What Makes Tunable Resistive Pulse Sensing So Precise?

How tunable resistive pulse sensing (TRPS) enables precise EV measurements.

Izon Science’s tunable resistive pulse sensing (TRPS) platforms provide a true particle-by-particle approach to measuring EV size, concentration, and zeta potential. With TRPS, each particle is measured only once as it passes through the nanopore; once it has passed through the pore, it is not measured again. This contrasts with bulk measurement techniques known as ‘ensemble techniques’ which instead measure many particles in the sample at the same time.<super-script>1,2,3<super-script> With ensemble techniques, some particles may be measured more than once (and others could be missed), thereby contributing a disproportionate amount of information towards the overall ensemble-averaged measurement. This can be likened to taking multiple snapshots of the same particles. Furthermore, as measurements using dynamic light scattering are weighted towards larger particles, particle size distribution can easily be distorted.<super-script>4<super-script>

True single-particle measurements are needed to provide insightful, high-resolution information. For TRPS size measurements, there is a linear relationship between the particle volume and magnitude of the resistive pulse generated, therefore particle diameters can be determined with high accuracy.<super-script>5<super-script> Similarly, zeta potential can be measured for each individual particle by inspecting pulse duration, while concentration is derived from blockade frequency.

The ability to test a blank buffer provides confidence

When the solution is devoid of particles (i.e., the measurement electrolyte has been applied) there should be no blockades showing against the background current. Whereas contaminated buffer or other similar issues would not be detected with ensemble instruments, TRPS will detect the presence of individual particles in a solution.

Users can therefore proceed with confidence knowing that blockades are truly representative of particles in the sample. As information on size, concentration and zeta potential are all derived from interpreting information from the blockades, this is highly important.

Standardised calibration particles provide consistency

TRPS measurement requires the use of standards which are analysed under the exact same conditions as the sample (including electrolyte characteristics). Izon-supplied calibration particles are negatively-charged carboxylated polystyrene particles (CPCs), which have been previously characterised against neutral NIST particles. CPC measurements are used in calculations of sample characteristics and allow all measurements to be fully traced back to a NIST standard.

Optimise and maintain conditions for calibration and measurement

Modern TRPS software provides a real-time measure of the root mean square (RMS) which is a strong indicator of the signal-to-noise ratio. RMS is the square root of the mean square, and is calculated using a defined set of current measurements over a defined time period. As the RMS value is constantly being updated, parameters can be tweaked until an acceptable RMS value has been obtained (<15 pA). This is then monitored to ensure it is maintained throughout the calibration and measurement process.

Prior to TRPS measurements, parameters can be adjusted to maximise the blockade size relative to the background current, thereby providing a further avenue towards high quality data. This can be achieved by reducing the stretch of the nanopore, increasing the voltage, and reducing the pressure. These strategies do have the potential to create unfavourable running conditions, therefore establishing TRPS running conditions is a delicate balancing act requiring the constant monitoring of parameters. Conditions can be further controlled by changing the molarity of the electrolyte.

Exoid design considerations for minimising noise

Recognising and addressing the sources of noise is a critical aspect of instrument design at every level, including hardware, software, mechanical design, and the choice and arrangement of individual components. Below are several examples.

Signal bandwidth: Current is a continuous signal, and it must be measured at discrete time points to obtain data for TRPS measurements. The sampling frequency used to obtain current measurements is known as the signal bandwidth, and has a critical impact on data quality and accuracy.<super-script>6<super-script> Optimising this sampling frequency, therefore, was an important aspect of TRPS development. The signal bandwidth can be thought of as the highest sampling frequency that can be used to obtain a signal; any lower, and important changes may be missed. Increasing the signal frequency is only beneficial to a point, beyond which there will be no further benefits – only more noise. Optimising the signal bandwidth therefore requires a delicate balance of these factors and was guided by the Nyquist–Shannon sampling theorem which states: to properly resolve a feature in your signal, you must sample with a frequency that is at least double that of your fastest change.

Nanopore material: Different material types will have different inherent levels of noise. This was a factor considered when choosing the nanopore material.

Use of high-quality components: Comparisons of noise contributions from relays, filters and various other components helped guide their selection.  

Arrangement of various components: Components are arranged in a way that minimises noise from the environment. Continuous metal housing surrounds fluid cells and cables, and the upper fluid cell has been re-designed to include a full Faraday cage.

Relevance of tunable resistive pulse sensing in therapeutic and diagnostic settings

TRPS enables precise measurements of particle size, concentration and zeta potential – characteristics which are highly relevant for the development of EVs and other nano-sized vesicles as therapeutic vehicles. The accurate profiling of these parameters is important for guiding the development of biotherapeutics – including vaccines – by enabling aggregate detection, accurate dosing, and assessments of particle stability. A recent study published in the Journal of Pharmaceutical Sciences describes TRPS as a promising technology for submicron particle analysis in biopharmaceuticals, following the accurate quantification of monoclonal antibodies in the range of 200-900 nm.<super-script>7<super-script>

EV size and concentration have been compared in many studies aimed at understanding EV-based differences under varying physiological and pathological conditions, including in glioblastoma<super-script>8<super-script>,spontaneous preterm birth<super-script>9<super-script> and myocardial infarction.<super-script>10<super-script> While TRPS enables the accurate measurement of these physical parameters in basic EV research, the method may also be used in some settings to further develop diagnostic tests that rely on the binding of antibodies or aptamers to EVs of interest. This has been applied in a similar situation by Billinge et al.(2013) who describe the use of TRPS to detect real-time changes in surface charge indicative of binding between a DNA aptamer to a particle surface.<super-script>11<super-script>Such an approach can support the validation of EV-based biomarkers by applying TRPS at different steps of assay optimisation.

Quality EV data starts with reproducible isolation

In computer science, there is a common saying:‘ garbage in, garbage out’, which refers to the concept that flawed or insufficient data input produces an unreliable and pointless output. Similarly, reproducible isolation is a prerequisite for accurate and meaningful EV analysis. To ensure downstream analysis always starts with a clean, size-controlled population, Izon recommends coupling TRPS (and other downstream analyses) to Izon’s qEV isolation platform, which utilises automation and size-exclusion chromatography to reliably maximise EV separation.

Honing in on the fine details: a microscopy analogy

In some ways, qEV isolation and TRPS can be likened to using a microscope. With microscopy, detailed morphological information can be obtained by first adjusting the coarse focus to rapidly adjust the position of the stage, followed by more refined adjustments with the fine adjustment knob. This approach enables biologists to identify components of interest and then zoom into the detail.

Similarly, qEV isolation is an effective method for cutting out the noise and isolating particles of interest. Once this has been achieved, biologists can narrow in on the EVs they are looking for, using TRPS and appropriate nanopore selection. Fine-tuning and noise reduction is then achieved through the inherent single-particle nature of TRPS, use of calibration particles, as well as strategies for optimising and maintaining running conditions.

Learn how TRPS is being applied across diverse areas of research


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