Developing Diagnostic Assays for Detection of Proteins, Pathogens and Nucleic Acids

The proliferation of nanoparticle-based bioanalytical sensors in recent years has driven the need for improved particle characterization techniques. Precise information on the properties of these particles, including validation of their specific functionality, undoubtedly speeds up assay development.

Balakrishnan Raja is a PhD student working in Professor Richard Willson’s research group in the Department of Chemical and Biomolecular Engineering, University of Houston. The group is focused on the development of several particle-based diagnostic assays for the sensitive detection of proteins, pathogens and nucleic acids using novel techniques for labeling and signal readout.

qNano has been used to characterize the size distribution and bio-functionality of a variety of nano- and microparticle labels, including magnetic microparticles, and unique non-spherical particles, such as modified M13 bacteriophage, silica-coated strontium aluminate phosphorescent nanoparticles and micro-retroreflector cubes.  Commerically available and in-house synthesized nano- and microparticles, ranging from 40 nm to 5 µm, are used as biosensing labels and reporters in novel diagnostic assays for proteins, viruses and bacteria.

An accurate size distribution of these nanoparticles, along with the validation of surface modification with antibodies, is essential in order to optimize synthesis and evaluate their viability for a particular assay format.

“The qNano measures particle concentration and provides more accurate size distributions than DLS, while surface charge distributions before and after surface modification validate antibody modification”

The analyte-induced aggregation of nano-phosphors, measured using the qNano, is being investigated as a possibly assay format based on the hypothesis that that the large polydispersity of the phosphors and their non-spherical shape may help form analyte-induced aggregates more efficiently than spherical nanoparticles of comparable sizes would. Initial results are promising, with the detection of biotinylated BSA as a model analyte down to low picomolar levels.

In the case of immuno-phage nanoparticles (M13 bacteriophage modified with analyte-specific antibodies and covalently tagged with HRP molecules), particles which are too small (~30 nm) to be directly counted using the qNano, can be detected by indirect protocols that only measure size changes induced by active immuno-phage. This allows evaluation of label stability and serves as a quality control measure.

“When compared to ensemble measurements using techniques such as DLS, the single-particle resolution of the qNano’s TRPS platform that simultaneously measures particle size, surface charge and concentration makes it easier to characterize our particulate labels and help enhance assay performance.”

Further reading:

Sapsford, K. E.; Tyner, K. M.; Dair, B. J.; Deschamps, J. R.; Medintz, I. L. Analytical chemistry 2011, 83, 4453-88.

Sherlock, T.; Nasrullah, A.; Litvinov, J.; Cacao, E.; Knoop, J.; Kemper, S.; Kourentzi, K.; Kar, A.; Ruchhoeft, P.; Willson, R. Journal of Vacuum Science & Technology B: Microelectronics and Nanometer Structures 2011, 29, 06FA01.