Classifications

• • 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
  • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
  • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
  • C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
  • C07K17/02—Peptides being immobilised on, or in, an organic carrier
  • C07K17/08—Peptides being immobilised on, or in, an organic carrier the carrier being a synthetic polymer
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
  • C08J7/12—Chemical modification

Definitions

• This invention relates to a process for modification and acti ⁇ vation of a polymer surface in order to immobilize proteins and other types of molecules to the polymer surface.
• the invention further relates to the polymers in modified form, and the polymers having immobilised on their surfaces molecules such as proteins.
• Immunosorbent analysis e. g. IRMA (Immuno Radio Metric Assay) , ELISA (Enzyme Linked Immuno Sorbent Assay) IFMA
• Polystyrene premodified during production with reactive groups such as -OH, -S0 3 H, or -NH-, are available on the market. However, with these it is necessary to separately produce the desired articles from the specific modified polystyrene.
• This invention in its first aspect relates to a novel and efficient process by which polymer surfaces upon treatment with a Ce(IV) salt and electromagnetic radiation are chemically modified in such a way that proteins and other ligands can be bound more tightly, possibly even covalently, to the surface, and thereby be immobilized.
• the invention in its second aspect relates to polymers in modified form prepared for the establishment of covalent bonds to proteins or other ligands in order to immobilize these ligands to the polymer surface. » In a third aspect the invention relates to the modified polymers of the invention whereto proteins or other ligands have been immobilized.
• the invention relates to the use of the method or the modified polymers in analytic techniques such as immuno sorbent analysis (IRMA, ELISA, IFMA/FIA, ILMA/LIA) , or more generally in polymer support technology.
• analytic techniques such as immuno sorbent analysis (IRMA, ELISA, IFMA/FIA, ILMA/LIA) , or more generally in polymer support technology.
• the invention relates to a package compri ⁇ sing the unmodified polymer formed in a desired configuration for a specific desired end use in combination with a set of chemicals suitable for modifying the polymer surface in order to prepare it for immobilization of proteins or other ligands.
• Fig. 1 shows the effect of activation with a Ce(IV) salt and electromagnetic radiation, and the effect of adding Berol ® or ethanolamine on the binding of a protein to a polymer surface.
• Fig. 2 shows the relation between the irradiation time and the relative binding of a ligand to a polymer surface.
• Fig. 3 shows the relation between the concentration of a Ce(IV) salt and the relative binding.
• Fig. 4 shows the binding at a fixed concentration of enzyme conjugated antibody as a function of the log of the concentra ⁇ tion of competing unlabelled species to be bound added to microtiter wells, and the effect of adding Berol ® and ethanol ⁇ amine.
• Fig. 5 shows the binding of biotinamine (sometimes abridged as BioNH 2 ) to a polystyrene surface.
• Fig. 6 shows the binding of spermidine to a number of polymer surfaces activated in accordance with the invention as a function of irradiation time.
• Fig. 7 shows the binding of 15 I-labelled protein as a function of activation treatment.
• Fig. 8 shows the effect of divinyl sulfone treatment on the binding of a protein to a CAN activated polymer surface.
• Fig. 9 shows the saturation of avidin binding to biotinamine coated CAN plates.
• a constant amount of avidin-peroxidase con ⁇ jugate and various amounts of avidin was used, x-x-x: Biotin coated, CAN-activated plates, ⁇ - ⁇ - ⁇ : CAN-activated plates 0-0-0: untreated plates.
• Fig. 10 shows the influence of the concentration of CAN used in the activation on the binding of avidin peroxidase conjugate on CAN-activated plates (D-O-D) and on biotin-coated CAN-plates (x-x-x) .
• Fig. 11 shows the effect of spermine on the binding of biotin ⁇ amine to CAN activated plates.
• the plates were (1) CAN-ac ⁇ tivated, (2) incubated with the indicated concentration of spermine, (3) washed, (4) coated with biotinamine, and (5) assayed with avidin peroxidase conjugate.
• Fig. 12 shows the effect of mercaptoethanol on the binding of biotinmercaptane to CAN-activated plates. Experiment as in Fig. 11 except biotinmercaptane was exchanged for biotinamine and 2- mercaptoethanol for spermine.
• Fig. 13 shows the effects of a mixture of spermine and mercap ⁇ toethanol on the binding of biotinamine or biotinmercaptane to CAN activated polystyrene plates. Experiments as in Figures 11 and 12 using 100 ⁇ g/ml spermine and 100 mg/ml(10%) 2-mercaptoethanol.
• Fig. 14 shows the stability of CAN-activated plates. The plates were activated on day 0 and kept humid in closed plastic bags at 4°C (X) or 20°C ( ). At the times indicated the plates were coated with biotinamine and assayed with avidin-peroxidase conjugate.
• Fig. 15 shows an example of local irradiation dependent activation of a polystyrene surface.
• Fig. 16 shows the formula of varius biotin derivatives used.
• This invention in its first aspect relates to a method for modifying polymer surfaces, which method comprises a) application of a solution of a Ce(IV) salt to the surface of the polymer, whereby the surface is covered with the solution, b) irradiation of the solution covered surface with electromagnetic radiation, whereby the surface is activated, c) washing the activated surface once or repeatedly,
• polymers to be used are any suitable polymer, and may preferably be selected from the group comprising polystyrene, polyethylene, polypropylene, polyure- thane, polycarbonate, polyethylene terephthalate glycol, polyvinyl acetate, polyvinyl chloride, polyvinylpyrrolidone, polyacrylonitrile, polymethylmethacrylate, polytetrafluorethy- lene, butyl rubber, styrenebutadiene rubber, natural rubber, poly-4-methylpentylene, and polyesters.
• polystyrene polyethylene, polypropylene, polyure- thane, polycarbonate, polyethylene terephthalate glycol, polyvinyl acetate, polyvinyl chloride, polyvinylpyrrolidone, polyacrylonitrile, polymethylmethacrylate, polytetrafluorethy- lene, butyl rubber, styrenebutadiene rubber, natural rubber, poly-4-methylpent
• the cerium(IV) salt used can be any Ce(IV) salt, but pre- ferably complex salts with a cation of the Ce(IV)X 2 6+ type, where X is a suitable cation, such as NH 4 + , Li + , Na + , K + , or Cs + , pre ⁇ ferably NH 4 + in combination with the appropriate number of one or more suitable anions, which preferably is nitrate, but any other type of Ce(IV) salt capable of generating radicals upon being subjected to electromagnetic radiation is suitable.
• a special type of activator salt is "double" salts such as CsNH 4 [Ce(N0 3 ) 6 ] which are just as useful for this invention as the "pure" salts.
• any suitable solvent may be used. Suitable examples include water, dimethyl sulfoxide (DMSO) , acetonitrile, and acidic aqueous media.
• DMSO dimethyl sulfoxide
• acetonitrile acetonitrile
• acidic aqueous media are preferred.
• the acid should be chosen as one capable of stabilizing the radicals formed, and in connection with a nitrate salt a nitrogen containing acid, preferably HN0 3 , would be suitable.
• the electromagnetic radiation used is typically ultra violet visible light, preferably near visible UV light, but other types of electromagnetic radiation capable of generating radicals from the Ce(IV) salt is suitable. This could be X- rays or 7-radiation.
• the irradiation may be applied diffusely to the surface, but for certain uses local and/or directed application is pre ⁇ ferred, and in some instances even mandatory.
• liquids such as water, or buffers.
• an additional step (d) may be added to the above process comprising a chemical modification of the activated polymer surface.
• These further modifications of the activated surface are examplified by: i) hydrolysis reactions leading to alcohols, ii) elimination reactions resulting in alkenes, iii) oxidation reactions leading to epoxides, aldehydes, and carboxylic acids, and iv) addition reactions leading e.g. to halogenides.
• the modification reagents include inter alia bases (NaOH) , aacciiddss ((HH 22 SS00 44 )) ,, ppeerrooxxiiddeess ((HH 22 00 22 ,, RRCC00 22 OOHH)) ,, Cr 2 0 7 2" ' Mn0 4 " ' halogens (I 2 , Br 2 , Cl 2 ) and pseudohalogens (BrCN)
• the inventors have applied this type of photochemistry in a more general manner by treating polymer surfaces with Ce(IV) salts, and electro ⁇ magnetic radiation, and tested the properties of the resulting surfaces in terms of their capacity to bind protein and low molecular weight amines.
• the activated group may be coupled to an intermediate molecule, hereinafter designated spacer molecule or spacer, which in accordance with the intended use may be homofunctional, meaning that the spacer contains only reactive groups of one kind (e.g. -NH 2 , -OH, or -COOH) or heterofunctional, meaning that the spacer contains reactive groups of more than one kind (e.g. -NH 2 , -SH and -OH) .
• the spacer is usually of the so-called bifunctional type, meaning that the spacer contains two reactive groups, whereof one is intended to bind to the polymer surface function, and the other to the species to be immobilized.
• the spacer may also be multifunctional, meaning that one spacer is capable of binding more than one species to the polymer surface.
• the spacer is used for several purposes, such as stabilizing the highly reactive activated group in the polymer in order to control and regulate the formation of the covalent bond, or in the case of a bulky ligand the spacer may provide a solution to problems connected with steric hindrance for the ligand, and thereby give the ligand "access" to the polymer surface.
• a further modification of the activated groups and/or spacers can be obtained through the use of so-called linker reagents.
• Suitable homobifunctional linker reagents for the purpose of this invention may be: divinyl sulfone, o-phenylenedimaleimi- de, dimethyl adipimidate, glutaraldehyde, glutaconaldehyde, carbodiimides, tolylene-2,4-diisocyanate, disuccinimidyl suberate, bis-oxiranes, bis-N-hydroxysuccinimide esters, etc., preferably divinyl sulfone.
• Suitable heterobifunctional linker reagents may be maleimido- benzoic acid N-hydroxysuccinimide ester.
• a polystyrene surface such as the wells in an ELISA microtiter plate, is treated with CAN in aqueous nitric acid and radiated with UVA. It is believed that this generates nitrate radicals, which abstract hydrogen from the polymer resulting in nitrate ester formation, hydroperoxydation, hydroxylation, or other oxidations, etc.
• Adsorption of proteins to polystyrene is presumably mostly due to hydrophobic and electrostatic interactions which can be subdued by including a detergent in the coating mixture.
• a detergent for testing we chose Berol ® EMU-043 which is non-ionic and chemi ⁇ cally inert for this purpose, and as shown in Fig. 1 relating to Example 1 below, this detergent inhibits adsorption of protein (antibody) to non-treated polystyrene.
• ethanolamine which contains two of the functions (-NH 2 , -OH) which are primarily responsible for covalent protein binding. Ethanolamine has no inhibitory effect on protein adsorption to unmodified polystyrene (Example 1, Fig. 1) .
• treatment with a linker agent is included in preferred embodiments of the methods of the in ⁇ vention, meaning that the invention further relates to a method for modifying polymer surfaces, which method comprises a) application of a solution of a Ce(IV) nitrate and a nitrogen containing acid to the surface of the polymer, b) irradiation of the surface with long wavelength ultraviolet light, c) washing the surface once or repeatedly, e) incubation of the polymer surface with a linker agent, and f) washing the surface once or repeatedly.
• polymer surfaces are obtained that may be better suited for having proteins or other species immobi ⁇ lized thereto than the surfaces that are not treated with a linker agent.
• an additional step (d) may be added to the above process comprising a chemical modification of the activated polymer surface.
• the invention in a further aspect relates to methods of immobi ⁇ lizing proteins or other species to a polymer surface, which methods in addition to steps (a) to (c), (d) or (f) above comprise the following steps: g) application of a protein or other species to the activated, and washed surface, and h) washing the surface.
• the species used may be any type of molecule, such as a pro ⁇ tein (an enzyme, an antibody, an antigen) , a peptide, a nucleic acid (DNA or RNA) , a carbohydrate, a lipid, an amino acid, a nucleoside, an amine, a thiol, an alcohol, or whatever it is desired to immobilize on the polymer surface, including coupling of catalysts, fluorescent compounds, and/or inorganic moieties, possibly through chelating agents.
• a pro ⁇ tein an enzyme, an antibody, an antigen
• species to be immobilized is also meant to encompass cells, virus, microorganisms, and the like.
• the protein or other species which was applied in step (g) is thus immobilized on the polymer surface.
• nitrate radicals by irradiation of CAN can be detected by flash photolysis (R. W. Glass and T. W. Martin: “Flash Generation and Decay Kinetics of the Nitrate Radical in Aqueous Nitric Acid Solutions” J. Am. Chem. Soc. 9_2, 5084-5093 (1970)) .
• the modification of the invention can also be effectuated by treatment of the polymer surface with nitrate radicals in the vapour phase.
• biotinamine (6-biotinylaminohexane-l-amine) and biotinmercaptane (6-biotinylaminohexane-l-thiole)
• biotinamine (6-biotinylaminohexane-l-amine)
• biotinmercaptane (6-biotinylaminohexane-l-thiole)
• Fig. 2, 5, 9, and 12 avidin-peroxidase conjugate
• spermidine to polystyrene, polyethylene, polypropy ⁇ lene, or polymethylmethacrylate tubes (Fig. 6) .
• Fig. 5, column 5 the amount of avidin-peroxi ⁇ dase bound to CAN activated polystyrene via biotinamine (Fig. 5, column 5) equals that bound to untreated polystyrene via adsorption (in the absence of detergent; Fig. 5, column 1) .
• Fig. 8 shows that inclusion of the linker, divinylsulfone (DVS) , increases the Berol ® resistant (covalent) protein binding to CAN activated polystyrene by approximately a factor of two.
• the binding of avidin to biotin coated CAN- activated plates is saturated at elevated concentrations of avidin (Fig. 9) , and this saturation occurs at a concentration of -1 ⁇ g/ml, i.e. similar to that observed with IgG.
• the optimum CAN concentration for activation for biotinamine coating is also similar ( ⁇ 2 mM) (Fig. 10) to that determined for IgG coating indicating that the same underlying photochemistry is responsible for the activation in both cases.
• biotin derivatives (1) biotin-N-hydroxy succinimide (biotin active ester) , (2) biotinamine, (3)t-Boc-biotin amine, (4) biotinhexole, (5) biotinheptane, (6) biotinmercaptane, and (7) biotindisulfide shown in Fig. 16, where X is biotinyl, and the quenchers spermine and mercaptoethanol.
• biotin derivatives containing -NH 2 or -SH groups bind to the activated surface whereas derivatives containing -OH or protected -NH 2 groups do not. Accordingly, the binding of biotinamine is inhibited by spermine (Fig. 11) and the binding of biotinmercaptane by mercaptoethanol (Fig. 12) . It is noteworthy that the binding of biotinamine is more strongly inhibited by spermine than by mercaptoethanol and similarly that the binding of biotinmercap ⁇ tane is more strongly inhibited by mercaptoethanol than by spermine (Fig. 13) .
• PS Polystyrene
• AvPo Avidinperoxidase conjugate
• ⁇ Tween ® protein binding in the presence/absence of detergent Tween ® 20.
• CAN CAN activated plates.
• CAN/BioNH 2 CAN activated, biotin- coated plates.
• the CAN activated plates are stable for at least 30 days when kept humid at 4°C or room temperature as assayed by their ability to bind biotinamine (Figure 14) ,
• the IgG horse radish peroxidase (HRP) conjugated protein was quantitated by measuring the peroxidase activity in 100 mM sodium citrate buffer (Merck, reinst) pH 5.2, containing 0.03% H 2 0 2 , and 0.5 mg/ml 1,2-phenylenediamine, dihydrochloride (OPD) tablets (Dakopatts) as chromogenic substrate. 100 ⁇ l mixture was added to each well and the plate was incubated at 20°C for 10 minutes. The enzymatic reaction was terminated by addition of 100 ⁇ l 2 N H 2 S0 4 and the colour reaction was quantified by measuring E 492 using an automated ELISA scanner (EAR 400, SLT- LAB INSTRUMENTS, Austria).
• HRP horse radish peroxidase
• Figs. 2, 3, and 4 were constructed from data obtained in experiments parallel to this example by varying different parameters of the method of the invention.
• Fig. 3 indicates that the optimum transition group metal salt concentration (CAN) is about 4 mM, and Fig. 2 indicates that an optimum radiation time for that salt with the present equipment would be from 8 to 10 minutes.
• CAN transition group metal salt concentration
• Iodination was performed with Na 125 I (carrier free, Amersham) and iodo-beads (Pierce) according to the manufacturers (Pierce) recommendation.
• the specific activity of the labeled IgG was « 0.5 ⁇ Ci/ ⁇ g.
• 125 I-labelled protein was quantified by autoradiography of the microtiter plates using an Agfa Curix RP1 X-ray film and a 7 mm steel mask between the plate and the film.
• the autoradiogram was scanned at 550 nm with a Shimadzu CS930 densitometer scanner, and the results are shown in Fig. 7.
• Example l Activation of the plates was performed as in Example l, optionally omitting the divinyl sulfone step. Incubation with biotinamine (10 ⁇ g/ml in PBS) was done for 30 minutes at 20°C.
• Fig. 2 shows the effect of irradiation with ( ⁇ ) or without (Q) biotinamine treatment.
• Biotinmercaptane (6-biotinylaminohexane-l-thiol) to ELISA plates This was performed analogously to Example 3 except biotinmercaptane was exchanged for biotinamine.
• Fig. 6 shows the results in terms of 3 H-spermidine binding as a function of the time of irradiation.
• a mixture of a 32 P-end labeled DNA fragment (90 bp EcoRI-PvuII fragment of plasmid pUC19 (Nielsen et aJL. Biochemi- stry 27 . , (1988) 6338-6343) 100 ⁇ g calf thymus DNA and 10 ⁇ g psoralen-disulfide (Eisner et al Analytical Biochem. 149, (1985) 578-581) in 1 ml 10 mM Tris-HCl, 1 mM EDTA buffer was irradiated for 60 min with light a source used for CAN ac ⁇ tivation.
• a polymer surface such as a polystyrene surface
• a Ce(IV) salt such as a nitric acidic solution of cerium ammonium nitrate
• electromagnetic radiation such as long wavelength ultraviolet radiation
• the activation method of the invention drastically increases (>40 fold) the capacity of the treated surface to bind low molecular weight amines and mercaptanes.
• This type of surface modification could be useful for a variety of techniques based on solid support "chemistry” such as immunosorbent assays, affinity column chromatography, enzyme immobilization (e.g. in fermenting) , therapeutic instruments (e.g. dialysis) , etc.
• chemistry such as immunosorbent assays, affinity column chromatography, enzyme immobilization (e.g. in fermenting) , therapeutic instruments (e.g. dialysis) , etc.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Organic Chemistry (AREA)
• Genetics & Genomics (AREA)
• Immunology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Biomedical Technology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Biotechnology (AREA)
• Medicinal Chemistry (AREA)
• Microbiology (AREA)
• Molecular Biology (AREA)
• General Engineering & Computer Science (AREA)
• Hematology (AREA)
• Urology & Nephrology (AREA)
• Cell Biology (AREA)
• Food Science & Technology (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Biophysics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Polymers & Plastics (AREA)
• Peptides Or Proteins (AREA)
• Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=8099930

Family Applications (1)

Country Status (7)

Families Citing this family (5)

Family Cites Families (2)

• 1990
  • 1990-04-24 DK DK100490A patent/DK100490D0/da not_active Application Discontinuation
• 1991
  • 1991-04-24 WO PCT/DK1991/000107 patent/WO1991016377A1/en not_active Application Discontinuation
  • 1991-04-24 JP JP3508447A patent/JPH06506961A/ja active Pending
  • 1991-04-24 AU AU77728/91A patent/AU7772891A/en not_active Abandoned
  • 1991-04-24 EP EP91919024A patent/EP0526587A1/en not_active Withdrawn
• 1992
  • 1992-10-22 FI FI924792A patent/FI924792A0/fi not_active Application Discontinuation
  • 1992-10-23 NO NO92924118A patent/NO924118L/no unknown

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events