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WO1996030409A1 - Procede pour l'immobilisation de proteines et de polyelectrolytes sur la surface des produits solides - Google Patents

Procede pour l'immobilisation de proteines et de polyelectrolytes sur la surface des produits solides Download PDF

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Publication number
WO1996030409A1
WO1996030409A1 PCT/CZ1996/000010 CZ9600010W WO9630409A1 WO 1996030409 A1 WO1996030409 A1 WO 1996030409A1 CZ 9600010 W CZ9600010 W CZ 9600010W WO 9630409 A1 WO9630409 A1 WO 9630409A1
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WO
WIPO (PCT)
Prior art keywords
proteins
protein
washing
polyelectrolyte
adsorption
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CZ1996/000010
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English (en)
Inventor
Eduard Brynda
Milan Houska
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ustav Makromolekularni Chemie Akademie Ve^? C^?eske Republiky
Original Assignee
Ustav Makromolekularni Chemie Akademie Ve^? C^?eske Republiky
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ustav Makromolekularni Chemie Akademie Ve^? C^?eske Republiky filed Critical Ustav Makromolekularni Chemie Akademie Ve^? C^?eske Republiky
Publication of WO1996030409A1 publication Critical patent/WO1996030409A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • 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

Definitions

  • the invention relates to a novel method of immobilisa ⁇ tion of proteins and polyelectrolytes on surfaces of solids thereby forming surface films consisting of molecular layers of proteins and polyelectrolytes arranged according to an eligible architecture.
  • the irreversible physical adsorption is currently used for immobilisation of proteins capable of a strong interac- tion with surfaces.
  • the attachment is not usually very stable in the biological environment due to an exchange of the adsorbed protein for proteins in solution with a higher affinity for the surface.
  • a strong physical interaction of the adsorbed protein with the surface often decreases its activity.
  • a most frequently used method of immobilisation is a chemical (covalent) binding between an active group of protein (e.g. amino, phenyl, and other nucleophilic groups) and an active group on the substrate surface.
  • the surface binding via hydroxyl groups activated with bromcyanogen has been the most widely used method.
  • the disadvantage is a low stability of the bond, an ion exchanging character of the activated surface and a toxicity of the reagents used.
  • a more stable bonding via surface hydroxyl groups can be achieved by activation with chloroformates.
  • Another type of active surfaces is represented by materials with reactive epoxide groups. The rather high pH (ranging from 9 to 13) necessary for the activation of the epoxide groups often damages the reacting proteins.
  • a common disadvantage of systems for the immobilisation of proteins on rigid surfaces is a substantial degree of deactivation of proteins caused by the physical interaction with the underlying surface and a presence of spots of a bare surface remaining between the attached protein molecules.
  • a nonspecific adsorption of proteins from the surrounding solution as well as an activation of adverse reactions in the physiological environment may occur on the uncoated surface areas.
  • One way of a protein immobilisation on the inert rigid surfaces avoiding the above drawbacks is covering surfaces with a thick layer consisting of a crosslinked mixture of a hydrogel with the active protein.
  • a uniform film can be mechanically deposited only on some objects (it cannot be deposited on, e.g., porous materials).
  • Thick films adhere inadequately to substrates due to the contractions caused by changes in the degree of swelling.
  • the protein immobilised inside the film is not active if the corresponding reagents (analytes) are too big to diffuse into the interior of the thick crosslinked film.
  • the object of the invention is a method for the immobi- lisation of proteins and polyelectrolytes onto surfaces and formation of films consisting of molecular layers of proteins and polyelectrolytes which are arranged in an eligible architecture characterised in that (a) protein or polye- lectrolyte is immobilised on the surface by the physical adsorption or by the covalent binding followed (b) by the consecutive alternating adsorption of the protein and polyelectrolyte under the conditions of formation of an ionic bond between the protein and the polyelectrolyte so that a film composed of alternating molecules of proteins and polyelectrolytes is formed and (c) the film is subsequently fixed by a chemical crosslinking between active groups of protein or between active groups of protein and polyelectro ⁇ lyte; (d) the multilayer film of protein which does not contain another polylectrolyte can be formed by using polyelectrolyte without chemical groups reacting with crosslinker and by washing the polyelectrolyt
  • the immobilisation of the first layer is obtained by contacting the surface with an aqueous solution of a globular protein, e.g., albumin, fibrinogen and globulin, which adsorbs irreversibly via hydrophobic interaction on materials with a high free interfacial energy between their surfaces and water, i.e.
  • a globular protein e.g., albumin, fibrinogen and globulin
  • the physical adsorption is performed by a mere contact of an aqueous solution of the protein without any chemical activa ⁇ tion of the surface or protein.
  • the physical adsorption is not quite irreversible on neutral hydrogels consisting of movable polymer chains like cuprophane, polyhydroxyalkyl- acrylates and methacrylates, polyacrylates, agarosa, sepharo- sa, polyacrylamide.
  • This type of materials can be coated with the first protein layer by one of the known chemical methods.
  • a coating mediated by an ionic interaction can be applied only if the ionic bond is stable during the subsequent formation of the multilayer film and if a covalent attachment of the protein to the surface can be formed during the crosslinking step.
  • a polyelectrolyte can be immobilised by the physical adsorption via an ionic interaction with the opposite charged surface; a polyelectrolyte with reactive groups can be immobilised to an activated surface by a covalent bonding using the known methods.
  • the protein which adsorbs onto the immobilised polyelectrolyte in the subsequent steps must form a covalent bond with the surface during the final crosslinking.
  • collagen, gelatine and crosslinked albumin or fibrinogen can be used as solid substrates, b) The consecutive alternating adsorption of proteins and polyelectrolytes.
  • Additional layers of the protein and polyelectrolyte are consecutively adsorbed by alternating contacts of the surface with the protein and polyelectrolyte solutions.
  • solutions of different proteins and/or polyelectrolytes films are prepared in which different proteins and/or polyelectrolyte alternate in a required sequences.
  • the ionic strength and pH of solutions are chosen so that the protein and polyelectrolyte form ionic complexes.
  • a molecular layer of polyelectrolyte connects the neighbouring protein layers.
  • Polysaccharides, polypeptides and synthetic polymers soluble in water such as, e.g., polystyrene sulfonate, polyvinyl sulfonate, sulfonated cellulose, polymethacrylic acid and its copolymers, polyglutamates, polyvinylpyridine andpolyionenes can be used as polyelectrolytes.
  • a crosslinker e.g., glutaraldehyde, in which the ionic bonding between the protein and polyelectrolyte layers is maintained.
  • the crosslinker forms chemical bond between reactive groups of proteins (amino, phenyl and other nucleophilic groups). If the polyelectrolyte reacts with the crosslinker (e.g., via amino groups of some polysaccharides or synthetic polye- lectrolytes) , then the polyelectrolyte becomes an integral part of the covalent network. d) The washing out of polyelectrolyte. Polyelectrolytes which do not react with the crosslinker can be washed out of the multilayer with a solution in which ionic interactions between the protein and polyelectrolyte are cancelled so that a covalently crosslinked film composed only of proteins is prepared.
  • the novel method in comparison to known methods, is applicable to virtually any solid material which, with the exception of neutral hydrogels, need not to be chemically activated.
  • the protein films are formed on ready made devices by a mere contact with the water solutions of the components to be deposited. Dimensions and shapes of the devices to be coated are unlimited. Uniform films are deposited on materi- als like foils, tubes, fibres, hollow fibres, powders, porous particles etc. Contrary to a monomolecular protein layer, the multilayer film with a defined number of layers is more resistant to the erosion in the biological environment. The thickness of the films is several orders of magnitude smaller and more uniform than that of films prepared by casting. For an easier storage and handling the films on solids can be dried and reswollen just before the use.
  • Multilayer films composed of layers of different proteins can contain inner layers of an inactive protein (e.g. albumin) which forms an ideal transition from the solid surface to the outer layers of actively functioning protein.
  • an inactive protein e.g. albumin
  • Such inner film minimises interfacial tension between the surface and water, minimises nonspecific adsorption and does not initiate adverse reacti ⁇ ons of the organism.
  • Active protein molecules immobilised on the film are better accessible to big reactants.
  • the primary protein layers of e.g. albumin can serve as an anchor site for other proteins which do not adsorb directly on the surface. If the antigen or substrate for the immobilised protein can migrate through an albumin film, the albumin membrane covering the active protein layer can protect it.
  • Immobilised layers of different enzymes can be arranged into assemblies of mutually cooperating enzymes in which the product of one layer is a substrate for the enzyme in the neighbouring layer.
  • SA serum albumin
  • PSS sodium polystyrene sulfonate
  • PSS sodium polystyrene sulfonate
  • the washing removed from the crosslinked film residual PSS and glutaraldehyde and the final film contained 3,43 ⁇ g Fb/cm 2 .
  • PSS sodium polystyrene sulfonate
  • the adsorption of the second and third layer of IgG by repeating the sequence of the steps (2) to (4) and (6) .
  • the film of three IgG layers contained 2,4 ⁇ g IgG/cm 2 .
  • PSS sodium polystyrene sulfonate
  • the washing of the surface with CB contained 1 ⁇ g U/cm 2 .
  • the adsorption of PSS from 0,1 % PSS in CB.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Procédé d'immobilisation de protéines et de polyélectrolytes à la surface de produits solides sous forme de films composés de couches moléculaires de protéines et de polyélectrolytes disposées selon une architecture appropriée. Le procédé est caractérisé en ce qu'il consiste (a) à immobiliser la couche de protéines ou de polyélectrolytes par adsorption physique ou par une liaison chimique; (b) à alterner de manière consécutive l'adsorption des couches moléculaires de protéines et de polyélectrolytes portant des charges électriques opposées; (c) à réaliser par réticulation chimique la fixation du film complexe ainsi obtenu; et (d) à évacuer par lavage les constituants non réticulés.
PCT/CZ1996/000010 1995-03-27 1996-03-26 Procede pour l'immobilisation de proteines et de polyelectrolytes sur la surface des produits solides Ceased WO1996030409A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CZ1995761A CZ286845B6 (cs) 1995-03-27 1995-03-27 Způsob imobilizace bílkovin a polyelektrolytů na povrchu pevných objektů
CZPV761-95 1995-03-27

Publications (1)

Publication Number Publication Date
WO1996030409A1 true WO1996030409A1 (fr) 1996-10-03

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CZ (1) CZ286845B6 (fr)
WO (1) WO1996030409A1 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000003797A1 (fr) * 1998-07-15 2000-01-27 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Revetement polyelectrolytique de specimens biologiques
KR101046337B1 (ko) 2008-06-10 2011-07-05 충남대학교산학협력단 고분자 전해질 다층박막과 마이크로 컨택트 프린팅을이용한 단백질 고정화방법
US8092836B2 (en) 1998-03-19 2012-01-10 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Production of nanocapsules and microcapsules by layer-wise polyelectrolyte self-assembly
US8093039B2 (en) 2007-04-10 2012-01-10 The Trustees Of The Stevens Institute Of Technology Surfaces differentially adhesive to eukaryotic cells and non-eukaryotic cells
EP2504702A4 (fr) * 2009-11-24 2014-12-17 Argos Inc Dispositifs de détection de composés à analyser
CN105339791A (zh) * 2013-03-14 2016-02-17 赛维德恩特有限公司 固相上的分子网
US9733242B2 (en) 2012-10-07 2017-08-15 Sevident, Inc. Devices for capturing analyte
US9910040B2 (en) 2012-07-09 2018-03-06 Sevident, Inc. Molecular nets comprising capture agents and linking agents
WO2020201350A1 (fr) 2019-04-03 2020-10-08 Vib Vzw Moyens et méthodes de séquençage des peptides d'une seule molécule
WO2024184407A1 (fr) 2023-03-06 2024-09-12 Vib Vzw Procédé d'identification de o-glycopeptides de surface cellulaire spécifiques d'une tumeur

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3109123A1 (de) * 1981-03-11 1982-09-23 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V., 8000 München Verfahren zur kovalenten immobilisierung von biologischen detoxikationssystemen auf kuenstlichen oberflaechen
DE4141302A1 (de) * 1991-12-14 1993-06-17 Inst Molekularbiologie Ak Traegerfixierte biologisch aktive oder physiologisch wirksame proteine und verfahren zu ihrer herstellung

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3109123A1 (de) * 1981-03-11 1982-09-23 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V., 8000 München Verfahren zur kovalenten immobilisierung von biologischen detoxikationssystemen auf kuenstlichen oberflaechen
DE4141302A1 (de) * 1991-12-14 1993-06-17 Inst Molekularbiologie Ak Traegerfixierte biologisch aktive oder physiologisch wirksame proteine und verfahren zu ihrer herstellung

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
W. KONG ET AL.: "IMMOBILIZED BILAYER GLUCOSE ISOMERASE IN POROUS TRIMETHYLAMINE POLYSTYRENE BASED ON MOLECULAR DEPOSITION.", JOURNAL OF THE CHEMICAL SOCIETY, CHEMICAL COMMUNICATIONS, no. 11, 7 June 1994 (1994-06-07), LETCHWORTH GB, pages 1297 - 1298, XP002008268 *
Y. LVOV ET AL.: "LAYER-BY-LAYER ASSEMBLY OF ALTERNATE PROTEIN/POLYION ULTRATHIN FILMS.", CHEMISTRY LETTERS, no. 12, December 1994 (1994-12-01), TOKYO JP, pages 2323 - 2326, XP002008267 *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8092836B2 (en) 1998-03-19 2012-01-10 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Production of nanocapsules and microcapsules by layer-wise polyelectrolyte self-assembly
US8168226B2 (en) 1998-03-19 2012-05-01 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Production of nanocapsules and microcapsules by layer-wise polyelectrolyte self-assembly
US6699501B1 (en) 1998-07-15 2004-03-02 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften. E.V. Polyelectrolyte coverings on biological templates
WO2000003797A1 (fr) * 1998-07-15 2000-01-27 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Revetement polyelectrolytique de specimens biologiques
US8093039B2 (en) 2007-04-10 2012-01-10 The Trustees Of The Stevens Institute Of Technology Surfaces differentially adhesive to eukaryotic cells and non-eukaryotic cells
KR101046337B1 (ko) 2008-06-10 2011-07-05 충남대학교산학협력단 고분자 전해질 다층박막과 마이크로 컨택트 프린팅을이용한 단백질 고정화방법
EP2504702A4 (fr) * 2009-11-24 2014-12-17 Argos Inc Dispositifs de détection de composés à analyser
US10900962B2 (en) 2009-11-24 2021-01-26 Sienna Cancer Diagnostics Inc. Molecular nets and devices for capturing analytes including exosomes
CN107462707A (zh) * 2009-11-24 2017-12-12 赛维登特公司 用于检测分析物的装置
US9910040B2 (en) 2012-07-09 2018-03-06 Sevident, Inc. Molecular nets comprising capture agents and linking agents
US9733242B2 (en) 2012-10-07 2017-08-15 Sevident, Inc. Devices for capturing analyte
EP2972343A4 (fr) * 2013-03-14 2016-08-24 Sevident Inc Filets moléculaires sur phases solides
CN110068677A (zh) * 2013-03-14 2019-07-30 赛维德恩特有限公司 固相上的分子网
AU2014236090B2 (en) * 2013-03-14 2019-09-12 Inoviq Inc. Molecular nets on solid phases
CN105339791A (zh) * 2013-03-14 2016-02-17 赛维德恩特有限公司 固相上的分子网
WO2020201350A1 (fr) 2019-04-03 2020-10-08 Vib Vzw Moyens et méthodes de séquençage des peptides d'une seule molécule
WO2024184407A1 (fr) 2023-03-06 2024-09-12 Vib Vzw Procédé d'identification de o-glycopeptides de surface cellulaire spécifiques d'une tumeur

Also Published As

Publication number Publication date
CZ76195A3 (en) 1996-10-16
CZ286845B6 (cs) 2000-07-12

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