Advancement of Measurement Techniques for Tunable Resistive Pulse Sensing

<p>Tunable resistive pulse sensing (TRPS) is a particle-by-particle analysis technique combining the Coulter principle with size-tunable pores. TRPS can be used to characterize biological and synthetic particles 50 nm - 20 µm in diameter. Information is obtained from the resistive pulse signal, a transient change in ionic current observed when a particle passes through the pore. TRPS has been shown to provide excellent resolution and accuracy for measuring particle size and concentration as well as providing information about particle charge. TRPS is therefore applicable to many industrial and fundamental research areas involving aptamers, drug delivery particles, extracellular vesicles and other biological particle types. Advancement of this technology requires a better understanding of the technique, particularly in the area of particle surface charge measurement and this Thesis helps to provide that understanding. In this work, firstly particle ζ-potential measurement using TRPS was investigated. A number of different measurement methods are presented and the uncertainties associated with each method are outlined. The ζ-potential for a variety of particles with different surface charges were measured in a range of electrolytes. Particle ζ-potential measurements were then improved upon with the addition of streaming potential measurements to measure the pore surface charge. The ζ-potential of the pore surface, which makes a significant contribution to particle ζ-potential calculations, was measured using a set up which works alongside the qNano. Streaming potential measurements were also used to investigate changes in the pore surface charge following application of number of different chemical coatings. The volume of data collected and detail of analysis in this work (including uncertainties) is unprecedented in TRPS ζ potential measurements. Biphasic pulses arising from the charge on the particles were investigated. The pulse is conventionally resistive, but biphasic pulses which include both resistive and conductive components are significant for less than 50 mM salt concentrations when measuring 200 nm particles. The experimental variables investigated include the concentration of the electrolyte, particle charge, pore size, applied voltage, and the direction of particlemotion. Conductive pulse size was seen to decrease with increasing electrolyte concentration and pore size and increase with applied voltage. A linear relationship was found between conductive pulse magnitude and particle surface group density. The influence of direction of motion on conductive pulses was consistent with concentration polarization of an ion selective pore. Biphasic pulses were also seen to affect conventional TRPS particle size measurements. Finally, size distribution broadening due to varying particle trajectories was investigated. Pulse size distributions for monodisperse particles became broader when the pore size was increased and featured two distinct peaks. Relatively large pulses are produced by particles with trajectories passing near to the edge of the pore. Other experiments determined that pulse size distributions are independent of applied voltage but broaden with increasing pressure applied across the membrane.</p>

Advancement of Measurement Techniques for Tunable Resistive Pulse Sensing

<p>Tunable resistive pulse sensing (TRPS) is a particle-by-particle analysis technique combining the Coulter principle with size-tunable pores. TRPS can be used to characterize biological and synthetic particles 50 nm - 20 µm in diameter. Information is obtained from the resistive pulse signal, a transient change in ionic current observed when a particle passes through the pore. TRPS has been shown to provide excellent resolution and accuracy for measuring particle size and concentration as well as providing information about particle charge. TRPS is therefore applicable to many industrial and fundamental research areas involving aptamers, drug delivery particles, extracellular vesicles and other biological particle types. Advancement of this technology requires a better understanding of the technique, particularly in the area of particle surface charge measurement and this Thesis helps to provide that understanding. In this work, firstly particle ζ-potential measurement using TRPS was investigated. A number of different measurement methods are presented and the uncertainties associated with each method are outlined. The ζ-potential for a variety of particles with different surface charges were measured in a range of electrolytes. Particle ζ-potential measurements were then improved upon with the addition of streaming potential measurements to measure the pore surface charge. The ζ-potential of the pore surface, which makes a significant contribution to particle ζ-potential calculations, was measured using a set up which works alongside the qNano. Streaming potential measurements were also used to investigate changes in the pore surface charge following application of number of different chemical coatings. The volume of data collected and detail of analysis in this work (including uncertainties) is unprecedented in TRPS ζ potential measurements. Biphasic pulses arising from the charge on the particles were investigated. The pulse is conventionally resistive, but biphasic pulses which include both resistive and conductive components are significant for less than 50 mM salt concentrations when measuring 200 nm particles. The experimental variables investigated include the concentration of the electrolyte, particle charge, pore size, applied voltage, and the direction of particlemotion. Conductive pulse size was seen to decrease with increasing electrolyte concentration and pore size and increase with applied voltage. A linear relationship was found between conductive pulse magnitude and particle surface group density. The influence of direction of motion on conductive pulses was consistent with concentration polarization of an ion selective pore. Biphasic pulses were also seen to affect conventional TRPS particle size measurements. Finally, size distribution broadening due to varying particle trajectories was investigated. Pulse size distributions for monodisperse particles became broader when the pore size was increased and featured two distinct peaks. Relatively large pulses are produced by particles with trajectories passing near to the edge of the pore. Other experiments determined that pulse size distributions are independent of applied voltage but broaden with increasing pressure applied across the membrane.</p>

Nanoparticle Charge and Shape Measurements using Tuneable Resistive Pulse Sensing

<p>Accurate characterisation of micro- and nanoparticles is of key importance in a variety of scientific fields from colloidal chemistry to medicine. Tuneable resistive pulse sensing (TRPS) has been shown to be effective in determining the size and concentration of nanoparticles in solution. Detection is achieved using the Coulter principle, in which each particle passing through a pore in an insulating membrane generates a resistive pulse in the ionic current passing through the pore. The distinctive feature of TRPS relative to other RPS systems is that the membrane material is thermoplastic polyurethane, which can be actuated on macroscopic scales in order to tune the pore geometry. In this thesis we attempt to extend existing TRPS techniques to enable the characterisation of nanoparticle charge and shape. For the prediction of resistive pulses produced in a conical pore we characterise the electrolyte solutions, pore geometry and pore zeta-potential and use known volume calibration particles. The first major investigation used TRPS to quantitatively measure the zeta-potential of carboxylate polystyrene particles in solution. We find that zeta-potential measurements made using pulse full width half maximum data are more reproducible than those from pulse rate data. We show that particle zeta-potentials produced using TRPS are consistent with literature and those measured using dynamic light scattering techniques. The next major task was investigating the relationship between pulse shape and particle shape. TRPS was used to compare PEGylated gold nanorods with spherical carboxylate polystyrene particles. We determine common levels of variation across the metrics of pulse magnitude, duration and pulse asymmetry. The rise and fall gradients of resistive pulses may enable differentiation of spherical and non-spherical particles. Finally, using the metrics and techniques developed during charge and shape investigations, TRPS was applied to Rattus rattus red blood cells, Shewanella marintestina bacteria and bacterially-produced polyhydroxyalkanoate particles. We find that TRPS is capable of producing accurate size distributions of all these particle sets, even though they represent nonspherical or highly disperse particle sets. TRPS produces particle volume measurements that are consistent with either literature values or electron microscopy measurements of the dominant species of these particle sets. We also find some evidence that TRPS is able to differentiate between spherical and non-spherical particles using pulse rise and fall gradients in Shewanella and Rattus rattus red blood cells. We expect TRPS to continue to find application in quantitative measurements across a variety of particles and applications in the future.</p>

Nanoparticle Charge and Shape Measurements using Tuneable Resistive Pulse Sensing

<p>Accurate characterisation of micro- and nanoparticles is of key importance in a variety of scientific fields from colloidal chemistry to medicine. Tuneable resistive pulse sensing (TRPS) has been shown to be effective in determining the size and concentration of nanoparticles in solution. Detection is achieved using the Coulter principle, in which each particle passing through a pore in an insulating membrane generates a resistive pulse in the ionic current passing through the pore. The distinctive feature of TRPS relative to other RPS systems is that the membrane material is thermoplastic polyurethane, which can be actuated on macroscopic scales in order to tune the pore geometry. In this thesis we attempt to extend existing TRPS techniques to enable the characterisation of nanoparticle charge and shape. For the prediction of resistive pulses produced in a conical pore we characterise the electrolyte solutions, pore geometry and pore zeta-potential and use known volume calibration particles. The first major investigation used TRPS to quantitatively measure the zeta-potential of carboxylate polystyrene particles in solution. We find that zeta-potential measurements made using pulse full width half maximum data are more reproducible than those from pulse rate data. We show that particle zeta-potentials produced using TRPS are consistent with literature and those measured using dynamic light scattering techniques. The next major task was investigating the relationship between pulse shape and particle shape. TRPS was used to compare PEGylated gold nanorods with spherical carboxylate polystyrene particles. We determine common levels of variation across the metrics of pulse magnitude, duration and pulse asymmetry. The rise and fall gradients of resistive pulses may enable differentiation of spherical and non-spherical particles. Finally, using the metrics and techniques developed during charge and shape investigations, TRPS was applied to Rattus rattus red blood cells, Shewanella marintestina bacteria and bacterially-produced polyhydroxyalkanoate particles. We find that TRPS is capable of producing accurate size distributions of all these particle sets, even though they represent nonspherical or highly disperse particle sets. TRPS produces particle volume measurements that are consistent with either literature values or electron microscopy measurements of the dominant species of these particle sets. We also find some evidence that TRPS is able to differentiate between spherical and non-spherical particles using pulse rise and fall gradients in Shewanella and Rattus rattus red blood cells. We expect TRPS to continue to find application in quantitative measurements across a variety of particles and applications in the future.</p>

Coordinated Detection of Micro- and Nanoparticles using Tuneable Resistive Pulse Sensing and Optical Spectroscopy

<p>The detection and characterisation of micro- and nanoscale particles has become increasingly important in many scientific fields, spanning from colloidal science to biomedical applications. Resistive Pulse Sensing (RPS) and its derivative Tuneable Resistive Pulse Sensing (TRPS), which both use the Coulter principle, have proven to be useful tools to detect and analyse particles in solution over a wide range of sizes. While RPS uses a fixed size pore, TRPS uses a dynamically stretchable pore in a polyurethane membrane, which has the advantages that the pore geometry can be tuned to increase the device's sensitivity and range of detection. The technique has been used to accurately determine the size, concentration and charge of many different analytes. However, the information obtained using TRPS does not give any insight into the particle's composition. In an attempt to overcome this, an experimental technique was developed in order to obtain simultaneous, time-resolved, high-resolution optical spectra of particles passing through the pore. Due to the ordered and controllable fashion in which the particles are guided through the sensing region, this approach has an advantage over diffusion based optical techniques. The experimental setup for the coordinated electrical and optical measurements involves many underlying physical phenomena, e.g. microuidics, electrokinetic effects, and Gaussian beam optics. A significant proportion of this work was therefore devoted to the development and the optimisation of the experimental setup by adapting a commercial TRPS device and a spectrometer with an attached microscope. Methods to engineer the spot size of a Gaussian beam to account for the different pore diameters, and the development of algorithms to filter, analyse and coordinate the recorded data are essential to the technique. The results using fluorescently labelled polystyrene particle sets with diameters from 190nm to 2 µm show that matching rates between the electrical and optical measurements of over 90% can repeatedly be achieved. Mixtures of particle species with similar diameters but with different fluorescent labels were used to demonstrate the technique's capability to characterise the analyte on a particle-by-particle basis and extend the information that can be obtained by TRPS alone. It was also shown that the data acquired with the electrical and optical measurements complement each other and can be used to better understand the TRPS technique itself. The influence of experimental parameters, such as the particle velocity, the beam size and the optical detection volume, on the intensity of the optical signals and the matching rates was studied intensively. These studies showed that the technique requires a careful experimental design to achieve the best results. Overall, the developed technique enhances the particle-by-particle specificity of conventional RPS measurements, and could be useful for a range of particle characterization and bio-analysis applications. Alongside the experiments, semi-analytic modelling and simulations using the Finite Element Method (FEM) were used to understand the particle motion through the pores, to interpret the experimental data, and predict the optical signals. The models were also used to assist the design and the optimization of the experiments. The FEM models were implemented with increasing physical detail and show superior understanding of the TRPS signals compared to the semi-analytic model, which is conventionally used in the TRPS field. The physical phenomena considered included o -axis trajectories, particle-field interactions for both fluid and electric fields, and the non-homogeneous distribution of ions close to the charged membrane and particle interfaces. Several effects which have been observed experimentally could be explained, including the intrinsic pulse height distribution, the current rectification, and the occurrence of bi-phasic pulses, demonstrating the benefits of FEM methods for RPS.</p>

Coordinated Detection of Micro- and Nanoparticles using Tuneable Resistive Pulse Sensing and Optical Spectroscopy

<p>The detection and characterisation of micro- and nanoscale particles has become increasingly important in many scientific fields, spanning from colloidal science to biomedical applications. Resistive Pulse Sensing (RPS) and its derivative Tuneable Resistive Pulse Sensing (TRPS), which both use the Coulter principle, have proven to be useful tools to detect and analyse particles in solution over a wide range of sizes. While RPS uses a fixed size pore, TRPS uses a dynamically stretchable pore in a polyurethane membrane, which has the advantages that the pore geometry can be tuned to increase the device's sensitivity and range of detection. The technique has been used to accurately determine the size, concentration and charge of many different analytes. However, the information obtained using TRPS does not give any insight into the particle's composition. In an attempt to overcome this, an experimental technique was developed in order to obtain simultaneous, time-resolved, high-resolution optical spectra of particles passing through the pore. Due to the ordered and controllable fashion in which the particles are guided through the sensing region, this approach has an advantage over diffusion based optical techniques. The experimental setup for the coordinated electrical and optical measurements involves many underlying physical phenomena, e.g. microuidics, electrokinetic effects, and Gaussian beam optics. A significant proportion of this work was therefore devoted to the development and the optimisation of the experimental setup by adapting a commercial TRPS device and a spectrometer with an attached microscope. Methods to engineer the spot size of a Gaussian beam to account for the different pore diameters, and the development of algorithms to filter, analyse and coordinate the recorded data are essential to the technique. The results using fluorescently labelled polystyrene particle sets with diameters from 190nm to 2 µm show that matching rates between the electrical and optical measurements of over 90% can repeatedly be achieved. Mixtures of particle species with similar diameters but with different fluorescent labels were used to demonstrate the technique's capability to characterise the analyte on a particle-by-particle basis and extend the information that can be obtained by TRPS alone. It was also shown that the data acquired with the electrical and optical measurements complement each other and can be used to better understand the TRPS technique itself. The influence of experimental parameters, such as the particle velocity, the beam size and the optical detection volume, on the intensity of the optical signals and the matching rates was studied intensively. These studies showed that the technique requires a careful experimental design to achieve the best results. Overall, the developed technique enhances the particle-by-particle specificity of conventional RPS measurements, and could be useful for a range of particle characterization and bio-analysis applications. Alongside the experiments, semi-analytic modelling and simulations using the Finite Element Method (FEM) were used to understand the particle motion through the pores, to interpret the experimental data, and predict the optical signals. The models were also used to assist the design and the optimization of the experiments. The FEM models were implemented with increasing physical detail and show superior understanding of the TRPS signals compared to the semi-analytic model, which is conventionally used in the TRPS field. The physical phenomena considered included o -axis trajectories, particle-field interactions for both fluid and electric fields, and the non-homogeneous distribution of ions close to the charged membrane and particle interfaces. Several effects which have been observed experimentally could be explained, including the intrinsic pulse height distribution, the current rectification, and the occurrence of bi-phasic pulses, demonstrating the benefits of FEM methods for RPS.</p>

Resistive Pulse Sensing with Micro-fabricated Nanopores (NP-RPS) for Sub-micron Biomolecule and Bionanoparticle Analysis

Biomolecules and bionanoparticles, such as nucleic acids, proteins, microorganisms and extracellular vesicles (EVs), are recognized as important targets for fundamental research, clinical diagnostic and therapeutic applications. To gain detailed information of those bionanoparticles, we demonstrate an electroosmotic (EO) driven transport behavior in silicon and silicon nitride-based nanopore, towards an accurate measure of concentration and sizing of sub-micro particles for a general biological interest.