Concentration and Preservation of Very Low Abundance Biomarkers in Urine, such as Human Growth Hormone (hGH), by Cibacron Blue F3G-A Loaded Hydrogel Particles

Abstract

Urine is a potential source of diagnostic biomarkers for detection of diseases, and is a very attractive means of non-invasive biospecimen collection. Nonetheless, proteomic measurement in urine is very challenging because diagnostic biomarkers exist in very low concentration (usually below the sensitivity of common immunoassays) and may be subject to rapid degradation. Hydrogel nanoparticles functionalized with Cibacron Blue F3G-A (CB) have been applied to address these challenges for urine biomarker measurement. We chose one of the most difficult low abundance, but medically relevant, hormones in the urine: human growth hormone (hGH). The normal range of hGH in serum is 1 to 10 ng/mL but the urine concentration is suspected to be a thousand times less, well below the detection limit (50 pg/mL) of sensitive clinical hGH immunoassays. We demonstrate that CB particles can capture, preserve and concentrate hGH in urine at physiological salt and urea concentrations, so that hGH can be measured in the linear range of a clinical immunometric assay. Recombinant and cadaveric hGH were captured from synthetic and human urine, concentrated and measured with an Immulite chemiluminescent immunoassay. Values of hGH less than 0.05 ng/mL (the Immulite detection limit) were concentrated to 2 ng/mL, with a urine volume of 1 mL. Dose response studies using 10 mL of urine demonstrated that the concentration of hGH in the particle eluate was linearly dependent on the concentration of hGH in the starting solution, and that all hGH was removed from solution. Thus if the starting urine volume is 100 mL, the detection limit will be 0.1 pg/mL. Urine from a healthy donor whose serum hGH concentration was 1.34 ng/mL was studied in order detect endogenous hGH. Starting from a volume of 33 mL, the particle eluate had an hGH concentration of 58 pg/mL, giving an estimated initial concentration of hGH in urine of 0.175 pg/mL. The nanotechnology described here appears to have the desired precision, accuracy and sensitivity to support large scale clinical studies of urine hGH levels.

Figure 1

Amplification strategy for immunological detection of human growth hormone (hGH) in urine. Particles are mixed with urine, subsequently capture hGH, and are separated via centrifugation. Elution of hGH from particles is performed with an acetonitrile (ACN)-NH4OH buffer. Immunoassay is performed on hGH eluted from the particles

Figure 2

Chemical reactions for the synthesis of particles: (a) allylamine functionalized particles are created; (b) Cibacron Blue F3G-A dye is covalently incorporated into the particles via the amino group

Figure 3

Characterization of Cibacron Blue F3G-A loaded particles: (a) atomic force microscopy (AFM) image showing size homogeneity; (b) picture of 2 mL vials of NIPAm/CB and NIPAm/AA particles

Figure 4

Photon correlation spectroscopy analysis of dependence of particle size on temperature: (a) plot of average values of light scattering measurements of particle diameter at different temperatures. Hydrogel particles are thermoresponsive; (b) histograms showing the amplitude of the particle size distribution at each temperature

Figure 5

Evaluation of size exclusion properties of NIPAm/CB particles by SDS-PAGE. Particles were incubated with a mixture of proteins with a molecular weight range between 66 and 6.5 kDa, dissolved in Surine. Lane 1 protein mixture; Lane 2 supernatant (Out), Lane 3 particle content (In). Only proteins with a molecular weight lower than 45 KDa were trapped by the particles; ovalbumin and BSA were completely excluded

Figure 6

SDS-PAGE analysis of particles carrying different baits and their ability to harvest hGH. Lane 1 hGH control; Lane 2 Cibacron Blue 5% loaded particle supernatant (Out); Lane 3 Cibacron Blue 5% loaded particle content (In); Lane 4 Cibacron Blue 10% loaded particle supernatant (Out); Lane 5 Cibacron Blue 10% loaded particle content (In); Lane 6 allylamine particle supernatant (Out); Lane 7 allylamine particle content (In); Lane 8 beta-cyclodextrin loaded particle supernatant (Out); Lane 9 beta-cyclodextrin loaded particle content (In); Lane 10 acrylic acid particle supernatant (Out); Lane 11 acrylic acid particle content (In). Cibacron Blue 5% and 10% had the best affinity for hGH of all the particles tested and both completely depleted the supernatant and achieved a high degree of concentration of the hGH

Figure 7

SDS PAGE analysis of particles incubated with solutions of different pH values, in the presence of urea and salts. (a) Study of the effect of pH, urea and KCl on particle uptake of hGH. Lane 1 hGH control; Lane 2 0.01 mg/mL hGH solution at pH 4; Lane 3 NIPAm/CB particle supernatant at pH 4 (Out); Lane 4 NIPAm/CB particle content at pH 4 (In); Lane 5 0.01 mg/mL hGH solution at pH 5; Lane 6 NIPAm/CB particle supernatant at pH 5 (Out); Lane 7 NIPAm/CB particle content at pH 5 (In); Lane 8 NIPAm/CB particle supernatant at pH 6 (Out); Lane 9 NIPAm/CB particle content at pH 6 (In); Lane 10 NIPAm/CB particle supernatant at pH 7 (Out); Lane 11 NIPAm/CB particle content at pH 7 (In); Lane 12 NIPAm/CB particle supernatant at pH 8 (Out); Lane 13 NIPAm/CB particle content at pH 8 (In); Lane 14 0.01 mg/mL hGH solution at pH 10; Lane 15 NIPAm/CB particle supernatant at pH 10 (Out); Lane 16 NIPAm/CB particle content at pH 10 (In); Lane 17 NIPAm/CB particle supernatant in presence of urea (Out); Lane 18 NIPAm/CB particle content in presence of urea (In); Lane 19 NIPAm/CB particle supernatant in presence of KCl (Out); Lane 20 NIPAm/CB particle content in presence of KCl (In). Uptake of hGH is higher at lower pH and it is not hindered by the presence of urea or KCl. (b) SDS PAGE analysis of NIPAm/CB particles incubated with hGH diluted in synthetic urine (Surine) at different pH values. Lane 1 hGH control; Lane 2 0.01 mg/mL hGH solution in Surine; Lane 3 NIPAm/CB particle supernatant at pH 4 (Out); Lane 4 NIPAm/CB particle content at pH 4 (In); Lane 5 NIPAm/CB particle supernatant at pH 5 (Out); Lane 6 NIPAm/CB particle content at pH 5 (In); Lane 7 NIPAm/CB particle supernatant at pH 6.7 (Out); Lane 8 NIPAm/CB particle content at pH 6.7 (In); Lane 9 NIPAm/CB particle supernatant at pH 8 (Out); Lane 10 NIPAm/CB particle content at pH 8 (In). Particles successfully sequestered hGH from solution, and the efficiency was higher at lower pH

Figure 8

SDS PAGE analysis of time studies. (a) Uptake time course. Lane 1 hGH control; Lane 2 NIPAm/CB particle supernatant after 1 min incubation (Out); Lane 3 NIPAm/CB particle content after 1 min incubation (In); Lane 4 NIPAm/CB particle supernatant after 5 min incubation (Out); Lane 5 NIPAm/CB particle content after 5 min incubation (In); Lane 6 NIPAm/CB particle supernatant after 20 min incubation (Out); Lane 7 NIPAm/CB particle content after 20 min incubation (In); Lane 8 NIPAm/CB particle supernatant after 40 min incubation (Out); Lane 9 NIPAm/CB particle content after 40 min incubation (In); Lane 10 NIPAm/CB particle supernatant after 60 min incubation (Out); Lane 11 NIPAm/CB particle content after 60 min incubation (In). The sequestration process was very fast, with the majority of hGH being captured by particles after one minute. (b) Preservation time course. Lane 1 hGH control; Lane 2 NIPAm/CB particle supernatant after 1 h (Out); Lane 3 NIPAm/CB particle content after 1 h incubation (In); Lane 4 NIPAm/CB particle supernatant after 4 h incubation (Out); Lane 5 NIPAm/CB particle content after 4 h incubation (In); Lane 6 NIPAm/CB particle supernatant after 24 h incubation (Out); Lane 7 NIPAm/CB particle content after 24 h incubation (In); Lane 8 NIPAm/CB particle supernatant after 48 h incubation (Out); Lane 9 NIPAm/CB particle content after 48 h incubation (In). Once captured by particles, hGH remains stable up to at least 48 h

Figure 9

SDS PAGE analysis of cadaveric hGH spiked in Surine and incubated with particles. Lane 1 0.01 mg/mL cadaveric hGH solution in Surine; Lane 2 NIPAm/CB particle supernatant (Out); Lane 3 NIPAm/CB particle content (In). Particles sequestered all the three major isoforms of hGH (22, 20, and 17 kDa) and increased the concentration of the minor isoforms, so that they could be detected by silver staining

Figure 10

SDS-PAGE analysis of NIPAm/CB particles incubated with IL-8 diluted in Surine. Lane 1) IL-8 Control, Lane 2 NIPAm/CB particle supernatant (Out), Lane 3 NIPAm/CB particle content (In)

Figure 11

SDS PAGE analysis for optimizing the elution conditions.(a) Lane 1 hGH control; Lane 2 0.01 mg/mL hGH solution in Surine; Lane 3 NIPAm/CB particle content after elution at pH 7 (In); Lane 4 NIPAm/CB particle content eluted at pH 7 (Eluate); Lane 5 NIPAm/CB particle content eluted at pH 7 (Eluate); Lane 6 NIPAm/CB particle content after elution at pH 8 (In); Lane 7 NIPAm/CB particle content eluted at pH 8 (Eluate); Lane 8 NIPAm/CB particle content eluted at pH 8 (Eluate); Lane 9 NIPAm/CB particle content after elution at pH 10 (In); Lane 10 NIPAm/CB particle content eluted at pH 10 (Eluate); Lane 11 NIPAm/CB particle content eluted at pH 10 (Eluate). Partial elution of hGH from particles was obtained with buffers at pH 7, 8, and 10. (b) Lane 1 hGH control; Lane 2 NIPAm/CB particle supernatant (Out); Lane 3 NIPAm/CB particle content after elution with acetonitrile/NH4HCO3(In); Lane 4 NIPAm/CB particle content eluted with acetonitrile/NH4HCO3 (Eluate); Lane 5 NIPAm/CB particle content eluted with acetonitrile/NH4HCO3(Eluate); Lane 6 NIPAm/CB particle supernatant (Out); Lane 7 NIPAm/CB particle content after elution with acetonitrile/NH4OH (In); Lane 8 NIPAm/CB particle content eluted with acetonitrile/NH4OH (Eluate); Lane 9 NIPAm/CB particle content eluted with acetonitrile/NH4OH (Eluate). Complete elution of hGH from particles was obtained with acetonitrile/NH4OH

Figure 12

Immulite assay readings for NIPAm/CB particles incubated with two different urine models containing hGH and Surine with and without interfering proteins. The efficiency with which the particles concentrated hGH in Surine is not affected by the presence of proteins that may compete with the hormone for binding to the CB bait

Figure 13

Particle dose response for capture of hGH. (a) Recombinant hGH was spiked in Surine and increasing amounts of particles were incubated with the analyte containing solution. The plateau is reached at about 600 μg. (b) Recombinant hGH was spiked in human urine and increasing amounts of particles were added to the solution. The plateau was reached at about 600 μg. (c) Cadaveric hGH was spiked in human urine and recovered with particles

Figure 14

Effective amplification of urine hGH immunoassay sensitivity. Particles raised the concentration of recombinant hGH above the detection limit of the Immulite and greatly amplified the concentration of a solution in the linear range. UD means undetectable (below the detection limits of the Immulite assay)

Figure 15

Effective amplification of urine hGH immunoassay sensitivity. Dose response study on recombinant hGH spiked in 10 ml of Surine. 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)