EP1421376A2 - Surface d'identification bioanalytique a densite d'elements d'identification optimisee - Google Patents

Surface d'identification bioanalytique a densite d'elements d'identification optimisee

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Publication number
EP1421376A2
EP1421376A2 EP02797619A EP02797619A EP1421376A2 EP 1421376 A2 EP1421376 A2 EP 1421376A2 EP 02797619 A EP02797619 A EP 02797619A EP 02797619 A EP02797619 A EP 02797619A EP 1421376 A2 EP1421376 A2 EP 1421376A2
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EP
European Patent Office
Prior art keywords
detection surface
detection
analytes
surface according
binding
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.)
Withdrawn
Application number
EP02797619A
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German (de)
English (en)
Inventor
Michael Pawlak
Gert L. Duveneck
Ning-Ping Huang
Susan Margaret De Paul
Marcus Textor
Rolf Hofer
Eveline SCHÜRMANN-MADER
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.)
Bayer AG
Original Assignee
Zeptosens AG
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Filing date
Publication date
Application filed by Zeptosens AG filed Critical Zeptosens AG
Publication of EP1421376A2 publication Critical patent/EP1421376A2/fr
Withdrawn legal-status Critical Current

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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
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic

Definitions

  • the invention relates to a detection surface on a support with an optimal (in terms of area) binding capacity for the detection and binding of one or more analytes from one or more samples brought into contact with this surface, characterized in that a) said detection surface is a mixture of specific ones biological or biochemical or synthetic recognition elements for the recognition and binding of said analytes with components which are “neutral” with respect to these analytes, ie components that do not bind these analytes, and b) said specific recognition elements, based on the entire recognition surface or any partial area thereof, less than a complete one Take monolayer.
  • the invention also relates to a method for the qualitative and / or quantitative detection of one or more analytes in one or more samples, characterized in that said samples and optionally further reagents are brought into contact with a detection surface according to the invention and from the binding of the analyte or further to Analyte detection of used detection substances resulting changes in optical or electronic signals can be measured.
  • recognition elements which are biological, biochemical or synthetic
  • the solid support can be both a macroscopic type with a surface area of square millimeters to square centimeters and also a microscopic type, for example in the form of so-called beads, ie approximately spherical particles with typical diameters in the micrometer range.
  • the surface of such a solid Carrier with recognition elements immobilized thereon will hereinafter be referred to as a "recognition surface”.
  • the present invention solves the problem of already providing a detection surface with a maximum binding capacity and at the same time minimizing the extent of unwanted non-specific binding to the surface.
  • the mixture of specific recognition elements and components which are “neutral” with respect to the analytes, that is to say components which do not bind these analytes, furthermore has a promoting effect on the binding ability of said specific recognition elements by denaturing said recognition elements on the surface of the support (which impair or even cancel the binding ability)
  • this mixture promotes a uniform distribution of the detection elements within the detection surface, for example by preventing the detection elements from accumulating in clusters after application in liquid solution and evaporation of the solution.
  • the first object of the invention is a detection surface with an optimal (in terms of area) binding capacity for the detection and binding of one or more analytes from one or more samples brought into contact with this surface, characterized in that a) said detection surface is a mixture of specific biological or biochemical or synthetic recognition elements for recognizing and binding said analytes with components which are “neutral” with respect to these analytes, ie components which do not bind these analytes, and b) said specific recognition elements, based on the entire recognition surface or any partial area thereof, less than a complete monolayer , take in. Said specific recognition elements, based on the entire recognition surface or any partial area thereof, preferably form one tenth to one half of a complete monolayer. In addition, it is preferred that said specific recognition elements and components which are “neutral” to the analytes, based on the entire recognition surface or any partial area thereof, together form at least two thirds of a complete monolayer.
  • a second object of the invention is accordingly a structured detection surface with an optimal (in terms of area) binding capacity for the detection and binding of one or more analytes from one or more samples brought into contact with this surface, characterized in that a) said detection surface in discrete , spatially separated measuring ranges, a mixture of specific biological or biochemical or synthetic recognition elements for the recognition and binding of said analytes with components which are “neutral” with respect to these analytes, ie, those analytes that do not bind, and b) said specific recognition elements, based on the area of the discrete ones Take up measuring ranges less than a complete monolayer.
  • said specific recognition elements based on the entire recognition surface or any partial area thereof, form a tenth to a half of a complete monolayer.
  • spatially separated measuring areas are to be defined by the area, the biological or biochemical or synthetic detection elements immobilized there for the detection of an analyte from a liquid sample and those with the detection elements mixed, "neutral" molecules compared to said analytes.
  • These surfaces can have any geometry, for example the shape of points, circles, rectangles, triangles, ellipses or lines. It is possible that in a two-dimensional arrangement up to 1,000 000 measuring ranges are arranged, whereby a single measuring range can take up an area of 10 "4 mm 2 - 10 mm 2 .
  • the density of the measurement areas can be more than 10, preferably more than 100, particularly preferably more than 1000 measurement areas per square centimeter.
  • a detection surface according to the invention is usually generated on a solid support.
  • the application and subsequent adhesion can take place through electrostatic interaction or, more generally, through physical adsorption.
  • the orientation of the recognition elements is then generally statistical.
  • this adhesion-promoting layer is transparent at least at one excitation wavelength.
  • the thickness of such an optional adhesion-promoting layer is preferably less than 200 nm, but particularly preferably less than 20 nm.
  • the optional adhesion-promoting layer can, for example, be a chemical compound from the groups of silanes, functionalized silanes, epoxides, functionalized, charged or polar polymers and "self-organized passive or functionalized monolayers or multilayers ".
  • discrete (spatially separated) measurement areas are generated by spatially selective application of biological or biochemical or synthetic detection elements on a surface of a carrier or on an adhesion-promoting layer additionally applied to a carrier surface, preferably using an or several methods from the group of processes involving "inkjet spotting", mechanical spotting using a pen, pen or capillary, "micro contact printing”, fluidic contacting of the measuring areas with the biological or biochemical or synthetic recognition elements by their supply in parallel or crossed microchannels, under the influence of pressure differences or electrical or electromagnetic potentials as well as photochemical or photolithographic immobilization processes.
  • the biological or biochemical or synthetic recognition elements can be selected from the group consisting of proteins, for example mono- or polyclonal antibodies and antibody fragments, peptides, enzymes, aptamers, synthetic peptide structures, glycopeptides, oligosaccharides, lectins, antigens for antibodies (e.g. Biotin for streptavidin), with additional binding sites functionalized proteins ("tag proteins", such as "histidine tag proteins") and their complex formation partners.
  • Another group of compounds, which are also preferred as recognition elements include nucleic acids (for example DNA, RNA, oligonucleotides) and nucleic acid analogs (for example PNA) and their derivatives with artificial bases.
  • a third preferred group of compounds as recognition elements comprises soluble, membrane-bound proteins isolated from a membrane, such as, for example, receptors and their ligands.
  • Said “neutral” components, which do not bind the analyte or analytes, can also be selected from the groups consisting of albumin, in particular bovine serum albumin or human serum albumin, casein, nonspecific, polyclonal or monoclonal, foreign species or empirically unspecific antibodies for the analyte (s) to be detected (especially for immunoassays), detergents - such as Tween 20 -, fragmented natural or synthetic DNA that does not hybridize with polynucleotides to be analyzed, such as an extract of herring or salmon sperm (especially for polynucleotide hybridization assays), or also uncharged but hydrophilic polymers , such as polyethylene glycols or dextrans.
  • WO 00/65352 describes coatings with graft copolymers (“graft copolymers”) with a polyionic main chain, for example binding to a support (electrostatically) and “non-interactive” (adsorption-resistant) side chains, for coating bioanalytical sensor platforms or Implants for medical applications.
  • graft copolymers with a polyionic main chain, for example binding to a support (electrostatically) and “non-interactive” (adsorption-resistant) side chains, for coating bioanalytical sensor platforms or Implants for medical applications.
  • a preferred embodiment of a detection surface according to the invention is characterized in that the detection elements are bound to the free end or near the free end of a fully or partially functionalized, "non-interactive" (adsorption-resistant, uncharged) polymer, said non-interactive "(adsorption-resistant, uncharged) ) Polymer is bound as a side chain to a charged, polyionic polymer as the main chain and forms together with this a polyionic, multifunctional co-polymer.
  • a large group of embodiments of a detection surface according to the invention is characterized in that the polyionic polymer main chain is cationically (positively) charged at approximately neutral pH.
  • the polyionic polymer main chain is cationically (positively) charged at approximately neutral pH.
  • it can be selected from the group of polymers which comprises amino acids with a positive charge at approximately neutral pH, polysaccharides, polyamines, polymers of quaternary amines and charged synthetic polymers.
  • the cationic polymer main chain can also comprise one or more molecular groups from the group consisting of lsysine, histidine, arginine, chitosan, partially deacetylated chitin, amine-containing derivatives of neutral polysaccharides, polyaminostyrene, Polyamine acrylates, polyamine methacrylates, polyethyleneimines, polyaminoethylenes, polyaminostyrenes and their N-alkyl derivatives.
  • polyionic polymer main chain is anionically (negatively) charged at approximately neutral pH.
  • the cationic main chain can be selected from the group of polymers comprising amino acids with attached groups with negative charge at approximately neutral pH, polysaccharides and charged synthetic polymers with negatively charged groups.
  • the cationic polymer main chain can comprise one or more molecular groups from the group which comprises polyaspartic acid, polyglutamic acid, alginic acid or their derivatives, pectin, hyaluronic acid, heparin, heparin sulfate, chondroitin sulfate, dermatan sulfate, dextran sulfate, polymethyl methacrylic acid, oxidized cellulose, carboxymic acid, carboxymic acid, maloxymic acid, carboxymethyl acid, maloxymic acid, carboxymethyl acid, maloxic acid, carboxymethyl acid.
  • non-interactive (uncharged) polymer as the side chain can be selected from the group comprising poly (alkylene glycols), poly (alkylene oxides), neutral water-soluble polysaccharides, polyvinyl alcohols, poly-N-vinylpyrrolidones, phosphorylcholine derivatives, non-cationic poly ( meth) acrylates and their combinations.
  • the biological or biochemical or synthetic recognition elements are bound to the “non-interactive” side chain at its free end or close to its free end via reactive groups: it is particularly preferred that said reactive groups are selected from the group comprising hydroxy (-OH), carboxy (-COOH), ester (-COOR), thiols (-SH), N-hydroxysuccinimide, maleimidyl, quinone, vinyl sulfone, nitrilotriacetic acid ("nitrilotriacetic acid", NTA) and combinations thereof.
  • reactive groups are selected from the group comprising hydroxy (-OH), carboxy (-COOH), ester (-COOR), thiols (-SH), N-hydroxysuccinimide, maleimidyl, quinone, vinyl sulfone, nitrilotriacetic acid ("nitrilotriacetic acid", NTA) and combinations thereof.
  • a detection surface according to the invention is applied to an essentially optically transparent carrier.
  • essentially optically transparent is to be understood to mean that a layer characterized thereby is at least 95% transparent, at least at the wavelength of a light irradiated by an external light source, for its optical path perpendicular to said layer, provided that the layer is not reflective
  • “essentially optically transparent” is understood to mean that the sum of transmitted, reflected and optionally coupled into a layer and guided therein is at least 95% of the incident light at the point of incidence of the incident light.
  • excitation light an excitation light emitted by a carrier in the direction of the detection surface and from the opposite side, if appropriate by a medium radiated above the detection surface in the direction of the detection surface.
  • This excitation light can be used, for example, for excitation luminescence, or specific fluorescence or phosphorescence.
  • the essentially optically transparent carrier comprises a material from the group comprising moldable, sprayable or millable plastics, metals, metal oxides, silicates, such as, for. B. glass, quartz or ceramics.
  • a possible embodiment is characterized in that the detection surface according to the invention is applied to an adhesion-promoting layer applied to the essentially optically transparent carrier, which is also essentially optically transparent.
  • a special embodiment is characterized in that recesses for the production of sample containers are structured in the surface of said carrier.
  • This Recesses typically have a depth of 20 ⁇ m to 500 ⁇ m, preferably from 50 ⁇ m to 300 ⁇ m.
  • the essentially optically transparent carrier comprises a continuous optical waveguide or divided into individual waveguiding regions. It is particularly advantageous if the optical waveguide is an optical layer waveguide with a first, essentially optically transparent layer (a) facing the detection surface on a second, essentially optically transparent layer (b) with a lower refractive index than layer (a).
  • said optical layer waveguide is substantially planar.
  • Suitable planar optical layer waveguides and their modifications are, for example, in patent applications WO 95/33197, WO 95/33198, WO 96/35940, WO 98/09156, WO 99/40415, PCT / EP 00/04869 and PCT / EP 01 / 00605. The content of these patent applications is therefore fully introduced as part of this description.
  • this layer is in optical contact with one or more optical coupling elements from the group consisting of prism couplers, evanescent couplers with matched optical waveguides with overlapping evanescent fields, End face couplers are formed with focusing lenses, preferably cylindrical lenses, arranged in front of one end face of the wave-guiding layer, and grating couplers.
  • optical coupling elements from the group consisting of prism couplers, evanescent couplers with matched optical waveguides with overlapping evanescent fields, End face couplers are formed with focusing lenses, preferably cylindrical lenses, arranged in front of one end face of the wave-guiding layer, and grating couplers.
  • the excitation light is coupled into the optically transparent layer (a) with the aid of one or more grating structures (c) which are pronounced in the optically transparent layer (a).
  • Another component of the invention is a method for the qualitative and / or quantitative detection of one or more analytes in one or more samples, characterized in that said samples and optionally other reagents are brought into contact with a detection surface according to the invention according to one of the aforementioned embodiments and from the Binding of the analyte or other changes in optical or electronic signals resulting from the detection substances used to detect the analyte can be measured.
  • the invention also relates to a method for the qualitative and / or quantitative detection of one or more analytes in one or more samples, characterized in that said samples and optionally further reagents are brought into contact with and out of a structured detection surface according to one of the above-mentioned embodiments the binding of the analyte or other changes in the detection substances used for the detection of analytes resulting from optical or electronic signals emanating from the discrete measuring ranges are measured in a spatially resolved manner
  • the one or more samples are preincubated with a mixture of the various detection reagents for determining the analytes to be detected in said samples and these mixtures are then brought into contact with a detection surface according to the invention in a single addition step.
  • the detection of the one or more analytes is based on the determination of the change in one or more luminescences.
  • the excitation light from one or more light sources can be radiated in an incident light excitation arrangement. It can also be irradiated in a transmission light excitation arrangement.
  • a method is preferred which is characterized in that the detection surface, optionally mediated via an adhesion-promoting layer, is arranged on an optical waveguide, which is preferably essentially planar, that the one or more samples are brought into contact with the one or more analytes to be detected therein and optionally further detection reagents sequentially or after mixing with said samples in a single step with said detection surface and that the excitation light from one or more light sources in the optical waveguide, analogously is coupled in as described above for the optical layer waveguide.
  • Characteristic of a special embodiment of the method according to the invention is that the detection of the one or more analytes on a detection surface above a grating structure (c) or (c ') formed in the layer (a) of an optical layer waveguide on the basis of the result of the binding of the analyte and / or further detection reagents, on the immobilized biological or biochemical or synthetic recognition elements, resulting changes in the resonance conditions for coupling an excitation light into the layer (a) of a carrier designed as a layer waveguide or for coupling out light carried in the layer (a).
  • a variant of the method according to the invention is particularly preferred, which is characterized in that said optical waveguide is designed as an optical layer waveguide with a first optically transparent layer (a) on a second optically transparent layer (b) with a lower refractive index than layer (a), that excitation light continues to be coupled into the optically transparent layer (a) with the aid of one or more grating structures which are pronounced in the optically transparent layer (a) and is guided as a guided wave to the measuring areas (d) thereon, and that the evanescence continues Field of said guided wave generated luminescence of luminescent molecules is detected with one or more detectors and the concentration of one or more analytes is determined from the intensity of these luminescence signals.
  • (1) the isotropically emitted luminescence or (2) coupled into the optically transparent layer (a) and coupled out via a grating structure (c) or (c ') coupled luminescence or luminescence of both components (1) and (2) can be measured simultaneously , It is part of the method according to the invention that a luminescent dye or luminescent nanoparticle is used as the luminescent label to generate the luminescence, which can be excited and emitted at a wavelength between 300 nm and 1100 nm.
  • the luminescence label be bound to the analyte or in a competitive assay to an analog of the analyte or in a multi-stage assay to one of the binding partners of the immobilized biological or biochemical or synthetic recognition elements or to the biological or biochemical or synthetic recognition elements.
  • Another embodiment of the method is characterized in that a second or even further luminescence label with the same or different excitation wavelength as the first luminescence label and the same or different emission wavelength is used.
  • the second or even more luminescent label can be excited at the same wavelength as the first luminescent dye, but emit at other wavelengths.
  • a variant of the method consists in that charge or optical energy transfer from a first luminescent dye serving as donor to a second luminescent dye serving as acceptor is used to detect the analyte.
  • Another embodiment of the method is characterized in that, in addition to the determination of one or more luminescences, changes in the effective refractive index on the measurement areas are determined.
  • a further development of the method is characterized in that the one or more luminescences and / or determinations of light signals are carried out polarization-selectively at the excitation wavelength. It is preferred that the one or more luminescences are measured with a different polarization than that of the excitation light.
  • Part of the invention is a method according to one of the aforementioned embodiments for the simultaneous or sequential, quantitative or qualitative determination of one or more analytes from the group of proteins, such as antibodies or antigens, receptors or ligands, chelators, with additional binding sites functionalized proteins ("Tag Proteins ", such as” histidine tag proteins ") and their complex formation partners, oligonucleotides, DNA or RNA strands, DNA or RNA analogs, enzymes, enzyme factors or inhibitors, lectins and carbohydrates.
  • samples to be examined are, for example, naturally occurring body fluids such as blood, serum, plasma, lymph or urine or tissue fluids or egg yolk.
  • sample to be examined is an optically cloudy liquid, surface water, a soil or plant extract, a bio- or synthesis process broth.
  • samples to be examined are prepared from biological tissue parts or cell cultures.
  • the invention furthermore relates to the use of a method according to the invention for quantitative or qualitative analyzes for determining chemical, biochemical or biological analytes in screening methods in pharmaceutical research, combinatorial chemistry, clinical and preclinical development, for real-time binding studies and for determining kinetic parameters in affinity screening and in research, on qualitative and quantitative analyte determinations, in particular for DNA and RNA analysis and the determination of genomic or proteomic differences in the genome, such as single nucleotide polymorphisms, for measuring protein-DNA interactions, for determining control mechanisms for the m-RNA expression and for protein (bio) synthesis, for the preparation of toxicity studies and for the determination of expression profiles, in particular for the determination of biological and chemical marker substances such as mRNA, proteins, peptides or low-molecular organic (messenger) substances, as well as for the detection of antibodies , Antigens, pathogens or bacteria in pharmaceutical product research and development, human and veterinary diagnostics, agrochemical product research and development, symptomatic and presymptomatic plant diagnostic
  • Poly (L-lysine) hydrobromide (molecular weight approx. 20 kDa), streptavidin from Streptomyces avidinii (molecular weight approx. 60 kDa), protein avidin (molecular weight approx. 66 kDa), biotinylated goat anti-rabbit (ie bound to biotin) Immunoglobulin (anti-R-IgG biotin, molecular weight approx. 150 kDa) and biotinylated bovine serum albumin (BSA biotin, molecular weight approx. 66 kDa) were obtained from Sigma-Aldrich (Buchs, Switzerland).
  • N-hydroxysuccinimidyl ester of methoxy-poly (ethylene glycol) propionic acid (MeO-PEG-SPA, molecular weight 2 kDa) and the ⁇ -biotin- ⁇ -N-hydroxysuccinimidyl ester of poly (ethylene glycol) carbonate (biotin-PEG-CO 2 -NHS , Molecular weight 3.4 kDa) were obtained from Shearwater Polymers Inc. (Huntsville, USA).
  • Rabbit immunoglobulin (anti-human albumin) R-IgG, molecular weight approx. 150 kDa
  • rabbit anti-BSA rabbit anti-bovine serum albumin
  • a thin-film waveguide designed as a grating coupler (TiO-SiO 2 solgel as a waveguiding layer on a glass substrate, period of the coupling grating in the waveguiding layer: 417 nm) (micro vacuum Ltd., Budapest, Hungary) with a sputtered thereon, 12 nm thin Nb 2 O 5 layer.
  • these supports, with Nb 2 O 5 as the top layer were sonicated in 0.1 M HC1 for 10 minutes, thoroughly rinsed with ultrapure water, blown dry with nitrogen and subsequently with oxygen plasma in a PDC plasma cleaner / sterilizer for 2 hours -32G (Harrick, Ossining, USA).
  • Figure 1 shows schematically the synthesis of PLL-g-PEG.
  • N-Hydroxy-succinimidyl esters of both biotinylated and non-biotinylated poly (ethylene glycol) (“PEG”) are reacted with poly (L-lysine) ("PLL”) in a stoichiometric ratio to produce the desired product.
  • PEG polyethylene glycol
  • PLL poly (L-lysine)
  • PLL-g-PEG derivatives includes the molecular weights of the polymer partial chains of the copolymers, the grafting ratio and the percentage of biotinylated PEGs. Accordingly, “PLL (20) -g / " 5.57-PEG (2) / PEG-Biotin (3.4) 30%” a polymer formed from a main chain made of poly (L-lysine) with a molecular weight of 20 kDa and side chains, 70% consisting of poly (ethylene glycol) with a molecular weight of 2 kDa and 30% of biotinylated poly (ethylene glycol) with a molecular weight of 3.4 kDa.
  • the grafting ratio of 3.5 means that on average two out of every seven lysine groups (lysine units) are biotinylated or non-biotinylated PEG chains are bound. Since all of the polymers mentioned in this example are produced from similar precursors as an alternative to "PLL-g-PEG / PEG-Biotin30%", the abbreviation "PPB30" should also be used. Abbreviations are used for other percentages of biotinylated PLL-g-PEGs.
  • PLL-HBr Poly (L-lysine) hydrobromide
  • STBB sodium tetraborate buffer
  • PLL-HBr Poly (L-lysine) hydrobromide
  • STBB sodium tetraborate buffer
  • the solution is stirred and then filtered (0.22 ⁇ m Durapore membrane, sterile Millex GV, Sigma-Aldrich, Buchs, Switzerland) and filled into a sterile culture tube.
  • MeO-PEG-SPA powder is then added in a suitable amount according to the stoichiometric ratio while stirring the solution evenly.
  • the solution is transferred to a dialysis tube (Spectr / Por dialysis tube, molecular weight limitation (cut-off) 6-8 kDa, Sochochim, Lausanne, Switzerland). Dialysis is carried out for 24 hours in one liter of phosphate buffered saline ("PBS", 10 mM, pH 7.0), followed by 24 hours of further dialysis in one liter of deionized water. The product is then kept at a temperature of -50 ° for 48 hours C and a pressure of 0.2 mbar freeze-dried.
  • PBS phosphate buffered saline
  • Biotinylated PLL-g-PEG is synthesized in a manner similar to that previously described.
  • Biotin-PEG-CO 2 -NHS powder is slowly added in an appropriate amount according to the stoichiometric ratio to the filtered solution of PLL-HBr solution and stirred for one hour.
  • MeO-PEG-SPA is then added in an amount appropriate to the stoichiometric ratio, and the resulting solution is stirred for a further five hours.
  • the further steps of dialysis and product recovery are the same as described above.
  • the grafting ratio and the percentage of biotin in the biotinylated PEG derivatives are estimated using 1H-NMR.
  • the lyophilized polymers are dissolved in D 2 O and the spectra recorded with a 300 MHz NMR spectrometer. The values determined from this are summarized in Table 1.
  • Table 1 Grafting ratio and percentage of biotin in biotinylated PEG derivatives, determined using 1H-NMR.
  • the mass of adsorbed polymer on the Nb 2 ⁇ 5 surfaces is determined on the basis of the difference in the coupling conditions for light coupling into a grating coupler sensor before and after application of the respective polymer layers.
  • the principle of operation of a grating coupler sensor is described, for example, in US Pat. No. 4952056.
  • a grid coupler structure (BIOS I, ASI AG, Zurich, Switzerland) was used as the measuring instrument.
  • a carrier pretreated according to section 2 of this example is equilibrated in HEPES-1 buffer (10 mM HEPES, pH 7.4) for at least five hours before an experiment, then inserted into the lattice coupler measuring instrument and there in HEPES-1 for another hour - Buffer equilibrated until a stable baseline, ie a stable resonance angle for coupling the excitation light into the highly refractive waveguiding layer using the coupling grating has been achieved.
  • the polymer-coated supports are sequentially under continuous flow (flow rate: 1 ml / h) with solutions of streptavidin (100 ⁇ g / ml), anti-R-IgG-biotin (100 ⁇ g / ml) and finally R-IgG (200 ⁇ g / ml) incubated.
  • streptavidin 100 ⁇ g / ml
  • anti-R-IgG-biotin 100 ⁇ g / ml
  • R-IgG 200 ⁇ g / ml
  • BSA biotin 100 ⁇ g / ml
  • anti-BSA rabbit anti-bovine serum albumin
  • anti-BSA 200 ⁇ g / ml
  • anti-BSA rabbit anti-bovine serum albumin
  • a thin-film waveguide (TiO 2 -SiO 2 sol gel as a waveguiding layer on a glass substrate, period of the coupling grating in the waveguiding layer: 417 nm) with a 12 nm thin Nb 2 O 5 layer applied thereon serves as a carrier.
  • Nb 2 O 5 -coated surfaces It is believed that the strong adsorption of polymers, which include PLL as an essential component, on Nb 2 O 5 -coated surfaces is based primarily on electrostatic interaction between this metal oxide surface and the polymer as a multi-charged adsorbate.
  • the aim of applying a mixture of PLL-g-PEG and PLL-g-PEG / PEG-Biotin is to achieve an optimal binding capacity of the polymer-coated surface by adjusting the mixing ratio and at the same time to minimize non-specific binding.
  • Biotin, bound as a recognition element in the polymer PLL-g-PEG / PEG-biotin serves as a specific recognition element for molecules such as, for example, avidin or streptavidin, to which "biotinylated" molecules (ie with Biotin-linked molecules), such as anti-RIgG-biotin, can be bound, which in turn can serve as recognition elements for an analyte (in this example, R-IgG).
  • the mass of adsorbed polymer on the Nb 2 ⁇ 5 surfaces is determined on the basis of the difference in the coupling conditions for light coupling into the lattice coupler before and after application of the respective polymer layers. From this, values of 167 +/- 8 ng / cm 2 adsorbed polymer for pure PLL-g-PEG and 213 +/- 13 ng / cm 2 for pure PPB20 are determined. Taking into account the molecular weights and the grafting ratio determined by means of NMR, the surface concentrations of the adsorbed polymers are determined for each mixing ratio used. Within the experimental accuracy there is a uniform value of 2.5 +/- 0.1 pmol / cm 2 , which leads to the conclusion is that the mixing ratio of the polymers on the surface is the same as before in solution.
  • the ratio of bound streptavidin to surface-immobilized biotin is 1: 6.5.
  • the relatively high excess of biotin sub-molecules as immobilized recognition elements compared to bound streptavidin, which was added in an excess, which should lead to the saturation of all available binding sites in terms of quantity, can be explained by the fact that a part of the biotin molecule is not accessible on the surface, but rather could be hidden in the PEG sublayer.
  • one streptavidin molecule could also bind to two or more biotin molecules.
  • Figure 2 shows the amount of anti-R-IgG biotin bound as a function of the concentration of surface bound biotin.
  • the amount of bound anti-R-IgG biotin also initially increases.
  • a surface concentration (density) of approx. 11.2 pmol / cm 2 of biotin bound via PEG / biotin corresponding to a concentration of 1.68 pmol cm bound streptavidin (or x% of a complete monolayer)
  • a maximum of the amount of bound anti-R -IgG-biotin reached about 0.43 pmol / cm.
  • the amount of bound anti-R-IgG biotin decreases again.
  • the decrease in the binding capacity for anti-R-IgG biotin can be explained by steric hindrance of the available binding sites on streptavidin. It should also be taken into account that the anti-R-IgG-biotin molecule with a size similar to that of anti-R-IgG (from 14.3 nm x 5.9 nm x 13.1 nm (HD Kratzin, W. Plam, M. Stangel, WE Schmidt, J. Friedrich, N. Hilschmann, Biol. Chem. HS 370 (1989) 263 - 272)) occupy an approximately 2.5 times larger footprint than streptavidin if one has a "foot print" of size 14.3 nm x 5.9 nm.
  • the smaller protein BSA-biotin (molecular weight approx. 150,000) is used instead of anti-R-IgG-biotin (molecular weight approx. 150,000). 50,000), followed by the delivery of the anti-BSA antibody in the subsequent step.
  • the “chip” previously brought into contact with anti-R-IgG-biotin is brought into contact with R-IgG as analyte, followed by rinsing with buffer.
  • R-IgG the binding behavior for R-IgG very closely follows the trend of the binding curve of anti-R-IgG-biotin, as described above for the binding of anti-R-IgG-biotin.

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Abstract

L'invention concerne une surface d'identification située sur un support présentant une capacité de liaison optimale (par rapport à la surface), servant à identifier et à fixer un ou plusieurs analytes contenus dans un ou plusieurs échantillons mis en contact avec cette surface. L'invention est caractérisée en ce que a) ladite surface d'identification présente un mélange constitué d'éléments d'identification biologiques ou biochimiques ou synthétiques spécifiques, servant à identifier et à fixer lesdits analytes, et de composants « neutres » par rapport à ces analytes, c'est-à-dire ne fixant pas ces analytes, et b) lesdits éléments d'identification spécifiques occupent moins d'une monocouche complète, par rapport à la totalité de la surface d'identification ou d'une partie quelconque de cette dernière. L'invention concerne également un procédé pour la détection qualitative et/ou quantitative d'un ou de plusieurs analytes dans un ou plusieurs échantillons. Ce procédé est caractérisé en ce que lesdits échantillons et éventuellement d'autres réactifs sont mis en contact avec une surface d'identification selon l'invention et en ce que des variations de signaux optiques ou électroniques, résultant de la fixation de l'analyte ou d'autres substances de détection utilisées pour détecter l'analyte, sont mesurées.
EP02797619A 2001-08-27 2002-08-24 Surface d'identification bioanalytique a densite d'elements d'identification optimisee Withdrawn EP1421376A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
CH15852001 2001-08-27
CH158501 2001-08-27
PCT/EP2002/009489 WO2003021253A2 (fr) 2001-08-27 2002-08-24 Surface d'identification bioanalytique a densite d'elements d'identification optimisee

Publications (1)

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EP1421376A2 true EP1421376A2 (fr) 2004-05-26

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US (1) US20040253596A1 (fr)
EP (1) EP1421376A2 (fr)
AU (1) AU2002361223A1 (fr)
WO (1) WO2003021253A2 (fr)

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US8372614B2 (en) * 2005-02-07 2013-02-12 The United States Of America, As Represented By The Secretary Of Agriculture Ethanol production from solid citrus processing waste
EP3222142A1 (fr) 2006-02-28 2017-09-27 Kodiak Sciences Inc. Conjugués de polymères contenant de l'acryloyloxyéthylphosphorylcholine et leur préparation
US8288157B2 (en) 2007-09-12 2012-10-16 Plc Diagnostics, Inc. Waveguide-based optical scanning systems
US9528939B2 (en) 2006-03-10 2016-12-27 Indx Lifecare, Inc. Waveguide-based optical scanning systems
US9423397B2 (en) 2006-03-10 2016-08-23 Indx Lifecare, Inc. Waveguide-based detection system with scanning light source
US9976192B2 (en) 2006-03-10 2018-05-22 Ldip, Llc Waveguide-based detection system with scanning light source
GB2461026B (en) 2008-06-16 2011-03-09 Plc Diagnostics Inc System and method for nucleic acids sequencing by phased synthesis
JP5757535B2 (ja) 2009-04-29 2015-07-29 ピーエルシー ダイアグノスティクス， インコーポレイテッド 走査光源による導波管に基づく検出システム
US8765432B2 (en) 2009-12-18 2014-07-01 Oligasis, Llc Targeted drug phosphorylcholine polymer conjugates
ES2819217T3 (es) 2013-09-08 2021-04-15 Kodiak Sciences Inc Conjugados de polímeros iónicos dipolares y factor VIII
US10018566B2 (en) 2014-02-28 2018-07-10 Ldip, Llc Partially encapsulated waveguide based sensing chips, systems and methods of use
US9840553B2 (en) 2014-06-28 2017-12-12 Kodiak Sciences Inc. Dual PDGF/VEGF antagonists
EP3207128B1 (fr) 2014-10-17 2022-07-27 Kodiak Sciences Inc. Conjugués polymères zwittérioniques de butyrylcholinestérase
US11181479B2 (en) 2015-02-27 2021-11-23 Ldip, Llc Waveguide-based detection system with scanning light source
RU2744860C2 (ru) 2015-12-30 2021-03-16 Кодиак Сайенсиз Инк. Антитела и их конъюгаты
AU2020364071A1 (en) 2019-10-10 2022-05-26 Kodiak Sciences Inc. Methods of treating an eye disorder

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AU2002361223A1 (en) 2003-03-18
US20040253596A1 (en) 2004-12-16
WO2003021253A3 (fr) 2003-11-20
WO2003021253A2 (fr) 2003-03-13

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