DE10127220A1 - Carrier for analyzing chemical and biological samples, e.g. for diagnosis, on which binding reaction produces structure that can be read by optical methods - Google Patents

Abstract

Carrier (A), generates structures that can be read by optical or magneto-optical techniques used in information technology. Carrier (A), for analyzing chemical or biological samples in which information about binding/non-binding of sample at a particular site, is generated by creating a structure (S) at the locus of reaction between a substance in the sample and carrier-bound binding agent. The size of (S) is selected such that its properties are functionally equivalent to the primary information structural elements of an optical or magneto-optical, high capacity memory used in information technology. Independent claims are also included for; (1) Apparatus for preparing (A); (2) Apparatus for analyzing molecular binding reactions on (A); and (3) Kit that contains substances for an analysis on (A).

Description

Introduction and description

Analyzes of biological samples can be advantageous using so-called biochips be performed. Chemical or biological substances are applied to metal, Glass, membrane or plastic surfaces, especially polycarbonate supports, applied. These substances then react with other substances from one applied sample. The site-specific binding of these substances then enables one Evidence of the molecular interaction and usually also a statement about the bound Amount or substance composition and / or strength of interaction. According to the state of the art, the bonds are mostly created by creating an optical one Signal detected. Usually a microscope or a functional one analog device, such as a CD read head, used. The information about a Successful or non-successful binding at a certain point is determined by image evaluation obtained, which in biochips of higher density typically by computer software after an analog-to-digital conversion. In one embodiment, the  Information about a successful or non-successful binding at a certain point by the deposition of granules ("beads") at the point of reaction of a substance from a applied sample marked with the carrier-bound substance (see e.g. Taton, Mirkin, Letsinger [2000] Science 289, pp. 1757-1760). According to the state of the art bound beads but either identified by the image analysis described above or they bring about a chemical reaction which is essentially photometric Methods (color change, dye precipitate) is detected. A successful one Color change or a dye precipitate then again requires an image analysis Generation of the information which is used for further computer analysis can.

The method described here considerably simplifies this process in that the information about a successful or non-successful binding at a certain point is generated by a chemical reaction for the production of detectable structures, in particular granules ("beads") or layers of Precipitates, at the site of the reaction of a substance from an applied sample with the carrier-bound substance in a manner which directly generates a binary signal in a suitable readout unit. This signal no longer requires image processing in the above sense to be used. According to the invention, this is done in an advantageous embodiment variant by producing silver grains at the site of the bond, which form on an initiator, in particular an electron donor such as a metal molecule or granule, which in turn is connected to the sample to be detected ( FIG. 1). The binary reading is ensured in that the size of the precipitate is chosen so that its properties are essentially similar to the information-carrying structures of an optical, magneto-optical or electromagnetic mass storage device known from information technology. In another advantageous embodiment, the resulting detectable structure can also be generated by initiating a chemical reaction from a substance previously applied only at certain points on the carrier, in particular in the form of signal-emitting surface structures with properties defined for the detector, which are referred to below as pits , For this purpose, in addition to the molecule applied to the carrier, which is to interact with the sample to be analyzed, a precursor of another substance or a mixture which is suitable for this and which can be converted into a detectable structure is applied. The applied area corresponds essentially to a pit. After binding of a sensor molecule, this causes a reaction in this substance or the mixture of substances, which changes the optical properties of the coated structure, in particular by a reaction which spreads and breaks off in an essentially monomolecular manner on the surface up to the edges of the pit.

The binary information of such a mass storage device can be the position information provide the assignment of the by chemical or biochemical binding of a Sensor signal obtained binary signal to a defined substance enables which according to the prior art by printing, lithographic synthesis or others Suitable methods are applied to this position information in a defined arrangement has been.

As a carrier, polymer plastics with different, the analytical Task adapted physico-chemical properties, but also glass, Semiconductors, metals, metal alloys, ceramics, hybrid materials or combinations made of these materials. An advantageous application uses carriers made of polycarbonate of optical quality as well as data structures as they are in CD and DVD Find technology application.

The invention provides that a large number of different molecules is advantageous (Molecular libraries or molecules for several different individual tests) on the Can apply substrate surface. Different molecules become spatial for this limited applied within ordered structures. The surfaces, in particular less than 10 × 10 µm, advantageously less than 2 × 2 µm and with particular Advantage less than 1 × 1 µm expansion.  

The molecules applied to the substrate surface can be substances which are useful for chemical, biochemical or medical evidence. This include sugar, steroids, hormones, lipids, proteins, especially mono- or polyclonal or recombinant antibodies, peptides, antigens of all kinds, microorganisms or parts thereof, preparations and extracts from biological materials, Metabolism metabolites, DNA, RNA as well as natural and artificial derivatives thereof, in particular aptamers and PNA, but also organic chemical libraries, as they e.g. B. used for pharmacological developments, or haptens.

In addition to these molecules, others can be used according to a predefined arrangement Molecules or signaling elements can be applied to neighboring pits, which are used for Calibration or standardization of the analyzes are used. These can be on surfaces the same or similar order of magnitude and in particular spatially from the actual molecules used analytically by surfaces free of molecules (Empty spaces).

In its simplest form, the analysis carrier thus contains a pattern of pits which contain such molecules, and in between, but not necessarily in a regular arrangement, empty spaces which separate the pits from one another. Several such parallel tracks as well as linear, curved, radial, spiral, circular or other geometrical arrangements of pits are possible and form part of the invention. Several methods according to the prior art can be used to apply the sensor molecules to geometric patterns of pits, in particular:

• 1. A liquid bath (simple immersion of the carrier after selective activation of Pits).
• 2. Wetting using microchannels or microfluidic networks.
• 3. Stamp processes, in particular lithographic processes such as soft lithography.
• 4. spotting techniques.  
• 5. Ink-jet process.
• 6. Mechanical transfer of substances using rotating rollers (e.g. Laser printer, photocopier).
• 7. spin coating.

All substances that are useful for chemical, biochemical or medical detection can serve as sensor molecules. These include sugar, steroids, hormones, lipids, proteins, especially mono- or polyclonal or recombinant antibodies ( Fig. 1), peptides, all kinds of antigens, microorganisms or parts thereof, preparations and extracts from biological materials, metabolites, DNA ( Fig. 3), RNA and natural and artificial derivatives thereof, in particular aptamers and PNA, but also organic chemical drug libraries, such as those used for. B. used for pharmacological developments, or haptens.

Furthermore, the invention comprises a reader optimized for the wearer, which in advantageous embodiment a semi-automatic or automatic positioning and Allow the carrier to be read out. A downstream computer system requires Evaluation of the analyzes housed on the carrier special software, which on the respective analysis medium is tailored. This software can be beneficial Execution in the data portion present on the carrier. Therefor can therefore data readers such as CD-ROM or DVD player or comparable devices and whose successors are used, with which the carrier is compatible in principle. The analysis software can also be run on databases optionally available on the carrier access the standard values required in the context of the specific analysis in the Keep access.

The invention can be particularly advantageous in biochemical or biomedical Detection methods apply. Because of the practical handling  exemplary, in particular, evidence in biochemical laboratories, in laboratories of medical practices and hospitals as well as in all areas of environmental protection, quality control, of biotechnology, forensics, agricultural and plant hybrid analysis, and the molecular biological, pharmaceutical and medical research and development possible. Other ways of using an inventive Analysis platforms are also possible and thus the subject of the invention. The Invention comprises the necessary substance combinations ("kits"), which Contain agents for the inventive production of the detectable structures.

embodiments

• 1. A carrier made of polycarbonate with certain defined applied Position information in the form of "pits", essentially identical to a CD, is cleaned with 100% ethanol. After that, an antigen becomes a pathogenic Microorganism using a stamp from PDMS on certain areas of the Printed carrier. Stamp and polycarbonate carrier are made using a Bracket arranged to each other so that an exact positioning with each other accuracy of at least 1 micron is guaranteed. The carrier will after Printing for 40 min in a solution of 1% bovine serum albumin (BSA) incubated. The carrier is then washed with distilled water and washed with human sample serum incubated for 15 min at 37 ° C. After washing 2 × 5 min in PBS (20 mM Na-phosphate, 140 mM NaCl) the carrier with a 1: 350 Dilution in PBS of a goat anti-human IgG antibody coupled to Snm colloidal gold particles, incubated for 20 min at room temperature. After again a silver solution is added twice with PBS. This consists of an advantageous embodiment of 110 mg silver lactate, 850 mg hydroquinone, 2.55 g Citric acid monohydrate, 2.35 g trisodium citrate ad 100 ml water, which immediately before the reaction is freshly prepared. After 2-4 minutes in the dark the reaction is complete Washing stopped with distilled water and then with 2.5% (w / v) Sodium thiosulfate fixed in water. In a further advantageous embodiment, instead, a commercial one known to those skilled in immunohistology "Silver enhancement" solution (eg from Sigma-Chemie, Munich) can be used.

After washing again with distilled water, the carrier is in an im read the essential commercially available CD drive.

• 1. Biotinylated antibodies against a searched protein are mixed with a patient's serum sample, which was previously diluted 1: 3 in PBS (see above). The mixture is 10 min. Centrifuged at 13,000 × g and the supernatant applied to a detection support which is coated at certain points in a process analogous to that described in Example 1 with a further antibody which binds another epitope on the protein to be detected from the serum. After thirty minutes of incubation at room temperature and washing three times in PBS, a mixture of core streptavidin and biotinylated ferritin freshly prepared in PBS cooled to 4 ° C. is applied to the detection support. The ferritin was biotinylated using a prior art kit such that an average of 4-10 biotin molecules are bound per ferritin tetramer. Within an incubation period of typically 30 minutes, at a certain content of the protein sought, large cross-linked complexes form at the locations where the support is coated with the corresponding antibody ( FIG. 4). These complexes are to be optically detected, their arrangement is subsequently read out and verified analogously to Example 1.

Description of the figures

Fig. 1 shows a schematic representation of a document according to the invention a binding reaction of a sensor molecule (1) (in this case an antibody) to its antigen (2), which is bound to a support (3). A colloidal gold particle of 1 nm average diameter ( 4 ) attached to the sensor molecule ( 1 ) leads to the deposition of a silver grain ( 5 ).

Fig. 2 image of fine lines in the micrometer range, which is based on an antibody-antigen reaction with downstream silver deposition according to Fig. 1. Rabbit immunoglobulin was used as the antigen, and a goat antibody against rabbit immunoglobulin was used as the detection agent, which antibody is coupled to colloidal gold particles. The detectable structure was deposited by silver precipitation. A: Analog image taken with a CD reading head; B: In an advantageous embodiment, a binary signal can already be generated in the read head of the device; C: Analog image line from A, characterized by the horizontal, dashed line in A; D: binary signal of the same image line, formed by means of a threshold criterion in the analog signal, characterized by the horizontal line in C.

Fig. 3 example of a detection of a DNA hybridization to a DNA array using the agent of the invention. Streptavidin ( 6 ) is bound to a support ( 3 ). Non-specific binding sites are saturated by a block substance ( 7 ). Nucleic acid molecules ( 9 ) coupled to biotin ( 8 ) are bound. A sample containing a sensor oligonucleotide ( 11 ) provided with a "tag" ( 10 ) is then added. Its binding is finally detected by a colloidal gold particle ( 13 ) coated with a tag binding molecule ( 12 ) that binds the tag ( 10 ), which leads to a deposition of a metal ( 14 ).

Fig. 4 is a detectable component structure according to Example 2. A on a carrier (3) applied receptor (15) binds a ligand (16) attached to a hapten, in this case biotin (17) is coupled. After adding a mixture of streptavidin ( 18 ) and biotinylated ferritin ( 19 ), detectable molecular complexes ( 20 ) are formed.

Claims (19)

1. Carrier for the analysis of chemical or biological samples, characterized in that the information about a successful or non-successful binding of the chemical or biological sample at a specific point is generated in that a structure at the location of the reaction of a substance with an applied sample of the carrier-bound substance is produced, the size of the structure being formed being chosen such that its properties are essentially functionally equivalent to the primary information structure elements of an optical or magento-optical mass storage device known from information technology. 2. Carrier according to claim 1, characterized in that the individual generated detectable structures an area of less than 10 microns × 10 each Cover the micrometer. 3. Carrier according to claim 1 or 2, characterized in that the structure produced a binary information unit on the disk essentially functional equivalent. 4. Carrier according to at least one of claims 1 to 3, characterized in that a structure created by binding a single molecule to a binary Functional information bit on the data carrier essentially corresponds. 5. Carrier according to one of claims 1 to 4, characterized in that the binary Information can be read out with the aid of optical methods, in particular by resonant or non-resonant optical effects such as reflection, refraction, plasmon Resonance, scattering, diffraction, fluorescence, phosphorescence, luminescence,  Change in the level of polymerisation, change in absorption or transmission, Phase shift or quenching. 6. Carrier according to one of claims 1 to 4, characterized in that the binary Information can be read using electromagnetic methods, in particular through magnetic effects, electrostatic effects, change in resonance frequency Mass change, Hall effect or conductivity, resistance or Changes in capacitance. 7. Carrier according to at least one of claims 1 to 6, characterized in that the structures produced essentially from metal or one of its oxides, There are complexes or salts. 8. Carrier according to at least one of claims 1 to 7, characterized in that the formation of the structures is initiated by an electron donor, in particular by a precious metal such as copper, nickel, zinc, platinum, silver or Gold, typically as bodies with a diameter smaller than 100 nm, or by a single atom or cluster of these metals, typically in chelate or complex form. 9. Carrier according to at least one of claims 1 to 8, characterized in that the detectable structure is precipitated from a solution which is a Contains metal compound, especially silver or gold salts such as their nitrates, Lactates or halides. 10. A carrier according to at least one of claims 1 to 9, characterized in that the structure is generated by a chemical or biological conversion, especially through a reaction with other substances.   11. A carrier according to at least one of claims 1 to 10, characterized in that the structure through chemical or biological catalysis using enzymes, in particular alkaline phosphatase, horseradish peroxidase, glucose oxidase, Beta-galactosidase, lactate dehydrogenase or other enzymes is generated, by reacting a substrate from the solution in particular so that a Precipitate is generated. 12. Carrier according to at least one of claims 1 to 11, characterized in that the detectable structure by a chemical or biological polymer tion reaction is generated. 13. A carrier according to at least one of claims 1 to 12, characterized in that the detectable structure through the assembly of several molecules is not necessarily exclusively of biological origin, which in particular from antibodies, avidin, streptavidin or one of them Derivatives, as well as from their natural binding partners or from haptenized, especially with biotin, digoxigenin, phenoxazolone or FITC haptenized substances, especially haptenized ferritin, horseradish Peroxidase, cytochrome or other metalloproteins or colored proteins, is effected. 14. Carrier according to at least one of claims 1 to 13, characterized in that the detectable structure by initiating a chemical reaction from one beforehand only at certain points on the wearer, especially in the form of defined ones Areas like pits, applied substance is created. 15. Device for producing a carrier according to one of claims 1 to 14, characterized characterized that the steps, but not necessarily all steps, which have led to the formation of the binary detectable structure, under defined  Ambient conditions are carried out and preferably semi-automatically or be carried out automatically. 16. Device for the analysis of molecular binding reactions on a support after a of claims 1 to 14, characterized in that the binary reading with the help Optical method is carried out, in particular resonant or non-resonant optical effects such as reflection, refraction, plasmon resonance, scattering, Diffraction, fluorescence, phosphorescence, luminescence, change in the Polimersi station level, change in absorption or transmission, phase shift or Quenching. 17. Device for the analysis of molecular binding reactions on a support after a of claims 1 to 14, characterized in that the binary reading with the help electromagnetic method is carried out, in particular by magnetic Effects, electrostatic effects, resonance frequency change with mass change, Hall effect or changes in conductivity, resistance or capacitance. 18. Device for the analysis of molecular binding reactions on a support after a of claims 1 to 14, in particular for use in biochemical laboratories, in Laboratories of medical practices and hospitals as well as in all areas of the Environmental protection, quality control, biotechnology, forensics, agricultural and Plant hybrid analysis, as well as molecular biological, pharmaceutical and medical research and development. 19. Kit containing the essential substances for carrying out one or more Analyzes with the supports described in claims 1 to 14.