Multifunctional Core–Shell Nanoparticles: Discovery of Previously Invisible Biomarkers

Abstract

Many low-abundance biomarkers for early detection of cancer and other diseases are invisible to mass spectrometry because they exist in body fluids in very low concentrations, are masked by high-abundance proteins such as albumin and immunoglobulins, and are very labile. To overcome these barriers, we created porous, buoyant, core–shell hydrogel nanoparticles containing novel high affinity reactive chemical baits for protein and peptide harvesting, concentration, and preservation in body fluids. Poly(N-isopropylacrylamide-co-acrylic acid) nanoparticles were functionalized with amino-containing dyes via zero-length cross-linking amidation reactions. Nanoparticles functionalized in the core with 17 different (12 chemically novel) molecular baits showed preferential high affinities (KD < 10–11 M) for specific low-abundance protein analytes. A poly(N-isopropylacrylamide-co-vinylsulfonic acid) shell was added to the core particles. This shell chemistry selectively prevented unwanted entry of all size peptides derived from albumin without hindering the penetration of non-albumin small proteins and peptides. Proteins and peptides entered the core to be captured with high affinity by baits immobilized in the core. Nanoparticles effectively protected interleukin-6 from enzymatic degradation in sweat and increased the effective detection sensitivity of human growth hormone in human urine using multiple reaction monitoring analysis. Used in whole blood as a one-step, in-solution preprocessing step, the nanoparticles greatly enriched the concentration of low-molecular weight proteins and peptides while excluding albumin and other proteins above 30 kDa; this achieved a 10,000-fold effective amplification of the analyte concentration, enabling mass spectrometry (MS) discovery of candidate biomarkers that were previously undetectable.

Application of Analyte Harvesting Nanoparticle Technology to the Measurement of Urinary HGH in Healthy Individuals

“…A key element of the hydrogel nanoparticles is the incorporation of the bait [9,10], which captures HGH with high affinity. We screened a series of nine chemical baits for HGH affinity within a urine matrix.…”

“…The nanoparticles are buoyant open mesh (>95% solute) hydrogel spheres ( Figure 3A) containing a core with an immobilized high affinity chemical bait. When mixed with the urine, the nanoparticles remain in colloid suspension to achieve extremely rapid access [10,11] and sequestration of the HGH target analyte. The bait binds HGH with high affinity (K D <10 −12 M, Figure 1), such that no detectable HGH can be measured outside the nanoparticles in the fluid phase.…”

“…Bait functionalized hydrogel nanoparticles [10,11] are a cost effective, user friendly technology that, in this study, permitted the reliable measurement of a low abundance protein analyte (HGH) in urine with a clinical grade immunoassay that until now is used for serum/plasma based analysis. Once the correct bait chemistry is matched to optimal analyte capture, this nanoparticle technology can be applied to the measurement of any analyte in any body fluid using any appropriate measurement platform ranging from immunoassays to mass spectroscopy-based multiple reaction monitoring [10].…”

“…Nanoparticles are added to the urine samples to concentrate the analyte into a small volume, which can then be eluted and measured by a clinical grade standard immunoassay designed for serum ( Figure 1). The nanoparticles can rapidly harvest, protect from degradation, and concentrate, low abundance analytes from biologic fluids while actively excluding high abundance proteins such as albumin [10][11][12][13].…”

“…In this study, we applied analyte harvesting hydrogel nanoparticles [10,11] to reduce the LLD of the Immulite clinical serum immunoassay for HGH. The Immulite Growth Hormone (HGH) (Recombinant 98/574) is a standard two site sandwich immunoassay which reports a lower of detection of 0.050 ng/mL.…”