Nanoparticle technology: amplifying the effective sensitivity of biomarker detection to create a urine test for hGH

Abstract

Several clinical-grade immunoassays exist for the specific measurement of hGH or its isoforms in blood but there is an urgent need to apply these same reliable assays to the measurement of hGH in urine as a preferred 'non-invasive' biofluid. Unfortunately, conventional hGH immunoassays cannot attain the sensitivity required to detect the low concentrations of hGH in urine. The lowest limit of sensitivity for existing hGH immunoassays is >50 pg/mL, while the estimated concentration of urinary hGH is about 1 pg/m-50 times lower than the sensitivity threshold. We have created novel N-isopropylacrylamide (NIPAm)-based hydrogel nanoparticles functionalized with an affinity bait. When introduced into an analyte-containing solution, the nanoparticles can perform, in one step, (1) complete harvesting of all solution phase target analytes, (2) full protection of the captured analyte from degradation and (3) sequestration of the analyte, effectively increasing the analyte concentration up to a hundredfold. N-isopropylacrylamide nanoparticles functionalized with Cibacron Blue F3GA bait have been applied to raise the concentration of urinary hGH into the linear range of clinical grade immunoassays. This technology now provides an opportunity to evaluate the concentration of hGH in urine with high precision and accuracy.

Figure 1

Schematic representation of hydrogel nanoparticle functioning. Adapted from Longo and colleagues.[42]

Figure 2

Chemical formula of Cibacron Blue F3GA (CB). Sulfonate groups, which are thought to play a fundamental role in CB-hGH complex formation, are highlighted with a red circle.

Figure 3

Atomic force microscopy (AFM) images of NIPAm/CB nanoparticles show that the size distribution of NIPAm/CB nanoparticles is very uniform. Aqueous nanoparticle suspension was deposited under humid atmosphere at room temperature on freshly cleaved mica. After 15 minute incubation, water was removed under nitrogen flow. AFM images were acquired on particles using an NSCRIPTOR™ DPN® System (NanoInk). Images were acquired under AC mode using a silicon tip with a typical resonance frequency of 300 kHz and a radius smaller than 10 nm.

Figure 4

SDS PAGE analysis shows that NIPAm/CB nanoparticles completely sequestered and concentrated pituitary hGH (the three major isoforms 22, 20, and 17 kDa are clearly visible by silver staining) and recombinant hGH from synthetic urine solution. Adapted from Fredolini and colleagues.[41]

Figure 5

Our new strategy for immunological detection of human growth hormone (hGH) in urine involves three steps 1) NIPAm/CB nanoparticles are briefly incubated with urine and separated via high speed centrifugation. 2) hGH that was captured by NIPAm/CB nanoparticles is eluted from the nanoparticles with an acetonitrile (ACN)-NH4OH buffer. 3) The elution process does not compromise the immunogenicity of hGH, which is measured by means of a clinical grade immunoassay (Immulite 1000, Siemens). Adapted from Fredolini and colleagues.[41]

Figure 6

Left panel: Interfering proteins do not diminish the concentration capabilities of NIPAm/CB nanoparticles. Solutions of synthetic urine (Surine) containing hGH alone and hGH mixed with interfering proteins were incubated with NIPAm/CB nanoparticles. The nanoparticles captured and concentrated hGH 30 fold with no interference by competing proteins, showing a very high binding capacity. Right panel: The sensitivity of hGH immunoassay is amplified. hGH is spiked in 10 mL of synthetic urine and mixed with NIPAm/CB nanoparticles. All solution phase hGH is rapidly sequestred within the nanoparticles. hGH concentration in the particle eluate is linearly dependent on hGH concentration in the original urine solution. UD means undetectable (below the detection limits of the Immulite, 50 pg/mL) Adapted from Fredolini and colleagues.[41]

Figure 7

The concentration of hGH in the urine can be calculated from the concentration of hGH in NIPAm/CB nanoparticle eluate by means of a calibration curve. Solutions obtained by dissolving rhGH in synthetic urine at decreasing concentration from 830 to 0.83 pg/mL were incubated with NIPAm/CB nanoparticles. Standard deviation of the concentration of hGH in NIPAm/CB nanoparticle eluate is always below 10% of the average value (3 replicates). Linearity between the concentrations in urine and particle eluate is evident, and the relationship can be mathematically described by y= 19.41x+ 0.28 (^r= 0.999). In the blue square a magnification of the curve for the low concentration range (83 pg/mL - 0.8 pg/mL). For hGH concentration in the original urine solution of 0.8 pg/mL a concentration of 0.3 ng/mL was measured by Immulite in the NIPAm/CB nanoparticle eluate.