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.

Fig. (1)

Summary of bait chemistry.

Fig. (2)

Schematic representation of particle structure and function. A. Particles are constituted by a bait containing core, surrounded by a sieving shell. Candidate biomarkers can be associated with high abundance carriers such as albumin. B. When introduced into a complex solution, such as serum, core-shell particles affinity capture all molecular species below the molecular weight cut off of the shell, strip the target biomarkers away from the excluded carrier protein by affinity competition, and preserve the captured molecules from degradation.

Fig (3)

Affinity capture and exclusion of albumin. SDS PAGE analysis of particles incubated with the following chemokines, mucosae-associated epithelial chemokine (MEC/CCL28), stromal cell-derived factor-1 beta, (SDF-1β/CXCL12b), and eotaxin-2 (CCL24), in the presence of bovine serum albumin (BSA) to which the chemokines asssociate. Solutions of the chemokines and BSA are shown in lanes 1, 4, and 7. After incubation with the particles, no chemokine was left in the supernatant (S, lane 2, 5, and 8) and all the chemokine solution phase molecules were captured by the particles (P, lanes 3, 6, and 9), while albumin was totally excluded by particles.

Fig (4)

SDS PAGE analysis of particles incubated with the following chemokines, mucosae associated epithelial chemokine (MEC/CCL28), stromal cell-derived factor-1 beta, (SDF-1β/CXCL12b), and eotaxin-2 (CCL24), in presence of high concentrations of trypsin. The starting solution of the chemokines (control) are shown in lanes 1, 4, and 7. Chemokines incubated with particles (Lane 3, 6, and 9) are protected from tryptic degradation whereas chemokines not incubated with particles (Lane 2, 5, and 8) are susceptible to proteolytic digestion.

Fig. (5)

Schematic representation of the concentration effect of nanoparticles. When incubated with a given volume of blood in which the majority of proteins are high molecular weight, high abundance proteins (depicted by the letter A), the particles capture all the solution phase biomarkers (depicted by the letter B) within a few minutes. The particles are then separated from the bulk solution and the captured proteins are eluted and collected in a small final volume. The ratio between the initial incubation and the final volume provides the concentration factor.

Fig. (6)

A. Effective amplification of native platelet derived growth factor (PDGF) in serum. Hydrogel NIPAm/AAc particles incubated with human serum. ELISA readings on the serum, the supernatant and the particle eluate. B. Effective amplification of human growth hormone (hGH) in urine. Dose response study using recombinant hGH spiked in 10 mL of synthetic urine. Supernatants are depleted and hGH concentration in the particle eluate is linearly dependent on hGH concentration in Surine. UD means undetectable (below the detection limits of the Immulite assay).

Fig. (7)

Examples of atomic force microscopy images of NIPAm/AAc and NIPAm/CB particles. The average diameter is 0.80 microns. The core shell nanoparticles we have used to capture and preserve biomarkers have a size of approximately 0.8 microns and have been successfully studied and characterized by traditional flow cytometry.

Fig. (8)

The envisioned application of particles directly in the Vacutainer device for blood collection.