By AZoNano.com Staff Writers
Tunable Resistive Pulse Sensing (TRPS) is a superior resolution technique which can precisely measure the size, charge, and concentration of extracellular vesicles (EVs). Moreover, it can analyze individual particles on particle-by-particle basis when they move across a pore-based sensor. Instruments such as the Izon qNano and qViro-X are the examples for tunable resistive pulse sensors.
The Izon devices demonstrate a superior level of analysis detail, accuracy, and precision than competitive products. Although relatively new, TRPS technique has already made an impact in EV research through the characterization of a many different exosome and microvesicle samples.
EV research is a dynamic and rapidly advancing field which shows promise to better understand cellular interactions and disease progression. For this purpose, techniques which can precisely determine the physical properties of EVs, including concentration, size and charge, are needed.
This article discusses the capability of the TRPS technique to measure EV size and concentration. Moreover, the article also covers the particle-by-particle charge analysis capability of this technique that may provide researchers with a novel tool to gain new insights and characterize EVs.
The Izon qNano and qViro-X particle characterization platforms are capable of accurately and precisely measuring the size, charge, and concentration of a variety of synthetic and biological particles. These devices feature a fluid cell isolated by an elastic membrane which has a single hole or ‘pore’ punctured in it.
Particles like microvesicles or exosomes are measured as a change in the electrical resistance when they move across the pore, as illustrated in Figure 1A. The resulting ‘resistive pulse’ signal produced by each particle is depicted in Figure 1B.
The data extracted from the resistive pulse signal can be utilized to measure the size and electrophoretic mobility (surface charge) of individual particle by relating the signal back to a suite of calibration particles with known properties.
This ensures the repeatability of the results and makes them objectively comparable as they are independent of sample variables provided by users. Moreover, the capability to calculate hundreds to thousands of particles within a sample within a few minutes provides a precise measurement of particle distribution. The sensitivity of the TRPS technique is demonstrated in Figure 1C.
Figure 1. TRPS at a glance. A is an image of the Izon qNano TRPS instrument. The magnified area is an illustration of particles passing through the tunable pore sensor. B is a close up of the resistive pulse signal generated by a particle passing through the pore. C illustrates the sensitivity of the TRPS signal to small differences in particle size.
Particle concentration is linearly proportional to the count of resistive pulses per minute, which is the rate at which particles move across the pore. Hence, TRPS devices provide a comprehensive measurement of the size, charge, and concentration of EV and nanoparticle samples. Moreover, as a high resolution technique, TRPS is capable of measuring minute differences in the properties of individual particles.
The comparison of TRPS and nanoparticle tracking analysis (NTA) of a trimodal mixture of 220, 330 and 410 nm polystyrene particles is illustrated in Figure 2. The results clearly show the inability of NTA to resolve the three particle populations in the sample. Moreover, NTA has a distribution that shifts towards the more numerous 220 nm particles.
On the other hand, the higher resolution analysis of TRPS is able to easily and precisely resolve all three particle populations and provides measurements of defined peaks that demonstrate the precision of the technique and the requirement for the high resolution analysis to give confidence in measured values.
Figure 2. Comparison of the sensitivity and accuracy of TRPS to NTA when measuring complex and polydisperse samples, such as a ‘multimodal’ sample composed of 220, 330 and 410 nm particles.
EVs are typically polydisperse, which means that they can have a wide range of sizes from 50 to 1000 nm, and their size is often utilized as a means to classify them. Besides classifying EVs, size has been potentially related to their origin.
Hence, EV size and polydispersity need to be accurately measured in their identification and application as an effective disease diagnostic. The analysis capabilities and versatility of Izon TRPS instruments are illustrated by measuring the size distribution of EVs separated from human urine and plasma in addition to cell culture media. Each case involved the analysis of at least 500 EVs, and the mean, mode and ranges obtained are depicted in Figure 3.
Figure 3. TRPS measurement of the size distribution of EVs isolated from A) human urine, B) human plasma and C) cell culture media. Histograms are the result of the analysis of a minimum of 500 individual EV events. The mean, mode and span of the EVs were found to be dependent on the sample media and isolation procedure. Importantly, the size distribution can be correlated back to a % of the measured population or the concentration (particles / mL) of that sized particle in the sample.
Characterizing particle aggregation is significant as it causes a decrease in overall particle concentration and an increase in measured particle size. Here, the measurement of the degree of aggregation of EVs extracted from foetal bovine serum (FBS) was performed with and without a non-ionic surfactant (Tween-20 at 0.0025% w/v in 3x PBS), as shown in Figure 4. The sample without surfactant has a broader range and more number of larger particles, nearly 16% representing over particles of over 250nm in size. On the other hand, the sample with surfactant has a narrow distribution.
Figure 4. TRPS measurement of exosome samples with and without the addition of the Tween-20 surfactant to reduce EV aggregation.
TRPS presents the particle size distribution as a function of the particle concentration. Hence, it is capable of measuring the concentration of each population in multimodal samples that has more than one distinct particle size population. In addition, the total vesicle concentration can be obtained from the sum of all the particle sizes.
It is necessary to precisely measure the concentration of EVs in a sample in order to better understand their function and ubiquity. EV concentrations are typically determined either by indirect spectroscopic measurements or on a particle-by-particle basis through flow cytometry that are usually relied on EV size, polydispersity and biochemical composition.
On the other hand, TRPS easily and directly measures EV concentration on a particle-by-particle basis by calculating the rate of particles traversing the pore over time. Since particle rate is independent, it is not affected by the size and biochemical composition of the particle as long as it is within the pore’s detection limits.
Moreover, with lower detection limit of ~40 nm and small sensing zone, TRPS is not affected from measuring multiple vesicles at the sample time, “swarming”, and can measure a wider range of particles, which would otherwise went unnoticed by other techniques.
The calculation of particle concentration from the count rate when calibrated against a known concentration standard is depicted in Figure 5. This linear relationship together with the counting of each particle only once when it traverses the pore makes TRPS to provide very sensitive and accurate measurement of particle number concentration.
Figure 5. TRPS measurement of exosome concentration. The count rate decreases linearly with sample dilution, i.e. particle concentration. The dotted lines represent 95% confidence intervals of the linear least squares fit of the data (green line).
Particle surface charge, typically described as ζ-potential, is a key characteristic of nano-and micro-sized particles. ζ-potential indicates particle stability and is used as a means of detecting the changes or differences in the surface properties of particles. Moreover, ζ-potential has recently been used an additional and orthogonal technique to characterize the homogeneity or differences in EV surface properties.
Unlike other methods, TRPS can simultaneously measure the size and ζ-potential of each particle when it moves across the pore. The simultaneous measurement of size and ζ-potential of three individual particle suspensions on a particle-by-particle basis is depicted in Figure 6. Here, each particle is differentiated from other particles on the basis of variations in their sizes as well as their ζ-potential. Besides having the potential to better understand, classify, and characterize EVs, TRPS shows promise ot be utilized as a diagnostic readout.
Figure 6. Simultaneous size and charge measurement of three nanoparticle populations of 200, 240 and 350 nm and -35, -8 and -20 mV, respectively. TRPS was able to simultaneously obtain size and zeta potential information of the three particle sets which clearly shows that these particles can be distinguished based on these characteristics.
From the results, it is evident that TRPS provides a high resolution, accurate, comprehensive and high throughput analysis tool to researchers for measuring and analyzing the size, charge, and concentration of EV samples. The novel technique is helpful in the fundamental characterization of EVs or bioassay platform development. Moreover, it is possible to apply the TRPS methodology to any particle suspension, including bacteria, viruses, nanobubbles, liposomes, emulsions, and any other particle system which is readily suspended in an aqueous electrolyte solution.
Izon Science has developed the world’s first nanopore based measurement system available for general use. Izon’s instruments are used for precise measurement and analysis of individual particles across a wide range of scientific fields including bionanotechnology, nanomedicine, vaccinology, microbiology, biomedical research, environmental science, and particle based nanoscience. Izon originated in New Zealand and now sells its products in 34 countries. It has its European headquarters in Oxford, UK and its US headquarters are in Cambridge, MA.
This information has been sourced, reviewed and adapted from materials provided by Izon.
For more information on this source, please visit Izon.