Particle Tracking on Exosomes ZetaView Multiparameter NTA sizing, counting and zeta potential C. Helmbrecht and H. Wachernig
Visual inspection of video 20130429_0009_269Exo9h_Dil10000 Agglomerates Approx. 70 nm (fast movement) > 100 nm (slow movement)
Particle size distributions Exosome A, B
Multiparameter analysis with the ZetaView Zeta potential Laser Counting Dispersed nanoparticles ZetaFocus Objective Video Camera Size distribution Cell channel cross section Automated particle tracking (APT): Autofocus Antivibration measures Multiposition (up to 11)
Operation of ZetaView Inject sample Visualize particles Collect data
Multiparameter NTA with ZetaView Parameter Principle Working range Exosomes Concentration The scattering of particles is detected. The number of particles corresponds to particle concentration. 0.5 10 5... 10 10 cm 3 10 6 10 10 cm 3 Particle size ASTM E283412 Nanoparticles move due to Brownian migration. The particle size distribution is computed by particle tracking. 20 nm 3 µm (Dependent on particle properties) 30 nm 200 nm Zeta potential ISO 130991,2,3 Particles bear surface charge and travel in an electrical field. The zeta potential, a measure for stability, is calculated from the particle velocity. 200mV 200mV 50 mv... 0 mv
Concentration Verification with size standards (NIST traceable) SiO 2, PS New algorithm increases accuracy even below 100 nm particle size!
Multiparameter NTA with ZetaView Parameter Principle Working range Exosomes Concentration The scattering of particles is detected. The number of particles corresponds to particle concentration. 0.5 10 5... 10 10 cm 3 10 6 10 10 cm 3 Particle size ASTM E283412 Nanoparticles move due to Brownian migration. The particle size distribution is derived by nano tracking analysis (NTA). 20 nm 3 µm (Dependent on particle properties) 30 nm 200 nm Zeta potential ISO 130991,2,3 Particles bear surface charge and travel in an electrical field. The zeta potential, a measure for stability, is calculated from the particle velocity. 200mV 200mV 50 mv... 0 mv
Particle size by brownian motion: Nanoparticle Tracking Analysis (NTA) Quantification of average mean square displacement per time interval = <, > 4 StokesEinstein equation = 6 AuNP 40 nm D = Diffusion coefficient <x,y²> = Mean square displacement k B = Boltzmann constant T = Temperature η = Viscosity r = Particle radius
Particle size by brownian motion: Nanoparticle Tracking Analysis (NTA) Quantification of average mean square displacement per time interval = <, > 4 StokesEinstein equation = 6 AuNP 40 nm D = Diffusion coefficient <x,y²> = Mean square displacement k B = Boltzmann constant T = Temperature η = Viscosity r = Particle radius
BMBO cell line derived exosomes Particle size distribution and kinetics with ZetaView Filtration (200 nm) Washing and enrichment by ultrazentrifugation Resuspension Sorensen buffer, ph 6.5
Multiparameter NTA with ZetaView Parameter Principle Working range Exosomes Concentration The scattering of particles is detected. The number of particles corresponds to particle concentration. 0.5 10 5... 10 10 cm 3 10 6 10 10 cm 3 Particle size ASTM E283412 Nanoparticles move due to Brownian migration. The particle size distribution is computed by particle tracking. 20 nm 3 µm (Dependent on particle properties) 30 nm 200 nm Zeta potential ISO 130991,2,3 Particles bear surface charge and travel in an electrical field. The zeta potential, a measure for stability, is calculated from the particle velocity. 200mV 200mV 50 mv... 0 mv
1807: Ferdinand F. Reuss noticed that the application of a constant electric field caused particles to migrate. Micro electrophoresis: a brief history 1997: CAD ZetaPhoremeter III Automated tracking 2006: ZetaView Multiparameter NTA Zeta potential Counting Sizing 1997: CAD ZetaPhoremeter IV
Principle of zeta potential determination Electrophoretic migration is captured for each single particle = Zeta potential ζ is obtained from the HelmholtzSmoluchowski equation = 4 ( ) PS 300 nm ε = Dielectric constant η = Viscosity of medium f(κa) = Debye function ζ = Zeta potential µ e =Εlectrophoretic mobility v = Velocity of particle in Efield E = Electrical field
Stability high salt concentration e.g. physiological NaCl low salt concentration e.g. deionized water Minimal distance electrical shielding Distance of aggregation (vdw force) corresponds to ζ removable ions
30 30 nm nm 180 nm Exosomes Sorensen buffer, ph7 1xBuffer BMBO cell derived exosomes Phosphate buffer ph7.0 Variation of conductivity by dilution with ultrapure water
Application of ZetaView Liposomes and micelles Drug delivery Proteine agglomeration Carbon nanotubes Beverage Virus samples Minerals & clay Emulsions Polymers Food and feedstuff Beverage emulsions Nanometals Nanobubbles Humic acids
Gold nanoparticles, 60 nm Due to compression of the diffusive layer agglomeration for ζ > 20 mv
Benefits o Multiparameter NTA provides zeta potential, concentration and sizing in one measurement (i.e. on the same sample) o Full control by seeing is believing principle for artefactfree results o In situ method for fragile samples o Autoalignment and autofocus after cell change o Multiple positions (up to 11) for statistic validity
Benefits o Multiparameter NTA provides zeta potential, concentration and sizing in one measurement (i.e. on the same sample) Relevant for bioparticles: o Full control by seeing is believing principle for Easy artefactfree and quick results measurements insitu (in comparison with electron microscopy) o In situ method for fragile samples Suited for low concentrated and polydisperse samples o Autoalignment (problematicand for DLS) autofocus after cell change o Multiple positions (up to 11) for statistic validity
Thank you very much for your attention Acknowledgement PD Dr. rer. nat. Bernd Giebel Dr. med. Payam Akhyari
ZP a closer look potential ζ potential difference
Deduction of stability from zeta potential 10 0 10 20 fast medium agglomeration agglomeration kinetics 30 no agglomeration 40 50 60 good excellent stability 70 adapted from Riddick 1968
Example 1 Saccharomyces cerevisiae
Particle Particle Interaction Attractive: vdw forces, (un)specific binding Repulsive: Electrostatic forces Zeta potential is a measure for stability Agglomeration/ aggregation Isolated particles
Electrophoretic migration is captured for each single particle Principle of zeta potential determination = Tracking algorithm measures velocity v parallel to Efield and calculates electrophoretic mobility µ e Zeta potential ζ is obtained from Helmholtz Smoluchowski equation = = 4 ( ) ζ = 12.9 µ e ε = Dielectric constant η = Viscosity of medium f(κa) = Debye function ζ = Zeta potential µ e =Εlectrophoretic mobility v = Velocity of particle in Efield E = Electrical field
Determination of zeta potential by electrophoresis Charged particles migrate in an electrical field Zeta potential is calculated from the particle velocity. Particle charge negative > anionic particles positive > cationic particles