Tunable Resistive Pulse Sensing (TRPS) to accurately characterize nano and microscopic particles

insights from industryHans van der VoornCEOIzon Science Ltd.

An interview with Hans van der Voorn conducted by James Ives, MPsych

Please give me an overview of Tunable Resistive Pulse Sensing (TRPS).

TRPS is the most powerful and accurate nanoparticle measurement and analysis method on the market. It’s a non-optical technique using precise single particle measurement to provide certainty detail on particle size distribution, surface charge and charge distribution, and particle concentration (count).

The fine scale accuracy of these core parameters enables a range of further analyses to be done, e.g. biological surface analysis using aptamers or direct aggregation analysis.

In TRPS a size tunable pore and tunable voltage and pressure are used to control and measure single particles suspended in electrolyte. Tunability applies to both pore size and the driving forces on the particles.

An electrical current through the pore is disrupted by each particle as it traverses the pore, creating a resistive pulse. These resistive pulses are analysed in detail and calibrated to provide content rich data with very high precision, repeatability and certainty.

The tunability of pressure and voltage enable precise control of particle flow which extends the range of measurement options and is what makes TRPS so powerful.

A pictorial explanation is shown here.

How are the size, charge and concentration of the samples measured? How accurate are these?

The volume of each particle passing through the pore is directly proportional to the depth or amplitude of the resistive pulse. The pulse size from the sample particle is directly measured against the pulse from a known calibration particle.

Because the particles are individually measured and calibrated the correct size distribution profile is given, elegantly solving the problem of laser methods being unable to provide a useful size distribution.

Each particle is typically measured to +/- 1nm of diameter. The physical diameter of the particle is measured, the same as is seen with TEM. The laser based methods (eg DLS, NTA) measure the hydrodynamic diameter, which includes a layer of liquid around the particle and therefore varies depending on the electrolyte.

Particle charge, usually approximated by the zeta potential, is derived from the electrophoretic mobility, which is the particle velocity created by an electric field. The width of the resistive pulse gives the time taken for the particle to travel between 2 known points.

TRPS uses a clever procedure to derive the electrophoretic mobility of each particle by analysing the pulse width or time of flight through the pore. TRPS is the only system that offers an accurate particle by particle charge distribution, which is now an important tool for nanoparticle research in nanomedicine and for nano-biologicals like exosomes.

The precision of TRPS charge measurement is such that changes in “drag” of particles, caused by, for instance, different configuration of DNA molecules on the particles’ surface, can be identified and measured even where the actual charge is the same.

Particle number (or count or concentration) is derived by measuring the particle flow rate (number/minute) under a range of pressures. Calibration against known particles is built into the TRPS method and is always part of the procedure.

The old-fashioned approach is to think of particle concentration as a single number, whereas the correct approach is to provide the number of particles in each of the size bands.

TRPS concentration measurements are accurate to +/- 10-20% on a linear scale, and significantly more accurate than NTA for instance, which only operates on a log scale.

How do you ensure quality and reliability of the measurements?

The instruments and consumables are all made to stringent quality assurance standards and certified to ISO 13485 Medical Devices.

The real quality assurance however comes from the underlying power of the TRPS technique and the way that calibration is automatically included in every measurement.

Does the system require any calibration, training or specialist software?

Yes, and all three are provided with the qNano instrument suite. The specialist software is an Izon product and has been purpose designed and developed over a 10-year period. It is a very powerful analytical system and is regularly upgraded to provide additional features and improved ease of use.

There is a training and certification system in place and an online knowledge base available to all users. Users are expected to be tested and certified, particularly for any QA work or published work.

Additional training is often provided for specialist applications such as different types of exosome analysis or new method development. Certified calibration particles, nanopores and reagents are provided as part of the system.

How does TRPS differ from other particle sizing techniques such as DLS, NTA or TEM?

TRPS is the most capable of all these methods. TEM has similar sizing accuracy to TRPS but can’t measure particle number or charge. DLS is the worst technique by a considerable margin.

TEM uses an electron beam, DLS and NTA use lasers and TRPS uses ionic current through a nano- or micro-scale pore to measure the particle properties. The DLS and NTA optical methods have low resolution so cannot provide an accurate size distribution for polydisperse particles. DLS is particularly poor in that respect.

TEM is often seen as the gold standard for particle size measurement. It provides detailed images with additional shape and surface features shown as well as size. TRPS uses monodisperse calibration standards that are originally measured by TEM but TRPS is more accurate and faster than TEM at measuring and resolving a high number of polydisperse particles.

With TEM, particles are placed on a grid and measured in a vacuum which can change their nature or cause sampling problems. TEM is a single particle technique so can generate a real size distribution but works best with uniform particles and calibration standards.

TEM is generally too expensive for everyday use so not really comparable with small bench top instruments and it can’t measure concentration or surface charge.

DLS is the most commonly used nanoparticle measurement technique and is by far the weakest in its capabilities. DLS, based on the analysis of laser light scattered out of a whole sample is a low-resolution technique really only applicable to monodisperse particles.

DLS based charge measurement has similar issues to its size measurement with polydispersity of either size or charge creating an insoluble set of data points. There is no concentration measurement capability. DLS should by now be considered obsolete in the biomedical fields that Izon operates in with TRPS.

NTA is a variation of DLS where the light scattered by the particles is tracked in 2-D on the surface over a short period of time. It uses the Stokes Einstein equation to determine the particle size by measuring the length of the diffraction track.

While more accurate than DLS it is still a low-resolution technique that is difficult to replicate or standardise between users and instruments. Each particle measurement has a variety of assumptions (eg a 2-D model for a 3-D case) and estimates implicit in the calculation, which is a fundamental limit to the precision and reliability.

As with DLS, NTA works best with monodisperse particle sets. NTA estimates particle number on a log scale, with 1-2 logs of accuracy.

TRPS provides the most detail and accuracy of the available methods in each of the main parameters of size, number and charge.

What are the most important applications of TRPS?

TRPS is best used where there is some complexity and a need for certainty. The main applications are therefore in biomedical research and development and in nano-biologicals research. Three fields of particular interest are nanomedicine, extracellular vesicles including exosomes, and viruses.

Applications under development for TRPS include its use in clinical diagnostics using particles as biomarkers and in nano-pharmaceutical QA. The combined use of charged probes like aptamers and precise charge measurement offer a range of novel applications that are just starting to be realised.

Nanomedicine is a field that particularly benefits from the precision and certainty of TRPS. Clinical adoption of nanomedicines is much slower than it should be because of the weakness of the DLS data that has been most commonly used.  

Exosome research is a rapidly emerging field based on highly complex and heterogeneous particles, ideally suited to TRPS.

What size of sample can be measured? How long are the processing times for these samples?

The standard sample size is 25-50 µL. Samples typically run for a minute or two, sometimes longer if more data points are required. Efficient set up allows a measurement to be done in as little as 15 minutes, with analysis and reporting more or less instantaneous.

In a daily work flow, around 50 samples can be accurately measured and analysed. An important breakthrough in exosome research was the development of SEC columns, which Izon sells under the brand name of qEV.

The use of qEVs has dramatically reduced preparation time for exosome research and provides a very clean sample for subsequent TRPS measurement and biological analysis

What does the future hold for TRPS and IZON Science?

Izon believes that TRPS will become the default particle measurement and analysis system for life sciences and biomedical applications because it the only method that can provide the accuracy, certainty and completeness of information that those fields need.

Izon’s qEV system is also rapidly becoming the gold standard for exosome and extracellular vesicle separation. Future developments are aimed at continually improving ease of use eventually leading to automated systems suitable for clinical diagnostics applications and routine quality assurance of pharmaceutical and life sciences products.

Izon will partner with diagnostics companies to develop streamlined solutions rather than become a diagnostics company itself.

The growth in adoption for TRPS and qEV systems is currently very high and is expected to continue for the foreseeable future. That will be good news for researchers, developers, users and manufacturers of nano- and nanobio-particle systems.

Where can readers find more information?

Izon’s website is at www.izon.com and the Izon online store is at store.izon.com

About Hans van der Voorn

Hans van der Voorn is the CEO and a founder of Izon Science Ltd. He originally trained as an engineer and believes that a combination of engineering and science offers the best solutions. Izon was founded in 2005 and Hans has been the CEO since 2007.

His main interest is in ensuring that the biomedical world understands and benefits from Izon’s TRPS and related products. Prior to entering the science world Hans worked in civil engineering and energy developments. Some of that knowledge has been applied to the nano world resulting in several patents and inventions.

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