Method for harvesting nanoparticles and sequestering biomarkers
Inventors
Luchini, Alessandra • 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-8382987-B2
Publication Date
2013-02-26
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 for harvesting analytes from solution comprising a polymeric matrix having a pore size that allows analytes to enter the capture particles, where the pore size of the capture particles are changeable upon application of a stimulus so that analytes of interest can be 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, and the particles may include an attractant (bait) either incorporated into or associated with the particle. The capture particles are used to isolate and identify analytes present in a mixture, to protect analytes which are subject to degradation upon harvesting, and to concentrate analytes of low abundance in a fluid.
The background identifies the problem that disease-relevant biomarkers in blood or body fluids often exist at exceedingly low concentrations within complex mixtures and can be masked by high-abundance species, and that degradation of protein biomarkers can occur immediately following collection due to endogenous or exogenous proteases. The invention addresses the need 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, enabling capture, protection from degradation, and amplification of low-abundance biomarkers (for example enabling measurement of low and labile human growth hormone in urine).
Claims Coverage
Overview: One independent claim is present and the main inventive features focus on (1) the NIPAm open-meshwork hydrogel particles bearing a covalently coupled analyte binding affinity molecule, (2) maintenance of pore size during uptake, and (3) affinity-driven concentration of analyte inside the particle.
NIPAm open-meshwork hydrogel capture particles with covalently coupled analyte binding affinity molecule
Mixing N-isopropylacrylamide (NIPAm) containing open-meshwork hydrogel capture particles that contain an analyte binding affinity molecule covalently coupled within a volume of the open-meshwork hydrogel capture particles with a fluid solution that contains an analyte of interest.
Invariant pore size during analyte uptake
There is no change in pore size of the open-meshwork hydrogel capture particles as the analyte of interest moves inside the open-meshwork hydrogel capture particles.
Affinity-driven concentration against a concentration gradient
The analyte binding affinity molecule captures 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.
Together the independent claim's inventive features claim NIPAm open-meshwork hydrogel particles bearing a covalently coupled affinity moiety that, without a change in particle pore size during uptake, sequester and concentrate analytes inside the particle via affinity binding to produce a higher internal analyte concentration.
Stated Advantages
Enables sequestering and harvesting of analytes from solution and isolation for analysis.
Protects captured analytes from degradation during subsequent sample handling and storage.
Concentrates low-abundance analytes to amplify their concentration in a fluid, enabling detection with standard analytical assays.
Changeable porosity (stimulus-responsive) permits analytes to enter under certain conditions and then to be sequestered inside the particle.
Facilitates one-step in-solution affinity and/or size-based separation, reducing need for multi-step conventional preparative procedures.
Captured biomarkers are preserved and reported to be stable at room temperature or at 37° C.
Particles can be easily isolated and separated from mixtures using physical or field-based methods or affinity handles.
Documented Applications
Harvesting, isolation and identification of biomarkers (nucleic acids, proteins, peptides, metabolites) from complex biological fluids such as blood, plasma, serum, and urine.
Protecting sequestered biomarkers from enzymatic degradation during collection, transport, and storage to preserve samples for downstream analysis.
Concentrating low-abundance biomarkers from urine (exemplified by human growth hormone) so they can be measured by standard clinical immunoassays, with application to anti-doping testing.
Use in kits and collection devices in which capture particles are filled into or coated on a collecting device for sample capture and later analysis.
Integration into microfluidic systems where capture particles sequester analytes and are then transferred for release and analysis.
Use as patches or coated support surfaces to capture analytes from skin or surfaces and as components to coat layers, slabs, meshes, or fabrics for sampling.
Application to diagnostic workflows for early-stage disease biomarker discovery, including cancer and other diseases where low-abundance, low-molecular-weight species are informative.
Environmental, food, and water testing for contaminants, toxicants, and small molecules by harvesting target analytes from environmental or food samples.
Metabolomic and targeted capture applications using particles bearing specific baits (e.g., for sugars, phosphopeptides, lipids, small molecules) to enrich defined molecular classes.
Potential use in blood-clearing applications such as dialysis or toxin removal by sequestering undesirable small molecules from blood or other fluids.
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