Assay File Examples

The Assay File will vary depending on the type of assay performed and protocol and technology type leveraged.  Provided below are examples of Assay Files for common types of assays identified across nanotechnology resources.

Physico-Chemical Characterization Assays

Size Assay

Size by Dynamic Light Scattering (DLS) Assay

Dynamic Light Scattering (DLS) is a type of spectroscopy that uses a laser beam to irradiate a sample containing particles in suspension resulting in light scattering. Rapid fluctuations in scattering intensity around a mean value at a certain angle occur because of particle diffusion and are dependent upon on particle size. The calculated correlation function yields a diffusion coefficient, for a given temperature and viscosity, that can be used to calculate particle size.

Zeta Potential Assay

Particle surface characteristics and charge play an important role in the particle’s physical state, stability in different media, agglomeration tendencies, and interaction with biological systems. Zeta potential measurement provides an indirect measure of the net charge and as a tool to test batch-to-batch consistency.

In Vitro Characterization Assays

Cytotoxicity Assay

Hepatocarcinoma Cytotoxicity MTT Assay

The MTT assay is a colorimetric assay that can assess the viability of cells by quantitation of the reduction of the yellow substrate MTT (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to a product that has a purple color. This assay can measure the cytotoxicity of a chemical or drug by determining the affect of treatment on cell viability. The Assay File represents an example for a MTT cytoxicity assay (MTT) performed on three nanoparticle samples, where 9 dilutions of each nanoparticle sample are exposed to porcine proximal tubule cells for three different times of exposure (6h, 2h, and 48 h).

Blood Contact Assays

Hemolysis Assay

Hemolysis refers to the breaking open of red blood cells causing release of hemoglobin into the plasma.  The Hemolysis assay measures the amount of hemoglobin in whole blood (TBH-Total Blood Hemoglobin) and the amount of hemoglobin released into plasma (PFH-Plasma Free Hemoglobin) when blood is exposed to nanoparticles.  Hemoglobin is oxidized to methemoglobin and when reacting with cyanide results in Cyanmethemoblobin (CMH). The hemolysis assay is used to evaluate the acute in vitro hemolytic properties of nanoparticles.

In Vivo Characterization Assays

Disposition and Pharmacokinetics Assay

The Disposition and Pharmacokinetics assay is performed to detect the nanoparticles (or their components) in tissues taken at various time points. A variety of methods can be used to detect and quantify nanoparticles in tissue.