monophosphates using a dual in-plane nanopore sensor made in a thermoplastic via replication.

We report the generation of ∼8 nm dual in-plane pores fabricated in a thermoplastic via nanoimprint lithography (NIL). These pores were connected in series with nanochannels, one of which served as a flight tube to allow the identification of single molecules based on their molecular-dependent apparent mobilities (i.e., dual in-plane nanopore sensor). Two different thermoplastics were investigated including poly(methyl methacrylate), PMMA, and cyclic olefin polymer, COP, as the substrate for the sensor both of which were sealed using a low glass transition cover plate (cyclic olefin co-polymer, COC) that could be thermally fusion bonded to the PMMA or COP substrate at a temperature minimizing nanostructure deformation. Unique to these dual in-plane nanopore sensors was two pores flanking each side of the nanometer flight tube (50 × 50 nm, width × depth) that was 10 μm in length. The utility of this dual in-plane nanopore sensor was evaluated to not only detect, but also identify single ribonucleotide monophosphates (rNMPs) by using the travel time (time-of-flight, ToF), the resistive pulse event amplitude, and the dwell time. In spite of the relatively large size of these in-plane pores (∼8 nm effective diameter), we could detect via resistive pulse sensing (RPS) single rNMP molecules at a mass load of 3.9 fg, which was ascribed to the unique structural features of the nanofluidic network and the use of a thermoplastic with low relative dielectric constants, which resulted in a low RMS noise level in the open pore current. Our data indicated that the identification accuracy of individual rNMPs was high, which was ascribed to an improved chromatographic contribution to the nano-electrophoresis apparent mobility. With the ToF data only, the identification accuracy was 98.3%. However, when incorporating the resistive pulse sensing event amplitude and dwell time in conjunction with the ToF and analyzed via principal component analysis (PCA), the identification accuracy reached 100%. These findings pave the way for the realization of a novel chip-based single-molecule RNA sequencing technology.

Quality and efficiency assessment of five extracellular vesicle isolation methods using the resistive pulse sensing strategy.

Extracellular vesicles (EVs) have attracted great interest due to their great potential in disease diagnosis and therapy. The separation of EVs from complex biofluids with high purity is essential for the accurate analysis of EVs. Despite various methods, there is still no consensus on the best method for high-quality EV isolation and reliable mass production. Therefore, it is important to offer a standardized method for characterizing the properties (size distribution, particle concentration and purity) of EV preparations from different isolation methods. Herein, we employed a NanoCoulter Counter based on the resistive pulse sensing (RPS) strategy that enabled multi-parameter analysis of single EVs to compare the quality and efficiency of different EV isolation techniques including traditional differential ultracentrifugation, ultrafiltration, size exclusion chromatography, membrane affinity binding and polymer precipitation. The data revealed that the NanoCoulter Counter based on the RPS strategy was reliable and effective for the characterization of EVs. The results suggested that although higher particle concentrations were observed in three commercial isolation kits and ultrafiltration, traditional differential ultracentrifugation showed the highest purity. In conclusion, our results from the NanoCoulter Counter provided reliable evidence for the assessment of different EV isolation methods, which contributed to the development of EV-based disease biomarkers and treatments.

Biosensing with Tailored Track-Etched Nanochannels

Inspired by the high sensitivity, efficiency and selectivity of biological ion channels, the development of solid-state nanochannel sensors has been at the forefront of nanotechnology research in the last years. Many researchers worldwide have concentrated their efforts on the development of artificial nanochannels that can mimic the features of biological ion channels. These synthetic nanostructures are based on abiotic materials which provide higher robustness, thermal stability, chemical versatility and mechanical resistance than their biological counterparts. Furthermore, synthetic nanochannels and specific surface functionalization strategies have enabled fine control over the geometry and charge distribution which yielded the ability to manipulate the flux of ions through the channel. This gave rise to the formation of nanochannel-based platforms with applications in (bio)sensing as well as other fields. The development of chemical- and biosensors requires a combination of reliable nanofabrication techniques and versatile surface modification strategies, which render high sensitivity and high selectivity, respectively [1].Tailored single polymer nanochannels are routinely fabricated by ion-track nanotechnology. Polymer films are first irradiated with individual swift heavy ions at the GSI UNILAC accelerator. Each ion generates a highly localized cylindrical damage zone along its trajectory. These so-called ion tracks are subsequently dissolved and enlarged in a chemical etching process. By selecting suitable etching conditions, geometry (cylindrical, conical or bullet-like) and size of the nanochannels can be adjusted. The nanochannel surface can then be functionalized by means of different chemical strategies to enhance the sensing capabilities.There are two main nanochannel sensing techniques, ion current rectification (ICR) and resistive-pulse sensing (RPS). Both are affected by the geometry and surface charge of the nanochannel and based on measuring the change in ion current flowing through the channel induced by the chosen analyte [2]. In both approaches, the presence of a target analyte triggers a variation in the physicochemical properties of the channel which can be related to measurable changes in the transmembrane current, and is proportional to the analyte concentration.In this contribution, we will present nanochannel sensing platforms based on several single polymer channels fabricated by ion-track nanotechnology and a selection of surface functionalization strategies applied to the nanochannels. We will then discuss selected examples recently obtained with these tailored functionalized nanochannels and demonstrate selective and sensitive sensing applications based on both the ICR and RPS phenomena.[1] Toum Terrones, Y., Cayón, V. M., Laucirica, G., Cortez, M. L., Toimil-Molares, M. E., Trautmann, C., ... & Azzaroni, O. (2022). Ion Track-Based Nanofluidic Biosensors. In Miniaturized Biosensing Devices: Fabrication and Applications (pp. 57-81). Singapore: Springer Nature Singapore.[2] Kaya, D., & Keçeci, K. (2020). Track-etched nanoporous polymer membranes as sensors: A review. Journal of The Electrochemical Society, 167(3), 037543.

Resistive Pulse Sensing on a Capillary-Assisted Microfluidic Platform for On-Site Single-Particle Analyses.

Capillary-assisted flow is valuable for utilizing microfluidics-based electrical sensing platforms at on-site locations by simplifying microfluidic operations and system construction; however, incorporating capillary-assisted flow in platforms requires easy microfluidic modification and stability over time for capillary-assisted flow generation and sensing performance. Herein, we report a capillary-assisted microfluidics-based electrical sensing platform using a one-step modification of polydimethylsiloxane (PDMS) with polyethylene glycol (PEG). As a model of electrical sensing platforms, this work focused on resistive pulse sensing (RPS) using a micropore in a microfluidic chip for label-free electrical detection of single analytes, and filling the micropore with an electrolyte is the first step to perform this RPS. The PEG-PDMS surfaces remained hydrophilic after ambient storage for 30 d and assisted in generating an electrolyte flow for filling the micropore with the electrolyte. We demonstrated the successful detection and size analysis of micrometer particles and bacterial cells based on RPS using the microfluidic chip stored in a dry state for 30 d. Combining this capillary-assisted microfluidic platform with a portable RPS system makes on-site detection and analysis of single pathogens possible.

Characterization of Extracellular Vesicles by Resistive-Pulse Sensing on In-Plane Multipore Nanofluidic Devices.

Extracellular vesicles (EVs) are cell-derived, naturally produced, membrane-bound nanoscale particles that are linked to cell-cell communication and the propagation of diseases. Here, we report the design and testing of in-plane nanofluidic devices for resistive-pulse measurements of EVs derived from bovine milk and human breast cancer cells. The devices were fabricated in plane with three nanopores in series to determine the particle volume and diameter, two pore-to-pore regions to measure the electrophoretic mobility and zeta potential, and an in-line filter to prevent cellular debris and aggregates from entering the nanopore region. Devices were tested with and without the channels coated with a short-chain PEG silane to minimize electroosmotic flow and permit an accurate measurement of the electrophoretic mobility and zeta potential of the EVs. To enhance throughput of EVs, vacuum was applied to the waste reservoir to increase particle frequencies up to 1000 min-1. The nanopores had cross-sections 200 nm wide and 200 nm deep and easily resolved EV diameters from 60 to 160 nm. EVs from bovine milk and human breast cancer cells had similar particle size distributions, but their zeta potentials differed by 2-fold, -8 ± 1 and -4 ± 1 mV, respectively.

Elucidating the Shape of Current Transients in Electrochemical Resistive-Pulse Sensing of Single Liposomes.

Electrochemical resistive-pulse (ERP) sensing with conductive carbon nanopipettes (CNPs) has recently been developed and employed for the detection of single liposomes and biological vesicles, and for the analysis of redox molecules contained in such vesicles. However, the origins of different shapes of current transients produced by the translocation of single vesicles through the CNP remain poorly understood. Herein, we report extensive finite-element simulations of both portions of an ERP transient, the current blockage by a vesicle approaching and passing through the pipet orifice and the faradaic current spike due to oxidation/reduction of redox species released from a vesicle on the carbon surface, for different values of parameters defining the geometry and dynamics of the vesicle/CNP system. The effects of the pipet geometry, surface charge, transport, vesicle trajectory, and collision location on the shape of current transients are investigated. The possibility of quantitative analysis of experimental ERP transients produced by translocations of liposomes and extracellular vesicles by fitting them to simulated curves is demonstrated. The developed theory can enable a more reliable interpretation of complicated ERP signals and characterization of the size and contents of single biological and artificial vesicles.

θ-Nanopipette for Single-Cell Resistive-Pulse Profiling of DNA Repair Proteins Accompanied by Drug Evaluation.

Single-cell analysis of the DNA repair protein is important but remains unachieved. Exploration of nanopipettte technologies in single-cell electroanalysis has recently seen rapid growth, while the θ-nanopipette represents an emerging technological frontier with its potential largely veiled. Here a θ-nanopipette is first applied for single-cell resistive-pulse sensing (RPS) of the important DNA repair protein O6-alkylguanine DNA alkyltransferase (hAGT). The removal of alkyl mutations by hAGT could restore the damaged aptamer linking with a structural DNA carrier, allowing the selective binding of the aptamer to thrombin with precisely matched size to produce distinct RPS signals when passing through the orifice. Kinetic analysis of hAGT repair was studied. Meanwhile, the device shows the simultaneous on-demand infusion of inhibitors to inactivate the hAGT activity, indicative of its potential in drug screening for enhanced chemotherapy. This work provides a new paradigm for θ-nanopipette-based single-cell RPS of a DNA repair protein accompanied by drug evaluation.

Metal-pad-enhanced resistive pulse sensor reveals complex-valued Braess paradox.

A resistive pulse sensor measures the electrical impedance of an electrolyte-filled channel as particles flow through it. Ordinarily, the presence of a nonconductive particle increases the impedance of the channel. Here we report a surprising experimental result in which a microfluidic resistive pulse sensor experiences the opposite effect: The presence of a nonconductive particle decreases the channel impedance. We explain the counterintuitive phenomenon by relating to the Braess paradox from traffic network theory, and we call it the complex-valued Braess paradox (CVBP). We develop theoretical models to study the CVBP and corroborate the experimental data using finite element simulations and lumped-element circuit modeling. We then discuss implications and potential applications of the CVBP in resistive pulse sensing and beyond.

Rapid Detection of Microparticles Using a Microfluidic Resistive Pulse Sensor Based on Bipolar Pulse-Width Multiplexing.

Rapid and accurate analysis of micro/nano bio-objects (e.g., cells, biomolecules) is crucial in clinical diagnostics and drug discovery. While a traditional resistive pulse sensor can provide multiple kinds of information (size, count, surface charge, etc.) about analytes, it has low throughput. We present a unique bipolar pulse-width, multiplexing-based resistive pulse sensor for high-throughput analysis of microparticles. Signal multiplexing is enabled by exposing the central electrode at different locations inside the parallel sensing channels. Together with two common electrodes, the central electrode encodes the electrical signal from each sensing channel, generating specific bipolar template waveforms with different pulse widths. Only one DC source is needed as input, and only one combined electrical output is collected. The combined signal can be demodulated using correlation analysis and a unique iterative cancellation scheme. The accuracy of particle counting and sizing was validated using mixtures of various sized microparticles. Results showed errors of 2.6% and 6.1% in sizing and counting, respectively. We further demonstrated its accuracy for cell analysis using HeLa cells.

High Sensitivity Extended Nano-Coulter Counter for Detection of Viral Particles and Extracellular Vesicles.

We present a chip-based extended nano-Coulter counter (XnCC) that can detect nanoparticles affinity-selected from biological samples with low concentration limit-of-detection that surpasses existing resistive pulse sensors by 2-3 orders of magnitude. The XnCC was engineered to contain 5 in-plane pores each with an effective diameter of 350 nm placed in parallel and can provide high detection efficiency for single particles translocating both hydrodynamically and electrokinetically through these pores. The XnCC was fabricated in cyclic olefin polymer (COP) via nanoinjection molding to allow for high-scale production. The concentration limit-of-detection of the XnCC was 5.5 × 103 particles/mL, which was a 1,100-fold improvement compared to a single in-plane pore device. The application examples of the XnCC included counting affinity selected SARS-CoV-2 viral particles from saliva samples using an aptamer and pillared microchip; the selection/XnCC assay could distinguish the COVID-19(+) saliva samples from those that were COVID-19(-). In the second example, ovarian cancer extracellular vesicles (EVs) were affinity selected using a pillared chip modified with a MUC16 monoclonal antibody. The affinity selection chip coupled with the XnCC was successful in discriminating between patients with high grade serous ovarian cancer and healthy donors using blood plasma as the input sample.

Particle counting immunoassay system using a low-aspect-ratio pore

We have developed a new particle counting immunoassay system integrated with a resistive-pulse sensor with a low-aspect-ratio pore. The particle counting immunoassay is based on the principle of latex aggregation. The target single-molecule concentration is measured by counting aggregated latex particles that form multimers over the reaction time. To enable highly accurate identification of the number of aggregated particles, we designed and fabricated a low-aspect-ratio pore device to perform quantitative structural analysis of particles. The aggregation numbers of the conjugated particles were classified based on blockade current profiles when the particles translocated through the pore. Time-dependent agglutination reaction monitoring demonstrated that the reaction speed increased for a solution containing a higher concentration of antigens. The results prove the feasibility of using the proposed immunoassay system to count particles while determining the aggregation number based on their current profiles.

Physicochemical characterization of lipid mixtures for liposomal formulations of polyenes.

The use of liposomes or lipid nanoparticles as a drug delivery systems have been under development for more than 40 years and they are up to now the most used targeted drug delivery system in clinical applications, e.g, Ambisome(R) (an amphotericin B liposomal formulation) and several of the COVID-19 vaccines. Among the main critical parameters that guarantee the successful use of these systems is the lipid composition, which will determine their size and surface charge. We recently developed a new liposome system that showed in previous preclinical studies the capability of delivering a polyene antimycotic in a safer and equally effective way than the direct IV administration of the polyene in solution. We present the preliminary results of characterizing the size and zeta potential of this formulation by means of Tunable Resistive Pulse Sensing as well as topographic and nanomechanical properties by Atomic Force Microscopy images of supported lipid bilayers of these liposomes. We compare these results with the existing liposomal formulation for amphotericin B. We believe that this liposomal system could be further used with other drugs that present low water solubility and/or toxic side effects. Funding: PAPIIT-100920, CONACyT-CF2019-74884.