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J Vis Exp. 2014 Aug 7;(90):e51789. doi: 10.3791/51789.

Hydrogel nanoparticle harvesting of plasma or urine for detecting low abundance proteins.

Author information

1
Center for Applied Proteomics and Molecular Medicine, George Mason University.
2
Ceres Nanosciences.
3
Center for Applied Proteomics and Molecular Medicine, George Mason University; vespina@gmu.edu.

Abstract

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

PMID:
25145492
PMCID:
PMC5783330
DOI:
10.3791/51789
[Indexed for MEDLINE]
Free PMC Article

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