Each nanoparticle formulation is unique, and each instrument or technique has it's optimal range and certain limitations. We chose particular technique for each nanoparticle sample taking into account what is the nature of that sample and what information that method can give us, and perform rigorous method validation and optional cross-validation using other techniques.
Atomic Force Microscopy is a powerful technique for resolving size and structure of smallest particles. Bruker Dimension Icon AFM instrument is used to provide the service and Laurell Spin Coater is used for sample preparation.
Thermo Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometery for identification and quantitation of proteins
Thermo Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometery for identification and quantitation of lipids
Biomarker profiling is an essential step in EV sample characterization. Traditional western can be challenging due to small sample volumes or low protein content.
Our Simple Western Service Features:
Small sample volume required (5 µl can be enough to test for 8 different antibodies)
Sample protein concentration as low as 0.2 mg/mL
Size-based and charge-based assays
EV proteins characterized must be those that are expected to be found in EVs, such as membrane proteins (CD63, CD9, CD81, integrins) and cytosolic proteins with membrane binding capacity (Annexins, Rabs, etc), as well as those, that belong to other cellular compartments and are not expected to be found in EV (GM130 from Golgi, Calnexinv from ER, cytochrome c from mitochondria).
Our high throughput system allows us to test for 8 different antibodies, achieve np tp pg sensitivity using as little as 5 µl of the sample, and get up to 96 data points in a single run. In addition to size-based separation, charge assays have the power to resolve phosphorylation isotypes and other post-translational modifications.
The results are presented as an electropherogram format with each peak displayed numerically. Electropherogram peaks are then digitally rendered as a “virtual blot".
Cryo-electron microscopy (Cryo-TEM or Cryo-EM) is the next evolution step of Electron Microscopy:
unaltered by drying in vacuum imaging, unlike conventional TEM
fast specimen freeze coverts water into transparent ice crystals
ice crystal transforms biomolecules in a rigid state allowing high-resolution imaging
sub 10 Å resolution (1 nm) allows imaging near-atomic structures
quick-fire imaging allows using a high power beam of 200 kV.
*Nanoparticle Tracking Analysis*
via Nanosight (Malvern) or Zetaview (Particle-Metrix) provides accurate nanoparticles size, size distribution and concentration measurement in the range 30-100nm. This technique is most widely used in exosome research. Unlike DLS (dynamic light scattering) Nanosight is capable of resolving sub-populations of particles with close size and provide particle by particle analysis. Turn around for orders under 100 samples - 3-5 business day.
While NTA technique is most commonly used to profile EVs and nanoparticles size, size distribution and concentration, the fNTA enables a whole new filed of research applications:
- fluorescent membrane labelling allows to asses EVs samples purity and sort out membranous vesicles out of total number of particles in solution
- biomarkers profiling via fluorescently labelled antibodies is a powerful tool to measure up to 4 different biomarker expression on the same sample, which saves you time and precious material
- RNA/DNA labelling for a perspective class of novel nano-formulations inspired by Moderna’s mRNA vaccine
- drug loading efficiency to validate number of cargo carrying particles
- drug release kinetics of the fluorescently labelled drug out of nanoparticles
The fNTA technique combines abilities of NTA, flow cytometry and fluorescent microscopy with advantage of nano-scale real-time analysis.
Analysis require a stock of your particles (pellet, lyocake, frozen solution) of at least 5E7 concentration. Upon your request, particles can be concentrated in the lab.
Laser diffraction analysis of size and size distribution of the particles in solution in the range 100nm-900um.
Potentiometric titration for determination of water content in solutions and dry powders.
Zeta-sizer Nano ZS (Malvern) or Nano Flex II (Particle-Metrix) DLS instruments are used to evaluate particles size and size distribution.
DLS is a classic way to report your particles size and is accepted format for your NDA FDA application. We recommend cross-validation by other technique, such as laser diffraction or TEM analysis.
Particles concentration of 1E10 is recommended with minimum volume of 1 mL for Zetasizer and 30 uL for Nano Flex II.
Transition Electron Microscopy (TEM) allows imaging of EVs and assessment of size and size distribution. Alpha Nano Tech provides statistically relevant data based on the imaging and analysis of thousands of particles per sample.
Immunogold TEM is a direct and powerful tool for the phenotypic characterization of EVs in addition to traditional TEM and protein analysis.
TEM is the best method to cross-validate particle size and size distribution data acquired from other methods, such as Nanoparticle Tracking Analysis (NTA), dynamic light scattering (DLS), or tunable resistive pulse sensing (TRPS).
Cryo-electron microscopy is another powerful tool to study nanoparticles, including exosomes. The samples are frozen in liquid nitrogen and thus the shape and microstructure are preserved better compared to the traditional TEM method.
Surface charge or Zeta-potential is one of a most important property of the nanoparticles, which defines their interaction with other molecules (such as e.g. albumin) stability, biodistribution, cell permeability and PK.
NTA-based (Zetaview, Particle-Metrix), DLS-based (Zetasizer, Malvern) and streaming potential-based (Stabino II, Particle-Metrix) are offered for analysis of Zeta-potential.
Method development, qualification and validation. Method development to include LOD, LOQ, Linearity, Accuracy, Precision.
From 1 nm to 1000 um: we use laser diffraction (Mastersizer by Malvern) to analyze particles, solutions of microparticles and DLS (Zetasizer Nano ZS by Malvern), NTA (Nanosight by Malvern), TRPS (qNano by Izon) for analysis of nanoparticles. Electron microscope by TEM/SEM is offered to confirm achieved data.
Exosomes and extracellular vesicles (EV) play important roles in myriad of biological processes and are widely studied. Biological matrix, isolation and purification steps, heterogeneity of particles’ size make characterization of exosomes considerably complex for most techniques available on the market. Over the past few years Nanoparticle Tracking Analysis (NTA) and Tunable Resistive Pulse Sensing (TRPS) technology by Izon were found to be the most suitable and reliable techniques for analysis of variety of nanoparticles, and found to be exceptionally suitable for exosomes characterization.
NTA sinultaneously identifies and tracks each individual particle moving under Brownian motion and relates movement trajectory to particle size. It results in exceptional capability to resolve and count particles of different size in the mixture.
TRPS is an impedance-based system and is the only technique that can deliver three fundamental parameters of nanoparticles:
A voltage is applied across a pore that is filled with electrolyte, resulting in an ionic current. As particles cross the pore they briefly increase electrical resistance, creating a resistive pulse, which is precisely proportional to particle volume. TRPS represents a rapid, convenient, accurate and reproducible method for nanoparticle analysis.
Both Nanosight and qNano are excellent complimentary tools to study and characterize exosomes. AlphaNanoTech operates with Nanosight NS200-HS (Malvern) and qNano (IZON). Cross-validation by Dynamic Light Scattering (DLS) and SEM/TEM imaging is available upon request.
AlphaNanoTech provides top level nanoparticle characterization services with following optional cross-validation by Tunable Resistive Pulse Sensing (TRPS) qNano technology by Izon, Nanoparticle tracking analysis by Malvern Nanosight, and SEM/TEM imaging.
Exosomes and Extracellular vesicles
Exosomes and microvesicles play important role in transmission of biological signals between cells. Pathophysiological role of exosomes is beginning to be realized in cancer, neurodegenerative diseases, infectious diseases and other conditions. They are also attracting significant interest as drug delivery and transfection vehicles.
Nanoparticles as drug delivery systems gained a lot of attention in past few decades. There is number of FDA approved nanomedicines on the market for treatment of cancer, infectious diseases, cardiac/vascular disorders, viral infections, and others. Nanoparticles are also used in vaccines, cosmetics, for imaging nad theranostics.
Aggregation state of viral particles or adjuvant, as well as their count is crucial to understand and optimize vaccine's biological activity and evaluate long term stability.
Jeol scanning and transmission electron microscopy to study your smallest particles and biological structures
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