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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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SDS-PAGE Analysis of PDGF incubated particles.(A) Lane 1) Starting solution containing BSA and PDGF (Control), 2) Supernatant (OUT); 3) Particle content (IN). Particles remove PDGF from carrier albumin with a total exclusion of albumin itself. (B) Lane 1) Starting solution containing PDGF, BSA, aprotinin (MW 6,500 Da), lysozyme (MW 14,400 Da), trypsin inhibitor (MW 21,500 Da), carbonic anhydrase (MW 31,000 Da), and ovalbumin (MW 45,000 Da) (Control), 2) Supernatant (OUT); 3) Particle content (IN). Particles harvest PDGF together with low molecular weight proteins and exclude proteins above ca 20,000 Da.
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pone-0004763-g004: SDS-PAGE Analysis of PDGF incubated particles.(A) Lane 1) Starting solution containing BSA and PDGF (Control), 2) Supernatant (OUT); 3) Particle content (IN). Particles remove PDGF from carrier albumin with a total exclusion of albumin itself. (B) Lane 1) Starting solution containing PDGF, BSA, aprotinin (MW 6,500 Da), lysozyme (MW 14,400 Da), trypsin inhibitor (MW 21,500 Da), carbonic anhydrase (MW 31,000 Da), and ovalbumin (MW 45,000 Da) (Control), 2) Supernatant (OUT); 3) Particle content (IN). Particles harvest PDGF together with low molecular weight proteins and exclude proteins above ca 20,000 Da.

Mentions: Human platelet derived growth factor (PDGF, MW 14,500 Da) was spiked in a solution containing bovine serum albumin (BSA, MW 66,000 Da) as carrier protein associated with the PDGF. Core-shell hydrogel nanoparticles added to the PDGF-BSA solution acted as a molecular sieve as evidenced by no detectable association of BSA with the particles, and the particles completely sequestered all the solution phase PDGF while completely excluding the BSA (Fig. 4A). This suggests that PDGF affinity for the bait was higher than that for carrier BSA. In order to further assess the molecular sieving properties of core-shell particles, a solution containing: PDGF, BSA, aprotinin (MW 6,500 Da), lysozyme (MW 14,400 Da), trypsin inhibitor (MW 21,500 Da), carbonic anhydrase (MW 31,000 Da), and ovalbumin (MW 45,000 Da), was used. The uptake of proteins by the particles was evaluated by SDS PAGE. Core-shell particles efficaciously captured and concentrated low molecular weight proteins with a weight less than 21,500 Da whereas proteins with high molecular weight remained excluded from the particles (Fig 4B).


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)

SDS-PAGE Analysis of PDGF incubated particles.(A) Lane 1) Starting solution containing BSA and PDGF (Control), 2) Supernatant (OUT); 3) Particle content (IN). Particles remove PDGF from carrier albumin with a total exclusion of albumin itself. (B) Lane 1) Starting solution containing PDGF, BSA, aprotinin (MW 6,500 Da), lysozyme (MW 14,400 Da), trypsin inhibitor (MW 21,500 Da), carbonic anhydrase (MW 31,000 Da), and ovalbumin (MW 45,000 Da) (Control), 2) Supernatant (OUT); 3) Particle content (IN). Particles harvest PDGF together with low molecular weight proteins and exclude proteins above ca 20,000 Da.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2651577&req=5

pone-0004763-g004: SDS-PAGE Analysis of PDGF incubated particles.(A) Lane 1) Starting solution containing BSA and PDGF (Control), 2) Supernatant (OUT); 3) Particle content (IN). Particles remove PDGF from carrier albumin with a total exclusion of albumin itself. (B) Lane 1) Starting solution containing PDGF, BSA, aprotinin (MW 6,500 Da), lysozyme (MW 14,400 Da), trypsin inhibitor (MW 21,500 Da), carbonic anhydrase (MW 31,000 Da), and ovalbumin (MW 45,000 Da) (Control), 2) Supernatant (OUT); 3) Particle content (IN). Particles harvest PDGF together with low molecular weight proteins and exclude proteins above ca 20,000 Da.
Mentions: Human platelet derived growth factor (PDGF, MW 14,500 Da) was spiked in a solution containing bovine serum albumin (BSA, MW 66,000 Da) as carrier protein associated with the PDGF. Core-shell hydrogel nanoparticles added to the PDGF-BSA solution acted as a molecular sieve as evidenced by no detectable association of BSA with the particles, and the particles completely sequestered all the solution phase PDGF while completely excluding the BSA (Fig. 4A). This suggests that PDGF affinity for the bait was higher than that for carrier BSA. In order to further assess the molecular sieving properties of core-shell particles, a solution containing: PDGF, BSA, aprotinin (MW 6,500 Da), lysozyme (MW 14,400 Da), trypsin inhibitor (MW 21,500 Da), carbonic anhydrase (MW 31,000 Da), and ovalbumin (MW 45,000 Da), was used. The uptake of proteins by the particles was evaluated by SDS PAGE. Core-shell particles efficaciously captured and concentrated low molecular weight proteins with a weight less than 21,500 Da whereas proteins with high molecular weight remained excluded from the particles (Fig 4B).

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