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The differentiating Power (Selectivity) of Particle Metrix Multi-Parameter NTA

Message carrying Nanoparticles like EVs and VLPs analyzed in Scattering and Fluorescence Mode

Sascha Raschke, Clemens Helmbrecht, Hanno Wachernig

For whom?

Extracellular vesicles (EVs) and virus like particles (VLPs) transport protein information in body liquids. These nanoparticles have numerous functions in physiology and pathology. Their diagnostic and therapeutic potential is of particular interest for biomedical research. Concentration and size distribution are key parameters to be monitored. For further differentiation the amount of surface charge expressed as zeta potential can be measured. Finally, the identification of their function is made using fluorescence (F-NTA).

 

Size is determined via Brownian motion by quantification of the average mean square displacement of the particles per unit timel. The scattered laser light is detected by a camera combined with a 10x microscope objective

Measure what you see

With the ZetaView® instrument, Brownian motion and classical micro-electrophoresis are presented in a modern laser scattering and laser induced fluorescence video microscope. Brownian diffusion and electrophoretic motion of individual nanoparticles are tracked either by the scattering of laser light (NTA, Z-NTA) or by tracking their fluorescent light (F-NTA) (see Figure: Brownian motion and USPs). This technique is suitable for analyzing the concentration, size and zeta potential in minutes. An outstanding feature identifies sub-populations by following characteristic parameters during the video capture of data. This is using the full potential of “measure what you see”.

 

The optical layout of the ZetaView® laser scattering video microscope with “ZetaFocus” allows the laser and objective to move independently

The method

Size and Zeta potential: The image of the laser-illuminated particles in the measurement cell is focused onto the video camera via the microscope objective. From the particle velocity under an applied electric field the electrophoretic mobility zeta potential is determined. With no field applied, Brownian motion is evaluated for particle size. By scanning the optics and/or pumping sub-volumes, robust averaging of sub-populations of particles is achieved. (see Figure: Optical layout).

Concentration: It is calculated as the number of detected particles divided by the focus depth. It is constantly displayed during the video analysis.

Fluorescence: The same measurands are obtained when an optical filter is placed in front of the video camera.

Size and concentration range: Minimum and maximum size range are dependent on sample and analysis type. It starts at 15 nm for gold and ends at 50 µm for non-sedimenting particles for zeta potential analysis. The Brownian motion size analysis is limited to maximum 1 µm size. The concentration ranges from 105 to 109 particles/mL.

 

For cleaning purposes, the cell cassette and the black measurement cell are easy to remove from the ZetaView® instrument. Cleaning of the cell is done in less than a minute

The advantage of using ZetaView®

Automation: The “Auto-Alignment” procedure keeps the system in focus for days, even after cell removal. By scanning multiple sub-volumes and acquiring many individual particles statistically robust results for concentration, size and zeta potential are obtained. The same holds for the fluorescent mode.

Salty samples: The conductivity of buffers like PBS is 13 mS/cm. The ZetaView® instrument is designed for making zeta potential analyses under such high salt concentrations. The conductivity is monitored with every measurement.

Simple cell handling: The cell channel is integrated in a „slide-in“ cassette  (see Figure Cell cassette). Cell cleaning takes a matter of a few minutes as required.

 

The „Number of particles versus Sensitivity”, or “NvS“ function, is a convenient tool giving information on the correct dilution

Fast and safe results: After finding the correct dilution with the „Number vs Sensitivity“-routine (see Figure NvS), the measurement mode – Scattering – Fluorescence – Size – Zeta Potential - is selected. Typically, eleven sub-volume scans are recorded with videos of a few seconds each. A statistical evaluation selects the positions with good image quality and averages their results. This has two advantages: A statistically viable result and a stress-free sample fill. Typically, 30 to 60 analyses per day are possible. Accuracy and repeatability are better than 5 % for standard samples at 100 nm and better than 5 mV for zeta potential.

Applications: A broad spectrum of nanoparticles can be measured. These may include extracellular vesicles, virus (like) particles, protein aggregates, liposomes, as well as inorganic particles like polystyrene beads, nano bubbles, Q-dots and gold particles.

 

Size distribution of EVs separated by a differential centrifugation followed by isolation via an isolation kit. 2 products were compared

Example: The preparation and purification of extracellular vesicles of high biological quality grade still requires a comprehensive multiple step procedure of separation and chemical treatment. Typically, differential centrifugation is followed by ultra-centrifugation or a separation step in an isolation kit. With the ZetaView® instrument the yield of various purification techniques can be qualified with regard to size distribution and concentration. (see Figure EVs isolated from fresh blood serum) In this example EVs were isolated from fresh blood serum by a 4-step differential centrifugation process followed by isolation with techniques defined by the two suppliers. In addition, the result may strongly depend on the manipulation of the sample. Differences in the isolation procedure may occur as well.

Facts, background information, dossiers
  • nanoparticles
  • liposomes
  • biomedical diagnosis
More about Particle Metrix
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