Method for harvesting nanoparticles and sequestering biomarkers
Inventors
Kunkel, Alessandra Luchini • Liotta, Lance • Petricoin, Emanuel • Bishop, Barney • Meani, Francesco • Fredolini, Claudia • Dunlap, Thomas M • Patanarut, Alexis
Assignees
CERES NANOSCIENCES Inc • Istituto Superiore di Sanita ISS • George Mason Research Foundation Inc
Publication Number
US-9383299-B2
Publication Date
2016-07-05
Expiration Date
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Abstract
Capture particles for harvesting analytes from solution and methods for using them are described. The capture particles are made up of a polymeric matrix having pore size that allows for the analytes to enter the capture particles. The pore size of the capture particles are changeable upon application of a stimulus to the particles, allowing the pore size of the particles to be changed so that analytes of interest remain sequestered inside the particles. The polymeric matrix of the capture particles are made of co-polymeric materials having a structural monomer and an affinity monomer, the affinity monomer having properties that attract the analyte to the capture particle. The capture particles may be used to isolate and identify analytes present in a mixture. They may also be used to protect analytes which are typically subject to degradation upon harvesting and to concentrate low an analyte in low abundance in a fluid.
Core Innovation
The invention provides capture particles comprising a polymeric matrix having a pore size that allows analytes to enter the capture particles and that is changeable upon application of a stimulus so that analytes of interest remain sequestered inside the particles. The polymeric matrix is made of co-polymeric materials having a structural monomer and an affinity monomer, the affinity monomer having properties that attract the analyte to the capture particle. The capture particles may be used to isolate and identify analytes present in a mixture, to protect analytes from degradation upon harvesting, and to concentrate low abundance analytes in a fluid.
The patent addresses the problems that disease-relevant biomarkers in blood or body fluids may exist in exceedingly low concentrations within a complex mixture and may be masked by high-abundance species, and that degradation of protein biomarkers can occur immediately following collection due to endogenous or exogenous proteases. The specification states a need in the art for particles that allow enrichment and encapsulation of selected classes of proteins and peptides from complex mixtures and protect them from degradation during subsequent sample handling. The invention further proposes that such particles would provide a powerful tool for discovery of novel biomarkers for early stage diseases.
The disclosure describes "smart" hydrogel capture particles whose porosity and overall size can be changed by changing the surrounding environment so biomarkers can enter under certain conditions and then be sequestered by changing conditions. The capture particles can include attractants (also termed baits or affinity monomers) either within the particle or as part of the particle material to provide affinity-based selection in addition to size-based sieving. The specification also describes use formats including solution-phase particles, kits with collection devices, patches, and integrated microfluidic systems, and notes that particles can be modified for facile separation by physical forces.
Claims Coverage
This patent text includes one independent claim from which three main inventive features are extracted.
Mixing with open-meshwork hydrogel capture particles
mixing a solution containing open-meshwork hydrogel capture particles that contain an analyte binding affinity molecule with a fluid solution that contains an analyte of interest;
Affinity-driven concentration within particles
further comprising the analyte binding affinity molecule capturing the analyte of interest against a concentration gradient resulting in a higher analyte of interest concentration within the volume of the open-meshwork hydrogel capture particles;
Elution by chemical treatment without changing pore size
further comprising eluting the analyte of interest, once captured, by a chemical treatment that dissociates the analyte of interest from the analyte binding affinity molecule within the volume of the open-meshwork hydrogel particles such that the pore size of the open-meshwork hydrogel capture particles does not change.
The independent claim covers (1) introducing open-meshwork hydrogel capture particles having an analyte-binding affinity molecule into a fluid containing an analyte, (2) affinity-driven accumulation of the analyte within the particle volume against a concentration gradient, and (3) chemical elution of the analyte without changing particle pore size.
Stated Advantages
Enrich and encapsulate selected classes of proteins and peptides from complex mixtures and protect them from degradation during subsequent sample handling.
Capture, protect from degradation, and amplify the concentration of low abundance biomarkers in urine so that labile and low abundance biomarkers can be measured by standard clinical immunoassays.
Select analytes by size and/or affinity and capture and/or release desired molecules in response to a physical or chemical treatment.
Enable rapid one-step, solution-phase partitioning (affinity and molecular sieving) in microvolumes, reducing the need for conventional multi-step procedures.
Provide room-temperature preservation and stabilization of captured biomarkers to mitigate degradation during transport and storage.
Allow facile separation of particles from mixtures through application of physical forces or by particle modifications (e.g., magnetic or electric attraction) to aid isolation.
Documented Applications
Harvesting, isolating, protecting, and concentrating biomarkers from biological fluids (e.g., blood, serum, plasma, urine) for discovery and diagnostic analysis.
Concentrating and preserving human growth hormone (hGH) in urine to enable measurement by routine clinical immunoassay and to address anti-doping testing limitations.
Kits for identifying an analyte in a mixture featuring a collection device filled or coated with the capture particles for subsequent analysis.
Microfluidic systems that incorporate capture particles to sequester analytes, transfer particles to a different location, release analytes, and perform downstream analysis.
Use of particles to protect captured proteins from enzymatic degradation and to concentrate low-abundance analytes, enabling downstream proteomic analyses (e.g., mass spectrometry).
Application to environmental and food testing: detection of contaminants in food, drinking water, and environmental samples as explicitly mentioned as sample types.
Particle-coated patches or support surfaces for capturing analytes from skin or other surfaces as a sample collection format.
Use of particles as a tool to clear toxins from blood, including potential dialysis applications and elimination of excess cholesterol, as stated in the disclosure.
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