Izon Science

Tunable Resistive Pulse Sensing

Tunable Resistive Pulse Sensing (TRPS) allows high-throughput single particle measurements as colloids and/or biomolecular analytes are driven through pores, one at a time. Particles crossing the nanopore are detected as a transient change in the ionic current flow, which is denoted as a blockade event with its amplitude denoted as the blockade magnitude.^[6][7] As blockade magnitude is proportional to particle size, accurate particle sizing can be achieved after calibration with a known standard. Analysis of individual particle translocation through the pore allows determination of particle electrophoretic mobility and calculation of individual particle charge, simultaneous with size, a unique feature of TRPS.^[8]

Nanopore-based Measurement

Fixed geometry micro-scale apertures for particle detection have been used in a wide range of industrial applications since the invention by W.H. Coulter in the 1950s.^[9] Quantitative resistive pulse sensing of nanoparticles using Coulter-type counters has been shown to hold promise as a fast and accurate alternative to established sizing methods.^[10][11] Fixed pore size enables sensitive size measurements, but also limits analysis size range, which has implications for the measurable sample polydispersity.

Izon’s instrumentation overcomes this limitation by using size-tunable pores that allow for the optimisation of the resistance pulse magnitude relative to the background current by matching the pore-size closely to the particle-size. As detection occurs on a particle by particle basis, the true average and polydispersity distribution can be determined, without the averaging effects inherent in other size analysis technologies, such as dynamic light scattering.^[12]

qNano Gold

The qNano Gold is a bench-top instrument for the practical implementation of TRPS for fluid-borne analysis of nanometre-scale particles.

The qNano Gold is a bench-top instrument for the practical implementation of TRPS for fluid-borne analysis of nano- and micro -scale particles. This instrument incorporates a sensitive manual control system for Izon’s dynamically adjustable nanopores, enabling tunable resistive pulse sensing over a wide particle size range (typically 50 nm – 20μm).^[15] Data acquisition software allows raw or filtered data to be viewed in real-time. TRPS using the qNano Gold enables particle-by-particle determination of the size, charge and concentration of a wide range of particle types.

Applications include:

• Particle-based drug delivery systems (e.g. liposomes, nano- and micro- bubbles, polymers, "Smart" drug delivery particles, etc)
• Analysis of extracellular vesicles (e.g. exosomes & microvesicles)
• Virus, VLP and bacterial analysis
• Biomedical diagnostics research (e.g. dye-doped nanoparticles, functionalized nanoparticles for immunodetection)
• Industrial research applications (e.g. paint pigments, food & beverage, magnetic & metallic particles, etc.)

The qNano Gold is sold as a full system ready for use including the base instrument, variable pressure module (VPM), fluid cell and a starter kit of nanopores, buffer solution and standard particle sets.

qViro-X

The qViro-X was launched in March 2012, as Izon's next generation virus analysis system, specifically designed for use in quality control environments such as viral vaccine manufacturing. Measurement involves pipetting 40 μL of sample into the upper fluid cell of the qViro-X. Detection occurs in real-time, allowing measurement of thousands of viral particles, with total analysis time per sample of 10 – 15 minutes.

Accurate viral particle size distribution output, determined on a particle-by-particle basis is useful for assessment of viral aggregation and stability during vaccine production. The number of viral aggregates can be reliably measured and displayed in relation to the total number of pure virus particles in the sample. As the qViro-X is capable of rapid counting the total number of virus particles present in a sample (vp / mL), when used in conjunction with infectivity assays this allows users to determine the relative concentrations of infective virus particles vs. total viral particles present.

Disposable elements of the instrument ensure that issues of contamination are handled easily. The stainless steel body of the instrument allows wash-down with harsh chemicals to meet stringent decontamination requirements. 21 CFR Part 11 compliant software provides a full data audit trail that meets FDA requirements.

Applications include:

• Vaccinology & Vaccine Production
• Virus Quantitation & Stability Assessment
• Gene Therapy Research - Quantitation and Analysis of Viral Vectors
• Microbiology & Virology Research (e.g. Virus-Cell Interactions)

qEV

Launched in 2015, Izon Science’s qEV Size Exclusion Columns enable the rapid isolation of extracellular vesicles (EVs) from cell culture supernatants and complex biological fluids. Each column removes background proteins, lipids, solutes, cell debris, and other particulates to improve the sensitivity and accuracy of downstream assays (e.g., TRPS, protein profiling, RNA profiling) while maintaining the biological properties of EVs.

Since the separation is based on size, vesicles flow through the column unretained and elute in the void volume. Proteins and other contaminants that are smaller than the pores of the stationary phase are retained by the column and elute later. Other isolation methods that are non-specific in nature require overnight incubation of vesicles with precipitation buffer. As a result, vesicular and non-vesicular particles are isolated together so additional steps are needed to separate EVs from contaminating particles. In contrast, isolations using qEV Columns take 15 minutes and removes 99% of contaminating background proteins and up to 95% of high-density lipoprotein (HDL) contaminates from samples.^[16]

Particle Sizing

The qNano Gold and qViro-X allow single particle measurement, and size distribution analysis of particle populations.

A discrete electrical signal is registered by the system each time a particle passes through the nanopore. Each individual particle signal is analysed and the make-up of the constituent populations is built up for the sample.

The magnitude of the electrical signal shows the volume of the particle (i.e. particle diameter). This allows rapid quantification of:

• Absolute size
• Size distribution and volume fraction analysis
• Resolving distinct populations within a sample mixture
• Assessment of polydispersity
• Aggregation and/or fragmentation levels
• Number and concentration of differently sized particles

Concentration

Evaluating the concentration of samples containing nano-sized particles is a key parameter for researchers in many fields. qNano and qViro can determine the concentration of a wide range of particle types including mono and polydispersed populations. It provides rapid and accurate determination of:

• Bacteria count
• Virus count
• Amount of drug
• Drug delivery dosage
• Amount of viral aggregates
• Amount of bacteria and cellular fragments

Reaction Dynamics

Reaction dynamics include – identification and analysis of particle-particle, particle-biomolecule, functionalization and aggregation interactions.

The beginning, middle and end of interactions can be effectively mapped by analysing subtle changes in particle size, surface charge and concentration simultaneously and on a particle-by-particle basis, allowing interaction dynamics and yields to be determined.

This can be applied to many different situations:

• Confirmation of binding interactions and functionalization
• Comparison of sample properties prior to and post interaction
• Small molecule detection and diagnostics – binding reactions between small molecules and the surface of particles allows detection of molecules that may otherwise be below the detection limit of the instrument
• Comparison of properties after exposure to different experimental conditions, such as pH, ionic strength, amount of added reagents and temperature
• Real time monitoring of binding interaction events, particle-by-particle, by combining reactants in the top fluid cell

The latest version of Izon’s Control Suite Software v2.1, allows improved analysis of reactions as they occur over time.

Nanopores

Izon's thermoplastic polyurethane nanopores are consumable items which fit to the qNano Gold and qViro-X instruments.

The nanopores are manufactured in a thermoplastic polyurethane cruciform using a proprietary technique. When fitted to the qNano Gold or qViro-X instruments, the cruciform can be stretched evenly along the x and y axes to affect nanometre scale adjustments to the nanopore.

Fluid Cells

Each fluid cell is divided into two sections, the top and bottom sections of the fluid cell being connected together after the nanopore cruciform has been positioned between the two halves. The minimum volume of sample fluid required is 40μL.

Direct access points to the fluid in the upper and lower fluid cell compartments allows for easy and safe handling of samples.

An electrical connection is made to the bottom of the fluid cell via a shielded SMA connector. Silver chloride electrodes are fitted into the fluid cell. The fluid cell is designed with shielding to minimize electrical noise.

Variable Pressure Module

The variable pressure module (VPM) provides pressure and vacuum control of sample flow, complementing the electrophoretic operation of the qNano and qViro instruments. The application of pressure as a dominant force allows charged and uncharged particles to be detected by the system, as electrophoresis is no longer required for driving sample flow.

VPM also allows accurate concentration determination.

The additional sample flow generated by the VPM adds two orders of magnitude to the lower detection limit of the qNano and qViro instruments. Sample concentrations down to around 10^5 particles per mL may be measured.

By finely controlling and balancing electrophoretic and pressure forces exerted on the particle, detailed mobility and charge information can be extracted in a wide range of electrolyte environments.

The VPM was initially sold as an add-on module but now comes standard with qNano or qViro instruments.