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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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Related in: MedlinePlus

Light scattering and atomic force microscopy characterization of nanoparticles.(A) At room temperature, core is approximately 360 nm in size whereas adding core-shell particles have a diameter of 700 nm at pH 4.5. Core and core shell particles follow a typical temperature dependent behavior. (B) Particle suspension in MilliQ water (pH 5.5, 1 µg/mL) was deposited on freshly cleaved mica under humid atmosphere at room temperature for 15 minutes and dried under nitrogen. Atomic force microscopy (AFM) image of nanoparticles was acquired. Particles have a diameter of approximately 800 nm and exhibit a homogeneous size distribution. The scale bar for particle height shows a maximum height of 168 nm. The AFM picture was acquired under dry status therefore the particles are distorted (flattened) from their spherical shape due to drying on the mica surface.
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pone-0004763-g003: Light scattering and atomic force microscopy characterization of nanoparticles.(A) At room temperature, core is approximately 360 nm in size whereas adding core-shell particles have a diameter of 700 nm at pH 4.5. Core and core shell particles follow a typical temperature dependent behavior. (B) Particle suspension in MilliQ water (pH 5.5, 1 µg/mL) was deposited on freshly cleaved mica under humid atmosphere at room temperature for 15 minutes and dried under nitrogen. Atomic force microscopy (AFM) image of nanoparticles was acquired. Particles have a diameter of approximately 800 nm and exhibit a homogeneous size distribution. The scale bar for particle height shows a maximum height of 168 nm. The AFM picture was acquired under dry status therefore the particles are distorted (flattened) from their spherical shape due to drying on the mica surface.

Mentions: For the particle architecture used in the present study, a NIPAm shell surrounds a NIPAm/AAc core, containing affinity bait moieties. The sieving capability of the NIPAm shell shields the core and its affinity bait groups from larger molecules that may be present and could compete with the intended low-abundance, low molecular weight molecular targets for binding to the affinity bait in the core. Light scattering characterization was conducted on the particles during synthesis and at the end of the process in order to compare the sizes of the core and the core-shell particles. The core diameter at 25°C and pH 4.5 is 364.7+/−4.3 nm whereas the diameter of the core-shell particles at the same conditions is 699.4+/−6.2 nm (Fig. 3A). This suggests that the thickness of the shell is about 170 nm. Following the characteristic behavior of AAc containing hydrogels, both the core and the core-shell particle size decreased with increasing temperature and decreasing pH (Fig. 3A). Particles were further characterized by means of atomic force microscopy (AFM). AFM particle images (Fig. 3B) confirm size homogeneity, and AFM particle diameter readings were consistent with those measured with light scattering. Particle concentration, as obtained by weighing the lyophilized particles, was 10 mg/mL, and the number of particles per milliliter was 230 million.


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)

Light scattering and atomic force microscopy characterization of nanoparticles.(A) At room temperature, core is approximately 360 nm in size whereas adding core-shell particles have a diameter of 700 nm at pH 4.5. Core and core shell particles follow a typical temperature dependent behavior. (B) Particle suspension in MilliQ water (pH 5.5, 1 µg/mL) was deposited on freshly cleaved mica under humid atmosphere at room temperature for 15 minutes and dried under nitrogen. Atomic force microscopy (AFM) image of nanoparticles was acquired. Particles have a diameter of approximately 800 nm and exhibit a homogeneous size distribution. The scale bar for particle height shows a maximum height of 168 nm. The AFM picture was acquired under dry status therefore the particles are distorted (flattened) from their spherical shape due to drying on the mica surface.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2651577&req=5

pone-0004763-g003: Light scattering and atomic force microscopy characterization of nanoparticles.(A) At room temperature, core is approximately 360 nm in size whereas adding core-shell particles have a diameter of 700 nm at pH 4.5. Core and core shell particles follow a typical temperature dependent behavior. (B) Particle suspension in MilliQ water (pH 5.5, 1 µg/mL) was deposited on freshly cleaved mica under humid atmosphere at room temperature for 15 minutes and dried under nitrogen. Atomic force microscopy (AFM) image of nanoparticles was acquired. Particles have a diameter of approximately 800 nm and exhibit a homogeneous size distribution. The scale bar for particle height shows a maximum height of 168 nm. The AFM picture was acquired under dry status therefore the particles are distorted (flattened) from their spherical shape due to drying on the mica surface.
Mentions: For the particle architecture used in the present study, a NIPAm shell surrounds a NIPAm/AAc core, containing affinity bait moieties. The sieving capability of the NIPAm shell shields the core and its affinity bait groups from larger molecules that may be present and could compete with the intended low-abundance, low molecular weight molecular targets for binding to the affinity bait in the core. Light scattering characterization was conducted on the particles during synthesis and at the end of the process in order to compare the sizes of the core and the core-shell particles. The core diameter at 25°C and pH 4.5 is 364.7+/−4.3 nm whereas the diameter of the core-shell particles at the same conditions is 699.4+/−6.2 nm (Fig. 3A). This suggests that the thickness of the shell is about 170 nm. Following the characteristic behavior of AAc containing hydrogels, both the core and the core-shell particle size decreased with increasing temperature and decreasing pH (Fig. 3A). Particles were further characterized by means of atomic force microscopy (AFM). AFM particle images (Fig. 3B) confirm size homogeneity, and AFM particle diameter readings were consistent with those measured with light scattering. Particle concentration, as obtained by weighing the lyophilized particles, was 10 mg/mL, and the number of particles per milliliter was 230 million.

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
Related in: MedlinePlus