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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 extremely dilute PDGF approximately 10-folds (1000 percent) as measured by ELISA assay.(A) ELISA readings of the starting solution of PDGF in Calibrator diluent RD6-3 (R&D Systems, animal serum with preservatives) at a concentration of 63.69+/−1.448 pg/mL and PDGF eluted from core-shell particles (491.14+/−4.818 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-g006: Core shell particles increase the concentration of extremely dilute PDGF approximately 10-folds (1000 percent) as measured by ELISA assay.(A) ELISA readings of the starting solution of PDGF in Calibrator diluent RD6-3 (R&D Systems, animal serum with preservatives) at a concentration of 63.69+/−1.448 pg/mL and PDGF eluted from core-shell particles (491.14+/−4.818 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: As shown in Fig. 5A, previously undetectable level of PDGF was recovered from the particles and successfully quantified by ELISA at concentrations ranging from 75 to 102 pg/mL. The value of PDGF concentration in the starting solution (18.92+/−4.313 pg/mL) reported in Fig. 5A was below the linear range of the ELISA immunoassay (minimum detectable PDGF dose = 30 pg/mL) and was estimated by using the optical density and extrapolated from the standard curve. Per manufacturer's instructions, the minimum detectable dose was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration. Therefore, core-shell particles, incubated with a PDGF solution at a concentration undetectable by ELISA, harvested and concentrated PDGF to a level higher than the detection limit of the assay. Saturation was reached with the minimum amount of particles when the PDGF solution was very dilute, as expected (Fig. 5B). A standard curve for PDGF ELISA assay was generated (Fig. 5C) in order to assess the quality of the procedure. A similar experiment was performed with a more concentrated PDGF solution. The concentration of PDGF in the starting solution was 63.69 (+/−1.448) pg/mL whereas the concentration of PDGF recovered from particles was 452.81 (+/−4.818) pg/mL yielding a concentration factor of about 700% (Fig. 6A). A PDGF solution was incubated with different volumes of particles and demonstrated that saturation was reached when the volume of particles was 200 µl (46 million particles, 1∶5 v/v particles∶PDGF solution ratio, Fig. 6B). The standard curve for PDGF ELISA assay was repeated (Fig. 6C).


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 extremely dilute PDGF approximately 10-folds (1000 percent) as measured by ELISA assay.(A) ELISA readings of the starting solution of PDGF in Calibrator diluent RD6-3 (R&D Systems, animal serum with preservatives) at a concentration of 63.69+/−1.448 pg/mL and PDGF eluted from core-shell particles (491.14+/−4.818 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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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2651577&req=5

pone-0004763-g006: Core shell particles increase the concentration of extremely dilute PDGF approximately 10-folds (1000 percent) as measured by ELISA assay.(A) ELISA readings of the starting solution of PDGF in Calibrator diluent RD6-3 (R&D Systems, animal serum with preservatives) at a concentration of 63.69+/−1.448 pg/mL and PDGF eluted from core-shell particles (491.14+/−4.818 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: As shown in Fig. 5A, previously undetectable level of PDGF was recovered from the particles and successfully quantified by ELISA at concentrations ranging from 75 to 102 pg/mL. The value of PDGF concentration in the starting solution (18.92+/−4.313 pg/mL) reported in Fig. 5A was below the linear range of the ELISA immunoassay (minimum detectable PDGF dose = 30 pg/mL) and was estimated by using the optical density and extrapolated from the standard curve. Per manufacturer's instructions, the minimum detectable dose was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration. Therefore, core-shell particles, incubated with a PDGF solution at a concentration undetectable by ELISA, harvested and concentrated PDGF to a level higher than the detection limit of the assay. Saturation was reached with the minimum amount of particles when the PDGF solution was very dilute, as expected (Fig. 5B). A standard curve for PDGF ELISA assay was generated (Fig. 5C) in order to assess the quality of the procedure. A similar experiment was performed with a more concentrated PDGF solution. The concentration of PDGF in the starting solution was 63.69 (+/−1.448) pg/mL whereas the concentration of PDGF recovered from particles was 452.81 (+/−4.818) pg/mL yielding a concentration factor of about 700% (Fig. 6A). A PDGF solution was incubated with different volumes of particles and demonstrated that saturation was reached when the volume of particles was 200 µl (46 million particles, 1∶5 v/v particles∶PDGF solution ratio, Fig. 6B). The standard curve for PDGF ELISA assay was repeated (Fig. 6C).

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