Nanoparticle Technology: Addressing the Fundamental Roadblocks to Protein Biomarker Discovery

Abstract

Clinically relevant biomarkers exist in blood and body fluids in extremely low concentrations, are masked by high abundance high molecular weight proteins, and often undergo degradation during collection and transport due to endogenous and exogenous proteinases. Nanoparticles composed of a N-isopropylacrylamide hydrogel core shell functionalized with internal affinity baits are a new technology that can address all of these critical analytical challenges for disease biomarker discovery and measurement. Core-shell, bait containing, nanoparticles can perform four functions in one step, in solution, in complex biologic fluids (e.g. blood or urine): a) molecular size sieving, b) complete exclusion of high abundance unwanted proteins, c) target analyte affinity sequestration, and d) complete protection of captured analytes from degradation. Targeted classes of protein analytes sequestered by the particles can be concentrated in small volumes to effectively amplify (up to 100 fold or greater depending on the starting sample volume) the sensitivity of mass spectrometry, western blotting, and immunoassays. The materials utilized for the manufacture of the particles are economical, stable overtime, and remain fully soluble in body fluids to achieve virtually 100 percent capture of all solution phase target proteins within a few minutes.

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

“…Hydrogel nanoparticles can be applied to various biological fluids, including whole blood, plasma, serum, cerebrospinal fluid, sweat, and urine. In one step, in solution, the nanoparticles perform a rapid (within minutes) sequestration and concentration of low molecular weight analytes 10,11,13,[15][16][17][18] . Proteins are subsequently eluted from the nanoparticles and detected using western blotting [19][20][21] , mass spectrometry 10,11,13,15,18,22,23 , immunoassays/ELISA 10,11,15,18 , or reverse phase protein microarray 16,24 assays.…”

“…The incorporation of the dye bait drives the uptake of molecules in solution and assures that captured molecules are preserved from degradation 13,15,18,25 . For this reason, protease inhibitors are not needed during sample pre-processing.…”

“…Despite the promise of serum proteomics, there are three fundamental and serious physiologic barriers thwarting biomarker discovery and translation to clinical benefit 10,11,16,25 .…”

Accessing to the minor proteome of red blood cells through the influence of the nanoparticle surface properties on the corona composition

“…23,24 Thus, the low-abundant protein compartment where diseaseassociated biomarkers are thought to reside is still unattainable. Various nanomaterial-associated strategies [25][26][27][28] have been integrated in MS in order to detect disease-associated biomarkers, and have been found extremely useful to improve protein enrichment and sample preparation, particularly NPassociated technology. [29][30][31] Finding biomarker for disease detection, stratification, as well as therapeutic monitoring is a biomedical priority.…”

Drug delivery to equine digital lamellar tissue

“…Novel diagnostic assays have been successfully formed by linking functionalised nanoparticles to biological molecules such as antibodies, peptides, proteins and nucleic acids (Driskell et al, 2005;Luchini et al, 2010). Coupled with spectroscopy, flow cytometry and histological methods these provide a powerful novel platform for mapping the molecular profiles associated with disease and infection and a highly sensitive, amplification-free method of pathogen detection .…”