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Core-shell hydrogel particles harvest, concentrate and preserve labile low abundance biomarkers.

Longo C, Patanarut A, George T, Bishop B, Zhou W, Fredolini C, Ross MM, Espina V, Pellacani G, Petricoin EF, Liotta LA, Luchini A - PLoS ONE (2009)

Bottom Line: Particle sequestered PDGF was fully protected from exogenously added tryptic degradation.Beyond PDGF, the sequestration and protection from degradation for a series of additional very low abundance and very labile cytokines were verified.We envision the application of harvesting core-shell nanoparticles to whole blood for concentration and immediate preservation of low abundance and labile analytes at the time of venipuncture.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Modena and Reggio Emilia, Modena, Italy.

ABSTRACT

Background: The blood proteome is thought to represent a rich source of biomarkers for early stage disease detection. Nevertheless, three major challenges have hindered biomarker discovery: a) candidate biomarkers exist at extremely low concentrations in blood; b) high abundance resident proteins such as albumin mask the rare biomarkers; c) biomarkers are rapidly degraded by endogenous and exogenous proteinases.

Methodology and principal findings: Hydrogel nanoparticles created with a N-isopropylacrylamide based core (365 nm)-shell (167 nm) and functionalized with a charged based bait (acrylic acid) were studied as a technology for addressing all these biomarker discovery problems, in one step, in solution. These harvesting core-shell nanoparticles are designed to simultaneously conduct size exclusion and affinity chromatography in solution. Platelet derived growth factor (PDGF), a clinically relevant, highly labile, and very low abundance biomarker, was chosen as a model. PDGF, spiked in human serum, was completely sequestered from its carrier protein albumin, concentrated, and fully preserved, within minutes by the particles. Particle sequestered PDGF was fully protected from exogenously added tryptic degradation. When the nanoparticles were added to a 1 mL dilute solution of PDGF at non detectable levels (less than 20 picograms per mL) the concentration of the PDGF released from the polymeric matrix of the particles increased within the detection range of ELISA and mass spectrometry. Beyond PDGF, the sequestration and protection from degradation for a series of additional very low abundance and very labile cytokines were verified.

Conclusions and significance: We envision the application of harvesting core-shell nanoparticles to whole blood for concentration and immediate preservation of low abundance and labile analytes at the time of venipuncture.

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Core shell particles increase the concentration of native PDGF in serum as measured by ELISA assay.(A) ELISA readings of the starting serum solution in Calibrator diluent RD6-3 (R&D Systems, animal serum with preservatives) at a concentration of 170.91+/−4.66 pg/mL and PDGF eluted from core-shell particles (1743.43+/−11.06 pg/mL). (B) PDGF concentration in core-shell particle eluate plotted against the quantity of particles utilized for the incubation, duplicate experiments. (C) ELISA standard curve of PDGF concentration versus absorbance. The standard curve was generated with two repeats for each PDGF calibrator concentration.
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pone-0004763-g007: Core shell particles increase the concentration of native PDGF in serum as measured by ELISA assay.(A) ELISA readings of the starting serum solution in Calibrator diluent RD6-3 (R&D Systems, animal serum with preservatives) at a concentration of 170.91+/−4.66 pg/mL and PDGF eluted from core-shell particles (1743.43+/−11.06 pg/mL). (B) PDGF concentration in core-shell particle eluate plotted against the quantity of particles utilized for the incubation, duplicate experiments. (C) ELISA standard curve of PDGF concentration versus absorbance. The standard curve was generated with two repeats for each PDGF calibrator concentration.

Mentions: A further experiment was performed in order to test the ability of core shell particles to sequester, concentrate and preserve native PDGF from human serum. We examined the effect of excess interfering proteins on the amount of particles necessary to reach saturation and complete depletion of native PDGF from serum. Serum was diluted 1∶10 in Tris HCl 50 mM pH 7 and incubated with increasing quantities of particles (200, 500, 1000, and 1500 µL). The value of PDGF in the starting serum solution was read as 170.92+/−4.66 pg/mL whereas the concentration of PDGF recovered from particles was 1743.43+/−11.06 pg/mL yielding a concentration factor of about 10-fold (1000 percent) (Fig. 7A). Saturation was reached at a value of 1000 µL (230 million particles, 1∶1 v/v particles∶serum solution, Fig. 7B). Given the fact that the starting concentration of PDGF in serum is higher than the concentration of PDGF in the solution of Fig. 6, we can conclude that the presence of serum, with its enormous protein content in the starting sample, requires less than double amount of particles to deplete the sample, thus confirming the extremely high binding capacity of particles, even in the presence of abundant serum proteins. The standard curve for the PDGF ELISA assay used in these studies is shown (Fig. 7C).


Core-shell hydrogel particles harvest, concentrate and preserve labile low abundance biomarkers.

Longo C, Patanarut A, George T, Bishop B, Zhou W, Fredolini C, Ross MM, Espina V, Pellacani G, Petricoin EF, Liotta LA, Luchini A - PLoS ONE (2009)

Core shell particles increase the concentration of native PDGF in serum as measured by ELISA assay.(A) ELISA readings of the starting serum solution in Calibrator diluent RD6-3 (R&D Systems, animal serum with preservatives) at a concentration of 170.91+/−4.66 pg/mL and PDGF eluted from core-shell particles (1743.43+/−11.06 pg/mL). (B) PDGF concentration in core-shell particle eluate plotted against the quantity of particles utilized for the incubation, duplicate experiments. (C) ELISA standard curve of PDGF concentration versus absorbance. The standard curve was generated with two repeats for each PDGF calibrator concentration.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2651577&req=5

pone-0004763-g007: Core shell particles increase the concentration of native PDGF in serum as measured by ELISA assay.(A) ELISA readings of the starting serum solution in Calibrator diluent RD6-3 (R&D Systems, animal serum with preservatives) at a concentration of 170.91+/−4.66 pg/mL and PDGF eluted from core-shell particles (1743.43+/−11.06 pg/mL). (B) PDGF concentration in core-shell particle eluate plotted against the quantity of particles utilized for the incubation, duplicate experiments. (C) ELISA standard curve of PDGF concentration versus absorbance. The standard curve was generated with two repeats for each PDGF calibrator concentration.
Mentions: A further experiment was performed in order to test the ability of core shell particles to sequester, concentrate and preserve native PDGF from human serum. We examined the effect of excess interfering proteins on the amount of particles necessary to reach saturation and complete depletion of native PDGF from serum. Serum was diluted 1∶10 in Tris HCl 50 mM pH 7 and incubated with increasing quantities of particles (200, 500, 1000, and 1500 µL). The value of PDGF in the starting serum solution was read as 170.92+/−4.66 pg/mL whereas the concentration of PDGF recovered from particles was 1743.43+/−11.06 pg/mL yielding a concentration factor of about 10-fold (1000 percent) (Fig. 7A). Saturation was reached at a value of 1000 µL (230 million particles, 1∶1 v/v particles∶serum solution, Fig. 7B). Given the fact that the starting concentration of PDGF in serum is higher than the concentration of PDGF in the solution of Fig. 6, we can conclude that the presence of serum, with its enormous protein content in the starting sample, requires less than double amount of particles to deplete the sample, thus confirming the extremely high binding capacity of particles, even in the presence of abundant serum proteins. The standard curve for the PDGF ELISA assay used in these studies is shown (Fig. 7C).

Bottom Line: Particle sequestered PDGF was fully protected from exogenously added tryptic degradation.Beyond PDGF, the sequestration and protection from degradation for a series of additional very low abundance and very labile cytokines were verified.We envision the application of harvesting core-shell nanoparticles to whole blood for concentration and immediate preservation of low abundance and labile analytes at the time of venipuncture.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Modena and Reggio Emilia, Modena, Italy.

ABSTRACT

Background: The blood proteome is thought to represent a rich source of biomarkers for early stage disease detection. Nevertheless, three major challenges have hindered biomarker discovery: a) candidate biomarkers exist at extremely low concentrations in blood; b) high abundance resident proteins such as albumin mask the rare biomarkers; c) biomarkers are rapidly degraded by endogenous and exogenous proteinases.

Methodology and principal findings: Hydrogel nanoparticles created with a N-isopropylacrylamide based core (365 nm)-shell (167 nm) and functionalized with a charged based bait (acrylic acid) were studied as a technology for addressing all these biomarker discovery problems, in one step, in solution. These harvesting core-shell nanoparticles are designed to simultaneously conduct size exclusion and affinity chromatography in solution. Platelet derived growth factor (PDGF), a clinically relevant, highly labile, and very low abundance biomarker, was chosen as a model. PDGF, spiked in human serum, was completely sequestered from its carrier protein albumin, concentrated, and fully preserved, within minutes by the particles. Particle sequestered PDGF was fully protected from exogenously added tryptic degradation. When the nanoparticles were added to a 1 mL dilute solution of PDGF at non detectable levels (less than 20 picograms per mL) the concentration of the PDGF released from the polymeric matrix of the particles increased within the detection range of ELISA and mass spectrometry. Beyond PDGF, the sequestration and protection from degradation for a series of additional very low abundance and very labile cytokines were verified.

Conclusions and significance: We envision the application of harvesting core-shell nanoparticles to whole blood for concentration and immediate preservation of low abundance and labile analytes at the time of venipuncture.

Show MeSH