WO2014108322A1 - Détection de molécules à l'aide de matrices dotées de différentes molécules de capture à chaque position de matrice - Google Patents

Détection de molécules à l'aide de matrices dotées de différentes molécules de capture à chaque position de matrice Download PDF

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WO2014108322A1
WO2014108322A1 PCT/EP2013/078147 EP2013078147W WO2014108322A1 WO 2014108322 A1 WO2014108322 A1 WO 2014108322A1 EP 2013078147 W EP2013078147 W EP 2013078147W WO 2014108322 A1 WO2014108322 A1 WO 2014108322A1
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array
molecules
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capture
detection
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Rainer Hintsche
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • 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

Definitions

  • the invention relates a method for the identification of biomolecules for analytical purposes.
  • microarray technology The parallel and simultaneous detection of multiple biochemical analytes in a sample using microarray technology is a well-known method in molecular biology, and plays an important role in the detection of nucleic acids and proteins.
  • the capture or probe molecules bind target molecules by affinity binding. Often these capture- analyte complexes are further modified by complex chemical methods and prepared for detection.
  • those array positions on which an affinity complex has formed are identified, for example by DNA hybridization.
  • DNA hybridization For this position-specific identification of affinity complexes physical or physico-chemical devices are used (Vo-Dinh andCullum, "Biosensors and biochips: advances in biological and medical diagnostics” Fresenius' Journal of Analytical Chemistry, 2004, p 540-551).
  • oligonucleotides which may consist of sequences of about 10-20 or more nucleotides, are immobilized on one position of an array. They bind their respective target nucleic acids in a position-specific manner and can thus be identified ("Microarray multiplex assay for the simultaneous detection and discrimination of hepatitis B, hepatitis C, and human
  • Antibodies or antigens are bound to an array position as scavengers, which then bind to complementary target proteins.
  • Affinity complexes are often labeled by colored dye or fluorescent marker molecules. This is achieved by covalent bonding, by intercalation or by additional affinity bonds.
  • the subsequent optical identification is based on the direct measurement of the position-specific intensity at specific optical wavelengths, or the production or the color change of a specific dye or the site-specific emissions of colored or fluorescent marker molecules.
  • the target DNA itself was labeled with the dye Cy-5 by PCR amplification. It then serves as an optical indicator and points to the array position on which the complex formation has taken place. Thus it allows the identification of the analytical target.
  • the conversion of a product of a chemical reaction can be measured and related directly to the amount of an affinity complex on an array position.
  • Affinity-bound target molecules on the array can be effectively marked by enzyme labeling. Marker enzymes generate optically or electrically active products from otherwise inactive substrates. The measurement of optically active products uses the same principles as described above for optical labels. For electrically active products, a redox reaction at the electrode is usually used for signal generation.
  • the enzyme labeling of an affinity complex is usually based on the well-known ELISA technique.
  • affinity binding molecules or groups of molecules labeling is achieved by molecular "sandwich" structures.
  • complementary and complexing agents commonly used in molecular biology and immunology is applied.
  • the molecular pairs of biotin / streptavidin, digoxigenin / anti-digoxigenin or antibodies and anti-antibodies are useful for these labeling procedures.
  • Another example is a fluorescein-binding group together with anti- fluorescein antibodies as a conjugate enzyme with Peroxidase.
  • nucleotide binding groups with a complementary nucleotide-enzyme conjugate eg aequorin
  • one binding partner is bound the affinity complex, whereas the other partner is coupled to the marker enzyme.
  • the amount of products producedby a marker enzyme eg, ⁇ - galactosidase or alkaline phosphatase
  • optical or electrical methods for detection and measurement may be used.
  • the electrically active species p-aminophenole,produced by the label enzymes ⁇ -galactosidase or alkaline phosphatase after the position specific formation of affinity complexes, is measured with interdigitated electrodes on each array position.
  • All known applications of array based techniques, independent of label type or detection principle, have in common that only one species of scavenger molecules is immobilized on each array position. Thus only one affinity complex is associated with each position.
  • To increase the number of target molecules that can be identified with an array up till now, the number of array positions had to be increased.
  • High density microarrays which can be used to simultaneously asses a broad range of genetic information, have advanced very far in this direction (Maitra et al.
  • Biosensors and Bioelectronics 2005, 20, 2435-2453 They achieve a higher array density by integrating CMOS circuits under the array positions of silicon chips. This decreases the area occupied by individual positions and therefore increases the overall number of array positions. All of the biochemical arrays described in the art do not allow the detection of multiple analyte species on one array position.
  • the objective of the present invention is to provide an efficient array-based method to detect multiple analytes on an array position without increasing the physical number of array positions. This objective is attained by the subject matter of the independent claims.
  • a method for the detection of multiple biochemical affinity complexes in an array device according to the invention compromises the following steps:
  • biochemical arrays can only detect one target molecule per position.
  • the aim of the present invention is to enhance common biochemical arrays to allow detection of two or more target molecules on each position.
  • mixtures of two or more capture probes are immobilized on a given array position. After capture of their respective targets, they are labeled through biochemical complexation and can thus be separately identified from the mix.
  • the present invention is the first realization of a biochemical multiplexing process. On a given array, it multiplies the number of identifiable analytical targets, without changing the existing apparatus.
  • a measuring device may be used to sequentially detect the different labels on its array positions. It is well suited for the application in flow- through systems, e.g. cartridges, in which the steps according to the inventive method can be carried out quickly in sequence.
  • the method of the invention is performed on common analytical arrays, on which various positions are arranged on so-called supports or carrier locations, which are spatially separated from one another.
  • any material is suitable on which two or more array positions may be arranged and may be registered by measurement.
  • the carrier consists of silicon, silicon compounds, glass, ceramic, metal, paper, one or more polymers, or combinations or layers thereof. Additionally, the carrier may be coated over its entire surface, or parts thereof. For example, some areas may be delimited by hydrophobic-hydrophilic treatments which support a localized immobilization of capture molecules on the array. Patterned coatings applied from metals such as gold, or polymers or biopolymers are also suitable.
  • arrays having surfaces or layers with additional optical properties for the acquisition or generation of optical signals.
  • carriers with array positions that are defined by mechanical barriers.
  • thin-film metal electrodes may be circular or patterned gold surfaces, such as the inter digitated electrodes.
  • the immobilization of the probes or capture molecules is performed, on a carrier, on two or more separated array positions.
  • the localized coating of the positions is achieved by applying the dissolved or dispersed probes or capture molecules in the form of drops to the predetermined array locations.
  • a suitable application is achieved by printing techniques or photolithographic methods.
  • Two or more types of capture or probe molecules are immobilized as a mixture on the same position. If present in the sample, the capture molecules then specifically bind two or more different target analytes at the same position. Since all array positions carry two or more receptor molecules, the analytical capacity increases in accordance with occupancy. On a multiply occupied array, the total number of identifiable analytical objectives grows to twice or more times the number of physically existing array positions.
  • probes or capture molecules All chemical and biochemical molecules that form affinity complexes can in principle be utilized as probes or capture molecules. These are the well-known complexing nucleic acids and oligonucleotides or proteins or peptides or haptens, or combinations thereof.
  • the immobilization of the probe or capture molecules is achieved by chemically binding them to the array positions.
  • binding is achieved by covalent chemical bonds.
  • binding is achieved by chemical interactions such as, but not limited to, Van der Waals forces, polar interactions or hydrogen bonds.
  • intermediate layers of inorganic or organic molecules or polymers or biopolymers may be used as a binding agents or adhesive layers.
  • compounds with reactive aldehyde or carboxyl or amino groups can, for example, be applied as adhesion-promoting layers to which the probes or capture molecules chemically bind.
  • Another way of immobilization is the binding of the capture molecules by means of thiol groups on structured gold surfaces.
  • the application of as little as a drop of a thiol-modified molecule on gold-coated positions will lead to spontaneous binding to such an array position.
  • proteins on gold such as antibodies, their intrinsic sulfur groups and hydrophobic moieties can be utilized.
  • the method of the invention uses two or more different capture molecules that are bound on the same position having the same reactive groups for immobilization.
  • the amounts of different species of capture or probe molecules in the mixture can be equal or can differ from one another.
  • the binding of target molecules to the probes also called complex formation, in some embodiments occurs according to the well-known lock-and-key principle of biomolecular interaction.
  • the complementary potential fields of the involved molecules bind the reaction partners to each other in order to form highly specific affinity complexes.
  • the arrays are brought into contact with analytical samples in aqueous solutions. If targets are contained in the samples, they exclusively form affinity complexes with their respective biochemical capture molecules on a position of the array.
  • targets are contained in the samples, they exclusively form affinity complexes with their respective biochemical capture molecules on a position of the array.
  • the specific complex formation takes place by the well- known process of hybridization, where double strands are formed from probe and target strands.
  • the recognition and binding principle is preferably based on the known antigen-antibody interactions.
  • small molecules such as haptens, which can be used both as a scavenger as well as a target molecules, complementary binding domains of proteins (such as of hapten-specific antibodies) or nucleic acids (e.g., aptamers) are used as binding sites .
  • the resulting affinity complexes are labeled with specific markers.
  • the marker molecules are bound either directly or with the aid of complexing agents to the affinity complexes composed of scavenger and analyte.
  • the labeling of the affinity complexes of scavengers and target protein or from scavengers and target nucleic acids uses various well-known biochemical reactions that are singularly used in the ELISA technique or in histology and other known analytical methods with molecular markers.
  • a major feature of the invention is to combine these in well-known and proven labeling methods in a new manner with the array based detection method.
  • a plurality of analytes in the mixture may be detected on a single physical array position. If two types of capture molecules per array position are immobilized in mixture, two different markers are used selectively, each for one scavenger.
  • the number of analytes per array position can be further increased by using a third scavenger per array position and linking the corresponding analyte with an additional complexing agent.
  • the method can be further expanded to four or more analytes per array position by adding more scavengers in the same way. These are then linked to additional complexing agents such as specific nucleotide sequences with 10-30 bases.
  • the method of the invention is only limited by the availability of different marker molecules in combination with selective labeling methods. With increasing numbers the scavengers will be diluted. This of course, has to correlated with the sensitivity of the detection method. If necessary, it may be compensated by increasing the detection area.
  • the method according to the invention is suitable to be uses with any type of molecule, molecular residues or molecular complexes as labeling groups— if they are distinguishable on any one array position and can thus be specifically assigned to an analyte.
  • Particularly suitable are different dyes or different enzymes.
  • the separate detection according to the invention can, for example, be achieved by measuring their optical absorption or emission at different wavelengths.
  • Suitable for the labeling of two to five different affinity complexes in the mixture are, for example, fluorescent dyes Cy2 (emission at 506 nm), Cy3 (emission at 570 nm), Cy5 (emission at 667 nm) and Cy7 (emission at 767 nm).
  • fluorescent dyes Cy2 emission at 506 nm
  • Cy3 emission at 570 nm
  • Cy5 emission at 667 nm
  • Cy7 emission at 767 nm
  • the presence and amount of a Cy2 dye-labeled affinity complex with an extincion maximum at 490 nm and an emission maximum at 508 nm is determined, and then the presence and amount of a dye-labeled Cy5 affinity complex with an excitation maximum at 650 nm and an emission maximum at 674 nm. In this way both dyes can safely be distinguished on the same array position.
  • affinity complexes are achieved through different marker enzymes such as peroxidases, catalases and pseudoperoxidases that catalyze distinct
  • a different kind of labeling is also possible by binding of different enzymes to the analytes on each array position.
  • known principles of the ELISA technique are used for complex formation.
  • Enzymes suitable for the use as a biochemical marker are catalase and oxidase enzymes, such as alkaline phosphatase and ⁇ -galactosidase and horseradish peroxidase.
  • the identification of the analytes after labeling with these enzymes is achieved by measuring their different products. This is realized by bringing the whole array into contact first with a substrate for an enzyme A (e.g.
  • Enzyme A produces another product than enzyme B which makes both enzymes distinguishable by their detection and the sequential order of measuring steps. Therefore it can be assigned to it's respective analyte.
  • Enzymes can be distinguished very well electrochemically. The current measured at each array electrode correlates linearly with the number of enzyme products produced at the electrode surface of the array position. As an alternative to electrochemical methods, the enzyme-analyte complexes can be distinguished by enzyme products with different optical properties, for example in the visible or ultraviolet optical spectrum. Also the different substrates are supplied to the array subsequently and separated by wash steps. The products formed in each case are measured spectroscopically and their position is specifically identified.
  • alkaline phosphatase usually p- aminophenyl phosphate is provided as a substrate. After cleavage of the phosphate group it reacts as p-amino-phenol to quinone at the electrodes and is measured amperometrically.
  • An other suitable marker enzyme in accordance with the invention is ⁇ -galactosidase, for which a variety of substrates is available. When used for electrical detection methods it catalazes the reaction from the electrically inactive p-nitrophenyl-P-D-galactopyranoside to p-amino-phenol, which is electrically detected in the same manner as described above for the phosphatase.
  • optical detection methods for example, o-nitrophenyl-P-D-galactopyranosidase, naphthol- AS-BI-P-D-galactopyranosidase and 4-methyl-P-D-umbelliferyl galactopyranosidase are used.
  • oligonucleotides can be used.
  • the oligonucleotides are labeled in advance with marker molecules such as dyes, enzymes or chelating molecule residues. They bind to a pre- defined complementary site of the respective target analytes by hybridization. This is achieved for example by the incorporation of chelating molecule residues such as digoxigenin or biotin.
  • the molecular residues are incorporated during an amplification of the target analytes by PCR reactions.
  • the PCR reaction mixtures (so-called master mix) are admixed with modified nucleosides which carry such molecular residues (e.g. digoxigenin modified deoxyuridine triphosphate (dUTP)).
  • dUTP digoxigenin modified deoxyuridine triphosphate
  • the detection of the digoxigenin is then achieved by an anti- digoxigenin antibody which is coupled to a marker enzyme as described above.
  • the different detectable enzyme products are made as described above.
  • biotin uses its strong affinity to streptavidin, which has multiple binding sites for biotin.
  • the procedure is analogous as for digoxigenin described.
  • proteins, such as antigens or antibodies or polypeptide complexes are detectable in accordance with the invention.
  • a large number of alternative labeling methods are known in the art. For example, after the formation of affinity complexes of captures on the array and the
  • a second specific antibody also called detection or secondary antibody
  • a marker enzyme or a dye conjuggated
  • a labeled antibody binds complementary to the analyte in the affinity complex, while unbound antibody is washed off.
  • the enzyme is provided with its substrate, which reacts as described above.
  • the dye or the electrically active product is formed locally, where the immunochemical reaction has taken place and thus identifies the target analyte on the specific array position.
  • the labeling methods are well-known and all of the reagents mentioned above are commercially available.
  • the different labeling on an array position uses, as described above, rinsing or washing steps to activate and identify the different markers on each array position.
  • the execution of this process is carried out in flow-through devices according to certain embodiments. There, the respective array is brought into contact with the changing liquids, so that sequential and consecutive reactions can be realized.
  • the procedure can be performed in any vessels in which liquid wets an array. To the required fluids are changed sequentially and manually or automatically.
  • optical or electrical methods can be used.
  • Particularly suitable for the process according to the invention are conventional optical transducers and sensors that register the optical properties of molecules on substrates in array formats.
  • Very suitable are also planar arrays with optically active coatings that allow, by interaction with neighboring molecules, for example, the site-specific optical identification by means of evanescent waves or surface plasmons.
  • measuring instruments with diodes and/or transistors, and/or lasers and other optical devices are used. These are capable of site-specific measurements on the arrays such that each position can be identified and the labels used are distinguishable.
  • a highly suitable measuring instrument is, for example, a beam guided laser, which allows to locate affinity complexes on conventional arrays which are labeled, for example with fluorescent molecule residues and, optionally, to determine their amounts.
  • measuring devices with CCD or CMOS sensors. Highly sophisticated detectors may detect different labels with distinguishable optical properties, eg. different optical wavelengths simultaneously in one run.
  • the arrays are formed in this case, for example, as gold or carbon electrodes.
  • the array items are individually detected by amperometric or voltammetric or impidimetric instruments. Even so- called “imaging”, ie, the simultaneous mapping of all the items of the array in the resolvable image is usable.
  • the capture or probe molecules are bound or immobilized spontaneously or by means of linkers.
  • the electrochemical signals of marker molecules and / or site-specifically formed products of enzymes are particularly advantageous measured with external measuring arrangements or by means integrated circuits in the same chip.
  • the number of analyte molecules correlates linearly with the measured electrical redox current.
  • Fig.la, lb, lc schematically show the basic idea of the method with the stepwise procedure at two exemplary positions of an analytical array.
  • Fig.la shows an arrangement for carrying out the first step with the immobilized nucleotide sequences as capture molecules on an array.
  • array position 1 On array position 1 the mixture of the nucleotide sequences 3 + 4 and on array position 2 the mixture of the nucleotide sequences 5 + 6are immobilized.
  • As a carrier of the array in particular glass or silicon chips, or polymers are used.
  • the array positions 1 and 2 have a structured coating with gold.
  • nucleotide sequences as capture molecules 3, 4 , 5 , 6 bind via their thiol group located at the end of the chain molecules spontaneously on gold surfaces. Thereby, mixtures of 3 + 4 are dropped or spotted on the array position 1. The same happens with the mixture of 5 + 6 to array position 2.
  • Fig. lb shows the arrangement with the immobilized nucleotide sequences as in la after recognition and binding of the complementary analytes from an analysis solution.
  • the capture nucleotide sequences selectively are selectively bound only to its target analyte, if present in the specially buffered solution analysis. If present in the sample, the capture nucleotide 3 selectively bind the target analyte 7 , capture nucleotide 4 the target analyte 8 , capture nucleotide 5 the target analyte 9, capture nucleotide 6 the target analyte 10 .
  • analyte have been previously covalently coupled with complexing molecule residues. This was performed by a conventional PCR, which is also used to amplify the analyte molecules. The respective complexing residues are bound to nucleotides and introduced by adding it to the master mix of the PCR reaction.
  • each of the analytes of one array position carries another complexing agent.
  • these are the target analyte 7 linked with covalently linked biotin 11 and the target analyte 8 linked with covalently complexing digoxigenin 12 .
  • the same complexing agents are used analogous to the analytes of all other array positions. So target analyte 9 is bound to 11 and the target analyte 10 to 12 .
  • Fig.lc shows the same arrangement of hybridized nucleotide analyte complexes as in Fig. lb plus the marker enzymes bound.
  • streptavidin is the complementary molecule 13, which is covalently linked to marker enzyme 14.
  • ⁇ -galactosidase as the marker enzyme 14 is commercially available as a conjugate with streptavidin.
  • the marker enzyme 14 binds only to biotin-linked molecules.
  • anti-digoxigenin antibodies are used as
  • Fig. la, lb, lc it is not important whether, or how many analytes are present in a solution to be detected. The absence of any or all analytes results only that the respective capture molecules are not complexing and remain empty and are not involved in the following reaction and detection steps.
  • Fig. 2a and 2b schematically show the electrical detection of complex molecules on the array positions, such as in Fig. lc are shown.
  • the enzyme labeling and detection of mixtures of capture reagent / analyte molecules on the array positions is reached by means of two successive steps of the detection.
  • Fig. 2a shows the method step A, the electric identification of the enzyme-labeled capture / analyte complexes as shown in Fig. lc.
  • a solution containing an enzyme substrate 14ES added over the entire array.
  • marker enzyme 14 it creates its product 14EP.
  • the electrode active product p- quinone-imine is liberated from the substrate 4-aminophenyl ⁇ -D-galactopyranoside by the ⁇ - galactosidase.
  • the electrical array detector serves a multipotentiostat, which detects the position specific slew rate of the currents on each array position. This 14EP is not detectable on the adjacent electrode array 2E because the distance for the diffusion is too long. Vice versa any marker enzyme 14 to array position 2 or on other array positions do not interfere the detection at position 1.Thus the analyte 7 on array position 1 is selectively identified.
  • a possible co-existing complex of captures 4 and analyte 8 marked with marker enzyme 16 on the array position 1 remains without reaction, because the enzyme is capable of reacting only with its specific substrate, and 14ES is not produced.
  • the array detector measures electrode 2E on the array position 2 performing the so-called electrical multiplexing. If analyte 9 is bound to capture 5 its marker enzyme 14 also liberates 14EP from 14ES. Than a current at electrode 2E is measured and thus unequivocally the analyte 9 identified as being present. Also on position 2 a co-existing complex of captures 6 and analyte 10 marked with marker enzyme 16 remains without reaction.
  • Fig.2b shows the method step B, the electrical identification of the enzyme-labeled capture / analyte complexes as described in Fig. lc .
  • step A the entire reaction liquid is removed by rinsing washing solution over the array. Than, a solution with substrat 16ES is flown over the array.
  • enzyme marker 16 the alkaline phosphatase is converting its substrate 16ES 4-aminophenyl-phosphate monosodium hydrate into its product 16EP the p-quinone imine. All molecular complexes with the marker enzyme 16 perform this reaction.
  • the amperometric current atelectrode IE and then in the following multiplexing with electrode E2 are measured indicating positions where 16EP is produced. In this way, at array position 1 the capture of 4 bound analyte 8 and array position 2 the capture of 6 bound analyte 10 unequivocally may be identified.
  • step B existing molecule complexes are labeled with marker enzyme 14 without reaction.
  • ⁇ -galactosidase as a marker enzyme 14 and alkaline phosphatase as marker enzyme 16 is carried out in a simple manner with conventional reagents for the enzyme labeling.
  • biotin as a complexing 11
  • digoxigenin as complexing 12 .
  • the streptavidin as the complementary molecule 13 is covalently linked to ⁇ -galactosidase, and only binds to biotin ( 11 ).
  • the anti-digoxigenin as a
  • label enzymes ⁇ -galactosidase 14 and alkaline phosphatase 16 react specifically with their own substrate.
  • the products 14EP and 16EP are principally the same molecules and the numbering indicates the different source and detection step.
  • the detected electrical responses can be assigned to ensure each analyte on each array position.
  • Fig.3 shows the principle of the invention applied to proteins or peptides or haptens as target analytes.
  • the exemplary embodiment shows the use of immobilized antibodies as capture molecules and different optical chromophores as labels of the affinity complexes.
  • Fig.3a shows an array of capture molecules, where on position 1 a mixture of antibodies 17 + 18 and array position 2 a mixture of antibodies 19 + 20 are immobilized.
  • Fig.3b shows the immobilized capture antibodies as in 3a, which have their complementary analytes bound.
  • the antibody binds 17 the target analyte 21 , the antibody 18 the target analyte 22 , the antibody 19 the target analyte 23 and antibody 20 the target analyte 24 .
  • Fig.3c shows the immobilized antibodies with affinity-bound analytes as in Fig.3b, which are specifically labeled.
  • the analyte 21 is complexing with the detection antibody 25, which is conjugated with complexing biotin 11.
  • Analogously analyte 22 is bound to detection antibody 26, which is conjugated with digoxigenin 12.
  • Analyte 23 in turn forms complexes with the detection antibody 27 biotin 11 conjugate and analyte 24 respectively forms complexes with the detection antibody 28 digoxigenin conjugate 12 .
  • Fig. 3d shows the same arrangement as in 3c complexing label enzymes.
  • the complexing agents 11 biotinand 13 streptavidin arecovalently linked with label enzyme 14 ⁇ - galactosidase and label enzyme 16 alkaline phosphatase respectively.
  • the detection of the target analytes are performed analogous to the process scheme as in 2a and 2b shown for nucleic acid complexes.
  • the ⁇ -galactosidase liberates the electrode active enzyme product of 14EP p-quinone imine from the substrate 4-aminophenyl ⁇ -D-galactopyranoside 14ES.
  • the analytes 21 and 23 sequentially detected.
  • the alkaline Phosphatase liberates their product the product p-quinone imine 16EP from 16ES the substrate 4-aminophenyl phosphate, mono-sodium hydrate.
  • the analytes 22 and 24 are sequentially detected. Since, in this embodiment, in analogy to Fig. 2 the label enzymes ⁇ -galactosidase and alkaline phosphatase react only with their corresponding substrates step A and B the detection of responsesof each capture molecule and each array position assign secure.
  • Figt.4 shows an embodiment for the application of optical detections and using an array of immobilized nucleotide sequences as in Fig. la.
  • Essential to this embodiment is that the analytes have been previously covalently linked to specific chromophoric molecule groups. This is achieved by means of polymerase chain reaction. Adding the corresponding nucleotides carrying the different chromophores in the master mix of these PCR reactions they are
  • the chromophore 29 the fluorescent dye Cy 2 with an excitation maximum at 490 nm and an emission maximum at 508 nm
  • the dye Cy5 with an excitation at 650 nm and an emission maximum at 674 nm
  • Fig.5 shows the measurement for the optical identification of analyte mixtures in the array comprising an array of molecules as shown in Fig.4.
  • the two dyeson one array position may be excited and determined and sequential as follows:
  • excitation and detection of emission may be done in parallel or sequentially.
  • the given method allows, in this exemplary embodiment, to double the number of analytical targets compared to the number of array positions.
  • optical measuring methods are particularly suitable to increase the number of catures and analytes per array position with additional chromophores, because the number of chemical steps are small and the optical measurement method is expandable with less effort.
  • the inventive method may be performed on arrays dived in conventional chemical vessels or flow-through devices.
  • Particularly suitable are flow-through devices, such as chambers, tubes, cartridges or hoses, which allow to realize fluidic changes fast and very effectively.
  • Fig.6 shows an apparatus for carrying out the method according to the invention on a planar array carrier 31 in a flow-through device 32 is placed.
  • the exchange of fluids 33 for chemical and biochemical reactions according to the invention is carried out by turning on and discharging the reaction solutions by common technique. Accordingly, an electrochemical detection is easily carried out by, for example electrode recordings are taken out from liquid-carrying parts.

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Abstract

La présente invention concerne un procédé de détection d'une pluralité d'espèces de substances à analyser issues d'un échantillon, comprenant les étapes consistant : fournir une matrice en phase solide comprenant une pluralité de positions de matrice, chaque position de matrice ayant une pluralité de molécules de capture molécules immobilisées sur elle, et chaque molécule de capture pouvant se lier spécifiquement à une espèce de substance à analyser particulière ; mettre ledit échantillon en contact avec ladite matrice en phase solide dans des conditions permettant que lesdites molécules de capture se lient spécifiquement à ladite espèce de substance à analyser, et la détection de la présence ou de l'absence d'une espèce de substance à analyser, et/ou son identification, à chaque position de matrice.
PCT/EP2013/078147 2013-01-08 2013-12-30 Détection de molécules à l'aide de matrices dotées de différentes molécules de capture à chaque position de matrice Ceased WO2014108322A1 (fr)

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DE201310000682 DE102013000682B3 (de) 2013-01-08 2013-01-08 Verfahren zur Detektion von Molekülen
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