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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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Immunoblot analysis showing that core-shell particles protect captured PDGF from tryptic degradation.(A) Sypro ruby total protein staining and (B) Immunoblot analysis with anti-PDGF antibody of the same PVDF membrane are presented. Lane 1) control PDGF+BSA solution; 2) content of particles incubated with PDGF+BSA (IN); 3) supernatant of particles incubated with PDGF+BSA (OUT); 4) content of particles incubated with BSA+PDGF+trypsin (IN+TRYPSIN); 5) supernatant of particles incubated with BSA+PDGF+trypsin (OUT+TRYPSIN); 6) BSA+PDGF+trypsin without particles incubated for 40 minutes (+TRYPSIN 40′); 7)) BSA+PDGF+trypsin without particles incubated for 20 minutes (+TRYPSIN 20′); 8)) BSA+PDGF+trypsin without particles incubated for 10 minutes (+TRYPSIN 10′); 9)) BSA+PDGF+trypsin without particles incubated for 0 minutes (+TRYPSIN 0′).
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pone-0004763-g009: Immunoblot analysis showing that core-shell particles protect captured PDGF from tryptic degradation.(A) Sypro ruby total protein staining and (B) Immunoblot analysis with anti-PDGF antibody of the same PVDF membrane are presented. Lane 1) control PDGF+BSA solution; 2) content of particles incubated with PDGF+BSA (IN); 3) supernatant of particles incubated with PDGF+BSA (OUT); 4) content of particles incubated with BSA+PDGF+trypsin (IN+TRYPSIN); 5) supernatant of particles incubated with BSA+PDGF+trypsin (OUT+TRYPSIN); 6) BSA+PDGF+trypsin without particles incubated for 40 minutes (+TRYPSIN 40′); 7)) BSA+PDGF+trypsin without particles incubated for 20 minutes (+TRYPSIN 20′); 8)) BSA+PDGF+trypsin without particles incubated for 10 minutes (+TRYPSIN 10′); 9)) BSA+PDGF+trypsin without particles incubated for 0 minutes (+TRYPSIN 0′).

Mentions: Degradation of biomarkers by endogenous and exogenous proteases is a major source of biomarker performance bias, and hinders the discovery and measurement of candidate biomarkers. Immunoblot analysis was used to evaluate the particles ability to protect PDGF from enzymatic degradation. Trypsin action on PDGF in the absence of particles was evident after 10 minutes and almost complete after one hour, as indicated by nearly undetectable PDGF bands at 14,000–17,000 Da (Fig. 9A and Fig. 9B Lanes 6–8). In marked contrast, PDGF incubated with trypsin and core-shell particles generated a single species band that was not diminished in staining intensity and was not fragmented, suggesting that particles successfully preserved PDGF from proteolysis (Fig. 9A and Fig. 9B Lane 4). The PDGF band for the particles loaded with PDGF without trypsin (Fig. 9A and Fig. 9B Lane 2) was identical to that for the particles loaded with PDGF and trypsin (Fig. 9A and Fig. 9B Lane 4) further suggesting that no PDGF protein was lost because of enzymatic degradation.


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)

Immunoblot analysis showing that core-shell particles protect captured PDGF from tryptic degradation.(A) Sypro ruby total protein staining and (B) Immunoblot analysis with anti-PDGF antibody of the same PVDF membrane are presented. Lane 1) control PDGF+BSA solution; 2) content of particles incubated with PDGF+BSA (IN); 3) supernatant of particles incubated with PDGF+BSA (OUT); 4) content of particles incubated with BSA+PDGF+trypsin (IN+TRYPSIN); 5) supernatant of particles incubated with BSA+PDGF+trypsin (OUT+TRYPSIN); 6) BSA+PDGF+trypsin without particles incubated for 40 minutes (+TRYPSIN 40′); 7)) BSA+PDGF+trypsin without particles incubated for 20 minutes (+TRYPSIN 20′); 8)) BSA+PDGF+trypsin without particles incubated for 10 minutes (+TRYPSIN 10′); 9)) BSA+PDGF+trypsin without particles incubated for 0 minutes (+TRYPSIN 0′).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2651577&req=5

pone-0004763-g009: Immunoblot analysis showing that core-shell particles protect captured PDGF from tryptic degradation.(A) Sypro ruby total protein staining and (B) Immunoblot analysis with anti-PDGF antibody of the same PVDF membrane are presented. Lane 1) control PDGF+BSA solution; 2) content of particles incubated with PDGF+BSA (IN); 3) supernatant of particles incubated with PDGF+BSA (OUT); 4) content of particles incubated with BSA+PDGF+trypsin (IN+TRYPSIN); 5) supernatant of particles incubated with BSA+PDGF+trypsin (OUT+TRYPSIN); 6) BSA+PDGF+trypsin without particles incubated for 40 minutes (+TRYPSIN 40′); 7)) BSA+PDGF+trypsin without particles incubated for 20 minutes (+TRYPSIN 20′); 8)) BSA+PDGF+trypsin without particles incubated for 10 minutes (+TRYPSIN 10′); 9)) BSA+PDGF+trypsin without particles incubated for 0 minutes (+TRYPSIN 0′).
Mentions: Degradation of biomarkers by endogenous and exogenous proteases is a major source of biomarker performance bias, and hinders the discovery and measurement of candidate biomarkers. Immunoblot analysis was used to evaluate the particles ability to protect PDGF from enzymatic degradation. Trypsin action on PDGF in the absence of particles was evident after 10 minutes and almost complete after one hour, as indicated by nearly undetectable PDGF bands at 14,000–17,000 Da (Fig. 9A and Fig. 9B Lanes 6–8). In marked contrast, PDGF incubated with trypsin and core-shell particles generated a single species band that was not diminished in staining intensity and was not fragmented, suggesting that particles successfully preserved PDGF from proteolysis (Fig. 9A and Fig. 9B Lane 4). The PDGF band for the particles loaded with PDGF without trypsin (Fig. 9A and Fig. 9B Lane 2) was identical to that for the particles loaded with PDGF and trypsin (Fig. 9A and Fig. 9B Lane 4) further suggesting that no PDGF protein was lost because of enzymatic degradation.

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