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WO2009064389A2 - Particules d'hydrogel intelligentes ; récolte de biomarqueurs ; purification d'affinité en une étape, exclusion diffusion et protection contre la dégradation - Google Patents

Particules d'hydrogel intelligentes ; récolte de biomarqueurs ; purification d'affinité en une étape, exclusion diffusion et protection contre la dégradation Download PDF

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WO2009064389A2
WO2009064389A2 PCT/US2008/012666 US2008012666W WO2009064389A2 WO 2009064389 A2 WO2009064389 A2 WO 2009064389A2 US 2008012666 W US2008012666 W US 2008012666W WO 2009064389 A2 WO2009064389 A2 WO 2009064389A2
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particles
nipam
solution
aac
proteins
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WO2009064389A3 (fr
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Alessandra Luchini
David H. Geho
Emanuel Petricoin Iii
Lance A. Liotta
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles

Definitions

  • Biomarkers are nucleic acids, proteins, protein fragments or metabolites indicative of a specific biological state, that are associated with the risk of contraction or presence of diseased Biomarker research has revealed that low-abundance circulating proteins and peptides present a rich source of information regarding the state of the organism as a whole 4 .
  • the concentration of proteins and peptides comprising the complex circulatory proteome ranges from 10 ⁇ 12 mg/mL to 10 ⁇ 3 mg/mL, spanning ten orders of magnitude, with a few high molecular weight proteins such as albumin and immunoglobulins accounting for 90% of total protein content 5 .
  • the low abundance and low molecular weight proteins and metabolites also present in the blood provide a wealth of information and have great promise as a source of new biomarkers.
  • Conventional methods, such as two dimensional gel electrophoresis do not have the sensitivity and resolution to detect and quantify low abundance low molecular weight proteins and metabolites.
  • MS mass spectrometry
  • Hydrogels are three-dimensional cross-linked polymeric networks that can imbibe large amounts of water 16 . They are usually formed through monomer polymerization in the presence of a cross-linking agent, which is typically a monomer with at least two polymerizable functional moieties.
  • Gels can be categorized as non-responsive (simple polymeric networks dramatically swell upon exposure to water) or responsive gels (have added functionality and display changes in solvation in response to certain stimuli such as temperature 17 pH, 18 ' 19 ionic strength 20 ' 21 light 22 ' 23 and electric field 24 ).
  • Poly (N-alkyl acrylamides) have been extensively studied with respect to their thermoresponsivity 16 ' 25 with poly(N-isopropylacrylamide) (NIPAm) being one of the most strongly explored temperature sensitive hydrogels within this group.
  • NIPAm containing particles are highly appealing for their potential biotechnological applications, because of their stability, uniformity, and versatility with regard to the ease of making physical-chemical modifications in the particles.
  • NIPAm particles have been investigated for drug delivery slow release and targeted release, for solute desorption 26"36 , interaction with cells 27 , and coupling with oligodeoxyribonucleotides (OND) as a solid phase for hybridization 28 . Since the size and porosity can be controlled by temperature, the use of temperature treatment to control uptake and release of chemicals has been one of the most extensively characterized application of NIPAm particles as vectors for controlled drug delivery 30'36 .
  • hydrogel particles containing an affinity bait and a defined porosity were developed and demonstrated to a) rapidly and in one step sequester the low molecular weight fraction of serum proteins, peptides and metabolites, b) remove and concentrate the target molecules from solution, and c) protect captured proteins from enzymatic degradation.
  • NIPAm based particles have been chosen because their high water content, broad range of tunable porosities, consistency and uniformity following synthesis, functional reconstitution following freeze-drying, and potential biocompatibility. By changing the percentage of cross linking agent and temperature, it is possible to control the particles size and the effective porosity.
  • a significant advantage for the application studied here is the ability of these particles to rapidly uptake molecules because of their open structure, high water content, dual hydrophobic and hydrophilic chemical moieties that can be substituted in the polymer, and large surface area. This is a critical requirement for the goal of rapid harvesting of labile small proteins in solution and protecting the proteins from degradation.
  • the small size, uniformity of particle dimension, and reproducibility from batch to batch, of NIPAm provide special advantages for applications in flow cytometry.
  • NIPAm N-isopropylacrylamide
  • Particle synthesis chemistry is described in the Supplementary Information.
  • PCS Photon Correlation Spectroscopy
  • NIPAm particle size decreased with increasing temperature (Figure 2A), which is a distinctive characteristic of thermo responsive hydrogels 30"35 .
  • the NIP Am/ AAc particles showed a similar temperature dependence with respect to particle size, however they also demonstrated a pH dependent behavior ( Figure 2B).
  • AAc groups are protonated and NIPAm/AAc particle size dependence on temperature is similar to underivatized particles.
  • AAc groups are partially deprotonated and the average particle size increases, likely due to Coulombic interaction between polymeric chain and osmotic pressure resulting from counter ion ingress in particles ' .
  • Figure 2 Particle characterization.
  • A Light scattering measurement of NIPAm particle size as a function of temperature (diameter decreases as temperature increases).
  • B Plots of correlation of the size of NIPAm/AAc particles with temperature (diameter decreases as temperature increases) and pH (diameter decreases as pH decreases).
  • Particles were further characterized by atomic force microscopy (AFM) using an NSCRIPTORTM DPN ® System (Nanolnk). Images were acquired under AC mode using a silicon tip with a typical resonance frequency of 300 kHz and a radius smaller than 10 nm. Aliquots of 1% w/v particles (50 ⁇ L) were deposited on freshly cleaved mica; samples were incubated for ten minutes in humid atmosphere at room temperature to allow deposition, and then dried under nitrogen flow. AFM images of these particles ( Figure 2C and 2D) show them to be homogeneous in size, and with particle diameters consistent with those measured with light scattering.
  • AFM atomic force microscopy
  • NIPAm particles were tested for their molecular sieve performance in solution as schematically presented in Figure 3; the goal being to create particles that could capture proteins and small molecules with molecular weights less than 20,000 Da since the peptidome is thought to contain a rich source of biomarkers 13"15 .
  • FIG. 1 Schematic drawing of molecular sieving of particles in solution. Low molecular weight proteins are harvested; high molecular weight proteins are excluded.
  • This size range contains informative proteins, peptides and metabolites that are difficult, if not impossible, to separate from complex protein mixtures (such as serum or plasma) with adequate yield using 2-D gel electrophoresis or column chromatography.
  • the degree of cross-linking within the particle enabled exclusion of albumin and other high abundance large molecules while capturing molecules with sizes smaller than the cut-off pore size of the particles. Particles with varied degrees of cross-linking were investigated until one was identified that demonstrated an effective 20,000 Da exclusive pore size. These particles were further studied in order to evaluate their sieving efficiency and nonspecific binding of excluded molecules to the particle surface. Because serum albumin is present in large excess (10 6 -10 9 fold) relative to the proteins and peptides of interest, it was necessary to examine the efficiency and completeness of albumin exclusion.
  • FIG. 4 Flow cytometry analyses of FITC-incubated particles.
  • A Uptake is dose dependent.
  • B Uptake rapidly reaches saturation with a FITC concentration of 20 ⁇ M.
  • NIPAm particles were also incubated with FITC-labeled bovine serum albumin (BSA), MW 66,000 Da, with a dyermolecule ratio of 1 :1 (FITC-BSA, Sigma), FITC labeled insulin MW 3,500 Da, with a dye:molecule ratio of 1 :7 (Invitrogen), or FITC labeled myoglobin MW 17,000 Da, with a dye:molecule ratio of 1.36.
  • BSA bovine serum albumin
  • FITC-BSA FITC labeled insulin MW 3,500 Da
  • dye:molecule ratio of 1 :7 Invitrogen
  • FITC labeled myoglobin MW 17,000 Da with a dye:molecule ratio of 1.36.
  • Myoglobin (Sigma) was FITC labeled by means of the HOOK - Dye Labeling Kit (G Bioscience) in accordance of the vendor ' s instructions. Concentrations of all fluorescent species were adjusted in order to equalize the fluorescence signal.
  • NIPAm particles incubated with FITC and FITC-labeled proteins Flow cytometry measurements of (A) BSA and insulin, (B) myoglobin and free FITC.
  • C SDS-PAGE of particles incubated with insulin: Lane 1) insulin solution (Control), 2) NIPAm supernatant (Out, substance excluded from the particles), 3) wash 1, 4) wash 2, 5) NIPAm particles (In, substance captured by the particles).
  • D SDS-PAGE of NIPAm particles incubated with BSA and myoglobin: 1) BSA and myoglobin (Control), 2) NIPAm supernatant (Out), 3) wash 2, 4) NIPAm particles (In). BSA is totally excluded.
  • a negatively-charged moiety was selected as a bait for proteins and molecules that have a positive net charge. Incorporation of a negatively-charged bait within the particles would allow the particles to preferentially sequester and concentrate positively-charged proteins, peptides and other biomolecules. Therefore, particles were prepared based on a NIP Am/ AAc copolymer, which carries a large net negative charge at pH values greater than 3.5. As shown schematically in Figure 6, the presence of charged bait, in principle, should enhance substantially the K eq and thereby achieve a significantly higher concentration of the target protein inside the particle compared to the solution outside the particle.
  • FIG. Schematic depiction of affinity-based sequestering.
  • NIPAm/AAc particles concentrated analytes from solution with substantially greater efficiency relative to underivatized NIPAm particles.
  • Suspensions of NIPAm and NIPAm/AAc particles (10 mg/mL) were incubated for 1 hour with myoglobin (MW 17,000 Da, 20 ⁇ M in water).
  • myoglobin MW 17,000 Da, 20 ⁇ M in water.
  • Figure 7A significant levels of myoglobin remained in the bulk solution with some protein being bound by the particles, which lack the anionic affinity bait.
  • NIPAm/AAc particles, which contain the anionic affinity bait all of the myoglobin had been captured by the NIPAm/AAc particles, with no detectable myoglobin remaining in bulk solution.
  • FIG. 7 Protein sequestering by NIPAm/AAc particles (+ bait ) versus NIPAm particles (- bait), SDS-PAGE analysis of (A) Myoglobin (aqueous solution, pH 5.5) sequestration by particles + and - bait.
  • Lane 1 myoglobin, 2) NIPAm supernatant (Out), 3) NIPAm particle (In), 4) NIPAm/AAc supernatant (Out), 5) NIPAm/AAc particle (In), 6) NIPAm/AAc particles 1 :64, 7) NIPAm/AAc particles 1 :32, 8) NIPAm/AAc particles 1 :128. and 9) NIPAm/AAc particles 1 :256.
  • BSA and myoglobin sequestration by particles+bait (NIPAm/AAc) at two pH values.
  • NIPAm/AAc affinity baited particles The efficiency of NIPAm/AAc affinity baited particles to bind and concentrate proteins and peptides with MW less than ca. 20,000 Da is illustrated in Figure 8A.
  • NIPAm/AAc and NIPAm particles were each incubated for 1 hour with lysozyme (20 ⁇ M) and BSA (20 ⁇ M) in Tris (pH 7, 50 mM). The particles then were washed with three 1 mL volumes of water, and the captured proteins were electro-eluted onto an SDS polyacrylamide gel.
  • NIPAm/AAc particles appeared to have captured all of the lysozyme present in the solution, there was no indication that BSA had been bound non-specifically by the particles. As was observed with myoglobin, NIPAm particles, lacking the affinity bait, did not appear to significantly concentrate lysozyme.
  • FIG. 8 (A) SDS-PAGE analysis of particles- and +bait incubated with BSA and lysozyme: Lane 1) BSA and lysozyme solution prior to particle introduction. 2) NIPAm (- bait) supernatant (Out), 3) wash 3, 4) NIPAm (- bait) particles (In), 5) wash 2, 6) wash 1 , 7) NIPAm/AAc (+ bait) supernatant (Out), 8) wash 3, and 9) NIPAm/AAc (+ bait) particles (In).
  • MWCO molecular weight cut off
  • the solution consisted of 0.5 mg/mL of each of the following proteins: aprotinin (MW 6,500 Da, Sigma- Aldrich), lysozyme (MW 14,400 Da, Sigma- Aldrich), trypsin inhibitor (MW 21,500 Da, Invitrogen), carbonic anhydrase (MW 31,000 Da, Sigma- Aldrich), ovalbumin (MW 45,000 Da, Sigma-Aldrich), and BSA (MW 66,000 Da, Fisher Scientific) dissolved in Tris (pH 7. 50 mM).
  • aprotinin MW 6,500 Da, Sigma- Aldrich
  • lysozyme MW 14,400 Da, Sigma- Aldrich
  • trypsin inhibitor MW 21,500 Da, Invitrogen
  • carbonic anhydrase MW 31,000 Da, Sigma- Aldrich
  • ovalbumin MW 45,000 Da, Sigma-Aldrich
  • BSA MW 66,000 Da, Fisher Scientific
  • NIP Am/ AAc baited particles incubated with the protein solution, effectively captured and concentrated all protein molecules with MW less than ca. 21,500 Da, and did not bind any proteins with MW greater than 21,500 Da.
  • the MWCO resolution achieved with NIP Am/ AAc and NIPAm particles compares favorably, or exceeds, that associated with standard molecular sieving chromatography 41 .
  • NIP Am/ AAc particles were incubated with platelet derived growth factor B (0.003 mg/mL, 14,500 Da, Cell Signaling) and BSA (0.067 mg/mL) in Tris (100 mM pH 7) for one hour. Washing procedure was the same as described before. SDS PAGE in Figure 9 shows complete PDGF uptake and BSA exclusion.
  • PDGF is a representative model for low abundance low molecular weight protein present in the blood and PDGF-B concentration in blood is 3.3 ng/mL 42 .
  • FIG. 9 SDS PAGE analysis of particles + bait incubated with PDGF B and BSA. Lane 1) BSA and PDGF B, 2) NIP Am/ AAc supernatant (Out), 3) NIP Am/ AAc particles (In).
  • Figure 10 Flow cytometry analysis of (A) NIPAm and (B) NIP Am/ AAc particles incubated with FITC-labeled insulin aqueous solution and FITC-labeled insulin spiked in serum, respectively.
  • Figure 12. Uptake time course study (A) Mean values of the percentage, relative to the initial amount, of lysozyme in solution incubated with two quantities of NIP Am/ AAc particles as measured by RPPAs (three replicate analyses and standard deviation shown). (B) SDS-PAGE analysis of a lysozyme and BSA solution incubated with NIP Am/ AAc particles. Lane 1) BSA and lysozyme solution. 2-1 1) alternating supernatant (Out) and particles (In) for each of 5, 10, 20, 30. and 60 minutes incubation times. Lysozyme uptake is rapid and complete, while BSA exclusion is total.
  • NIPAM and NIPAm/AAc particles were evaluated by incubating the particles with a 1 : 10 v/v dilution of serum in water for 1 hour.
  • the trapped proteins were electrophoretically eluted from the particles under denaturing conditions and then trypsin digested.
  • the particles were heated in SDS sample buffer for 5 minutes at 100 °C and loaded on a 4-20 % Tris Glicine gel (Invitrogen). Bands below 30 kDa were cut and in-gel trypsin digestion was performed ' ' .
  • LC/ESI MS liquid chromatography/electrospray ionization tandem mass spectrometry
  • LTQ-Orbitrap mass spectrometer Thermo Fisher.
  • Reverse phase column was slurry-packed in-house with 5 ⁇ m, 20 A pore size Cl 8 resin (Michrom BioResources, CA) in 100 mm i.d. x 10 cm long fused silica capillary (Polymicro Technologies, Phoenix, AZ) with a laser-pulled tip.
  • the column was washed for 5 minutes with mobile phase A (0.1% formic acid) and peptides were eluted using a linear gradient- of 0% mobile phase B (0.1 % formic acid, 80% acetonitrile) to 50% mobile phase B in 50 minutes at 200 nl/min. then to 100% B in an additional 5 minutes.
  • the LTQ mass spectrometer was operated in a data-dependent mode in which each full MS scan was followed by five MS/MS scans were the five most abundant molecular ions were dynamically selected and fragmented by collision-induced dissociation (CID) using a normalized collision energy of 35%.
  • CID collision-induced dissociation
  • MS/MS data were matched against the NCBI (National Center for Biotechnology Information) human protein database with the program SEQUEST (Bioworks software. Thermo) using full tryptic cleavage constraints.
  • the list of identified proteins demonstrated that albumin and other high abundance serum proteins were not present in the particles.
  • the list of identified proteins indicates that the particles sequestered rare and small-sized serum proteins and peptides.
  • haptoglobin-related protein 4558 0723.0 1.12E-11 9.76E-01 10.22 39004.70
  • haptoglobin [Homo sapiens] 4826762.0 1.12E-11 3.70E+00 40.21 45176.59
  • alpha 1 globin [Homo sapiens] 4504347.0 1.12E-1 1 9.62E-01 10.16 15247.92
  • beta globin [Homo sapiens] 4504349.0 1.12E-11 9.36E-01 10.17 15988.29
  • pro-platelet basic protein 450598I.0 3.55E-14 5.61E+00 60.25 13885.42 precursor [Homo sapiens]
  • complement component 3 4557385.0 3.55E-14 9.80E-01 10.20 187045.30 precursor [Homo sapiensj small nuclear ribonucleoprotein polypeptide 4507129.0 3.55E-14 9.15E-01 10.19 10796.64
  • albumin precursor [Homo 450 2027.0 3.55E-14 9.28E+00 100.22 69321.63 sapiens]
  • A-gamma globin [Homo 28302131.0 3.55E-14 9.44E-01 10.14 16118.27 sapiens] platelet factor 4 (chemokine
  • H4 histone family member J 45043 15 0 3 55E . 14 L51 E+00 2(U 3 ⁇ 36(U 8 [Homo sapiens]
  • alpha 1 globin [Homo sapiens] 4504347.0 3.55E-14 9.15E-01 10.14 15247.92
  • CDK5 regulatory subunit associated protein 1 isoform b 28872784.0 3.55E-14 9.05E-01 10.12 56187.84
  • G-type receptor 1 [Homo 7656967.0 3.55E-14 1.56E+00 20.14 329276.70 sapiens] procollagen, type in, alpha 1 4502951 0 3 55E . 14 9 12E . O 1 1 (u 5 1384 70.20
  • Protein Sequestration by Particle Blocks Protease Degradation One of the major problems associated with biological fluids is the potential for sample degradation during collection, transport, storage and analysis. Endogenous clotting cascade enzymes, enzymes released from damaged cells, or exogenous enzymes (from contaminating bacteria) can contribute to the degradation of diagnostically important proteins.
  • Figure 13 Schematic drawing illustrating the ability of particles to protect proteins from enzymatic degradation.
  • NIPAm/AAc particles were incubated at 37°C in a pH 7 NH 4 HCO 3 (100 mM) solution containing lysozyme (0.5 mg/mL) and trypsin (0.05 mg/mL, Promega). Trypsin was selected for these studies based on its small size and the fact that the tryptic digestion of lysozyme would produce very characteristic cleavage products. The conditions used in this experiment would allow both lysozyme and trypsin to enter the particle.
  • NIPAm/AAc particles (+ bait) protect bound proteins from degradation by enzymes that may be present.
  • A NIPAm/AAc particles incubated with a solution containing lysozyme and trypsin for 1 hr: Lane 1) lysozyme, 2) lysozyme incubated with trypsin, 3) NIPAm/AAc supernatant (Out), 4) NIPAm/AAc particles (In), 5) BSA and lysozyme, 6) BSA and lysozyme + protease, 7) NIPAm/AAc particles supernatant (Out), and 8) NIPAm/AAc particles (In).
  • NIPAm/AAc particles incubated overnight with BSA, lysozyme, and trypsin Lane 1) lysozyme, 2) trypsin, 3) lysozyme + trypsin, 4) NIPAm/AAc supernatant (Out), 5) NIPAm/AAC particles (In), 6) lysozyme and BSA, 7) BSA and lysozyme + protease, 8) NIPAm/AAc supernatant (Out), and 9) NIPAm/AAc particles (In).
  • NIPAm/AAc particles were incubated at 37 0 C with a combination of BSA (0.5 mg/mL), lysozyme (0.5 mg/mL) and trypsin (0.05 mg/mL) in 100 raM NH 4 HCO 3 (pH7).
  • BSA 0.5 mg/mL
  • lysozyme 0.5 mg/mL
  • trypsin 0.05 mg/mL
  • the reaction was analyzed using SDS-PAGE after incubating 1 hr and overnight.
  • the majority of BSA had been digested after 1 hr and the band corresponding to full-length BSA had disappeared after incubating overnight (Figure 14B).
  • the NIPAm/AAC particles efficiently sequestered both lysozyme and trypsin, and protected lysozyme from proteolysis by trypsin.
  • the particles did not bind BSA, and the presence of low molecular weight bands in the supernatant after 1 hour and overnight incubation accompanied by the decrease in intensity of the band corresponding to full-length BSA indicates that BSA was not protected from degradation by trypsin.
  • Suppression of proteolytic activity by enzymes small enough to enter the particles, such as trypsin, may occur because immobilization of the enzymes by the charge-bait particle prevents them from binding substrate proteins or may be the result of steric hindrance associated with trapping of the substrate by the affinity-bait groups in the particle thus preventing enzymes from productively binding target proteins inside the particle.
  • the functional state of the proteins sequestered by the charge-bait may be similar to that of proteins arrested using a precipitating fixative treatment.
  • NIPAm particles lane 1) NIPAm particles supernatant (Out), 2) NIPAm particles (In), 3) + trypsin NIPAm supernatant (Out), 4) + trypsin NIPAm particles (In), 5) + trypsin, 6) BSA + reduced and alkylated protein solution incubated with NIPAm particles, supernatant (Out), 7) NIPAm particles (In).
  • hydrogel bait-containing particles as a new tool for harvesting and concentrating small molecule analytes and biomarker candidates from biological fluids, allowing high throughput analysis of low- abundance and low molecular weight components.
  • These nanoparticles present a rapid and straightforward workflow for direct utility in raw body fluids, while the work herein described the particles with a negative charge that preferentially bind cationic species, positively charged particles such as a NIPAm/al IyI amine copolymer could be used to selectively harvest and concentrate anionic species from biological fluids.
  • hydrophobic metabolites could be captured for comprehensive metabolomic studies by using more hydrophobic particles such as NIPAm/styrene copolymers.
  • Analyte-specific chemical or protein or nucleic acid affinity baits can be incorporated.
  • boronate-containing particles which are known to bind saccharides, would be utilized to sequester glycoproteins from solution 4 . Consequently, NIPAm-allylamine copolymers are currently being synthesized that contain a bait for anionic proteins.
  • p-vinylphenylboronic acid (VPBA) is under consideration as a copolymer for harvesting of sugars and nucleic acids.
  • affinity baits such as triazinil-based reactive dyes (that have affinity towards proteins), hexadecylamine (for lipids uptake) and cyclodextrins (able to associate small molecules) are being noncovalently or covalently immobilized within the particles.
  • affinity baits such as triazinil-based reactive dyes (that have affinity towards proteins), hexadecylamine (for lipids uptake) and cyclodextrins (able to associate small molecules) are being noncovalently or covalently immobilized within the particles.
  • the bait chemistry described above to harvest the following small metabolites L-Dopa, homogentisic acid. Dopamine, Dopac and 5-hydroxyindoleacetic acid. This extends the utility of the technology to the realm of metabolomics.
  • NiPam particles by temperature changes retained their native conformational state 47 Consequently possible means of eluting native proteins from the particles include modifying the temperature or pH of the solution, increasing the ionic strength, or electroeluting the proteins under non denaturing conditions, in the absence of detergent.
  • SUPPORTING INFORMATION AVAILABLE Available in the Supplementary Information are details on particles synthesis protocol, SDS PAGE analysis on molecular sieving properties and enzymatic degradation, and tables (Table Sl and S2) listing proteins (with peptide coverage lists) identified via LC-MS/MS (ESI) on material electroeluted from NIPAm and NIP Am/ AAc particles. This material is available free of charge via the Internet at http://pubs.acs.org.
  • NIPAm N-isopropylacrylamide
  • BIS BIS
  • SDS 0.057 g
  • the solution was placed in a round bottom 250 ml 3 -neck flask fitted with a condenser and thermometer at a medium stir rate (Coning magnetic stirrer). The solution was heated to 70 0 C for 1 hour under a nitrogen atmosphere.
  • NIP Am/acrylic acid (AAc) particles were fabricated using the same reaction condition as NIPAm particles above. The initial monomer solution was obtained by dissolving NIPAm (1.3 g), BIS (0.10 g), and AAc (0.072 g) in 150 ml water. All particles were purified via dialysis (Spectra/Por 7 dialysis membranes, MWCO 10,000, VWR) against frequent changes of stirring water for 2 weeks at 4 0 C.
  • a core containing affinity bait moieties
  • a NIPAm shell is surrounded by a NIPAm shell.
  • the sieving capability of the NIPAm shell will shield 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.
  • a shell solution was prepared by dissolving NIPAm, 0.02 molar equivalents each of BIS and SDS in H 2 O and filtering the solution through a membrane filter. The solution was degassed under vacuum for several minutes and then purged with nitrogen for 2 h at room temperature with stirring.
  • the core solution was prepared by dissolving NIPAm, 0.08 molar equivalents of AAc and 0.02 molar equivalents of BIS in H?O and then the solution was filtered. The core solution was then degassed and purged with nitrogen at 70 0 C as described for the preparation of the NIPAm particles. Once the solution had equilibrated at 70°C and stirred under nitrogen for 1 hour, APS (0.005 molar equivalents) was added to the core solution. After the NIPAm/AAc core reaction had been allowed to incubate for 3 h at 70°C under nitrogen, the shell solution was added to the reaction flask followed by and additional aliquot of APS.
  • the reaction was then allowed to stir at 70 0 C under nitrogen for an additional 3 h. At which point, the reaction was removed from heat and allowed to stir overnight under nitrogen at room temperature. The particles were then collected and washed in the same fashion as described for the NIPAm particles.
  • Core shell particles have the same molecular sieving cut off as NIPAm/AAc. Light scattering measurement of core shell particles diameter gave a value of 1048 run for the NIPAm/AAc core and 1198 ran when the NIPAm shell was added. In order to determine if core shell particles had the same molecular weight cut off (MWCO) as NIPAm/AAc particles, a solution of protein molecular weight markers was used.
  • MWCO molecular weight cut off
  • the solution consisted of 0.5 mg/mL of each of the following proteins: aprotinin (MW 6,500 Da, Sigma-Aldrich), lysozyme (MW 14,400 Da, Sigma- Aldrich), trypsin inhibitor (MW 21,500 Da, Invitrogen), carbonic anhydrase (MW 31,000 Da, Sigma-Aldrich), ovalbumin (MW 45,000 Da, Sigma-Aldrich), and BSA (MW 66,000 Da, Fisher Scientific) dissolved in Tris (pH 7, 50 mM). Incubation time was 1 hour and particles were washed as described in the manuscript. SDS PAGE analysis reported in Figure Sl shows a substantial agreement in MWCO values for the two types of particles.
  • Core shell particles have the same molecular weight sieving as NIPAm/AAc particles.
  • Control protein solution was incubated with NIPAm/AAc and core shell particles.
  • Core shell particles protect lysozyme from chymotrypsin enzymatic degradation.
  • Lysozyme digestion was performed in 100 mM Tris HCl containing 10 mM CaC12, pH 7.8, at 30 0 C for 3 hours. Core shell particles were incubated with lysozyme and trypsin in the digestion conditions described above.
  • Lane 1 lysozyme, 2) chymotrypsin, 3) lysozyme + chymotrypsin, 4) lysozyme + chymotrypsin incubated with core shell particles, supernatant (Out), 5) lysozyme + chymotrypsin incubated with core shell particles, particles (In), 6) lysozyme + BSA + chymotrypsin, 7) lysozyme + BSA + chymotrypsin incubated with core shell particles, supernatant, 8) lysozyme + BSA + chymotrypsin incubated with core shell particles, particles (In), 9) lysozyme + BSA.

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Abstract

L'invention porte sur un procédé pour récolter des biomarqueurs suivant lequel une molécule appât d'affinité est introduite dans du N-isopropylacrylamide pour produire une particule qui effectuera au moins trois fonctions indépendantes en l'espace de quelques minutes. Ceci induit un criblage de dimension moléculaire, une capture d'affinité de toutes les molécules cibles en phase de solution, et une protection complète de protéines récoltées vis-à-vis d'une dégradation enzymatique, les analytes capturés pouvant alors être facilement électro-élués pour analyse.
PCT/US2008/012666 2007-11-09 2008-11-10 Particules d'hydrogel intelligentes ; récolte de biomarqueurs ; purification d'affinité en une étape, exclusion diffusion et protection contre la dégradation Ceased WO2009064389A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114306376A (zh) * 2022-01-04 2022-04-12 北京理工大学 一种花生过敏原蛋白亲和吸附试剂、制备方法及其应用

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* Cited by examiner, † Cited by third party
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GB0311664D0 (en) * 2003-05-21 2003-06-25 Univ Manchester Polymeric hollow nanospheres
GB0515625D0 (en) * 2005-07-29 2005-09-07 Univ Manchester Hydrogel particle
WO2007038523A2 (fr) * 2005-09-27 2007-04-05 Center For Applied Proteomics And Molecular Medicine Methode d'isolement d'analytes provenant d'un echantillon

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114306376A (zh) * 2022-01-04 2022-04-12 北京理工大学 一种花生过敏原蛋白亲和吸附试剂、制备方法及其应用
CN114306376B (zh) * 2022-01-04 2023-08-08 北京理工大学 一种花生过敏原蛋白亲和吸附试剂、制备方法及其应用

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