REFERENCE TO RELATED APPLICATIONS

This application claims an invention which was disclosed in Provisional Application Number 61/218,670, filed 6/19/2009, entitled "Bait Chemistries in Hydrogel Particles for Serum Biomarker Analysis". The benefit under 35 USC §119(e) of the United States provisional application is hereby claimed, and the aforementioned application is hereby incorporated herein by reference.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under Grant No. DE-FC52- 04NA25455, awarded by the Department of Energy. The government has certain rights in the invention.

FIELD OF THE INVENTION

The invention pertains to the incorporation of dye affinity baits into hydrogel particles used to concentrate, purify and protect small and labile analytes from degradation in blood and other body fluids. This invention describes the identification of novel non- triazine organic dye chemistries that can be used as affinity baits to capture proteins and other biomolecules useful in the fields of medical diagnostics, environmental science, toxicology, and infectious disease. Incorporation of unique affinity dye compounds within hydrogel capture particles improves analyte yield and preanalytical precision, and stabilizes the analyte against degradation, while increasing measurement sensitivity. The particles in this invention can be used for routine clinical testing as well as for discovery of low abundance disease biomarkers. Example hydrogel particles containing new high affinity bait chemistries were used to identify a new set of human serum biomarkers.

BACKGROUND OF THE INVENTION There has recently been a surge of interest in the value and clinical potential of proteomic biomarkers. A general belief in the medical community is that the earlier a disease is treated, the more successful the therapeutic outcome. Consequently, the routine clinical availability of biomarker tests specific for early- stage diseases has tremendous potential to dramatically improve public health, even using currently utilized therapeutic modalities. For example, clinical oncologists expect that biomarker detection of pre- metastatic solid tumors of the breast, lung, ovary, and colon could lead to a significant improvement in survival. Unfortunately, despite the urgent clinical need, in the past ten years, for all disease categories combined, only a handful of novel biomarkers have graduated to routine clinical use. The slow biomarker pipeline persists despite considerable efforts within diagnostics research. The reasons for this failure stem from fundamental technical and biologic roadblocks spanning the biomarker development pipeline from biomarker identification and measurement to initial clinical validation. Two of these major roadblocks are:

Low Abundance: Disease-relevant biomarkers may exist in exceedingly low concentrations within a complex mixture of body fluid proteins containing high abundance proteins such as albumin.

Instability: Immediately after the blood or other body fluid is collected (e.g. byvenipuncture), degradation of proteins can occur, which is mediated by endogenous or exogenous proteinases.

SUMMARY OF THE INVENTION

Hydrogel particles are used to concentrate, partially purify and protect small and labile analytes from degradation in blood and other body fluids. Protein analytes extracted from the particles are analyzed by ELISA, western blotting, reverse phase protein arrays, and mass spectrometry. This invention contains new bait strategies that can extend the classes of analytes captured by the particles and a workflow involving N-isopropylacrylamide particles with a set of affinity bait examples, gel electrophoresis results for common protein biomarkers as well as mass spectrometry for the screening of sera from patient.

BRIEF DESCRIPTION OF THE DRAWING

Figure 1. A general description of bait chemistries and corresponding target classes of molecules.

Figure 2a -2b. Low abundance clinical protein biomarkers captured and enriched by particles. The table was constructed by incubating clinically relevant low abundance labile proteins with hydrogel particles containing 17 different examples of unique affinity baits. SDS-PAGE and silver-staining experiments were used to determine the ability of each dye to bind these proteins. The included table indicates the affinity of each bait for specific proteins.

Figure 3a-3c.Unique set of chemical affinity baits incorporated in the particle matrices demonstrate affinity capture of a subset of analytes. Particles contain the following chemical affinity baits 1 Alizarin Blue Black B; 2 Disperse Blue 3; 3 Remazol Brillan Blue R; 4 Pigment Red 177; 5 Acid Black 48; 6 Disperse Yellow 3; 7 Naphtol Blue Black B (AbI); 8 Disperse Orange 3; 9 Disperse Yellow 9; 10 Rhodamine 123; 11 Toluidine Blue O; 12 Acryic Acid, 13 Pararosaniline Base, 14 Brillant Blue R; 15 Acid Blue 22, 16 Vinyl Sulfonic Acid; 17 Cibacron Blue F3GA

Figure 4a-4b: New proteins never sequenced in serum before (not present in the HUPO Plasma Proteome Project comprehensive list, ttp://www.hupo.org/research/hppp/) were sequenced by nanospray tandem mass spectrometry after serum processing with particles, particles carrying the following baits 1 Alizarin Blue Black B; 2 Disperse Blue 3; 3 Remazol Brillan Blue R; 4 Pigment Red 177; 5 Acid Black 48; 6 Disperse Yellow 3; 7 Naphtol Blue Black B (AbI); 8 Disperse Orange 3; 9 Disperse Yellow 9; 10 Rhodamine 123; 11 Toluidine Blue O; 12 Acryic Acid, 13 Pararosaniline Base, 14 Brillant Blue R; 15 Acid Blue 22, 16 Vinyl Sulfonic Acid; 17 Cibacron Blue F3GA were used. Aliquots of 100 μL of nanoparticles (1 mg/mL) were incubated with 100 μL of serum for 30 minutes, separated by centrifugation and chemically eluted. Proteins listed in this table were found in at least one of the affinity bait loaded particles. All proteins shown were selected to have a p value (probability of randomized identification) <= 0.05.

Figure 5 : Protein sequenced by nanospray tandem mass spectrometry after serum processing with particles. Aliquots of 500 μL of particles (1 mg/mL) containing Cibacron Blue F3GA affinity bait were incubated with 500 μL of serum for 30 minutes, separated by centrifugation and eluted with 70% ACN - 10% ammonium hydroxide. Proteins listed in column 1 were found in the sample processed with Cibacron Blue F3GA functionalized particles and not in raw serum. All proteins shown are selected to have a p value (probability of randomized identification) <= 0.01.

Figure 6: Protein sequenced by nanospray tandem mass spectrometry after serum processing with particles. Aliquots of 500 μL of particles (1 mg/mL) containing Cibacron Blue F3GA affinity bait were incubated with 500 μL of serum for 30 minutes, separated by centrifugation and eluted with 70% ACN - 10% ammonium hydroxide. Proteins listed in column 1 were found only in the Cibacron Blue F3GA loaded particles and not in the particles containing alternative chemical affinity dyes. All proteins shown are selected to have a p value (probability of randomized identification) <= 0.01.

Figure 7: Protein sequenced by nanospray tandem mass spectrometry after serum processing with particles. Aliquots of 500 μL of particles (1 mg/mL) containing Disperse Yellow 9 affinity bait were incubated with 500 μL of serum for 30 minutes, separated by centrifugation and eluted with 70% ACN - 10% ammonium hydroxide. Proteins listed in column 1 were found in the sample processed with Disperse Yellow 9functionalized particles and not in raw serum. All proteins shown are selected to have a p value (probability of randomized identification) <= 0.01.

Figure 8: Protein sequenced by nanospray tandem mass spectrometry after serum processing with particles. Aliquots of 500 μL of particles (1 mg/mL) containing Disperse Yellow 9 affinity bait were incubated with 500 μL of serum for 30 minutes, separated by centrifugation and eluted with 70% ACN - 10% ammonium hydroxide. Proteins listed in column 1 were found only in the Disperse Yellow 91oaded particles and not in the particles containing alternative chemical affinity dyes. All proteins shown are selected to have a p value (probability of randomized identification) <= 0.01.

Figure 9: Protein sequenced by nanospray tandem mass spectrometry after serum processing with particles. Aliquots of 500 μL of particles (1 mg/mL) containing Disperse Yellow 3 affinity bait were incubated with 500 μL of serum for 30 minutes, separated by centrifugation and eluted with 70% ACN - 10% ammonium hydroxide. Proteins listed in column 1 were found in the sample processed with Disperse Yellow 3functionalized particles and not in raw serum. All proteins shown are selected to have a p value (probability of randomized identification) <= 0.01.

Figure 10: Protein sequenced by nanospray tandem mass spectrometry after serum processing with particles. Aliquots of 500 μL of particles (1 mg/mL) containing Disperse Yellow 3 affinity bait were incubated with 500 μL of serum for 30 minutes, separated by centrifugation and eluted with 70% ACN - 10% ammonium hydroxide. Proteins listed in column 1 were found only in the Disperse Yellow 3 loaded particles and not in the particles containing alternative chemical affinity dyes. All proteins shown are selected to have a p value (probability of randomized identification) <= 0.01.

Figure 1 Ia-I Ib: Protein sequenced by nanospray tandem mass spectrometry after serum processing with particles. Aliquots of 500 μL of particles (1 mg/mL) containing remazol brilliant blue R affinity bait were incubated with 500 μL of serum for 30 minutes, separated by centrifugation and eluted with 70% ACN - 10% ammonium hydroxide. Proteins listed in column 1 were found in the sample processed with remazol brilliant blue R functionalized particles and not in raw serum. All proteins shown are selected to have a p value (probability of randomized identification) <= 0.01.

Figure 12a- 12b: Protein sequenced by nanospray tandem mass spectrometry after serum processing with particles. Aliquots of 500 μL of particles (1 mg/mL) containing remazol brilliant blue R affinity bait were incubated with 500 μL of serum for 30 minutes, separated by centrifugation and eluted with 70% ACN - 10% ammonium hydroxide. Proteins listed in column 1 were found only in the remazol brilliant blue R loaded particles and not in the particles containing alternative chemical affinity dyes. All proteins shown are selected to have a p value (probability of randomized identification) <= 0.01.

Figure 13: Protein sequenced by nanospray tandem mass spectrometry after serum processing with particles containing a mixture of remazol brilliant blue R, disperse yellow 3, and disperse yellow. Aliquots of 500 μL of particles (1 mg/mL) containing Cibacron Blue F3GA affinity bait were incubated with 500 μL of serum for 30 minutes, separated by centrifugation and eluted with 70% ACN - 10% ammonium hydroxide. Proteins listed in column 1 were found in the sample processed with the mixture of functionalized particles and not in raw serum. All proteins shown are selected to have a p value (probability of randomized identification) <= 0.01. Figure 14: Protein sequenced by nanospray tandem mass spectrometry after serum processing with particles containing a mixture of remazol brilliant blue R, disperse yellow 3, and disperse yellow. Aliquots of 500 μL of particles (1 mg/mL) containing the mixture affinity bait were incubated with 500 μL of serum for 30 minutes, separated by centrifugation and eluted with 70% ACN - 10% ammonium hydroxide. Proteins listed in column 1 were found only in the mixture loaded particles and not in the particles containing alternative chemical affinity dyes. All proteins shown are selected to have a p value (probability of randomized identification) <= 0.01.

Figure 15: NIPAm/Remazol Brilliant Blue R particles, incubated with 30 mL of solution of human growth hormone (hGH) (0.242 ng/mL) in synthetic urine, sequestered all hGH present in solution and increased the concentration of hGH more than 60 times (14.9 ng/mL).

Figure 16. NIP Am/ Acid Black 48 particles completely deplete the supernatant and amplify > 30 fold the concentration of hGH spiked in synthetic urine at a concentration of 1 ng/mL. Particles were incubated with the hGH solution, separated by centrifugation, and supernatant and particle content was measured by Immulite 1000.

Figure 17. NIPAm/ Acid Black 48 particles have a yield of 95% of recovering hGH in synthetic urine. Particles were incubated with 1 mL of synthetic urine containing 8.74 ng/mL hGH, separated by centrifugation, the particle content was eluted, reconstituted in the same starting volume. Initial solution and particle eluate were measured by Immulite 1000.

Figure 18: NIP Am/Disperse Yellow 9 particles have a yield of 95% of recovering interleukin 6 (IL 6) in synthetic perspiration. Particles were incubated with 50 μL of synthetic perspiration containing 7912.5 pg/mL IL6 and separated by centrifugation; the particle content was eluted and reconstituted in the same starting volume. IL6 concentration in the eluate was 7875 pg/mL. Initial solution and particle eluate were measured by Immulite 1000.

Figure 19: SDS PAGE analysis showed that particles functionalized with two affinity baits in the core and in the shell acquire a behavior that is the sum of the two baits, and the external, shell bait does not interfere with the binding of proteins to the inner, core bait. Lane 1, serum; 2 vinyl sulfonic acid particles; 3 cibacron blue F3GA particles; 4 N- isopropylacrylamide shell - cibacron blue F3GA core particles; 5 cibacron blue F3GA core - vinylsulfonic acid shell particles.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy.

Example 1. Particle synthesis and dye coupling

Hydrogel particles capture particles containing novel affinity bait can be synthesized as both core and core-shell structures. The outer shell, included in the core-shell structure allows for size-sieving of higher molecular weight proteins in complex biofluids. In the subject invention both the core and the shell can contain the immobilized dye molecules or derivatives thereof.

PoIy(NIPAm-CO-AAc) Core particle synthesis N-isopropylacrylamide (NIPAm; Sigma-Aldrich) (4.750 g, 0.042 mol), N ₅ N methylenebis-acrylamide (BIS Sigma-Aldrich;) (0.400 g, 0.0026mol), and Acrylic acid (AAc; Sigma-Aldrich); (.525 μL), 0.0073 mol) were dissolved in 500 mL of H ₂ O and then filtered using a nitrocellulose membrane disc filter ( pore size 0.45 μm, Millipore) and transferred in a three neck round bottom flask. The solution was purged with nitrogen for 1 hour at room temperature , at medium stir rate, and then heated to 7O ⁰ C. The basis for this specific step in the polymerization method of the poly(NIPAm-co-AA) core can be found elsewhere. Potassium Persulfate (KPS, Sigma-Aldrich) ( 0.276 g, 0.00 lmol) was dissolved in 5 mL of H ₂ O and was added to the solution to initiate polymerization. The reaction was maintained at 7O ⁰ C under nitrogen for 6 h. Particles were washed five times via centrifugation (19000 rpm, 50 minutes, at Room Temperature) to eliminate un- reacted monomer and then re-suspended in 600 ml of H ₂ O.

PoIy(NIPAm-Co-AAc) Core-Shell particle synthesis

N-isopropylacrylamide (NIPAm; Sigma-Aldrich) (1.55 g, 0.013 mol), ,bis- acrylamide (BIS; Sigma-Aldrich) (162 mg,0.0010 mol) and KPS(0.092 g,,0.0003 mmol) were dissolved in 150 mL of H ₂ O , and then passed through a 0.45 μm filter. The solution was purged with nitrogen for 2 h at room temperature and medium stir rate. 200 mL of p-NIPAm-co-AAc core particles, were heated at 7O ⁰ C and purged under nitrogen for 1 hour. Then 25 ml the solution containing NIPAm and BIS was added to the particles and allowed to react for 15 min at 7O ⁰ C, after which the remaining 125 mL of shell solution was added in 25 mL aliquots over a period of two hours and 30 min. The reaction was maintained at 7O ⁰ C under nitrogen for 3 hrs, then cooled overnight and washed as previously described. Washed particles were re-suspended in 200 mL of H ₂ O.

Synthesis of NIPAm particles, functionalized with amino-dyes-baits in water media. (Pigment Red 177; Orange 3; Pararosanilina; Acid blue 22; Acid black 48; Remazol brilliant blue; Alizarin blue black B; Brilliant Blue; Toluidine Blue; Rhodamine 123; Disperse yellow 3; Disperse Blue 3 ).

Dye molecules containing primary amine group were coupled via condensation to the carboxylic group of acrylic acid group, bonded covalently to NIPAm particles.

10 mL of NIPAm /AAc particles were centrifuged (16.1 rcf,25 C, 15 minutes) and re- suspended in 10 mL OfNaH ₂ PO ₄ at pH 5. The particles, 1 mL of SDS 1% (w/v), 824 mg of EDC (N-(3Dimethylaminopropyl)N' ethyl carbodiimide hydrochloride; Fluka Analytical) and 612 mg of NHS (N-Hydroxy succinimide; Sigma- Aldrich) were mixed in a three-neck round bottom flask, and allowed to react at room temperature and medium stir rate. After 15 min the reaction was stopped by centrifugation and re-suspension in 20 mL OfNa ₂ HPO ₄ 0.2 M (pH > 8). An excess amount of dye was dissolved in 180 mL Na ₂ HPO ₄ 0.2 M, filtered via a 0.22 μm CA filter (Corning, NY, USA) and added to the activated particles, the reaction was maintained at room temperature at a medium stir rate overnight. The amount of dye required for the reaction was calculated so that the ratio of dye moles/ AAc moles was 10:1, while the number of acrylic acid moles was 0.0000121. Figure 3a-3c indicates the amount of dye used for the reaction. Particles were washed with water to eliminate un-reacted dye followed by centrifugation at 16.1 rcf, 25uC, 15 minutes. Supernatant was discarded and particles resuspended in 10 mL of water.

Synthesis of NIPAm particles 13.80% AAc, functionalized with amino-dyes-baits in Dimethylformamide (DMF) media.

(Orange 3; Pararosanilina; Disperse yellow 9,Pigment Red 177)

Amino dyes were coupled with NIP Am/ AAcNP by phosponium/uranium activation in Dimethylformamide media. 10 mL of NIPAm /AAc was freeze-dried and re-suspended in 10 rnL DMF (Dimethylformamide; Sigma- Aldrich). The particle solution was purged with nitrogen for 15 min at room temperature and medium stir rate. Then 68.84 mg of HBTU, 24.52 mg of HOBT and 40 μL of Nmm (N-Methylmopholine; Fluka Biochemika) were added to the particle solution and allowed to react at room temperature at medium stir rate for 5 minutes (the amount of HBTU (Peptides International) and HOBt (GL Biochem, Shanghai) was calculated considering a ratio reagent /AAc of 1.5, while the number of acrylic acid moles was 0.0000121). The reaction was maintained under nitrogen. An excess amount of dye was dissolved in DMF and added to the three-neck round bottom flask. The amount of each dye required for the reaction was calculated so that the ratio of dye moles/ AAc moles was 10:1. The reaction was maintained under nitrogen for 6 hours. Particles were washed five times with decreasing concentrations of DMF, to eliminate un-reacted dye and to favor particle re-hydration, via centrifugation at 16.1 rcf, 25uC, 15 minutes. Supernatant was discarded and particles were re-suspended in 10 mL of water

Synthesis of NIPAm/ Vinyl Sulfonic Acid Particles

N-isopropylacrylamide (NIPAm; Sigma-Aldrich) (0.475 g, 0.0042 mol), N ₅ N methylenebis-acrylamide (BIS Sigma-Aldrich;) (0.040 g, 0.026mol), and Vinyl Sulfonic Acid (VSA; Sigma-Aldrich); (.80.4 μL), 0.00073 mol) were dissolved in 60 mL of H ₂ O and then filtered using a nitrocellulose membrane disc filter ( pore size 0.45 μm, Millipore) and transferred in a three neck round bottom flask. The solution was purged with nitrogen for 1 hour at room temperature , at medium stir rate, and then heated to 7O ⁰ C. KPS ( 0.028 g, 0.000 lmol) was dissolved in 2.5 mL of H ₂ O and was added to the solution to initiate polymerization. The reaction was maintained at 7O ⁰ C under nitrogen for 6 h. Particles were washed five times as previously described and then re-suspended in 60 ml of H ₂ O.

Synthesis of boronic acid containing particles. Particles containing boronic acid were obtained by precipitation polymerization. 0.93 g of NIPAm and 0.028 g of BIS were dissolved in 30 mL of water, vacuum filtered and thoroughly purged with nitrogen. 0.066 g of vinylphenyl boronic acid (VPBA) was dissolved in 2 mL of ethanol and added to the water solution, which was further purged with nitrogen under constant stirring. Temperature was raised to 75°C and kept constant for 15 minutes. 0.005 g of potassium persulfate (KPS) was added to the solution to initiate the reaction. The reaction was maintained at 75°C for 4 hours under nitrogen and continuous stirring. Particles were studied with light scattering and showed temperature and pH sensitivity suggesting the formation of hydrogel and the incorporation of VPBA. Boronic acid content of the particles will be quantified via the carmine assay.

Example 2. Dyes bind different low abundance protein by SDS PAGE.

As shown in Figure 2a-2b specific classes of dyes with specific chemical side chain substitutions preferentially bind clinically relevant analytes. Some dye classes bind a limited set of analytes while others have larger spectrum of binding affinities. The chemical structure and the choice of the substitutions strongly determine the binding affinity and selectivity of the dye baits.

Aliquots of particles were incubated with a set of clinical proteins shown in Figure 2a-2b for 30 minutes room temperature. After incubation, the particles were centrifuged (7 min, 25 ⁰ C, 16 100 rcf), the supernatant was saved and the particles were washed three times by resuspending the pellets in 1 mL of water and centrifuging (7 min, 25 ⁰ C, 16 100 rcf). The particles and supernatants were then resuspending in 2% sample buffer and loaded directly on a 4-20% Tris-Glycine gel. The particles were retained in the stacking region of the gel while the proteins were electro-eluted and resolved in the gel. Proteins were detected using silver staining.

Hydrogel particles containing the following dyes: 1 Alizarin Blue Black B; 2 Disperse Blue 3; 3 Remazol Brilliant Blue R; 4 Pigment Red 177; 5 Acid Black 48; 6 Disperse Yellow 3; 7 Naphtol Blue Black B (AbI); 8 Disperse Orange 3; 9 Disperse Yellow 9; 10 Rhodamine 123; 11 Toluidine Blue O; 12 Acrylic Acid, 13 Pararosaniline Base, 14 Brilliant Blue R; 15 Acid Blue 22, 16 Vinyl Sulfonic Acid; 17 Cibacron Blue F3GA were used for the SDS page binding studies (Figure 2a-2b). A summary of the protein binding was used to characterize the resulting binding. Complete binding resulted when all of the specific protein of interest was captured and eluted from the particle. Partial binding resulted when a fraction of the protein remained in the supernatant while the remaining fraction of the protein was captured by the particles. "Not-captured" resulted when the particles did not capture any protein. All of the protein remained in the supernatant.

EXAMPLE 3. Particles increase the sensitivity of clinical grade immunoassay.

Aliquots of particles were incubated with aliquots of synthetic urine and synthetic sweat for 30 minutes room temperature under slow rotation. After incubation, the particles were centrifuged (7 min, 25 ⁰ C, 16 100 rcf), the supernatant was saved and the particles were washed three times by resuspending the pellets in 1 mL of water and centrifuging (7 min, 25 ⁰ C, 16 100 rcf). The particles were then incubated with elution buffers (70% acetonitrile, 10% ammonium hydroxide). Incubations with 30 mL of Surine were performed with 50 mL centrifuge tubes (Nalgene) and particles were separated from urine by centrifugation (45 min, 25 ⁰ C, 18 000 revolutions per minute (rpm)) and washed as described above.

Elution of captured analytes from the particles

Hydrogel affinity bait capture particles can harvest target analytes, as demonstrated for a variety of analytes, a variety of chemical baits, and a variety of biologically relevant fluids such as urine, sweat and serum. The washed pellet of particles was incubated for 15 min at room temperature with proper amounts of elution buffers. After incubation, the particles were centrifuged (7 min, 25 ⁰ C, 16 100 rcf) and the eluate was saved. The elution step was repeated twice and the eluates were pooled together. Eluate was freeze-dried.

The concentration of hGH eluted from particles was measured using the Immulite 1000 Growth Hormone System (Siemens Medical Solution Diagnostic). Eluates were diluted in GH Sample Diluent, and assayed according to the manufacturer's instructions.

Results

Aliquots of 1 mL of synthetic urine containing human growth hormone at a concentration of 8.7 ng/mL were incubated with 1 mL of N-isopropylacrylamide/acid black 48. Particles were eluted with 70% acetonitrile-10% ammonium hydroxide and the eluted proteins were resuspended in the same volume as the initial solution (1 mL). The supernatant was completely depleted from the target analyte (hGH). The overall yield of the process approached (95%). (Figure 1)

In order to verify that the efficiency of the capture -elution process was not dependent on one particular analyte and one particular body fluid, 50 μL of artificial perspiration spiked with interlukin-6 at a concentration of 7912.5 pg/mL were processed with 50 μL of N-isopropylacrylamide/disperse yellow 9 particles (1 mg/mL). Proteins were eluted with 70% acetonitrile-10% ammonium hydroxide and the eluted proteins were resuspended in the same volume as the initial solution. The overall yield for this process was greater than 95%. (Figure 2a-2b) N-isopropylacrylamide - Acid black 48 particles increase the sensitivity of a clinical grade immunoassay (Immulite 1000). Human growth hormone was spiked in 10 mL of synthetic urine and the solution was incubated with 1 mL of acid black 48 functionalized particles. Particles were separated by centrifugation and washed with water. Proteins were chemically eluted and freeze dried. Proteins were resuspended in a volume of 300 μL and analyzed with Immulite 1000. The concentration factor achieved exceeded 30x the starting concentration (Figure 3a-3c).

Remazol Brilliant Blue-R particles were incubated with 30 mL of synthetic urine containing human growth hormone at a concentration of 0.242 ng/mL. Proteins eluted from the washed particles were resuspended in 300 mL. The concentration factor achieved was higher than 60 times (Figure 4a-4b).

EXAMPLE 4. Mass Spectrometry - Bait loaded particle incubation with serum.

The novel bait chemistries can be used to sequester, concentrate and preserve low abundance serum proteins. Application of the subject invention to serum biomarker discovery using mass spectrometry shows utility for the discovery of a large set of proteins of diagnostic relevance that were previously unknown to exist in blood.

Aliquots of 500 μL of serum were diluted 1 :2 with 50 mM TrisHCl pH 7 and incubated with 500 μL of bait loaded particles listed in Figure 2a-2b. Particle-serum incubation time was 30 minutes. Particles were separated by centrifugation (16.1 rcf, 25 C, 10 minutes) and washed with 1 mL of 0.5 X PBS. After centrifugation (16.1 rcf, 25 C, 10 minutes) the supernatant was discarded and the pellet resuspended in 0.5 X PBS / 20% acetonitrile. The suspension was centrifuged again (16.1 rcf, 25 C, 10 minutes) and the supernatant discarded. The pellet was incubated for 5 minutes at 100 ⁰ C with 600 μL of elution buffer constituted by 1% TFA, 0.1 M Urea 80% ACN and then centrifuged (16.1 rcf, 25 C, 10 minutes). The elution step was repeated twice and the eluates were combined.

NIPAm/CB particles were washed twice with 0.25M NaSCN and eluted twice with 70% ACN, 10% NH4OH.

NanoRPLC-MS/MS analysis

Eluates from the particles were analyzed by mass spectrometry. Proteins dried with SpeedVac were reconstituted in 8 M urea, reduced by 10 mM DTT, alkylated by 50 mM iodoacetamide, and digested by trypsin at 37 ⁰ C overnight. Tryptic peptides were further purified by Zip-Tip (Millipore) and analyzed by reversed-phase liquid chromatography nanospray tandem mass spectrometry (LC-MS/MS) using an LTQ- Orbitrap mass spectrometer (ThermoFisher). After sample injection by autosampler, the Ci8 column (0.2x50mm, Michrom Bioresources, Inc.) was washed for 2 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 500 nanoliter/min, then to 100% mobile phase B for 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 where the five most abundant molecular ions were dynamically selected for collision-induced dissociation (CID) using a normalized collision energy of 35%. Tandem mass spectra were searched against NCBI human database with SEQUEST using tryptic cleavage constraints. High-confidence peptide identifications were obtained by applying the following filter criteria to the search results: Xcorr versus charge >= 1.9, 2.2, 3.5 for 1+, 2+, 3+ ions; ΔCn > 0.1; probability of randomized identification <= 0.01.

Results

Particles carrying the following baits were screened: 1 Alizarin Blue Black B; 2 Disperse Blue 3; 3 Remazol Brilliant Blue R; 4 Pigment Red 177; 5 Acid Black 48; 6 Disperse Yellow 3; 7 Naphtol Blue Black B (AbI); 8 Disperse Orange 3; 9 Disperse Yellow 9; 10 Rhodamine 123; 11 Toluidine Blue O; 12 acrylic Acid, 13 Pararosaniline Base, 14 Brilliant Blue R; 15 Acid Blue 22, 16 Vinyl Sulfonic Acid; 17 Cibacron Blue F3GA (3). Aliquots of 100 μL of particles were incubated with 100 μL of serum diluted 1 :2 with 50 mM Tris HCl pH 7. Particles were allowed to incubate with serum for 30 minutes and then separated by centrifugation. Protein captured by the particles were chemically eluted, trypsin digested and sequenced by mass spectrometry.

Results reported in Figure 4a-4b demonstrated that most of the dyes functionalized particles allowed sequencing of new proteins never sequenced in serum before (not present in the HUPO Plasma Proteome Project comprehensive list, http://www.hupo.org/research/hppp/)

Particles carrying the following chemical affinity bait remazol brilliant blue R, disperse yellow 3, disperse yellow 9, acrylic acid, cibacron blue F3GA, and mix = mixture of remazol brilliant blue R, disperse yellow 3, and disperse yellow 9 were selected. Aliquots of 500 μL of particles (1 mg/mL) were incubated with 500 μL of serum for 30 minutes, separated by centrifugation and chemically eluted. Proteins were trypsin digested and sequenced by mass spectrometry (Figure 5- 14)

Results demonstrated that particles carrying different dyes bind different groups of proteins. Protein sequestration is dependent on the chemical structure of the affinity bait.

EXAMPLE 5 Cibacron Blue F3GA core - Vinylsulfonic acid shell particle preparation.

Core shell bait containing particles have been prepared which contain two different classes of dye bait in the shell and the core. This double bait design provides a novel capture affinity profile. Unwanted contaminating albumin is EXCLUDED by the dye in the outer shell while at the same time the desired analyte is CAPTURED by the core (Figure 5).

N-isopropylacrylamide (NIPAm)/Allylamine (AA) particles were prepared containing 10% of AA with respect to the total monomer. NIPAm (0.89 g, 7.83 mmol) and N,N'-methylenebisacrylamide (BIS, 0.042 g, 0.27 mmol) were dissolved in 30 mL of water and then passed through a 0.2 μm filter nylon membrane. The solution was purged with nitrogen for 15 min at room temperature with a medium stirring rate before AA (0.051 g, 0.90 mmol) was added. The solution was purged with nitrogen for another 15 min and then heated to 75 ⁰ C. KPS (0.0070 g, 0.025 mmol) in 1.0 mL of water was added to the solution to initiate polymerization. The reaction was maintained at 75°C under nitrogen for 3 hours. The react ion was subsequently allowed to cool to room temperature and stirred overnight. The reaction mixture was then transferred to 2 mL microcentrifuge tubes and the particles harvested and washed via centrifugation (Eppendorf 5415R centrifuge). After centrifuging the particle suspension for 20 min at 23 ⁰ C and 16 100 relative centrifugal force (rcf), the supernatant from each tube was decanted and the particles were redispersed in 1.0 mL water. This concentration/redispersion process was repeated for a total of five washes. To obtain NIPAm/Cibacron Blue F3GA (CB) particles, CB (0.76 g, 0.90 mmol) was dissolved in 10 mL of 0.1 mol/L aqueous sodium carbonate. The NIP Am/ AA particle suspension (10 mL volume) was purged with nitrogen for 15 min with a medium stirring rate in a 100 mL three-neck round-bottom flask, after which solid sodium carbonate (0.106 g, 1.0 mmol) was added to the suspension. The suspension was then stirred at room temperature under nitrogen for about 1 min. The CB solution was then added to the NIPAm/ AA particle suspension, and the combined reaction mixture was then stirred at room temperature under nitrogen for 48 h. The resulting NIPAm/CB particles were harvested and washed using centrifugation as described earlier in the preparation of NIPAm/ AA particles. Dye loading was determined via spectrophotometry (Thermo Spectronic 20+). A calibration curve was constructed using CB stock solutions of known concentrations. The supernatants from the entire concentration/redispersion process were combined, and their absorbance at a wavelength of 608 nm was determined. The CB concentration of the combined supernatants was estimated using the calibration curve. NIPAm/CB particle diameter at room temperature is 312 nm, as measured with Submicron Particle Size Analyzer, Beckman Coulter. The measurement of particle diameter was performed using water as diluent (refractive index (RI) = 1.333, diluent viscosity=0.890 cP). The test angle was 90°. Average values were calculated for three measurements using a 200 s integration time, and the solutions were allowed to thermally equilibrate for 10 min before each set of measurements. Measured values were then converted to particle sizes via the Stokes Einstein relationship.

In order to build a vinylsulfonic acid (VSA) shell on the NIPAm/CB core, 20 mL of NIPAm/CB particle suspension, obtained as described before, were heated at 70 ⁰ C and purged with nitrogen for 1 hour. NIPAm (0.156 g, 1.38 mmol), BIS (0.013 g, 0.084 mmol), vinylsulphonic acid (26 ul, 0.334 mmol) and KPS (0.092 g, 0.328 mmol) were dissolved in water and passed through a 0.2 μm filter nylon membrane. After one hour, 3 mL of this solution were added to NIPAm/CB particle suspension and the remaining 17 mL of solution were added in aliquots of 3 mL every five minutes. Particle diameter after the shell synthesis reaction was 368 nm, as measured with Submicron Particle Size Analyzer.

Results:

In order to prove the utility of a structure comprising two different baits in the core and in the shell compartment, particles with comprised of cibacron blue F3GA core and containing vinyl sulfonic acid shell were obtained. Particles with the following properties were experimentally compared: vinylsulfonic acid core particles, cibacron blue F3GA particles, cibacron blue F3GA - N-isopropylacrylamide shell, and cibacron blue

F3GA core - vinyl sulfonic acid shell. Aliquots of 100 μL of particles were incubated with 10 μL of serum, separated by centrifugation and washed two times with water. Particles were loaded on a 4-20% TrisGly gel. SDS PAGE analysis revealed that: 1) vinyl sulfonic acid functionalized particles do not uptake significant amount of albumin (lane 2), X) cibacron blue F3GA particles have a different and richer pattern of captured proteins with respect to the vinylsulfonic acid particles and capture a certain amount of albumin (lane 3), 3) cibacron blue F3GA core- N-isopropylacrylamide shell particles maintain similar behavior to cibacron blue F3GA core particles (lane 4), 4) cibacron blue F3GA core - vinylsulfonic acid shell particles do not uptake significant amount of albumin and maintain a similar pattern of harvested low molecular proteins as cibacron blue F3GA core. Therefore, particles containing two different baits in the core and in the shell acquire a behavior that is the sum of the characteristic of the two baits. In particular in this embodiment of the invention, vinyl sulfonic acid shell did not prevent low molecular weight proteins from interacting with cibacron blue F3GA core.

EXAMPLE 6

Influence of the chemical bait structure on affinity of particles towards protein analytes.

In order to investigate the effect of the chemical structure of the affinity bait on the binding properties of particles, structure number 5 (acid black 48) was selected as a starting structure. Acid black 48 contains two anthraquinone rings and a number of sulfonate side groups. Structures 1 to 4 were selected for their similarity to acid black 48.

Structures 1-4 contain anthraquinone rings, sulfonate substitution and other substitution groups such as methyl groups (structure 2), aryl groups (structure 1 and 3) or no substitution groups (structure 4). Particles carrying vinylsulfonic acid are labeled with number 16 in Figure 6. Particles carrying the baits described above were incubated with low abundance, low molecular weight protein analytes and analyzed by SDS PAGE. Results showed that changes in the structure of the chemical bait affect binding properties of the particles such that particles functionalized with different baits have different binding patterns. For example, the chemokine Eotaxin 2 was completely captured by particles containing structure number 1, 4, and 5, was partially associated to particles carrying structure 2 and was not captured by particles carrying structure 3.