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Fluorescence and electrophoresis NTA at

Biological nanoparticles, such as extracellular vesicles (EVs), exosomes, lipids, viruses or proteins can be found in almost all fluids of living tissue. The Particle Metrix ZetaView® NTA technology is capable to characterize them reliably in water or physiological buffers. You can simultaneously get information about particle size, particle concentration and surface charge with just one single measurement of a particle sample. Similarly, cluster analysis based on similar properties is possible.

The concentration of the particles is determined by the number of particles detected by the camera in a calibrated sample volume that is illuminated by the laser of the ZetaView® instrument. It is always apparent and will be displayed as "live read-out" as soon as a particle sample is introduced into the instrument, even if a measurement has not yet started. The color coding of the concentration display in the ZetaView® instrument shows whether the particles in the sample are in the optimum measuring concentration. Thus, a concentration adjustment of the sample can take place before the measurement.

Pattern parameters such as intensity, surface geometry or shape of the particles as well as their temporal fluctuations are also recorded at each exposure. These parameters can be used to distinguish subpopulations, documented in the fcs format.

Due to Brownian motion, small particles in liquid move much faster than large particles. Each individual particle in the field of view of the camera is detected and tracked in its movement in a two-dimensional direction. The measured change in location within a certain time interval t gives a specific diffusion coefficient D for each individual particle. Using the Stokes-Einstein relation, the ZetaView® calculates therefrom the hydrodynamic particle radius r and thus the diameter of each particle. The results are summarized and presented in a particle size distribution.

What can affect the surface charge of BNPs and how strong? It is known that proteins have negative or positively charged end groups depending on the pH. These proteins are partly responsible for the fact that EVs usually have a certain negative charge. This is shown as zeta potential in the range between -30 and -36 mV. Any change in surface charge, such as changes in protein levels, flip-flopping of phosphatidylserine between the inner and outer membrane, or attachment of oppositely charged antibodies, will alter the zeta potential. There are therefore interesting possibilities to use the zeta potential as a monitor for influences on the particle surface. The proof is given by an electrophoresis experiment in which charged particles move according to the influence of an electric field E v. The measured electrophoretic mobility μe = v / E of each individual particle in the electric field represents the basis for the determination of the zeta potential.

Biological nanoparticles, such as extracellular vesicles, can be conjugated to fluorescently labeled antibodies and then analyzed for size, concentration, and, if required, for zeta potential and other pattern parameters in ZetaView® fluorescence mode. The particles to be analyzed should be sufficiently well purified and the conjugated sample must be freed from macromolecular substances and remaining free fluorescence molecules as well as small protein particles and lipids. Both, membrane dyes and primary and secondary fluorescently labeled antibodies can be used. By using the TWIN-laser system even phenotyping experiments can be performed on extracellular vesicles with a double-stained sample. For an experiment like this, two different fluorescent dyes conjugated to different antibodies are used to bind specifically to tetraspanins at the membrane surface (e.g., CD63-Alexa405 & CD81-Alexa488).

Since the BNPs to be analyzed in the ZetaView® instrument are located longer in the laser beam (0.5-1 sec.) in contrast to flow cytometry, the selection of photostable fluorescent dyes is essential for the quantitative evaluation. In the following figure, some of the dyes are listed by their photostability.