Resistive Pulse Sensing Research Articles

Detection and phenotyping of extracellular vesicles by size exclusion chromatography coupled with on-line fluorescence detection

New methods for quantifying extracellular vesicles (EVs) in complex biofluids are critically needed. We report the development of a new technology combining size exclusion chromatography (SEC), a commonly used EV purification technique, with fluorescence detection of specifically labelled EVs. The resulting platform, Flu-SEC, demonstrates a linear response to concentration of specific EVs and could form the basis of a system with phenotyping capability. Flu-SEC was validated using red blood cell derived EVs (REVs), which provide an ideal EV model with monodisperse size distribution and high EV concentration. Microfluidic Resistive Pulse Sensing (MRPS) was used to accurately determine the size distribution and concentration of REVs. Anti-CD235a antibody, specific to glycophorin A, and the more general wheat germ agglutinin (WGA), were selected to label REVs. The results show the quantitative power of Flu-SEC: a highly linear fluorescence response over a wide range of concentrations. Moreover, the Flu-SEC technique reports the ratio of EV-bound and free-antibody molecules, an important metric for determining optimal labelling conditions for other applications. Flu-SEC represents an orthogonal tool to single-particle fluorescent methods such as flow cytometry and fluorescent NTA, for the quantification and phenotyping of EVs.

Peptide Nanocarriers for Detection of Heavy Metal Ions Using Resistive Pulse Sensing.

The use of nanocarriers within resistive pulse sensing facilitates the detection and quantification of analytes. To date the field has been dominated by polyionic carriers or nanomaterials. Together they combine the recognition elements of a ligand with a stable support, facilitating the sample handling, analysis times, and multiplex detection. Here we develop the use of peptide-functionalized superparamagnetic nanocarriers to extract and quantify metal ions in solution. The interaction between nickel and the peptide ligand is measured as a change in translocation velocity of the carrier. The magnitude of change is proportional to the concentration of the metal ions in solution. Unlike DNA aptamers where a change in the tertiary structure and the folding of the polyanionic backbone influences the carrier velocity, the peptides here had a lower net charge under the assay conditions. To try and enhance the signal we engineered charged groups within the peptide to explore the effects on the signal. In all cases the metal ion binding dominated the velocity of the carrier. The assay was shown to work across 3 orders of magnitude and can detect Ni2+ in the presence of some other heavy metal ions. We demonstrate this by quantifying Ni2+ in both tap and pond water. The work allows for future multiplexed sensing strategies using both peptides and DNA aptamers in resistive pulse sensors.

Microfluidic Time-Division Multiplexing Accessing Resistive Pulse Sensor for Particle Analysis.

Due to its simplicity and robustness, pore-based resistive pulse sensors have been widely used to detect, measure, and analyze particles at length scales ranging from nanometers to micrometers. While multiple pore-based resistive pulse sensors are preferred to increase the analysis throughput and to overcome the clogging issues, the scalability is often limited. In response, by combining the time-division multiple access technique in the telecommunication field with the microfluidics, we reported a microfluidic time-division multiplexing accessing (TDMA) single-end resistive pulse sensor, in which particles can be analyzed through a scalable number of microfluidic channels. With an eight-channel microfluidic device and polystyrene particles as proof-of-principle, we successfully demonstrated this multiplexed technology is effective in measuring the particle size and concentration, in analyzing the particle arriving dynamics, and in discriminating mixed populations. Importantly, the availability of multiple sensing pores provides a robust mechanism to overcome the clogging issue, allowing the analysis to continue even when some of the pores are clogged. We anticipate this TDMA approach could find wide applications and facilitate future development of multiplexed resistive pulse sensing from the microscale to nanoscale.

Microfluidic multiple cross-correlated Coulter counter for improved particle size analysis

Coulter counters (a.k.a. resistive pulse sensors) were widely used to measure the size of biological cells and colloidal particles. One of the important parameters of Coulter counters is its size discriminative capability. This work reports a multiple pore-based microfluidic Coulter counter for improved size differentiation in a mixed sample. When a single particle translocated across an array of sensing pores, multiple time-related resistive pulse signals were generated. Due to the time correlation of these resistive pulse signals, we found a multiple cross-correlation analysis (MCCA) could enhance the sizing signal-to-noise (SNR) ratio by a factor of n1/2, where n is the pore numbers in series. This proof-of-concept is experimentally validated with polystyrene beads as well as human red blood cells. We anticipate this method would be highly beneficial for applications where improved size differentiation is required.

Resistive-Pulse Sensing and Surface Charge Analysis of a Single Nanoparticle Collision at a Conical Glass Nanopore.

In this study, the collision of monodispersed single Au@SiO2 nanoparticles (NPs; ∼83.5 ± 6.5 nm) at the outer orifice of a smaller-sized (∼40 ± 8 nm) conical glass nanopore was systematically investigated by the resistive-pulse sensing technique under three different surface charge states. When NPs approach the nanopipette orifice and collide, the ionic current changes dramatically, and the collision current amplitude shows a linear relationship with the potential bias applied. In our system, the electroosmotic flow is found to be the main driving force for the collision, and the resistive-pulse collision signal depends on the relative electroosmotic, electrophoretic, and electrostatic forces being applied, which in turn can reflect the surface charge properties of the tested NP and nanopore wall used. By fixing the size of the nanopore and NPs, the surface charge information on the NPs and nanopore walls can be discriminated by the magnitude and duration of the collision ionic current signals. This study manifested the feasibility of exploring the nanopore-based single NPs collision platform for surface charge analysis of the colloidal nanoparticle system.

Resistive Pulse Sensing as a High‐Resolution Nanoparticle Sizing Method: A Comparative Study

AbstractOne of the main challenges of sizing methods for nanoparticle (NP) suspensions is to distinguish between particles and particle populations with very small size differences. This would be especially important to follow various surface functionalization processes of nanoparticles resulting in small alterations of their size. In this respect, methods involving the detection of single particles, such as resistive pulse sensing (RPS) or nanoparticle tracking analysis, are generally considered superior to ensemble measuring methods such as dynamic light scattering. However, to compare the exact capabilities of these methodologies require systematic investigations in optimized conditions for each method. Here, such a study is presented for a narrow size range of spherical latex nanoparticles (60–200 nm). It is concluded that the RPS methodology based on quartz nanopipets as single nanopore counters, is the only sizing method among those studied capable to fully resolve a ternary mixture of 70, 110, and 140 nm average diameter NPs. The practical usefulness of this size resolution is demonstrated by following the increase in diameter of latex nanoparticles after their surface modification with antibodies.

Additively Manufactured Flow-Resistive Pulse Sensors.

Resistive pulse sensors (RPSs) provide detailed characterization of materials from the nanoparticle up to large biological cells on a particle-to-particle basis. During the RPS experiment, particles pass through a channel or pore that conducts ions, and the change in the ionic current versus time is monitored. The change in current during each translocation, also known as a "pulse", is dependent on the ratio of the particle and channel dimensions. Here we present a facile and rapid method for producing flow-RPSs that do not require lithographic processes. The additively manufactured sensor has channel dimensions that can be easily controlled. In addition, the fabrication process allows the sensor to be quickly assembled, disassembled, cleaned, and reused. Furthermore, the RPS can be created with a direct interface for fluidic pumps or imaging window for complementary optical microscopy. We present experiments and simulations of the RPS, showing how the pulse shapes are dependent on the channel morphology and how the device can count and size particles across a range of flow rates and ionic strengths. The use of pressure-driven fluid flow through the device allowed a rapid characterization of particles down to concentrations as low as 1 × 10-3 particles per mL, which equated to one event per second.

A novel microfluidic resistive pulse sensor with multiple voltage input channels and a side sensing gate for particle and cell detection

Traditionally, a resistive pulse sensor (also known as Coulter counter) works by letting a particle pass through a small orifice in an electrolyte solution. The detection sensitivity mainly relies on the volume ratio of the particle to the orifice. This paper presents a novel resistive pulse sensor which has a sensing orifice located on the side wall of a microchannel. In this way, the sensor can detect and count particles (or cells) without requiring particles (or cells) passing through the sensing gate. An equation was derived to relate the magnitudes of the detected signals and the electrical resistances. Results show that the magnitudes of the detected signals can be increased by applying voltages from more than one voltage input channels simultaneously. Under the same conditions, the magnitudes of the detected signals become larger when the diameters of particles are larger. Higher detection sensitivity can be obtained simply by increasing either the magnitudes of the applied voltages or the number of the voltage input channels, or reducing the opening of the side sensing gate to a size that is even smaller than the diameter of the particle. Due to the high detection sensitivity, detection of 1 μm particles by a relatively large sensing gate of 5 × 10 × 10 μm (width × length × height) was successfully demonstrated with a signal to noise ratio (S/N) of approximately 3. This sensor was also applied to detect and count human red blood cells and lymphocyte cells. Results show that this method can clearly distinguish the cells with different sizes based on the pre-determined-thresholds. Because this sensor does not require cells to pass through the sensing gate, the channel clogging problem can be avoided. More importantly, the detection sensitivity can be tuned by applying different voltages without fabricating a smaller sensing gate.

Automatic detecting and counting magnetic beads-labeled target cells from a suspension in a microfluidic chip.

A novel microfluidic method of continually detecting and counting beads-labeled cells from a cell mixture without fluorescence labeling was presented in this paper. The detection system is composed of a microfluidic chip (with a permanent magnet inserted along the channel), a signal amplification circuit, and a LabView® based data acquisition device. The microfluidic chip can be functionally divided into separation zone and detection zone. By flowing the pre-labeled sample solution, the target cells will be sequentially separated at the separation zone by the permanent magnet and detected and counted at the detection zone by a microfluidic resistive pulse sensor. Experiments of positive separation and detection of T-lymphocytes and negative separation and detection of cancer cells from the whole blood samples were carried out to demonstrate the effectiveness of this method. The methodology of utilizing size difference between magnetic beads and cell-magnetic beads complex for beads-labeled cell detection is simple, automatic, and particularly suitable for beads-based immunoassay without using fluorescence labeling.

Theory of Transport-Induced-Charge Electroosmotic Pumping toward Alternating Current Resistive Pulse Sensing.

In this work, we study transport-induced-charge electroosmosis toward alternating current resistive pulse sensing for the next generation of biomedical applications. Transport-induced-charge electroosmosis, being a new class of electrokinetic phenomenon, occurs as a salt concentration gradient works in synergy with an electric field in ultrathin nanopores. Apart from the conventional electric double layer-governed electroosmotic flow in which the flow behavior is subject to the surface charge, it is found that the transport-induced-charge electroosmotic flow behaves independently of surface charge magnitude but can be linearly regulated by the bulk salt concentration bias. The reversal of the electric field simultaneously inverses the induced charge allowing the establishment of a unidirectional flow under the application of a periodic alternating current field. This unique phenomenon permits continuous water and nanoparticles pumping through a two-dimensional material nanopore in spite of the reversal of the electric field. Built upon this mechanism, we propose a theoretical prototype of alternating current resistive pulse sensing in a two-dimensional nanopore system.

Humic acids modify the pulse size distributions in the characterization of plastic microparticles by Tunable Resistive Pulse Sensing

Tunable Resistive Pulse Sensing, TRPS, is an emerging technique used in quantification and measuring the size (particle-by-particle) of viruses, exosomes and engineered colloidal spheres in biological fluids. We study the features of TRPS to enhance size characterization and quantification of submicron-sized microplastics, also called plastic microparticles, MP, in freshwater environments. We report alterations on the detection of the resistive pulses in the TRPS caused by humic acids, HA, during the size measurement of polystyrene microspheres used as MP surrogate. We discuss the alteration of the electric field in the measuring channel of the TRPS apparatus induced by the passage of HA. TRPS is a fast and precise technique for counting and size determination of MP but needs the evaluation of the influence of the organic matter on the current blockades. We show that statistical clustering models of the magnitude distribution of the resistive pulses can help to detect and quantify changes in the pulse size distributions induced by flocculation of humic acids. Conclusions of this study indicate that TRPS can be a valuable tool to improve the knowledge of the MP fate in surface waters, in the vadose zone and groundwater.

Critical Evaluation of Microfluidic Resistive Pulse Sensing for Quantification and Sizing of Nanometer- and Micrometer-Sized Particles in Biopharmaceutical Products

The objective was to evaluate performance, strengths, and limitations of the microfluidic resistive pulse sensing (MRPS) technique for the characterization of particles in the size range from about 50 to 2000 nm. MRPS, resonant mass measurement (RMM), nanoparticle tracking analysis (NTA) and dynamic light scattering were compared for the analysis of nanometer-sized polystyrene (PS) beads, liposomes, bacteria, and protein aggregates. An electrical conductivity of at least 3 mS/cm (equivalent to 25 mM NaCl) was determined as a key requirement for reliable analysis with MRPS. Particle size distributions of PS beads determined by MRPS, NTA, and RMM correlated well. However, counting precision varied significantly among the techniques and was best for RMM followed by MRPS and NTA. As determined by measuring single and mixed PS bead populations, MRPS showed the highest peak resolution for sizing. RMM and MRPS were superior over dynamic light scattering and NTA for the characterization of stressed protein samples. Finally, MRPS proved to be the only analytical technique able to characterize both bacteria and liposomes. In conclusion, MRPS is an orthogonal technique alongside other established techniques for a comprehensive analysis of a samples particle size distribution and particle concentration.

Gold Nanoparticles with Ligand/Zwitterion Hybrid Layer for Individual Counting of Influenza A H1N1 Subtype Using Resistive Pulse Sensing.

Resistive pulse sensing (RPS) is an analytical technique for detecting particles with nano- to micrometer diameters, such as proteins, viruses, and bacteria. RPS is a promising tool for diagnosis as it can analyze the characteristics of target particles individually from ion current blockades as pulse waveforms. However, it is difficult to discriminate analog targets because RPS merely provides physical information such as size, shape, concentration, and charge density of the analyte. Influenza A virus, which is 80-120 nm in diameter, has various subtypes, demonstrating the diversity of virus characteristics. For example, highly pathogenic avian influenza infections in humans are recognized as an emerging infectious disease with high mortality rates compared with human influenza viruses. Distinguishing human from avian influenza using their differing biological characteristics would be challenging using RPS. To develop a highly selective diagnostic system for infectious diseases, we combined RPS with molecular recognition. Gold nanoparticles (GNPs) that have human influenza A (H1N1 subtype) virus-specific sialic acid receptors on the surface were prepared as a virus label for RPS analysis. A sulfobetaine and sialic acid (ligand) hybrid surface was formed on the GNPs for the suppression of nonspecific interaction. The results show a size change of viruses derived from specific interactions with GNPs. In contrast, no size shift was observed when nonspecific sialic acid receptor-immobilized GNPs were used. Detection of viruses by individual particle counting could be a new facet of diagnosis.

An Examination of the Validity of MRPS Method for the Detection of Label-free E. Coli and Enterococci in Ships Ballast Water

To test for compliance with the D-2 standard for ballast water management convention of the International Maritime Organization, the bacteriological quality of ship’s ballast water is determined to evaluate the risk of invasion of non-indigenous species both onboard ship and in Port. In this article, hydrodynamic flow focusing particles viewed through a microfluidic resistive pulse sensor (MRPS) was used to detect the pathogenic bacteria from ship’s ballast water. Escherichia coli and Enterococci were addressed as bacterial indicators to determine of their characteristic and concentration. The system provided the individual particle-by-particle readout in rather large 20 μm x 10 μm x 8 μm (length x width x height) horizontal rectangular microchannel duct. The average volume flow rate was 4.5 μl/min. However, it returned online and real time results for a complete of 30 μl sample volume during 15 min. The system was low maintenance, high in sensitivity, good accuracy and reliable. Targeted bacteria needed neither labelling nor extracting DNA.

Novel analytical methods to assess the chemical and physical properties of liposomes

Liposomes are used in commercial pharmaceutical formulations (PFs) and dietary supplements (DSs) as a carrier vehicle to protect the active ingredient from degradation and to increase the half-life of the injectable. Even as the commercialization of liposomal products has rapidly increased, characterization methodologies to evaluate physical and chemical properties of the liposomal products have not been well-established. Herein we develop rapid methodologies to evaluate chemical and selected physical properties of liposomal formulations. Chemical properties of liposomes are determined by their lipid composition. The lipid composition is evaluated by first screening of the lipids present in the sample using HPLC-ELSD followed by HPLC-MSMS analysis with high mass accuracy (<5 ppm), fragmentation pattern and lipid structure databases searching. Physical properties such as particle size and size distribution were investigated using Tunable Resistive Pulse Sensing (TRPS). The developed methods were used to analyze commercially available PFs and DSs. As results, PFs contain distinct number of lipids as indicated by the manufacture, but DSs were more complicated containing a large number of lipids belonging to different sub-classes. Commercially available liposomes have particles with wide size distribution based on size measurements performed by TRPS. The high mass accuracy as well as identification lipids using multiple fragment ions aided to accurately identify the lipids and differentiate them from other lipophilic molecules. The developed analytical methodologies were successfully adapted to measure the physiochemical properties of commercial liposomes.

The Design and Characterization of Multifunctional Aptamer Nanopore Sensors.

Aptamer-modified nanomaterials provide a simple, yet powerful sensing platform when combined with resistive pulse sensing technologies. Aptamers adopt a more stable tertiary structure in the presence of a target analyte, which results in a change in charge density and velocity of the carrier particle. In practice the tertiary structure is specific for each aptamer and target, and the strength of the signal varies with different applications and experimental conditions. Resistive pulse sensors (RPS) have single particle resolution, allowing for the detailed characterization of the sample. Measuring the velocity of aptamer-modified nanomaterials as they traverse the RPS provides information on their charge state and densities. To help understand how the aptamer structure and charge density effects the sensitivity of aptamer-RPS assays, here we study two metal binding aptamers. This creates a sensor for mercury and lead ions that is capable of being run in a range of electrolyte concentrations, equivalent to river to seawater conditions. The observed results are in excellent agreement with our proposed model. Building on this we combine two aptamers together in an attempt to form a dual sensing strand of DNA for the simultaneous detection of two metal ions. We show experimental and theoretical responses for the aptamer which creates layers of differing charge densities around the nanomaterial. The density and diameter of these zones effects both the viability and sensitivity of the assay. While this approach allows the interrogation of the DNA structure, the data also highlight the limitations and considerations for future assays.

Particle detection on microfluidic chips by differential resistive pulse sensing (RPS) method

The resistive pulse sensing (RPS) method has been widely used for characterization of particles, cells, and biomolecules due to its merits of high sensitivity and resolution. This paper investigates working parameters involved in detecting submicron and micron-sized particles by the differential RPS method on microfluidic chips. Effects of particle-to-sensor size ratio, ionic concentration and pH of the electrolyte solution, and applied electric field are studied systematically by using polystyrene particles with a size range from 140 nm to 5 µm. The results show that both the amplitude and the signal-to-noise ratio (SNR) of the RPS signals increase with the particle-to-sensor size ratio as well as the ionic concentration of the electrolyte media. The amplitude of the RPS signals also increases with increasing applied voltage, while the SNR experiences an upslope at low voltages and a decline under the condition of high voltages. pH has little effect on the background noise of the differential RPS signals but reduces the amplitude of the RPS signals at high pH. Grouping of RPS signals is considered to be caused by interactions between the sensor walls and the particles. Nanoparticle detection by the differential RPS method can be enhanced by optimizing these working parameters.

Rapid Assessment of Site Specific DNA Methylation through Resistive Pulse Sensing.

Many diseases are defined by patterns of DNA methylation which result in aberrant gene expression. We present a rapid assay based upon resistive pulse sensing, RPS, to characterize sequence specific DNA methylation sites in genomic DNA. We modify the surface of superparamagnetic beads, SPBs, with DNA (capture probe). The particles are added to solution where they bind to and extract sequence specific DNA (target DNA). The target loaded SPBs are then incubated with antibodies which bind to the methylation sites, and the velocity of the SPBs through the nanopore reveals the number and location of the epigenetic markers within the target. The approach is capable of distinguishing between different methylation sites within a DNA promoter region. Crucially the approach is not dependent on accurate sequencing of assayed DNA, with genomic regions targeted through complementary probes. As such the number of stages and reagents costs are low and the assay is complete in under 60 min which includes the incubation and run times. The format also allows simultaneous quantification of number of copies of methylated DNA, and we illustrate this with a dose response curve.

Simple functionalization method for single conical pores with a polydopamine layer

Resistive pulse sensing (RPS) is an interesting analytical system in which micro- to nanosized pores are used to evaluate particles or small analytes. Recently, molecular immobilization techniques to improve the performance of RPS have been reported. The problem in functionalization for RPS is that molecular immobilization by chemical reaction is restricted by the pore material type. Herein, a simple functionalization is performed using mussel-inspired polydopamine as an intermediate layer to connect the pore material with functional molecules.

Detection of Individual Molecules and Ions by Carbon Nanotube-Based Differential Resistive Pulse Sensor.

This paper presents a new method of sensing single molecules and cations by a carbon nanotube (CNT)-based differential resistive pulse sensing (RPS) technique on a nanofluidic chip. A mathematical model for multichannel RPS systems is developed to evaluate the CNT-based RPS signals. Individual cations, rhodamine B dye molecules, and ssDNAs are detected successfully with high resolution and high signal-to-noise ratio. Differentiating ssDNAs with 15 and 30 nucleotides are achieved. The experimental results also show that translocation of negatively charged ssDNAs through a CNT decreases the electrical resistance of the CNT channel, while translocation of positively charged cations and rhodamine B molecules increases the electrical resistance of the CNT. The CNT-based nanofluidic device developed in this work provides a new avenue for single-molecule/ion detection and offers a potential strategy for DNA sequencing.