JP2003517156A - Compositions and methods for performing biological reactions - Google Patents

Abstract

(57) Abstract The present invention relates to an apparatus for performing a biological reaction. In particular, the present invention relates to an apparatus for performing a nucleic acid hybridization reaction on a substrate layer having a large number of oligonucleotide binding sites using a substrate having a flexible cover.

Description

Detailed Description of the Invention

This application is a US patent application Ser. No. 09/605, filed June 28, 2000.
No. 766 and US patent application Ser. No. 09/4644, filed Dec. 15, 1999
No. 90 is a continuation application.

TECHNICAL FIELD OF THE INVENTION The present invention relates to compositions and methods for conducting biological reactions. In particular, the invention is
The present invention relates to a method of performing a nucleic acid hybridization reaction on a substrate layer on which a plurality of oligonucleotide binding sites are arranged.

Recent developments in molecular biology have provided the opportunity to identify pathogens, diagnose pathologies and make forensic determinations using gene sequences that are unique to the desired purpose. Due to the achievement of genetic information, there is a need for a high-capacity analytical instrument for performing a molecular biology assay, particularly a nucleic acid hybridization assay. Most urgent is the need to miniaturize, automate, standardize, and simplify such assays. This need has been met by the adoption of these procedures in clinical fields such as diagnostics, forensic science, and other areas, while hybridization assays were originally developed in the laboratory using purified products and performed by highly skilled personnel. This has arisen from the fact that there has arisen a need for devices and methods that allow an unskilled operator to effectively perform assays in larger volumes and less stringent assay conditions.

Nucleic acid hybridization assays are advantageously accomplished using probe array technology. This probe array technology utilizes the binding of target single-stranded DNA to immobilized DNA (usually oligonucleotide) probes. The limit of detection of a nucleic acid hybridization assay is determined by the sensitivity of the detection device during hybridization and the amount of target nucleic acid utilized to bind the probe, which is typically an oligonucleotide probe.

Nucleic Acid Hybridization Chamber discloses Smyc
No. 5,100,755 to Zek et al. Rava et al., US Pat. No. 554
5531 discloses a hybridization plate consisting of a plurality of oligonucleotide arrays. Hunnell, US Pat. No. 5,360,741 discloses a gas heated hybridization chamber. Anderson et al., U.S. Pat. No. 5,922,591.
No. 6,058,049 discloses a miniaturized hybridization chamber for use with an oligonucleotide array. Bsesemer US Pat. No. 5,945,334 discloses oligonucleotide array packaging.

[0006] Currently utilized oligonucleotide array technology does not provide maximum hybridization efficiency. The existing nucleic acid hybridization assay device has a large number of members, and each member causes inefficiency and inaccuracy.

Hybridization using oligonucleotide arrays effectively anneals small amounts of target DNA or other nucleic acids to immobilized probes.
l) must be achieved in a volume that can be done. In diagnostic assays, target DNA molecules are obtained in minute amounts (picomoles). In practice, it takes several hours (10 hours) for hybridization to be substantially complete at the low nucleic acid levels useful for biological samples.

Further, array hybridization has traditionally been performed with active mixing.
) Without static hybridization chamber. Under such conditions, the probability of a particular target molecule being array-hybridized to a complementary oligonucleotide probe immobilized on the surface depends on the concentration of the target molecule, the diffusion rate, and the interaction between the target molecule and the complementary oligonucleotide. Determined by action statistics. Therefore, hybridization times and inefficiencies are increased because a large number of samples must be tested to obtain useful information.

Further, maintaining a minimal amount of user manipulation increases efficiency. As currently practiced, oligonucleotide array hybridization requires a great deal of operator attention and manipulation and is highly skilled in performing analyses. For example, hybridization is typically performed in an assay room and data collection and analysis (
It is done in a separate device (such as a laser scanner or fluorescence microscope). This device requires a significant amount of user handling, resulting in assay time wastage and user error.

The economics of parallel processing and data analysis are neglected, as it is a limitation of current methods to perform hybridization on one array at a time.

Other limitations, inefficiencies, and expense result from the structural features of existing equipment. Many existing devices are limited in the size of substrate they can contain. Other devices cannot be discarded and require extensive washing between operations to prevent sample contamination. Still other devices are bulky and do not allow the rapid heating and cooling required for effective hybridization. Still other instruments must use expensive optics for analysis of reaction products.

Easy to use with small reaction volume, active mixing of fluid components, parallel processing and minimal user intervention, especially for performing biological reactions such as diffusion hybridization Equipment is required. In addition, it can be easily manufactured in a variety of sizes, is disposable, minimizes sample contamination, allows the use of low-cost optical analysis instruments, and provides a small space for rapid heating and cooling of sample fluids. A device is required. Still further, there is a need for methods that can utilize such devices to increase hybridization efficiency, particularly for biochip arrays that are understood by those of skill in the art. In light of the enormous interest in biochip technology, the financial rewards of biochip technology investment and research, and the wide range of products resulting from such research, this requirement is particularly significant.

SUMMARY OF THE INVENTION In accordance with the foregoing objectives, the present invention provides an apparatus for conducting a biological reaction. The device comprises a substrate having a first side and a second side (typically facing each other for a flat substrate). The array of biomolecules is placed on the first side and the flexible layer is attached to the first side of the substrate by an adhesive layer. Here, the adhesive layer is disposed on the first surface and forms a reaction space. The substrate has at least a first port extending through the substrate from the first surface to the second surface, the port having a first opening and a second opening. The first opening of the port is provided in the area bounded by the adhesive layer and is covered by the flexible layer (eg reaction space). The second opening of the port is provided on the second surface of the substrate. Alternatively, the port is provided in the flexible layer of the reaction space.

Optionally, a removable cover disposed over the second opening of the port, such as a foil tape, and solid at the first temperature and liquid at the second elevated temperature, the first surface of the substrate. And a water-soluble compound layer disposed between the flexible layer and the flexible layer. Optionally, the flexible layer is a translucent plastic or gas permeable membrane.

The port may be a truncated cone having a small diameter end and a large diameter end, where the small diameter end is provided in a region on the first substrate surface bounded by an adhesive layer (reaction space) and The radial end is provided on the second substrate surface. The wall of the port in some embodiments forms an angle of 90 ° C. or less with the second substrate surface. Optionally, the port contains a sample fluid with a contact angle. The angle between the second substrate surface and the port wall is less than or equal to the contact angle of the sample fluid.

A second port can be included. In this embodiment, the second port extends through the substrate from the first surface to the second surface and has a first opening and a second opening. Here, the first opening of the outlet port is provided on the first substrate surface in the region bounded by the adhesive layer and covered by the flexible layer. The second opening of the outlet port is provided on the second substrate surface and is covered with a removable cover. Alternatively, the second port is provided on the flexible layer.

Optionally, the device of the present invention has a reflective layer and / or a resistive heater disposed between the array and the first substrate surface. The latter can be a reflective layer disposed between the array and the first substrate surface.

In addition, the device can have a passivation layer, such as parylene, or a scanner. The scanner contacts the flexible layer at a location above the array. The scanner can be a light pipe and covers the entire reaction space.

In a preferred feature, the device further comprises a sample preparation chip in contact with the second substrate surface. Here, the sample preparation chip has a port that matches the first port of the device.

In a further feature, the device further comprises a roller. Here, the roller contacts the flexible layer at a location on the array. For example, the roller can move longitudinally across the reaction space.

In a further feature, the device includes a case with a lid, a base with a space, and a carriage with a scanner and rollers. Here, the carriage is provided in the space, and the substrate is removably mounted on the carriage such that the first substrate surface is in operative contact with the carriage.

In an additional aspect, the invention provides a device for conducting a biological reaction. The apparatus is a glass microscope slide having a first surface and a second surface opposite the first surface; an array of biomolecules disposed on the first surface of the slide, each biomolecule in the array Is a biomolecule array fixed to the first side with a polyacrylamide gel pad; a polyvinylidene chloride layer attached to the first side of the slide with a double-sided adhesive layer, the adhesive layer being placed on the first side of the slide A polyvinylidene chloride layer surrounding the area; first and second ports extending through the slide from the first surface to the second surface, each port having a first opening and a second opening. A first opening of each port is provided in an area on the first surface of the slide bounded by an adhesive and is covered by a flexible layer, and a second opening of each port has a second surface of the slide. Located on the top, each port has a small diameter end and a large diameter end Has a shape of a truncated cone with a small diameter end being a first opening and a large diameter end being a second opening.
And a second port; a layer of foil tape disposed over the second opening of each port; a layer of polyethylene glycol disposed between the first side of the slide and a layer of polyvinylidene chloride; an array A reflective layer disposed between the first substrate surfaces; a layer of parylene disposed between the reflective layer and a layer of polyvinylidene chloride; and a resistive heater.

In a further aspect, the invention provides a device for performing a biological reaction. The device comprises a substrate having a first surface and a second surface, a plurality of biomolecules disposed on the first surface of the substrate, and a flexible layer attached to the first surface of the substrate by an adhesive layer. Here, the adhesive layer is disposed on the first surface of the substrate and surrounds the region above it. The reaction space is enclosed between the flexible layer and the first substrate surface in the area defined by the adhesive layer. There are first and second ports extending through the flexible layer and the adhesive layer into a space enclosed between the flexible layer and the first substrate surface in an area defined by the adhesive layer.

[0024] In an additional feature, each of the plurality of biomolecules is a separate site or continuous layer,
It is attached to a fixed structure such as a gel pad made of a polymeric material such as polyacrylamide.

In a further feature, the device has a marking layer attached to the flexible layer. Here, the first and second ports extend through the marker layer and the flexible layer. The sign layer has an adhesive surface and a non-adhesive surface, where the sign layer is attached to the flexible layer using the adhesive surface.
The marker layer has windows corresponding in size and position to the area bounded by the adhesive layer.
Here, the window allows visual inspection of the flexible layer and the space enclosed between the flexible layer and the first substrate surface in the area defined by the adhesive layer. Optionally, a reflective layer disposed between the array and the first substrate surface and / or a resistive heater is provided.

In a further aspect, the invention provides a method of detecting a target analyte using the device.

[0027] (Detailed Description of Embodiments of the Invention)   The presently preferred embodiments of the invention will now be described with reference to the accompanying drawings.

The present invention is directed to methods and compositions for conducting large volumes of biological reactions on a biochip consisting of a substrate having an array of biological binding sites. The present invention provides a reaction chamber such as a hybridization chamber where the biochip consists of nucleic acids. A substrate, an adhesive layer, and a flexible cover are formed in the hybridization chamber. This system utilizes ports provided on either the substrate or the flexible cover to supply samples and / or reagents.

Accordingly, the present invention provides a device used to detect a target analyte in a sample. "Target analyte" or "analyte" or grammatical equivalents means any molecule, compound, or particle that is detected. The target analyte is preferably bound to a binding ligand, as outlined below. As will be appreciated by those in the art, this method can be used to detect large numbers of detectants. Basically, any detectable substance for which the binding ligands described herein are made can be detected using the methods of the invention.

Suitable analytes include organic or inorganic molecules, including biomolecules. In a preferred embodiment, the detected substances are environmental pollutants (including pesticides, insecticides, toxins, etc.), chemicals (
Solvents, polymers, organic substances, etc.), therapeutic molecules (including therapeutic and abusive drugs, antibiotics, etc.), biomolecules (hormones, cytokins, proteins, lipids, carbohydrates, cell membrane antigens and receptors (nerves, hormones, and (Including cell surface receptors) or their ligands), whole cells (including prokaryotes (such as pathogens), eukaryotic cells including mammalian tumor cells, etc.), viruses (retroviruses, herpesviruses, Adenovirus, lentivirus and the like), spores and the like. Particularly preferred analytes are environmental pollutants, nucleic acids, proteins (enzymes, antibodies, antigens, growth factors,
(Including cytokin), therapeutic and abusive drugs, and viruses.

In a preferred embodiment, the target detective is a nucleic acid. "Nucleic acid" or "oligonucleotide" or grammatical equivalents are covalently linked to each other.
Means at least 2 nucleotides that have been deleted. Nucleic acids of the invention generally include phosphodiester bonds, but in some cases include nucleic acid analogs, as outlined below. It has an alternate bakbone. For example, phosphoramide (Beau
cage et al., Tetrahedron 49 (10): 1925 (1923) and references therein; Letsinger, J. Org. Chem. 35: 3800 (1970); Sprinzl et al., Eur. J. B
iochem. 81: 579 (1977); Letsinger et al., Nucl. Acids Res. 14:
3487 (1986); Sawal et al., Chem. Lett. 805 (1984); Letsinger
Et al., J. A. Chem. Soc. 110: 4470 (1988); Pauwels et al., Chemica S.
cripta26: 141 (1986)), sulfur phosphate (Mag et al., Nucleic Acid Res.
19: 1437 (1991); U.S. Pat. No. 5,644,048), disulfuric acid phosphate (
Briu et al., J. Am. Chem. Soc. 111: 2321 (1989)), O-methylpopolamide (Eckstein, Oligonucleotides and Analogues: A Practical Appr.
oach, Oxford University Press), and peptide nucleic acid backbones and chains (Egholm, J. Am. Chem. Soc. 114; 1895 (1992); Meier et al., Chem. In.
t. Ed. Engl. 31: 1008 (1992); Nielsen, Nature, 365: 566.
(1993); Carlsson et al., Nature 380: 207 (1996), which are incorporated herein). Other analog nucleic acids have a positive backbone (Denpcy et al., Proc
． Natl. Acad. Sci. USA 92: 6097 (1995); Nonionic main chain (US Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 521614).
1 and 4469863; Kiedrowshi et al., Angew. Chem. Intl. Ed. Engli
sh30: 423 (1991); Letsinger et al. Am. Chem. Soc. 110: 44
70 (1988); Letsinger et al., Nucleosides & Nucleotide 13: 1597 (1.
994); Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Mod
Certification in Antisense Research ”, Ed.YS Sanghui and P. Dan Cook; Mesm
aeker et al., Bioorganic & Medicinal Chem. Lett. 4: 395 (1994); Jeff
s et al. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37
: 743 (1996)), and US Pat.
Issue 6, Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Mod
Included in the definition of nucleic acids are nucleic acids containing one or more carbocyclic sugars (Jenkins et al. Chem. Soc. Rev. (1995) 16
9-176). Some Nucleic Acid Analogues, Rawls, C & E News Jun 1997 2
Day 35, p. In addition, the nucleic acid analog (analog) is "locked (lock
ed) nucleic acid ”. All of these references are incorporated herein by reference. Modification of the ribose phosphate backbone facilitates the addition of electron transfer moieties (moieties),
Increases the stability and half-life of such molecules in physiological environments.

As will be appreciated by those in the art, all nucleic acid analogs may be used in the present invention. Moreover, the mixing of nucleic acids and analogs occurs naturally. For example, analog structures may be used at the attachment sites of conductive oligomers or electron transfer moieties. Alternatively, different nucleic acid analogs may be mixed, naturally occurring nucleic acids and analogs may be mixed.

As outlined, the nucleic acid may be single stranded or double stranded, and may include portions of both double stranded or single stranded sequence. Nucleic acid may be DNA, or may be both genomic and cDNA, RNA or hybrid. Here, the nucleic acid is a combination of deoxyribonucleotide and ribonucleotide, uracil,
Includes base combinations including adenine, thymine, cytosine, guanine, inosine, xanine hypoxanine, isocytosine, isoguanine and the like. The term used here
"Nucleoside" includes nucleotides and modified nucleosides such as nucleosides, nucleotide analogs, amino modified nucleosides. In addition, "nucleoside" includes non-naturally occurring analog structures. For example, an individual unit of peptide nucleic acid comprises a base, referred to herein as a nucleoside.

In a preferred embodiment, the invention provides a method of detecting a target nucleic acid. "
"Target nucleic acid", "target sequence" and grammatical equivalents thereof refer to a nucleic acid sequence on a single-stranded nucleic acid. The target sequence is a part of gene, regulatory sequence, genomic DNA, cDNA, m
It may be RNA or the like including RNA and rRNA. Considering that the long sequence is specific, it may have any length. In some embodiments, it is preferred to cleave the sample nucleic acid into fragments of 100 to 10,000 base pairs, and in some embodiments fragments of about 500 base pairs are preferred. As will be appreciated by those in the art, complementary target sequences can take many forms.
For example, it may be included within a larger nucleic acid sequence. Ie gene or mRN
All or part of A, restriction fragment of plasmid or genomic DNA
fragment)).

Probes (including primers) are made and hybridized to the target sequence to determine the presence or absence of the target sequence in the sample, as outlined below. Generally speaking, this term will be understood by those skilled in the art.

The target array may also consist of different target domains that are close (ie continuous) or spaced apart. For example, when using a ligation chain reaction (LCR) technique, a first primer hybridizes to a first target domain and a second primer hybridizes to a second target domain. The domains may be contiguous or may be linked to the polymerase and dN as described in detail below.
They may be separated by one or more nucleotides in conjunction with the use of TP.
The terms "first" and "second" do not mean awarding an array of orientations to a target array of 5'-3 'orientations. For example, assuming a complementary target sequence in the 5'-3 'orientation, the first target domain may be located 5'up to the second domain, or 3'up to the second domain.

In a preferred embodiment, the target detective is a protein. As will be apparent to those of skill in the art, there are numerous protein target detectors that may be detected using the present invention. Here, “protein” or grammatical equivalents thereof means proteins, oligopeptides and peptides, derivatives and analogs, proteins containing non-naturally occurring amino acids and amino acid analogs, peptide-like structures. The side chain is (R) or (S)
Any of the arrangements may be used. In a preferred embodiment, the amino acids are in the (S) or L configuration. As explained below, it is desirable to utilize protein analogs to delay the degradation by sample contaminants when the protein is used as a binding ligament.

Suitable protein targeting agents include, but are not limited to: (1) Immunoglobulins, particularly lgEs, lgGs, lgMs, particularly therapeutically or diagnostically relevant antibodies such as, but not limited to, human albumin antibodies, apolipoproteins (including apolipoprotein E), human chorion stimulating hormone, cortisol, α
-Fetoprotein, thyroxine, thyroid stimulating hormone (TSH), antithrombin, drug antibody (antieptileeptic drug (phenytoin, primidone, carbariene zepine, ethosuximide, valproic acid, phenobarbitol)), cardioactive drug (digoxin, lidocaine, Procainamide and disopyramide), bronchodilators (theophylline), antibiotics (chloramphenicol, sulfonamide)
, Antidepressants, immunosuppressants, drugs of abuse (amphetamine, methamphetamine, cannabinoids, cocaine and opium), any number of viral antibodies (orthomyxovirus, (eg influenza virus), paramyxovirus (eg respiratory syncytial virus, parotid) Adenitis virus, missies virus), adenovirus, rhinovirus, coronavirus, reovirus, togavirus (eg rubella virus), parvovirus, poxvirus (eg variola virus, variola virus), enteric virus (eg polyvirus, Coxsackie virus), hepatitis virus (including A, B and C), herpes virus (eg herpes simplex virus, varicella zoster virus, giant cell virus, Epstein-Barr virus)
, Rotavirus, Norwalk virus, Hantavirus, Arenavirus, Rhabdovirus (eg rabies virus), Retrovirus (HIV, HTL
VI-II and II), papovavirus (e.g. papilloma virus), polyomavirus, and picornavirus), and bacteria (including a wide range of pathogenic and nonpathogenic prokaryotes, Bacillus; Vibrio, e.g. .Cholerae; Escherichia, eg enterotoxogenic E. coli; Shigella, eg S. dysenteriae
Salmonella, eg S. typhi; Mycobacterium, eg M. Tuberculosis
, M. leprae; Clostridium bacteria such as C. botulinum, C.tetani, CC.diff
icile, C. perfringens; cereal bacteria such as C. diphtheriae; Streptococcus,
For example, S. pyogenes, S.M. pneumoniae; Staphylococcus, for example S. aureus; Haemophilus, for example H. Influenzae; Neisseria, for example N. meningitidis
, N. gonorrhoeae; Yersinia, for example G. lamblia, Y. pestis; Pseudomonas, such as P. aeruginosa, P.A. putida; Chlamydia, C.I. trachomati
s; Bordetella, for example B. pertussis; Treponema fungus, such as T. palladium
etc). (2) Enzymes (and other proteins). Including but not limited to: Enzymes used as markers or therapeutic agents for heart disease; creatine kinase, lactate dehydrogenase, aspartate aminotransferase, troponin T
, Myoglobin, fibrinogen, cholesterol, triglyceride, thrombin, tissue plasminogen activator (tPA); pancreatic disease indicators including amylase, lipase, chymotrypsin and trypsin; cholinesterase, bilirubin,
Liver-functioning enzymes and proteins, including alkaline phosphatase; bacterial and viral enzymes such as aldolase, prostatic acid phosphatase, end-stage deoxynucleotidyl transferase, and HIV protease. (3) hormones and cytokins, many of which serve as ligands for cell receptors, such as erythropoietin (EPO), thrombotic poietin (TPO),
Interleukins (including IL-1 to IL-17), insulin, insulin-like growth factors (including IGF-1 to -2), epidermal growth factor (EGF),
Metamorphosis growth factors (including TGF-α and TGF-β), human growth hormone, transferrin, epidermal growth factor (EGF), low density lipoprotein, high density lipoprotein, leptin, VEGF, PDGF, trichome nerve tissue trophic factor , Prolactin, adrenocorticotropic hormone (ACTH), calcitonin, human chorion stimulating hormone, cotrisol, estradiol, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), leutinzing hormone (LH),
Progeterone and testosterone. (4) Other proteins (α-fetoprotein, cancer lung antigen CEA, cancer marker, etc.).

Furthermore, any biomolecule for which an antibody is detected can be directly detected. That is, virus or bacterial cells, drugs for treatment and abuse, etc. can be directly detected.

Suitable target detections include carbohydrates and are markers for breast cancer (CA15-3, C).
A549, CA27, 29), mucin-like carcinoma-associated antigen (MCA), ovarian cancer (CA
125), pancreatic cancer (DE-PAN-2), prostate cancer (PSA), CEA, and colorectal and pancreatic cancer (CA19, CA50, CA242), but are not limited thereto.

Suitable target analytes are metal ions, especially heavy metals and / or toxic metals, such as aluminum, arsenic, cadmium, selenium, cobalt, copper, chromium, lead,
Including but not limited to silver and nickel.

These target substances are blood, lymph, saliva, vaginal and anal secretions, urine, feces, body fluids including sweat and tears, solid tissues including liver, spleen, bone marrow, lungs, muscles, brain and the like. It may be present in any number of different sample types, including but not limited to.

Accordingly, the present invention provides a device for detecting a target analyte having a solid substrate. Solid substrates can be formed in a number of ways with a wide variety of materials, as described herein and will be apparent to those skilled in the art. Furthermore, a single device may consist of more than one substrate. For example, there may be a "sample processing" cassette that interfaces via interface with a separate "detection" cassette. The raw sample is added to the sample processing cassette and the sample is manipulated to prepare for detection. The sample is removed from the sample processing cassette and added to the detection cassette. There may be additional functional cassettes to which the device is compatible, such as heating elements that are placed in contact with the sample cassette to produce a PCR-like reaction. In some cases, a portion of the substrate may be removable, for example the sample cassette may have a removable detection cassette so that the entire sample cassette does not contact the detection device. For example, US Pat. No. 5,603,351, PCTUS 96/17116, and December 1, 2000.
"Multilayer microfluidic device for detection reaction" filed on the 1st (Filing date not received)
See. These are incorporated herein by reference.

The composition of the solid substrate depends on the technique used to form the device, the use of the device, the composition of the sample, the analyte to be detected, the size of the wells and microchannels,
It depends on various factors including the presence of electronic components. In general, the devices of the invention should be easily sterilizable.

In a preferred embodiment, the solid substrate is silicon such as a silicon wafer, silicon dioxide, silicon nitride, glass and fused silica, gallium arsenide, phosphorous indium, aluminum, ceramics, polyimide, quartz, plastics, resins and poly. Methyl methacrylate, acrylic acid, polyethylene, polyethylene terephthalate, polycarbonate, polystyrene and other styrene copolymers, polypropylene, polytetrafluoroethylene, superalloys, zircaloys, steel, gold, silver, copper, tungsten, molybdenum, tantalum, kovar (KOVAR)
, Kevlar (LEVLAR), Kapton (KAPTON), Mylar (MYLA)
It can be formed from a wide variety of materials including, but not limited to R), brass, sapphire, and the like. When the sample handling process requires light-based technology, high quality glasses such as highly transparent borosilicate and fused silica that are transparent to light are preferred. In addition, as outlined here, some of the interior surfaces of the device are varied as needed to reduce non-specific binding and allow attachment of binding ligaments for biocompatibility or fluid resistance. It may be covered with a coating.

In a preferred embodiment, the solid support is US Patent Application Serial No. 09/2350.
No. 81, No. 09/337086, No. 09/464490, No. 09/4920
No. 13, No. 09/466325, No. 09/460281, No. 09/4602
No. 83, No. 09/387691, No. 09/438600, No. 09/5061
No. 78, 09 / 458,534, which consists of ceramic materials, which are incorporated herein by reference in their entirety. In this embodiment, the green sheets are laminated and sintered together to produce a substantially monolithic monolith.
thic) structure is made from a green sheet layer. A green sheet is a composite material in which inorganic particles of glass, glass-ceramic, ceramic, or mixtures thereof are dispersed in a polymer binder and may include additives such as plasticizers and dispersants. The green sheet is preferably in the form of a sheet with a thickness of 50 to 250 microns. Ceramic particles are typically metal oxides such as aluminum oxide and zirconium oxide. Examples of green sheets containing glass-ceramic particles are described in E.I. I. Sold from Du Pont de Nemours and Company
AX951 ". An example of a green sheet containing aluminum oxide particles is Ferr
It is "ferro-alumina" sold by Corp. The composition of the green sheet may be tailored to the particular application. The greensheet layers are laminated together and burned to form a substantially monolithic multilayer structure. Manufacture, processing and application of ceramic green sheets are described by Richard E. Mistier, "Tape Casting: A Basic Process That Meets the Requirements of the Electronic Industry", Ceramic Bulle
tin, vol. 69, no. 6, pp. 1022-26 (1990) and U.S. Pat. No. 39.
91029, which are incorporated herein by reference.

The method of making the device (shown as devices 100 and 200 in FIGS. 27-30) begins by providing a sheet of green sheet that is preferably 50 to 250 microns thick. Sheets of greensheet are cut to the desired size, typically 6 "x 6" for conventional processing, although smaller or larger devices may be used if desired. Each greensheet layer is textured using various techniques to create vias in the final multilayer structure.
, Forming the desired structure, such as a groove or cavity.

Various techniques may be used to texture the greensheet layer. For example, a part of the green sheet layer may be perforated to form a bias or a groove. This operation can be performed using a conventional multilayer ceramic punch, such as the Pacific Trinetics Corp. model APS-8718 automatic punch. Instead of perforating a portion of the material, features such as grooves and wells can be used to create a negative image of the desired structure.
It may be embossed on the surface of the green sheet by pressing the green sheet against the embossing plate with e). Also, weaving (te
xturing) may be performed by a laser tool having a laser mediated system, such as Pacific Trinetics' LVS-3012.

A wide range of materials may then be applied to each woven greensheet layer, preferably in the form of a thick film paste. For example, a metal-containing thick film paste may be placed on the green sheet layer to provide an electrically conducting path. The thick film paste typically comprises the desired material, but it may also be a metal or a dielectric in the form of a powder dispersed in an organic vehicle. The paste is also designed to have a viscosity suitable for the desired deposition technique, such as screen printing. The organic medium may include a small amount of flux, such as glass frit, to facilitate sintering. Thick film technology is described in J. D. Provance, "Performance review of thick film materials", "Insula
tion / Circuits (April 1977) ”, Morton L. et al. Topfer, Thick Film Microelectronics, Fabrication, Design and Application (1977), pp. 41-59, which are incorporated herein by reference.

The porosity of the resulting thick film can be adjusted by adjusting the amount of organic medium present in the thick film paste. That is, the porosity of the thick film can be increased by increasing the percentage of organic medium in the thick film paste.
Similarly, the porosity of the greensheet layer can be increased by increasing the proportion of organic binder. Another way to increase the porosity of thick films and greensheet layers is to disperse within the organic medium or organic bonds other organic phases that are not soluble in the organic medium. Polymer microspheres can be used advantageously for this purpose.

To add electrically conducting passages, the thick film paste typically comprises metal particles such as silver, platinum, palladium, gold, copper, tungsten, nickel, tin, or alloys thereof. . Examples of suitable silver pastes are E.I. I. Du Pont de Ne
Silver conductor composition numbers 7025 and 771 sold by mours and Company
It is 3.

The thick film paste is preferably applied to the green sheet layer by screen printing. In the screen printing process, the thick film paste is pressed against the pattern silk screen and placed on the green sheet layer in the corresponding pattern. Typically, the silkscreen pattern is photographically formed by exposing the mask. In this way, conductive traces are applied to the surface of the greensheet layer. Bias present in the greensheet layer may be filled with thick film paste. When filled with a thick fill paste containing an electrically conductive material, the bias helps provide an electrical connection between the layers.

After forming a desired structure in each layer of the green sheet, an adhesive layer is applied to either surface of the green sheet. Preferably the adhesive is a room temperature adhesive. Such room temperature adhesives have a glass transition temperature at room temperature or about 20 ° C., which allows the substrates to be bonded together at room temperature. Further, rather than undergoing a chemical change, chemically reacting with the substrate, or dissolving the components of the substrate, the room temperature adhesive penetrates the surface of the substrate to bond the substrates together. Sometimes such room temperature adhesives are referred to as "pressure sensitive adhesives". Suitable room temperature adhesives are typically aqueous emulsion (
emulsion), Rohm and Haas, Inc. And Air Products, Inc. For example, the material sold by Air Products, Inc. as "Flexcryl 1653" has been found to be good.

The room temperature adhesive may be applied to the green sheet by conventional coating techniques. It is desirable to dilute the applied pressure sensitive adhesive in water to facilitate coating, depending on the coating technique used and the viscosity and solidity of the starting materials when loaded. After coating, the room temperature adhesive can be dried. The dry thickness of the room temperature adhesive film is preferably in the range of 1 to 10 microns and the thickness should be uniform over the entire surface of the greensheet. Film thicknesses above 15 microns are undesirable. When an adhesive having such a film thickness is used, a large amount of organic material has to be removed, which may cause voids or delamination during firing. Membranes less than about 0.5 microns thick when dried are too thin due to poor adhesion between layers.

Of the conventional coating techniques, spin coating and spraying are preferred methods. When using spin coating, 10 grams of "Flexcryl 165
It is preferred to add 1 gram of deionized water for 3 ". If a spray is used, higher dilution levels are preferred to facilitate spraying. Further, it is preferred to raise the greensheet to a temperature of about 60 to 70 ° C when spraying the room temperature adhesive. This causes the material to dry almost instantly when placed on the green sheet. This momentary drying results in a more uniform and homogeneous film of adhesive.

After applying the room temperature adhesive to the greensheet layer, the layers are stacked on top of each other to form a multilayer greensheet layer. The layers are preferably stacked in an alignment die to maintain the desired registration between each layer structure. When using a conditioning die, conditioning holes must be added to each greensheet layer. Typically, when using a room temperature adhesive, the stacking process alone is sufficient to bond the greensheet layers together. In other words, little or no pressure is required to bond the layers together. However, in order to bond the layers more firmly, it is preferred that the layers be stacked and then laminated to each other.

The laminating process involves applying pressure to the stacked layers. For example, in a conventional laminating process, a uniaxial pressure of about 1000 to 1500 psi is applied to the stacked green sheet layers and then heated to, for example, 70 ° C. for about 10 to 15 minutes for about 300 minutes.
Apply equilibrium pressure from 0 to 5000 psi. When using the conventional laminating process,
It is not necessary to use an adhesive to bond the greensheet layers together.

However, a pressure of 2500 psi or less is preferred for good control over a range of structures such as internal or external cavities and grooves. Lower pressures are preferred to allow the formation of longer structures such as cavities and grooves. For example, 2
When using a stacking pressure of 500 psi, the size of properly formed internal cavities and grooves is typically limited to approximately 20 microns or less. Therefore, 1000
Pressures below psi are preferred as such pressures can form structures having sizes above about 100 microns using some dimensional control means. Three
Pressures of 00 psi or less are preferred because such pressures can form structures having sizes of 250 microns or greater with some dimensional control. Pressures of 100 psi or less, referred to herein as "near zero pressure", are preferred because there are some limits on the size of internal and external cavities or grooves that can be formed in a multilayer structure.

[0059]   The pressure is preferably applied by a uniaxial press in the laminating step.

[0060]   Alternatively, a pressure of about 100 psi or less may be applied manually.

For the manufacture of semiconductor devices, each sheet may have many devices interposed.

Therefore, after lamination, the multi-layer structure may be cut using conventional green sheet dicing or sawing equipment and separated into individual devices. Due to the high level of delamination or shear resistance provided by the room temperature adhesive, very small edge delamination occurs during the dicing process. Even if some layers separate around the edge after dicing, manual application of pressure to the edge allows the layers to be easily re-laminated without harming the rest of the device.

The final processing step is the firing of the laminated multilayer greensheet structure from the "green" state to form the final substantially monolithic multilayer structure. The firing process occurs in two important stages as the temperature rises. The first important step is the binder bumout step, which occurs in the temperature range of about 250 to 500 ° C, during which the binder in the greensheet layer and the organic components in the applied thick film paste are Other organic materials such as are removed from the structure.

The next important step is the sintering step, which occurs at high temperature, at which the inorganic particles sinter together, resulting in a dense, substantially monolithic multilayer structure.
The sintering temperature used depends on the nature of the inorganic particles present in the greensheet.
For many types of ceramics, suitable sintering temperatures are about 950 to about 1600.
C range and depends on the material. For example, sintering temperatures between 1400 and 1600 ° C. are typical for green sheets containing aluminum oxide. Other ceramic materials such as silicon nitride, aluminum nitride and silicon carbide require higher sintering temperatures such as 1700 to 2200 ° C. 750 to 9 for green sheets with glass-ceramic particles
Sintering temperatures in the range of 50 ° C are typical. Glass particles are generally about 350 to 70
A sintering temperature in the range of 0 ° C is required. Finally, the metal particles require a sintering temperature of 550 to 1700 ° C., depending on the metal in question.

Typically, the device is fired for about 4 hours to about 12 hours or more, depending on the materials used. In general, the calcination should have sufficient duration to remove organic material from the structure and completely sinter the inorganic particles. In particular,
The polymer is present as a binder in the greensheet and room temperature adhesive. Baking must be of sufficient temperature and duration to decompose these polymers and remove them from the multilayer structure.

Typically, multi-layer structures suffer space reduction during the firing process. A small space reduction of about 0.5 to 1.5% is usually seen in the binder bumout stage. At higher temperatures, a further space reduction of about 14 to 17% is typically seen during the sintering stage.

On the other hand, the spatial change due to firing can be controlled. In particular, (1) particle size and (2) organic components such as binders removed during the firing process to match spatial changes in two materials such as green sheets and thick film pastes. The percentages of should be in harmony. Furthermore, this spatial variation does not have to be exactly matched, but mismatches cause internal stresses in the device.
However, a symmetrical treatment, i.e. placing the same material or structure at opposite locations of the device, can provide some compensation for the contracted and anharmonic material. Large discrepancies in sintering temperature or spatial variations result in defects or breakage in parts or all of the device. For example, the device may be separated into individual layers, or may be deflected or warped.

As mentioned above, any dissimilar material added to the greensheet layer is co-fired. Such dissimilar materials can be added as a thick film paste, or as another green sheet layer, or can be added after the manufacturing process, ie after sintering. The advantage of co-firing is that the added material is sintered into a greensheet layer to be integrated into a substantially monolithic microfluidic device. However, to be co-fireable, the added material must have a variation in sintering temperature and space that is consistent with the greensheet layer. Since the sintering temperature depends greatly on the material, proper selection of the material is necessary to match the sintering temperature. For example, silver is a preferred material for applying electrically conducting passages, but if the greensheet layer contains aluminum particles that require a sintering temperature in the range of 1400 to 1600 ° C, silver Due to the relatively low melting point (961 ° C.), other metals such as platinum must be used.

Alternatively, the addition of another substrate or the joining of two post-sintered components can be done using a wide range of bonding techniques as outlined herein. For example, the two "halfs" of the device may be glued or fused together. For example, a particular detection platform, a reagent mixture such as a hydrogel, or biological components that are not stable at high temperatures can be sandwiched between the two halves. Alternatively, a ceramic device with open trenches or wells may be fabricated, additional substrates or materials placed on the device, and sealed with other materials.

[0070]   A particularly preferred substrate is glass such as a microscope slide.

In a preferred embodiment, the solid substrate is formed to handle a single sample containing multiple target analytes. That is, a single sample may be added to the device and the sample may be divided into equal parts and processed in parallel to detect a detected substance, or continuously processed to detect individual target detected substances continuously. Good. In addition, the sample may be removed periodically or at different locations for in-line sampling.

In a preferred embodiment, the solid substrate is configured to handle multiple samples containing one or more target analytes. Generally, in this embodiment, each sample is handled individually. That is, it is preferable that the operation and the analysis are performed in parallel and there is no contact or contamination between them. As an alternative, there may be several steps in common. For example, it is desirable to process different samples separately, but to detect all of the target analytes on a single detection platform.

Furthermore, all discussion made here is directed to using a substantially flat substrate with microgrooves and wells, but other geometries can be used. For example, two or more flat substrates can be stacked to produce a three-dimensional device, which has microgrooves flowing in one plane or between those planes. Similarly, wells can allow a larger volume of sample to span between two or more substrates. For example, both sides of the substrate can be etched to form microgrooves. See, for example, US Pat. Nos. 5,603,351 and 5,681,484. These are incorporated herein by reference.

The biochip substrate of the present invention has capture binding ligands attached in an array format. By “array” or “biochip” herein is meant a plurality of capture binding ligands, preferably nucleic acids in array form, the size of the array depending on the composition and end use of the array. Most of the discussion made here is directed to the use of nucleic acid arrays with capture probes, but is not meant to limit the scope of the invention. Other types of capture binding ligands (such as proteins) can be used.

Nucleic acid arrays are known to those of skill in the art and can be sorted in a number of ways and aligned (
ordered) array (eg, resolving chemistries at discrete locations (reso
lve) ability) and a random array. Aligned arrays include, but are not limited to, those using photolithography (registered trademark Affymetrix GeneChip), spotting technology (Syntei and others), printing technology (Hewlette Packard and Rosetta), 3D "gel pad" arrays, etc. It is not something that will be done. The size of the array can vary. When arrays with 2 to many different capture probes are made, very large arrays are possible. Generally, an array is 2 to 100,
000, with about 400 to about 1000 being most preferred and about 1000 being especially preferred. Arrays can be classified as "addressable". This means that the individual elements of the array precisely define the xy coordinates, which allows a given array element to be pinpointed.

The present invention is advantageously used to perform assays using biochip 18. Biochips used in the art include substrates comprising arrays or microarrays of biomolecules consisting of one member of a biological binding pair, preferably aligned arrays, more preferably aligned and addressable arrays. Typically,
Such an array is an oligonucleotide array consisting of a nucleotide sequence complementary to at least one sequence expected to be present in a biological sample. Alternatively, peptides or other small molecules can be arrayed on a biochip for immunoassay (
Here the alignment molecule can be an antigen, or a biological receptor (where the alignment molecule is a gand, agonist or antagonist of said receptor) can be analyzed.

One beneficial feature of biochips is the method of attaching biomolecules to the surface of the biochip. In the past, such methods had multiple reaction steps and required chemical modification of the solid support itself. A hydrogel-like absorbent matrix (ad
In embodiments having sorption), chemical modification of the gel polymer is required to provide the chemical function capable of forming covalent bonds with biomolecules. The efficiency of attachment chemistry and the strength of the chemical bonds formed are critical to the fabrication and ultimate performance of the microarray.

Polymerized hydrogels and gel pads are used as tie layers that adhere to the surface of biomolecules. Biomolecules include, but are not limited to, proteins, peptides, oligonucleotides, polynucleotides and large nucleic acid fragments. Oligonucleotide probes may be attached to the surface of a continuous layer of hydrogel or an array of gel pads. Gel pads consisting of biochips for use with the devices of the invention are conveniently manufactured as thin sheets or slabs, typically
A solution of acrylamide monomer, a crosslinker such as methylenebisacryamide, and a solution of a catalyst such as N, N, N ', N'-tetramethylethylenediamide (TEMED), and ammonium persulfate for chemical polymerization, or An initiator such as 2,2-dimethoxy-2-phenyl-acetophenone (DMPAP) for photopolymerization,
Two glass surfaces using spacers (eg glass plate or microscope slide)
It is installed between the two to obtain a polymer gel having a desired thickness. Generally, the acrylamide monomer and crosslinker are prepared in a solution of about 4-5% acrylamide (having an acrylamide / bisacrylamide ratio of 19/1) in water / glycerol and a small amount of initiator is added. The solution is exposed to ultraviolet (UV) radiation (eg, at least about 15
Minutes, 254 nm, or other suitable UV conditions, collectively referred to as “photopolymerization”), or thermal initiation at elevated temperatures (eg, typically about 40 ° C.) to polymerize and crosslink. After polymerization and crosslinking, the top glass slide is removed from the surface and the gel removed. The pore size of the gel (ie “sieving propert”
y) ”) is controlled by varying the amount of crosslinker and the percent solids in the monomer solution. Pore size can also be controlled by changing the polymerization temperature.

In the manufacture of polyacrylamide embodiments of polymerized hydrogel arrays (ie, patterned gels) of the present invention for use as tie layers for biomolecules, the acrylamide solution is typically UV polymerized / Image through the mask during the crosslinking process. The upper glass slide is removed after polymerization and the unpolymerized monomer is rinsed (developed) with water, leaving a fine pattern of polyacrylamide hydrogels, which is used to make crosslinked polyacrylamide hydrogel pads. Furthermore, when applying the lithographic technology known in the semiconductor industry,
Irradiate individual locations on the surface of polyacrylamide hydrogels to activate these specific locations and provide solid support for oligonucleotides, antibodies, antigens, hormones, hormone receptors, ligands or polysaccharides (eg, international application publications). WO91 /
See 07087. It is incorporated herein by reference. A) surface (eg polyacrylamide hydrogel surface).

In hydrogel-based arrays using polyacrylamide, biomolecules (such as oligonucleotides) are derived polymers (deriva) that consist of chemical groups that are homologous to the biomolecule.
The electron pair is covalently attached by forming an amide, ester, or disulfide bond with the tized polymer). Covalent attachment of biomolecules to polymers is typically done after polymerisation and chemical crosslinking of the polymer is complete.

Alternatively, an oligonucleotide that supports the 5'-final acrylamide modification can be used. It effectively copolymerizes with acrylamide monomer,
Form DNA-containing polyacrylamide copolymers (Rehman et al., 1999, Nucl.
eic Acids Research 27: 649-655). Using this approach, the stable probe-containing layer is supported by a support having exposed acrylic groups (eg, microtiter (
It can be manufactured on microtiter plates or silanized glass). This approach utilizes a commercially available "Acrydite®" capture probe (available from Mosaic Technologies, Boston, MD). Ac
The rydite moiety is a phosporamidite that has an ethylene group capable of free radical copolymerization with acrylamide. Can be used to introduce.

Referring to FIG. 1, a hybridization chamber 10 including a biochip is provided, and the hybridization chamber 10 includes a substrate 11 having a first surface 12 and a second surface 13 facing the first surface 12. , A flexible layer 16 attached to the first substrate surface 12 by an adhesive layer 15. The area 14 is provided on the first surface 12 with an adhesive layer 15
Bound by and covered by the flexible layer 16. Flexible layer 16, adhesive layer 1
5 and the first substrate surface 12 define a volume 25. The ratio of space 25 to area 14 is preferably about 0.025 mL / mm ² to about 0.25 m.
L / mm ² , preferably about 0.1 mL / mm ² to about 0.25 mL / mm ² , and more preferably about 0.1 mL / mm ² to about 0.2 mL / mm ² .

As shown in FIG. 3, a plurality of biomolecules are arranged between the flexible layer 16 in the region 14 and the first substrate surface 12. In a preferred embodiment, the plurality of biomolecules comprises an array 17 of biomolecules, which array is preferably attached to the first substrate surface 12. Array 17 preferably further comprises a gel pad 22. In an alternative preferred embodiment, the array 17 is arranged in successive layers of polyacrylamide. Figure 2
FIG. 3 is an exploded cross-sectional view of a part of the array 17, showing the gel pad 22. Each gel structure 22 is preferably cylindrical in shape, more preferably about 113 microns in diameter and about 25 microns.
It has a thickness of micron. The distance between each site in each array 17 is preferably about 3
It is 00 microns.

The water-soluble compound layer 28 is about 30 to about 60 ° C., preferably about 35 to about 50 ° C.
More preferably, it has a melting point of about 35 to about 45 ° C., but is located in the space 25 bounded by the first substrate surface 12, the flexible layer 16 and the adhesive layer 15.
Preferably, the water-soluble compound is biocompatible, does not adhere to the flexible layer 16 and helps prevent mechanical damage to the gel pad 22. The compound may be composed of any number of materials, but polymers such as glycol polymers, dextran, sugars and other carbohydrates are preferred. In a preferred embodiment, the compound is polyethylene glycol, preferably polyethylene glycol 600. Compound 28 is set up to consist of compound 28 except where the entire space 25 is occupied by array 17.

The array 17 can be arranged on the surface 12 by providing markings. The markings, which are preferably holes or pits in the surface 12, act as reference or reference points on the surface 12 for the precise placement of the array 17. The presence of the reference points results in a plurality of arrays 17 in one or more areas 14 on surface 12.
Is particularly advantageous in the embodiment having Here, the correct placement of the array is necessary for proper placement and orientation of the array in the hybridization chamber.

In the preferred embodiment, the first port 19 and the second port 20 extend through the flexible layer 16 as shown in FIG. The first port 19 acts as an input port and the region 14 where the first opening 29 is bounded to the first surface 12 by the adhesive layer 15.
Is disposed on the flexible layer 16 so as to be provided on the flexible layer 16. The second port 20 acts as an outlet port and is provided in the flexible layer 16 such that the first opening 31 opens in the region 14 bounded by the adhesive layer 15 on the first surface 12. Port 19
The openings of 20 and 20 may be covered by a removable cover 21. In a preferred embodiment, the replaceable cover 21 is a stopper, gasket, tape, preferably foil tape. In these preferred embodiments, one or more first notches 70 are provided such that the first notches 70 are in direct communication with the region 14 on the first substrate surface 12 bounded by the first adhesive layer 15. 1 is cut into the adhesive layer 15. The second notch 72 is cut into the flexible layer 16 at a location corresponding to the size and location of the first notch 70 in the adhesive layer 15, thereby forming one or more ports. In a particularly preferred embodiment, a ring of adhesive layer 74 is arranged around each second notch 72. As a result, the inner periphery of the adhesive ring 74 is coextensive with the outer periphery of the second notch 72. Preferably, the first notch 70 and the second notch 72 are circular in shape and have a diameter equal to the inner diameter of the adhesive ring 74. Preferably, the inner and outer diameters of the adhesive ring 74 are selected to form a tight seal with the pipette tip. In an alternative preferred embodiment, the second adhesive layer 76 of the flexible layer 16 does not cover the cool air 14 on the first substrate surface 12 and does not define the first port 19 and the second port 20. It is located in part. In this embodiment, the device is further die cut in the same manner as the first adhesive layer 15 into the window 58.
So as to form a marker layer 57. The window corresponds in position to the region 14 on the first substrate surface 12 and is applied to the second adhesive layer 76. In this embodiment, one or more third notches 78 are cut into the second adhesive layer 76 so that the third notches 78 correspond in shape, size and position to the first notches 70 and the second notches 72.
The fourth notch 80 has a shape and position corresponding to the first notch 70, the second notch 72, and the third notch 78, but is cut into the marking layer 57. The diameter of the fourth notch 80 is preferably larger than the diameters of the first notch 70, the second notch 72, and the third notch 78, so that after the device is assembled, a part of the second adhesive layer 76 may be the fourth. It is exposed by the notch 80. Preferably, the exposure position of the second adhesive layer 76 corresponds to the shape and size of the pipette.

In an alternative embodiment of the invention, the first port 19 and the second port 20 are
Rather than 6, it extends through the substrate 11. The illustrated embodiment is described in co-owned and co-pending patent application 09 / 464,490, which is incorporated herein by reference. In a preferred embodiment of the invention, the region 14 on the first substrate surface 12 comprises two rounded edges of physical size at the diagonally opposite corners of the region 14 and the remaining diagonally opposite regions 14 of the region 14. Two 90s at the corner
A square or rectangle with an angle of °. Preferably, when the first port 19 or the second port 20 extends through the flexible layer 16, the first of the first adhesive layer 15 is first.
Notch 70 is cut at the sharp edge of region 14, as shown in FIG. These embodiments are particularly preferred as they consist of a bonded structure with corners eliminated,
It is beneficial to prevent the formation of bubbles in region 14.

The substrate 11 is made of any solid support material, the material of which is metal,
Includes, but is not limited to, ceramics and glasses. Preferably, the substrate 11 is made of silicon or glass (for accuracy and rigidity), molded plastic (reducing manufacturing costs and thermal inertia), or ceramic (for microfluidic elements that fit together integral heating elements). There is. Preferably the substrate is glass.

The adhesive layer 15 uses an adhesive suitable for forming a watertight bond between the substrate 11 and the flexible layer 16. The adhesives include, but are not limited to, high temperature acrylics, rubber based adhesives and silicone based adhesives. The shape of the adhesive layer 15 is array 17
To be formed. The adhesive layer 15 can be provided on the first substrate surface 12 in a pattern that forms the region 14 in a desired shape, but is preferably the elliptical region 14. The adhesive layer 15 can be installed by using inkjet printing or offset printing, or by die cutting a desired shape from a sheet of adhesive. Further, substantially the entire portion of the first substrate surface 12 may be coated with an adhesive, and those portions of the substrate where it is not desired to retain the adhesive may be cured, for example by UV curing. In these embodiments, the portion that retains the adhesive properties is defined using a mask and retains the adhesive properties necessary to adhere the flexible layer 16 to the surface 12. In embodiments that use a die-cut adhesive, the adhesive is a double sided adhesive tape, preferably a carrierless double sided adhesive tape. The adhesive layer 15 is preferably applied in a layer having a thickness of 1 to 100 mm, preferably 25 to 50 mm, and more preferably about 50 mm.

The flexible layer 16 is formed of any flexible solid material. Solid materials include, but are not limited to, polypropylene, polyethylene, plastics including polyvinylidene chloride (sold as Saran ™ wrap), rubber including silicone rubber, high temperature polyester, and porous Teflon ™. Not something. The flexible layer 16 is preferably biocompatible and preferably has low water permeability to prevent water from evaporating from the substrate contained between the flexible layer and the substrate. The flexible layer 16 is also preferably optically clear and must be able to withstand temperatures of 50 to 95 ° C. for 8 to 12 hours without shrinking. In a preferred embodiment, the flexible layer is a gas permeable membrane. Flexible layer 16 is about 5 m
It is preferred to cover an area of m ² to about 1400 mm ² , preferably about 5 mm ² to about 600 mm ² , and more preferably about 100 mm ² to about 600 mm ² .

In a preferred embodiment, the present invention has a marking layer 57, as shown in FIG. The marker layer 57 is die-cut like the adhesive to form the window 58 corresponding to the region 14 on the first substrate surface 12. The marking layer is preferably a thick film with an adhesive layer, preferably an Avery laser label. The marking layer is preferably applied to the outer surface of the flexible layer by vacuum lamination.

The array 17 included in the region on the first substrate surface 12 is coated with the water-soluble compound 28. This water-soluble compound 28 protects and seals the biochip prior to use, as well as prevents degeneration and other damage to the array. The water-soluble compound 28, which has a melting point of about 30 ° C. to about 60 ° C., preferably about 35 ° C. to about 50 ° C., and more preferably about 35 ° C. to about 45 ° C., forms a space 25 between the array 17 and the flexible layer 16. Advantageously used to meet. Preferably the compound is polyethylene glycol, preferably polyethylene glycol 600. A particularly preferred feature of the hybridization chamber 10 of the present invention is that the water-soluble compound 28 fills the entire space 25, preferably at least a portion of the input port 19. This prevents the formation of bubbles in the space 25. This is because compound 28 first melts and mixes with hybridization fluid 26 using rollers 40 without creating bubbles in hybridization fluid 26. The absence of air bubbles in space 25 minimizes the artifacts detected by scanner 36 or light pipe 37.

Ports or holes can be formed in substrate 11 by diamond drilling in glass substrate embodiments, stamping or molding in plastic substrate embodiments, or using ceramic preparation techniques outlined herein. . This facilitates standardization of the dimensions of the hybridization chamber, for example by forming a substrate in which the first port 19 and the second port 20 are formed in a single operation. The substrate 11 and the removable cover 21 can be installed as a strip or a large sheet, and can be rolled to prevent air bubbles from enclosing. The flexible layer 16 can be applied with vacuum lamination to prevent entrapment of air, or after treating the adhesive layer 15 and the water-soluble compound 28, spinning or flowing liquid plastic onto the substrate 11. Then, it can be installed by subsequently curing the flexible layer. The individual hybridization chambers 10 can be formed by stacking, for example, using a diamond saw as shown in FIG.

FIG. 6 shows a preferred arrangement for making the hybridization chamber 10. Here, an alternate layer including the flexible layer 16, the adhesive layer 15, the uncut substrate 11 and the removable cover 21 is installed, and the hybridization chamber is cut into the laminated layers using, for example, a diamond saw or an arbitrary manufacturing tool. Manufactured by The sealed space 25 protects the array from debris created during the cutting process.

An alternative embodiment of the hybridization chamber 10 of the present invention includes a plurality of arrays 17 defined in a plurality of regions 14 under the flexible layer 16. Each area is array 1
7 and is supplied with a first port 19 and optionally a second port 20. In these embodiments, the adhesive layer 15 is disposed on the first substrate surface 12 in a pattern that defines each region 14, and the flexible layer 16 abuts the adhesive layer 15 to encompass the region 14 on said surface. Has been done. In one embodiment of the invention, a hybridization chamber 10 is manufactured that includes array 17 or a plurality of arrays 17 as described herein. Here, a hybridization fluid 2 composed of one or more target molecules
The addition of 6 provides a hybridization chamber ready for use.
In an alternative embodiment, the hybridization chamber 10 is manufactured without the array 17 to allow the user to insert the array 17. In these embodiments, at least one edge of flexible layer 16 is not adhered to first substrate surface 12.

In use of the hybridization chamber 10 of the present invention, a first volume of hybridization fluid 26, preferably comprising a biological sample containing the target nucleic acid, is first provided.
Add to the hybridization chamber via port 19. Prior to applying the hybridization fluid 26 to the hybridization chamber, the space 25 is preferably heated to a temperature above or equal to the melting point of the water-soluble compound 28. Once melted, the hybridization fluid 26 can be added to the hybridization chamber and mixed with the water soluble compound as shown in FIG. The water-soluble compound 28 preferably does not affect the hybridization in the room. Further, an amount of compound 28 is selected such that when compound 28 is mixed with sample fluid 26, hybridization efficiency is improved.

In an embodiment of the hybridization chamber having the first port 19 rather than the second port 20, the hybridization fluid is introduced into the hybridization chamber after the compound 28 has melted, and the hybridization fluid is preferably a pipette. It is preferable to circulate it in and out of the hybridization chamber. Thereby, the hybridization fluid 26 and the compound 28 are completely mixed so that the hybridization fluid 26 is evenly distributed on the surface of the array 17 or mixed with the gel pad 22 according to an embodiment of the array. Alternatively, the hybridization fluid 26 may be evenly distributed over the surface of the array 17 by physically manipulating the flexible layer 16 as described in detail below, or the gel pad 2
Mix to 2. In these embodiments, after the hybridization is complete, the hybridization fluid 26 is removed and a sufficient volume of wash solution 27 is circulated in and out of the hybridization chamber, preferably using a pipette, as shown in FIG. Thus, the array 17 is washed. First port 19 and second port 20
In a hybridization chamber embodiment consisting of both, after the compound 28 has melted, the hybridization fluid is introduced into the hybridization chamber and preferably at least one pipette is used to move the hybridization fluid into and out of the hybridization chamber. Circulation is preferred. As a result, the hybridization fluid 26 and the compound 28 are completely mixed and the hybridization fluid 26 is evenly distributed on the surface of the array 17, or mixed with the gel pad 22 according to the embodiment of the biochip. Then incubate the hybridization chamber for a time and temperature sufficient to achieve hybridization.
By doing so, hybridization is performed. When the hybridization is complete, the hybridization fluid 26 is removed through the outlet port 20,
The biochip is washed by applying a sufficient amount of washing solution and circulating it preferably into and out of the hybridization chamber using a pipette. In these embodiments, the cleaning liquid is applied via the input port and removed via the outlet port,
The cleaning liquid can be continuously supplied. In certain embodiments, the biochip containing the hybridized array is removed from the hybridization chamber for development and further manipulation if necessary. In a preferred embodiment, the biochip containing the hybridization array is analyzed in situ as described below.

FIG. 1B shows a hybridization chamber 1 according to the invention with a heating element 33.
0 shows an advantageous embodiment of 0. The heating element 33 further comprises a heating surface 34 adapted to the shape of the hybridization chamber 10 so as to substantially cover the region 14 under the flexible layer 16. The heating element 33 may be any heating means, including, but not limited to, resistance heaters, thermoelectric heaters, microwave absorption heaters.

The hybridization chamber 10 of the present invention is
It is advantageous to have a thermocouple 35 or other temperature sensing or measuring element that measures the temperature of the hybridization fluid 26 of FIG. These temperature sensing elements heat the heating element 33 to control the temperature of the hybridization fluid 26 and the wash solution 27.
And can be used to calibrate other elements such as the temperature sensitive scanning device 36 described below.

In one embodiment of the invention, biochip 1 in which hybridization occurs
By creating a dye or other material that reflects visible light at the 8th part, the basic (positi
ve) Hybridization can be detected visually. In these embodiments, the dye or other material is preferably enzymatically produced using, for example, a hybridization-specific immunoreagent, such as an antibody linked to an enzyme that causes the production of the dye. Visual inspection can be used to detect sites of basic hybridization. More preferably, the biochip containing the hybridization array is preferably scanned using the scanner 36 described below.

Basic hybridization on biochip 18 is illuminated by light of a specific wavelength bound by fluorescence using labeled target molecules in the biological sample or by hybridized double-stranded DNA molecules. Detection is preferably by including in the hybridization liquid an intercalating dye that fluoresces when exposed to light. Suitable interventional dyes include, but are not limited to, ethidium bromide, Hoechst DAPI, Alexa Fluor dye. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, propidium iodide, Cy3 and Cy5 (Amersham). These can be incorporated into target molecules, such as in vitro propagated fragments, using labeled oligonucleotide primers.

10A-10C show an embodiment of the invention having a scanner 36. The scanner 36 is positioned above (or below) the flexible layer 16 and continuously in the area 14
And illuminating the array 17 arranged thereon, the region 14 moves from one end to the other. Prior to analysis of the hybridized array, place all fluids in space 2.
5 so that the flexible layer 16 is in contact with the array 17. Scanner 36
Excites the fluorescent dye with preferably short wavelength light, more preferably light having a wavelength of 250 nm to 600 nm. The scanner 36 collects the emitted light from a specific area. The amount of emitted light is used to determine the amount of fluorescent dye present in the area, ie the amount of label target.

A particular embodiment of the scanner and scanning device 36 is shown in FIGS. 11A-11E. A particularly advantageous feature of the hybridization chamber 10 is that the flexible layer 16 is translucent to light of the appropriate wavelength, including light in the ultraviolet and visible portions of the spectrum.
A further advantageous feature of the hybridization chamber 10 is that the very thin flexible layer 16 immediately comes into close contact with the biochip 18 after the hybridization fluid 26 or wash fluid 27 has been removed from the hybridization chamber 10. . This combination of features reduces and eliminates free surface reflections, internal reflections, divergence or scatter of illumination light from the scanner, thereby optimizing the incident light illuminating array 17. This arrangement is more economical than existing devices because it eliminates the need for highly polished and low scattering surfaces, complex and expensive lenses, and eliminates focus and depth problems in more complex optical detectors. is there.

In another embodiment, the waveguide 37 contacts the surface of the flexible layer 16, which immediately contacts the surface of the array 17, as shown in FIG. 11B. In these embodiments, the illumination light and the emitted light are propagated and collected by the waveguide 37. The waveguide is designed to be slightly flexible to fit the contoured surface of the flexible layer 16. The waveguide 37 is in contact with the flexible layer 16, which is in contact with the array 17, so that there is no surface reflection even in situations where the array 17 and the waveguide 37 are arranged at different heights. Contact. The waveguide 37 has a larger surface area than the array 17. This provides the maximum amount of illumination light to the array 17 and the array 1
The maximum amount of light emitted from 7 is collected by the waveguide 37. A further advantage of the waveguide 37 is the ability to detect air bubbles that are formed in the hybridization fluid 26 or the wash buffer 27, the result of which is detected by the roller 40 in the flexible layer 16.
Can be used as a signal to address and remove such bubbles. Removing air bubbles in the hybridization fluid 26 or wash buffer 27 reduces the frequency of non-specific binding and artifacts detected by the scanner 36.

In an additional embodiment of the invention, the area 14 defined by the adhesive layer 15 further comprises a reflective layer 38. The reflective layer substantially covers the entire area 14 and is arranged between the array 17 and the first substrate surface 12. In a preferred embodiment, the reflective layer 3
8 is made of aluminum, gold, silver, or platinum. In these embodiments, the reflected optical signal or the optical signal transferred to the photodetector of the scanner 38 increases to four-fold. In a further advantageous embodiment of the invention the reflective layer 3
8 is metal film resistance or RF induction heating. In these embodiments, the reflective layer 3
8 can heat the slide without the need for an additional heating element 33. This is a particularly desirable feature in the portable embodiment of the hybridization chamber 10 of the present invention.

A passivation layer 39 can be provided on the reflective layer 38 if desired. The passivation layer 29 is preferably a parylene layer having a thickness of several microns provided by vapor deposition. In these embodiments, the amount of illumination required, ie the amount of power required to operate the scanner 36, is reduced. This is particularly suitable for improving battery life in battery-powered embodiments such as portable devices. In addition, the passivation layer 39 reduces artifacts in the light emission data due to obscuring objects.

The hybridization chamber 10 is preferably provided with a roller 40 that removably contacts the flexible layer 16. This roller 40 is located in the area 1 on the first substrate surface 12.
4 can be moved longitudinally across. In a preferred embodiment, the surface of the roller 40 comprises a fabric pattern 41, preferably a spiral pattern, which allows the roller to effectively mix the hybridization fluid and the wash fluid across the region 14 and the array 17. One advantageous arrangement of roller 40 and hybridization chamber 10 is shown in Figure 11E. As shown, the roller 40 can be advantageously connected to a movable arm 42. The moveable arm 42 can be arranged such that the roller 40 is in contact with the flexible layer 16 when in the first position, and can be moved to a second position in which it does not contact the flexible layer 16. Preferably, the movable arm 4
2 has a pivot point 44, and movement around the pivot point 44 is preferably controlled by a solenoid. In addition to the movement of the roller into and out of contact with the hybridization chamber 10, the roller 40 or the hybridization chamber 10 or both are movable in the longitudinal direction. This allows the roller 40 to mix the hybridization fluid 26 or the wash liquid 27 in the space 25 bounded by the flexible layer 16, the adhesive layer 15 and the first substrate surface 12 within the region 14 containing the array 17. it can. In embodiments consisting of multiple regions 14 with multiple arrays 17, rollers 40 are disposed longitudinally across each of the multiple regions 14 and hybridize fluid 26 or hybridizing fluid 26 in any space 25 containing array 17. The cleaning liquid 27 can be mixed.

In an additional embodiment, a sample preparation chip 45 having a port 46 can be attached to the hybridization chamber 10 as shown in FIGS. 12A-12C. The port 46 of the sample preparation chip 45 coincides with the first port 19 of the hybridization chamber 10 and allows effective movement of the sample into the space 25. An additional fiducial reference can be used to exactly match the hybridization chamber 10 with the sample preparation chip 45. First port 19
Access is made through the second substrate surface 13 so that the array is scanned without interference from the attached sample preparation chip. In alternative embodiments of the invention, the sample preparation chip 45 can be bonded to the second substrate surface 13 (FIG. 12B) or can be formed as an integral part of the substrate 11 (FIG. 12C).

A preferred embodiment of the hybridization chamber 10 of the present invention is a portable embodiment, as shown in FIGS. 10A-10C, which further comprises a scanner 36. In these embodiments, the portable device 47 comprises a base 48, a lid 49, a carriage 50 embodying rollers 40, a scanner 36, a heating element 33 and a thermocouple 35. The carriage 50 is shown in FIG. 11A. The device 47 has a compartment for disposing the hybridization chamber 10 in the vicinity of the carriage 50.
The carriage 50 comprises movable means for moving the roller 40, the scanner 36 and the heating element 33 with respect to the hybridization chamber 10, as required for the operations described above. The carriage 50 and the lid 49 store the hybridization fluid 26 and the washing liquid 27 in the first port 19 and the second port 2 as required by the user.
It is arranged so that it can be introduced into and removed from the hybridization chamber via 0. Alternatively, the device 47 further has fluid connections 52 to each of the first and second ports to provide for sample introduction and array washing after sample hybridization. Device 47 is preferably battery powered,
AC adapters are also included within the scope of the invention. In a further preferred embodiment, the lid 49 further comprises a display device 56 for displaying the analysis result.

There are various ways to use the device. If necessary, the target sequence is adjusted using known techniques. For example, as is apparent to those of skill in the art, known lysis buffers, sonications, electropora.
treatment) to lyse the cells, and optionally purify and amplify. Furthermore, the reactions described herein can be performed in a variety of ways, as will be apparent to those of skill in the art. In the preferred embodiment described below, the components of the reaction may be added simultaneously or may be performed sequentially in any order. In addition, the reaction includes various other reagents included in the assay. These include reagents such as salts, buffers, neutral proteins such as albumin, detergents, which facilitate optimal hybridization and detection, and / or non-specific or background Reduce interactions. In addition, reagents such as protease inhibitors, nuclease inhibitors, antibacterial agents, etc. that improve the efficiency of the assay may be used depending on the sample preparation method and target purity.

Furthermore, in most embodiments, the double stranded target nucleic acid is denatured into a single strand, allowing hybridization of the primer to other probes of the invention. The preferred embodiment utilizes a heating step, generally by raising the reaction temperature to about 95 ° C, although PH modification and other techniques may be used.

As outlined herein, the present invention provides multiple capture probes that hybridize to certain portions or domains of the target sequence. The probes of the present invention are designed to be complementary to a target sequence (the target sequence of a sample, or other probe sequence used in, for example, sandwich assays known to those of skill in the art). This results in hybridization of the target sequence and the probe of the invention. As outlined below, this complementarity need not be perfect. Multiple base pair mismatches (mism) that interfere with hybridization between the target sequence and the single-stranded nucleic acid of the invention.
atch). However, if the number of mutations is high and hybridization does not occur even if the hybridization conditions are not stringent, the sequence is not the complementary target sequence. Thus, "substantially complementary" means that the probe is sufficiently complementary to the target sequence to hybridize under normal reaction conditions.

A variety of hybridization conditions may be used in the present invention, including high, medium and low stringency conditions. For example, Maniatis et al., Molecular Cloning: Laboratory Manual, 2nd Edition, 1989, and Short Protocols in Molecular Biology, ed. See Ausubel et al. These are incorporated herein by reference. Stringent conditions are sequence-dependent and will be different in different circumstances. An extensive guide to nucleic acid hybridization can be found in Tijssen, Techniques in Biochemistry and Molecular Biology Using Nucleic Acid Probes, "Overview of Hybridization Principles and Nucleic Acid Assay Strategies" (1993). Generally, stringent conditions are selected at a given ionic strength and pH of about 5-10 ° C below the melting point (Tm) for a particular sequence. The Tm is the temperature (at a given ionic strength, pH and nucleic acid concentration) at which 50% of the probe complementary to the target equilibrates to hybridize to the target sequence (at Tm the target sequence is in excess, so 50 % Probe is in equilibrium)
. Stringent conditions are salt concentrations less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salt) at pH 7.0 to 8.3. . The temperature is at least about 30 ° C. for short probes (eg 10 to 50 nucleotides) and at least about 60 ° C. for long probes (eg 50 or more nucleotides). Stringent conditions may be achieved with the addition of helical destabilizing agents such as formamide. Hybridization conditions may be varied when a non-ionic backbone or PNA is used. Further, a cross-linking agent may be added after the targeting to attach the double-stranded hybridization complex to the cross-linking, that is, sharing the electron pair.

[0114] Thus, the assay generally proceeds under stringent conditions that allow the formation of hybridization complexes only in the presence of the target. Stringent conditions can be controlled by changing process parameters that are thermodynamic variables. The thermodynamic variables are
These include, but are not limited to, temperature, formamide concentration, salt concentration, disturbing salt concentration, pH, organic solvent concentration and the like.

These parameters may be used to control specific binding, as outlined in US Pat. No. 5,681,697. Thereby, it is preferable to carry out the process at higher stringency conditions to reduce specific binding.

As described herein, there are many possible detection techniques that can be utilized with the present invention. In a preferred embodiment, photolabeling technology is used, as outlined herein. In a preferred embodiment, a label such as an optical dye (eg, a fluorescent dye) is added to the assay complex consisting of the target detective and the capture binding ligand. In some embodiments, for example, in the case of nucleic acids, the label can be added to the target by incorporation during amplification reactions such as PCR. For example, other labels such as fluorescent dyes or biotin can be added to PCR primers or dNTPs for enzyme mixing. As an alternative, as mentioned above, the intercalator (intercalator
) Can be used.

Alternatively, in a preferred embodiment, US application Ser. No. 09 / 458,553; 09 /
458501; 09/572187; 09/495992; 09/344217.
WO00 / 31148; 09/439889; 09/438209; 09/3
44620; 09/478727; PCTUS00 / 17422; WO98 / 2.
0162; WO98 / 12430; WO98 / 57158; WO99 / 5731
7; WO99 / 67425; PCT00 / 19889; WO99 / 57319, electron detection methods such as those described can be used. All of these are incorporated herein by reference. These embodiments use an array of microelectrodes on the substrate.

The following examples demonstrate specific embodiments of the invention and its uses. These are listed for illustrative purposes and are not a limitation of the present invention. The references cited herein are incorporated herein by reference.

Example 1 Assembly of Hybridization Chamber A method of assembling a hybridization chamber according to the present invention is shown in FIG.

502FL ultra-clean laminated adhesive film (
Four elliptical holes were formed in the 3M) layer. A similar pattern was punched in the Avery laser label 5663 used as the frame and marking layer. In the meantime, a sheet of polyvinylidene chloride film was stretched on a stainless steel frame at 100 ° C.
Annealed for 30 minutes. By vacuum laminating the Avery label in a vacuum laminating press, the Avery label was attached to one surface of the polyvinylidene chloride film. A vacuum of 15 psi was applied for 30 minutes, the vacuum was removed and a mechanical pressure of 15 psi was maintained for 1 minute. Adhesive was applied to the other side of the polyvinylidene chloride film in the same manner as the label.

Next, the adhesive-coated film was attached to a glass slide prepared in advance. The array of nucleic acid probes and gel pad was mounted on a glass slide by standard methods. A port was drilled in the slide using a diamond drill. A polyvinylidene chloride film was attached to the slide using a vacuum laminating press. A vacuum of 15 psi was maintained for 1 minute and then a mechanical pressure of 15 psi was maintained.

Each hybridization chamber was filled with polyethylene glycol 500 using a 10 mL pipette. A layer of 3M7350 polyester tape was applied to the slide to seal the port.

Example 2 Using the assembly die cutter of the upper charging hybridization chamber , 502FL ultra-clean laminated vinegar adhesive film (
Four elliptical holes were formed in 3M). A similar pattern was punched in the Avery laser label 5663 used as the frame and marking layer. Meanwhile, a sheet of polyvinylidene chloride film was stretched on a stainless steel frame and kept at 100 ° C for 3 hours.
Annealed for 0 minutes. By vacuum laminating the Avery label in a vacuum laminating press, the Avery label was attached to one surface of the polyvinylidene chloride film. 1
A vacuum of 5 psi was applied for 30 minutes, the vacuum was removed and a mechanical pressure of 15 psi was maintained for 1 minute. Adhesive was applied to the other side of the polyvinylidene chloride film in the same manner as the label.

Next, the adhesive-coated film was attached to a glass slide prepared in advance. The array of nucleic acid probes and gel pad was mounted on a glass slide by standard methods. A polyvinylidene chloride film was attached to the slide using a vacuum laminating press. 15p
A vacuum of si was maintained for 1 minute and then a mechanical pressure of 15 psi was maintained.

Each hybridization chamber was filled with polyethylene glycol 500 using a 10 mL pipette. A layer of 3M7350 polyester tape was applied to the slide to seal the port.

[Brief description of drawings]

1A-1D are diagrams of a preferred embodiment of the present invention showing the preparation of a reaction chamber.

FIG. 1A is a cross-sectional view of an apparatus showing a hybridization chamber prefilled with a water-soluble compound to contact a heating element.

FIG. 1B is a cross-sectional view of an apparatus showing mixing of a water-soluble compound with a biological sample liquid.

FIG. 1C is a cross-sectional view of the device showing a chamber filled with a sample liquid / water-soluble compound mixture, with the first and second ports covered by seals.

FIG. 1D is a plan view of the device showing the adhesion pattern defining the individual regions containing the array of oligonucleotide probes.

FIG. 2 is an exploded cross-sectional view of a chamber showing an array of gel pads of a preferred embodiment of the present invention.

FIG. 3 is an exploded perspective view showing an array of biomolecule probes showing positioning of a gel pad on a substrate according to a preferred embodiment of the present invention.

FIG. 4 is an exploded cross-sectional view of a port showing a conical port of a preferred embodiment of the present invention.

FIG. 5 is a perspective view of a marker layer, a flexible layer and an adhesive layer of a preferred embodiment of the present invention.

FIG. 6 is a sectional view of a laminating chamber according to a preferred embodiment.

7A-7E are plan views of layers of another preferred embodiment of the present invention having inlet and outlet ports extending through the flexible layer.

FIG. 7A is a plan view of a first adhesive layer.

FIG. 7B is a plan view of a flexible layer.

FIG. 7C is a plan view of a second adhesive layer.

FIG. 7D is a plan view of a marker layer.

7E is a plan view of the combined layers of FIGS. 7A-7D. FIG.

8A-8B are detailed views of a notch cut in a first adhesive layer and a marking layer of a preferred embodiment of the invention having inlet and outlet ports extending through the flexible layer.

9A-9C are cross-sectional views of a preferred embodiment of the present invention showing the process of analyzing an array after the reaction is complete.

FIG. 9A is a diagram showing the apparatus when the reaction is completed.

FIG. 9B illustrates removal of sample fluid from the chamber such that the flexible layer contacts the array.

FIG. 9C is a diagram showing a state in which an array is analyzed using a laser scanner.

10A-10C are diagrams illustrating a portable embodiment of the present invention.

FIG. 10A is a side view of a portable scanning system.

FIG. 10B is a perspective view of the preferred embodiment with a portable scanning device showing the flexible layer in contact with the carriage.

FIG. 10C is a diagram of a portable system showing a lid with a display device.

11-11E are cross-sectional views of the direct contact optical fiber scanner shown in FIG.

12A-12C are views of another embodiment showing an apparatus connected to a sample preparation chip.

FIG. 12A is a view showing an embodiment in which the sample preparation chip is detachably arranged on the second surface of the substrate.

FIG. 12B is a view showing an embodiment in which the sample preparation chip is fixed to the second surface of the substrate.

FIG. 12C illustrates an embodiment in which the sample preparation chip is incorporated into the substrate.

FIG. 13 illustrates a preferred embodiment assembly of the present invention and its use.

─────────────────────────────────────────────────── ─── Continuation of front page (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE , TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE , GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US , UZ, VN, YU, ZA, ZW [Continued summary]

Claims (7)

[Claims] 1. A device for performing a biological reaction, comprising: a) a substrate having a first surface and a second surface, b) an array of biomolecules arranged on the first surface, and c) an adhesive layer. A biological reaction device comprising: a flexible layer attached to the first surface to generate a reaction space; and d) a port extending from the second surface to the reaction space of the first surface. 2. A device for performing a biological reaction, comprising: a) a substrate having a first surface, b) an array of biomolecules arranged on the first surface, and c) an adhesive layer on the first surface. A biological reaction device comprising a flexible layer attached to generate a reaction space and having a first port to the reaction space. 3. The device according to claim 1, wherein the substrate is glass, silicon, ceramic or plastic. 4. The device according to claim 1, wherein the biomolecule is a nucleic acid. 5. The apparatus according to claim 1, wherein the reaction space is made of a water-soluble compound. 6. The device according to claim 1, wherein the biomolecule is attached to the substrate using a gel pad. 7. The apparatus according to claim 1, further comprising a scanner.