WO2005073363A1 - An analysis chip, an apparatus comprising the chip, the chip test kit and the measurement method thereof - Google Patents

Abstract

An analysis chip comprises of a top element, a base element, a reversible or irreversible isolation reaction structure, and one or more broadband cavity reactors made of any other structures. The reactor includes the broadband cavity whose width is more than 600µm, and a liquid inlet, a liquid outlet, and the said reaction isolation structure. The surface static water contact angle of the top and/or base element material layer is between 40- 80 degrees. An apparatus comprising a blank element used as the top and/or base element or one of them of the analysis chip is also provided. Futhermore, the chip test kit and the measurement method thereof are also provided.

Description

 —Analysis chip, packaged chip body, chip reagent

 ¾ ^ Field

 The present invention relates to a detection chip for qualitative and / or quantitative analysis of a sample, and particularly to a detection chip having a wetting phenomenon of a reaction medium. The present invention also relates to a device and a kit including the detection chip, and a qualitative and quantitative analysis method using the chip. Background

 The detection chip, also simply referred to as a chip in the present invention, is one of the most attractive detection devices currently developed. At present, the most commonly used chips are biochips, and the most commonly used in biochips are peptide chips and gene chips. The peptide chip is a biochip prepared by using a plurality of amino acid sequence structures (including proteins) as probes to be fixed on a substrate. The gene chip is a chip that hybridizes nucleic acids and nucleotides with complementary nucleic acids and nucleotide probes in the specimen to form a hybrid, or combines with specific antibodies, and then displays the detection results by color reaction. Biochip has a wide range of applications, including gene expression detection, gene screening, drug screening, disease diagnosis and treatment, environmental monitoring and governance, forensic identification and other fields.

 The core of the chip is the reactor on it. The chip reactor in the present invention refers to a place where a probe is sequentially fixed in a chip and specifically reacts with a target during detection, and other related structures communicating with it. In the present invention, a probe refers to a substance fixed on a solid-phase carrier to capture a target, and includes a biological probe, such as DNA, a polypeptide, a protein, a cell, a tissue, and other biological components. In the present invention, a substrate refers to a component in a chip that includes a solid-phase carrier holding a probe; a probe board refers to a substrate on which a probe is fixed.

In the present invention, a chip is defined as a single reactor chip (n = 1) and a multi-reactor chip (n is equal to or greater than 2) according to the number of reactors on the chip n. In the present invention, the reaction «is defined as a flow reactor and a non-flow reactor according to whether the liquid-phase medium added in the detection process can be directed to flow in the reactor; a biochip characterized by a flow reactor and a non-flow reactor It is divided into mobile chips and non-mobile chips. In the present invention, the reactor is divided into open and closed reactors according to whether the upper part of the reactor probe is opened during the entire detection process; the reactor is characterized by this The chips are defined as open and closed chips, respectively.

 The chip reactor usually has the characteristics of the above-mentioned several kinds of reactors at the same time. In the present invention, these reaction skins are defined as reactors characterized by all of their properties, and biochips characterized by these reactors are also defined in the same manner. For example, if there is an open structure without a cover above the probe array during the detection process, and the liquid medium added by the beaker can be directed to flow in the reactor, the reactor is defined as an open flow reactor, and the corresponding chip Defined as an open mobile chip. Others by analogy.

 The status of the chip is as follows:

 1. Non-mobile chip

 Non-mobile chips include closed non-mobile chips and open non-mobile chips. Currently, the most widely used are open non-mobile chips. Current open-flow chips include single-reactor open-flow chips and multi-reactor open-flow chips. An example of a single-reactor, open-flow, non-flowing chip is a chip that is based on a microscopic slide, activated, spotted, and has no additional structure. The closed-type non-flowing chip closes the reactor during the reaction, and removes the sealing layer during the addition and washing (refer to CN 1335501A).

 2.Mobile chip

 There are currently three types of flow chips: microfluidic chips, capillary chips, and microarray flow chips. Micro-fluidic chip, also known as micro-fluidic chip, refers to the use of micro-pipes (such as capillaries) as network samples such as pumps, micro-valves, micro-reservoirs, micro-electrodes, micro-detection elements, pre-treatment, and liquid transport The analysis function is integrated into a micro-full analysis system.

 The development of microfluidic chips is very active, such as 02353129.0, 03232582.7, 02353130.4, 98805243.1, 02247726.8, 02247727.6, 02145102.8, 99114512.7, 98813487.X and 03277112.6 Chinese patent applications; 6,643,010, 6,730,516, 6,4953,010, 6,432,290, and 6,303,288 U.S. Patent Nos .: 20020045265, 20040166504, 20040175298, 20040081583, 20030012697 and US Patent Nos. 20030000835.

Track size usually means less than 0.05 mm in width and less than 0.025 mm in depth. Generally, only one probe is fixed in each microchannel reactor of a microfluidic chip, and one target in a sample is detected at a time. The advantages of the micro-channel chip are high dexterity and speed. Due to the extremely small legs, the disadvantages are: 1) The probe array cannot be fixed; 2) the fluid flow resistance is large; 3) the cleaning is difficult; and 4) for some detections, such as the detection of fluorescent markers, the results cannot often be read directly with an ordinary chip scanner.

 The capillary chip includes a chip for fixing a probe on a wall of a capillary channel or a glass fiber structure inserted into a capillary. CN 2483395A can be seen. The advantage is that the preparation is simple, the disadvantages are: 1) the continuous operation cannot be performed; and 2) the probe array cannot be fixed in a capillary.

 A probe array is fixed in the reactor of the microarray chip. Enclosed microarray flow chip can be seen in Chinese patent ZL022229310. The irreversible closed microarray flow chip can be seen in CN 2559986Y, and the reversible closed microarray flow chip can be seen in Chinese patent ZL022229310. Their advantage is that the probe array can be fixed, but the disadvantage is that the wetting efficiency is lower and the reaction efficiency is lower.

 In fact, the factors that are considered in the design of existing closed-chamber flow chips are the probe-target reaction kinetics conditions and signal observation conditions. The movement of the liquid phase medium in the reactor, especially in the reaction chamber of the reactor, is one or one of the following: More than one combination to achieve. For the reaction chamber, these are all exogenous liquid transmission forces. Of course, the top surface, bottom surface and walls of the reaction chamber are often hydrophilic, but the endogenous liquid transmission power due to its hydrophilicity is weak. Even if the chip is perpendicular to the horizontal plane and the liquid inlet is placed below, the reactor has a certain degree of capillary phenomenon, but if the chip is perpendicular to the horizontal plane, it is difficult for the reaction chamber to be filled with the reaction medium. As a result, the distribution of the liquid medium in the reaction chamber of the current closed microarray chip in the detection chamber, especially the operation containing the conversion medium, is sometimes insufficient, for example, the presence of gas hinders the liquid phase. The distribution of the medium, which affects the detection results.

 In short, a kind of high reaction efficiency (such as high sensitivity), simple production, simple detection operation (such as no need for PCR), ordinary chip scanner (such as confocal light scanner) can be used to read the results, and the cost is low. Chips are still an important goal of research and development. Summary of the invention

According to an aspect of the present invention, there is provided an analysis chip including a top surface element and a bottom surface element. Pieces, reversible or irreversible reactor isolation structures and optional other structures forming one or more multiple broadband chamber reactors, wherein the reactor comprises a broadband cavity having a cavity width greater than 600 um, preferably greater than 1000 μm Chamber, liquid inlet structure, liquid outlet structure, and the reactor isolation structure, and A) the broadband chamber includes a liquid inlet, a liquid outlet, a chamber wall, a top surface and a bottom surface, and is fixed to the bottom surface ^ / And the probe on the top surface; B) the width of the cavity, the height of the wall of the chamber, and the static 7 contact angle of the bottom surface and the top surface are selected as [^, so that when the fixed The time required for the bottom surface ¾ with the probe to be parallel to the horizontal plane and the ion-free water added to the liquid inlet to move to fill the broadband chamber by itself is less than 2 seconds, preferably less than 1 second.

 According to another aspect of the present invention, there is provided a device including a top surface element, a bottom surface element, or a preparation element of the top surface element or the bottom surface element of the wet chamber chip as described above.

 According to another aspect of the present invention, there is provided a chip reagent kit including the above-mentioned wet chamber chip.

 According to still another aspect of the present invention, it provides a qualitative analysis method and a quantitative analysis method, which include adding a sample with or without a labeled reaction to the above-mentioned humidified chamber chip, and passing the sample with or without the labeled The target reacts with the probe. BRIEF DESCRIPTION OF THE DRAWINGS

 Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings and embodiments. In the drawings: FIG. 1 is a schematic top view of a bottom surface component of a reversible closed-type humidification chamber chip,

 FIG. 2A is a schematic bottom view of a top surface element of a reversible closed-humidity chamber chip, and FIG. 2B is a schematic top view of the top surface element shown in FIG. 2A.

 Figure 3 is a schematic diagram of an irreversible closed-humidity chamber chip,

 FIG. 4A is a schematic top view of an irreversible closed-type wet chamber chip bottom surface element, FIG. 4B is a schematic top view of a wet chamber chip top surface element shown in FIG. 4A, and FIG. 5 is a sub-array containing multiple probes Schematic diagram of Yuanlong from the bottom.

1. Bottom surface component 2. Bottom liquid adding area 3. Bottom liquid discharging area 4. Probe

 5. Broadband chamber bottom surface 6. Broadband chamber wall 7. Resilient material isolation structure of the reactor

8. Top surface element 9. Broadband cavity outlet 10. Broadband cavity inlet 11, Hydrophobic material isolation structure 12, Positioning structure 13, Reactor inlet structure

14. Reactor liquid exit structure 15. Reactor liquid inlet structure isolation structure

 16.Reactor liquid discharge structure isolation structure17.Reactor liquid inlet pool18.Reactor liquid outlet pool

 The purpose of the present invention is to provide a chip with low reaction efficiency, high production efficiency, simple fabrication, simple detection operation, and direct reading of results with an ordinary chip scanner.

 Therefore, the flow chip of the present invention is an analysis chip containing one or more multiple broadband chamber reactors formed by a top surface element, a bottom surface element, a reversible or irreversible reactor isolation structure, and optionally other structures. Wherein the reactor includes a broadband chamber having a chamber width greater than 600 μm, preferably greater than 1000 m, a liquid inlet structure, a liquid outlet structure, and the reactor isolation structure, and A) the broadband chamber includes a liquid inlet Port, liquid outlet, chamber wall, top and bottom surfaces, and probes fixed to the bottom surface and top surface; B) the cavity width, the height of the chamber wall, and the bottom surface ^ / and The static water contact angle of the top surface is selected as it [^, so that when the bottom surface where the probe is fixed and the top surface are parallel to the horizontal plane, the ion-free water added to the liquid inlet moves by itself to fill The time required for the broadband chamber is less than 2 seconds, preferably less than 1 second. The reaction medium includes an aqueous solution, and the material properties include hydrophilic and hydrophobic.

 The detection chip in the present invention, referred to as a chip, refers to a qualitative and / or quantitative miniaturized detection product. The principle is to sequentially fix a trace probe on the surface of a solid-state carrier substrate (abbreviated as substrate). To make it react specifically with the target molecule under detection conditions, and then the results of the reaction can be identified in an addressable manner. The chip according to the present invention includes, but is not limited to, chip concepts that are currently popular (such as Biochip, Microarray, Bioarray in English), and the form is not limited (can be rectangular, circular butterfly, etc.). The humidified chamber chip according to the present invention refers to a chip whose reaction chamber has a wetting phenomenon of a reaction medium.

In the present invention, we studied the reaction efficiency (represented by the absorption capacity improvement rate in Example 1) and the intensity of the wetting phenomenon (the time required for the deionized water to fill the broadband cavity in the horizontal direction by the wetting phenomenon To express the relationship between). We found that there is a characteristic intensity of the wetting phenomenon. When the intensity of the wetting phenomenon increases to a certain amount, for example, deionized water is filled by the wetting phenomenon in the horizontal direction. The time required for the broadband chamber is less than 2 seconds, and the ship is less than 1 second. The effect on the response efficiency is decisive, and it is expressed as a non-linear increase in the rate of increase in suction capacity (Table 1). Table 1

 Method Reference is made to Example 1. The bases used are epoxy based bases (static water contact angle of 47 degrees on the bottom surface), the probe is HCV antigen, the target is human serum HCV antibody, and the label is rhodamine-labeled secondary antibody

 ** Time required for deionized water to fill the broadband chamber in a horizontal direction by the phenomenon of wetting

*** Experiments with open non-flow control chips may have mentioned the application of capillary phenomenon in other chip-related inventions, but the very important relationship between the intensity of the wetting phenomenon and the reaction efficiency when working at the horizontal plane is not in the Mentioned in published chip literature. Although we do not intend to get into too much theoretical discussion, we also have some opinions on this. For the efficiency of reactions on a chip, such as the reaction between a probe and a target, a determining factor is the migration of the target to the probe. The fluid velocity and surface wetting are important factors determining this migration. We observed that within a certain range, the lower the flow rate of the reaction medium, the greater the importance of surface wetting. We found that in the usual reaction medium (linearity is less than 0.005—0.1 ml / min / cm ² ), the reaction efficiency (such as the spirit C¾ under a limited sample cake) is a function of the surface wetting or wetting phenomenon (such as the difficult to wet The time required to wet the entire reaction cell is expressed in fractions. It is particularly surprising that we found that when the intensity of the wetting phenomenon increased to a certain amount (for example (Reactor No. 5 in Table 1), which has a decisive influence on the reaction efficiency, thus providing an important basis for the high-efficiency chip of the present invention. An important difference from the existing flow chip is that the chip of the present invention, especially the chip of the crane scheme of the present invention, has a reaction efficiency in detecting the reaction when the wetness of the reaction medium reaches a certain value in the reactor. Decisive influence on the chip.

 The wide-band chamber reactor in the humidified chamber chip of the present invention is not a general wide-band chamber reactor (such as the above-mentioned capillary reactor, microfluidic reactor, flow reactor, etc.). Horizontal wet «, flat reactor with minimum width. In the wide-band chamber reactor of the present invention, the probes may be fixed in a non-array type or in an array type on a substrate included in the top surface element and the bottom surface element, but the crane is fixed in an array type. Leg array refers to an ordered and addressable probe arrangement, which includes a dot array (with probe points as the unit of arrangement) and a linear array (with probe segments or probe bands as the unit of arrangement). The simplest array type is a row X column arrangement, such as MXN, MX1, 1XN arrangement, and so on. In fact, depending on the use of the chip, there can be and should be different options for the orderly distribution of the probes. It is not necessary to be limited to a single array distribution. Although the wet chamber chip of the present invention can use a concave channel (such as an etched capillary groove leg channel) as the emblem channel, the crane solution does not use this type of complicated concave channel.

 In the wet chamber chip of the present invention, the material of the top surface and / or the bottom surface of the broadband chamber is selected from one or any two or more of the following: glass, silicon and silicon compounds, metal oxides, Metal and polymer materials and their respective derivatives and more. There is a solution in which the surface of the fixed-type probe array in the broadband chamber has a reaction-reactive medium property (for example, glass material), and the surface of the non-fixed probe array has a lyophobic property (for example, plastic, rubber).

In the humidified chamber chip according to the present invention, the static water contact angle between the top surface and the bottom surface is 40-80, and the cross-section of the broadband chamber is rectangular or a derivative with a rectangle as a boundary, and the The rectangular "width ratio" is greater than 3, preferably greater than 5, or even greater than 10. The aforementioned capillary reactor and microchannel reactor (essentially a capillary reactor) have laid the foundation for the research of capillary layer folding columns The broadband chamber reactor of the present invention is a derivative (such as a standard or approximate ellipse type) with a rectangular cross section or a rectangular outer boundary, and the ^ / width ratio is greater than 3, It is preferred that the reactor has a wet iim greater than 5. In the present invention, we have found that it is very important to provide a sufficient width to hold a probe array and to form a sufficiently strong wetting phenomenon. In the chip according to the present invention, the cavity width is 2000-7000 μηι. The uniformity of the flow reaction in a capillary reactor or microchannel is easily acceptable as it has been demonstrated by capillary chromatography. However, the uniformity of flow response over a wide area is easily questionable. In the present invention, we spotted a 200 μm antibody probe dot array on a wide surface (2000-7000 π), and reacted with a rhodamine-labeled secondary antibody with a reaction speed of 10 m. / min / mm ² . Through the experimental results, we found that within the width of this probe distribution area, the reaction efficiency of each probe point to the labeled secondary antibody (fluorescence scanner reading) was sufficiently parallel (the error was less than ± 20%).

 In the chip according to the present invention, the height of the cavity wall is 10-600 / m, preferably 50-400 μm, and more preferably 100-300 μm. Although the smaller the chamber wall height (often the height of the isolation structure), the stronger the wetting phenomenon may be. However, the too small height of the isolation structure increases the flow resistance and the flow time required for the same linear velocity. Through the experimental results, we found that when the cavity wall height is 100-300 μηι, the chip has better wetting reaction time / fluid pressure cleaning efficiency and comprehensive balance properties with chips with other cavity wall heights.

 In the chip according to the present invention, the increase rate of the adsorption capacity of the wide-band chamber reactor is greater than 200%, greater than 300%, and more than greater than ■%. Adsorption capacity is another ffl feature of chip reactors. Although the definition of a suitable adsorption capacity is still difficult for the time being, a definition of a relative adsorption capacity is meaningful as a term bond. In the present invention, the second improvement of the adsorption capacity (humidity chamber chip reaction «attachment capacity ^ photo opening«; the adsorption capacity of the moving chip) X 100%] wherein the measurement of the adsorption capacity is performed in the same manner as in Example 1.

In the chip according to the present invention, the probe is included in a nanostructure active region on the bottom surface and / or the top surface, and the nanostructure active region includes a conventional solid phase support and an active nanostructure fixed thereon, The active nanostructure includes a nanostructure unit and a probe fixed on the nanostructure unit, and the nanostructure unit includes a protruding height greater than 3 nm and a protruding half-height at least one-dimensional dimension of 1-500 nm, preferably 1-100. nano-convex bodies between nanometers, and the nano-convex # ¾ has a distribution density in the nano-structure region of more than 1 / μηι ² , preferably more than 10 / μ η ² . For chips containing active nanostructures, please refer to our other invention (PCT International Patent Application PCT / CN2004 / 000437), the contents of which are incorporated herein by reference.

Through Example 3, we found that the broadband nano-chamber reactor containing active nanostructures of the present invention, The combination of meso-nanostructures and moisturizing sulphur produces new properties (characterized by spiritual pulp), which are different from the properties of nanostructures and wetting phenomena, and even exceed the independent additive properties of nanostructures and wetting phenomena. One possible reason is that the combination of the wetting phenomenon and the nano-phenomenon has created a new phenomenon that the two are not only independent of each other, but also strengthen each other, so that the reactor has new characteristics (such as suction capacity), New functions (such as spirit. In the present invention, the nanostructure includes a structure containing one or more of the following nanostructures: silicon oxide, titanium oxide, alumina powder, and their respective derivative powders.

 In the chip according to the present invention, the isolation structure includes a reversible closed isolation structure. In the present invention, the isolation structure refers to a structure in the reactor that prevents the reaction medium from leaking or leaking. In the present invention, the reversible closed isolation structure means that the reaction medium is closed when it is necessary to prevent the reaction medium from leaking or leaking, and it is open when it is not necessary to prevent the reaction medium from leaking or leaking (for example, during scanning). Isolation structure. The reversible closed isolation structure forms or participates in forming the cavity wall. In one embodiment according to the present invention, a chip containing the reversible enclosed isolation structure is provided.

 The reversible closed isolation structure is one or more of the following structures: a reversible adhesive structure, a mechanical isolation structure, and a physical-chemical isolation structure. In the present invention, the reversible bonding refers to bonding with a reversible adhesive; the mechanical isolation refers to using a mechanical seal structure (for example, a seal coating, a seal sheet, a seal, etc.) on a top surface element and a bottom surface element. Pads, sealing linings, etc.); the physicochemical isolation refers to the isolation using the physical and chemical properties of the material (such as resistance to wetting). In particular, the reversible closed isolation structure of the present invention may be a combination of the above-mentioned isolation structures, such as a mechanical seal structure on the top element and a physical-chemical isolation seal structure on the bottom element, for example, in addition to the isolation structure as a chamber wall 1. High water-absorbing material structures on the top surface element and bottom surface element, air isolation area, hydrophobic material isolation area, pressure balance structure, etc.

The reversible adhesive structure includes a reversible adhesive glue layer; the mechanical isolation structure includes an elastic structure of a polymer material; and the physical-chemical isolation structure includes a wetting-resistant structure. The elastic structure of the polymer material includes a polymer material coating or sheet or tape containing a Shore hardness of 20-100 degrees; and the wetting-resistant structure includes a high hydrophobicity including a static water contact angle greater than 80 degrees, preferably greater than 100 degrees. Material coating or sheet or tape. The polymer materials with a Shore hardness of 20-100 degrees include one or more of the following polymer combinations: natural rubber and its derivatives; polyestrogens, such as nitrile rubber, butyl rubber, fluorine rubber, silicon rubber, Fluorosilicone rubber, etc .; various organic polymers, such as silicone polymers, fluoropolymers, polycarbonates, plastics. The highly hydrophobic material includes silicone, a fluoropolymer, and a hydrophobic nanomaterial.

 In the chip according to the present invention, the leakage element and the bottom surface element contain a reaction medium leakage monitoring structure. The leak monitoring structure includes a sealed fine-grain leak observation area.

 In the chip according to the present invention, the user isolation structure may further include a non-closeable isolation structure. The legs must not be closed. The closed-type isolation structure takes shape. When necessary, the tree JF of the Mil part or the ^ P M element ^ ^ surface element can be put (for example, vacuum suction of a partial cover ¾ t) to form an open ^ empty room. In one embodiment of the present invention, the chip containing the irreversible closed isolation structure is provided. Household M irreversible isolation structures include irreversible bonding.

 At this time, the thickness of the part of the wide-beam chamber reactor in the household element and the Li element is less than 0.5 mm, less than or equal to 0.15 mm, and the detection rate is greater than 90%. In the example of the present invention, an example of this component is a cover ¾it or a slide glass with an original height of 0.15mm.

 The broadband chamber reactor is preferably a reactor suitable for scanning and detecting a reaction result with a fluorescence scanner. In the examples, we were surprised to find that, contrary to the expectations of the relevant materials, when the cover glass of the top element is thin enough (^ 0.15mm), the chip of the present invention can be directly used in a light-concentrating fluorescence scanner without the need for There were no significant differences in the results obtained with or without the coverslip.

 In one embodiment of the chip according to the present invention, the user's liquid structure and the liquid discharge structure may include a square isolation structure. The open-cut concrete isolation structure includes sparse ice convex bodies, high sparse ice convex bodies and ¾7j convex bodies with a height of less than 1000 u rn and a crane less than 500 μm, and the ¾¾ liquid structure ^ and the effluent structure may contain a reactive medium The reaction medium is aspirated by mouth to aid flow.

In the chip according to the present invention, there are two or more ordered sub-arrays in the M »array, one of which contains one or more 酉 5¾», and the other sub-array contains one or A variety of accessories that fit the household's legs †. Households and ligands include legs and antibodies. Another solution is to connect two or more fiber devices in series, for example, a chip formed by connecting a broadband chamber capillary tube and a capillary reactor. In the embodiment, an example of ¾ kinds of capillary chips is given, in which at least one reactor is fixed with a leg, and the other reactor is fixed with an antibody corresponding to the antigen, with and with the immobilized antigen in between. And antibody ^ / and ¾¾ body structure. The probe is selected from a biological substance, and the biological substance is a substance selected from one or two or more of any combination of the following groups: an antigen, an antibody, a ligand, and a ligand index. Adaptation molecules, peptides, and single- or multi-stranded DNA, nucleotides, polynucleotides, sugars, coenzymes, cofactors, antibiotics, steroids, viruses, and cells screened by enhanced fibrillation technology.

 In the chip according to the present invention, other structures include components.

Sfcto is closed to the sub-reactor structure when the sample is loaded, and cannot be removed when the sample is loaded. The element contains one or more of the following: a film, sheet, metal-organic composite film, sheet, ¾J †.

 Preferably, the protection element and the reactor are connected by one or more of the following reversible or irreversible sealing structures: a heat-sealed structure, a chemically sealed structure, and a reversible or irreversible adhesive layer. More preferably, the protective element is pre-cut to facilitate de-closing.

 According to another aspect of the present invention, there is provided a device including a top surface element, a bottom surface element, or a preparation element of the top surface element or the bottom surface element of the wet chamber chip described above. In the present invention, the device refers to a device that can be used independently or a reaction or detection device, or a part of the reaction or detection device (eg, a component forming the chip).

 In a preferred embodiment, the device of the present invention comprises at least one of the bottom or top surfaces of the broadband chamber reactor described above and a partially or fully enclosed structure, and optionally contains more than one of said plurality of said probe arrays.

 According to still another aspect of the present invention, a chip kit is provided, which contains the above-mentioned wet chamber chip.

According to a preferred embodiment of the present invention, the probes on the chip include nanostructure active regions on the bottom surface and the top surface, and the nanostructure active regions include a conventional solid-phase carrier and active nanometers fixed thereon Structure, the active nanostructure comprises a nanostructure unit and a probe fixed on the nanostructure unit, the nanostructure unit includes a protruding height greater than 3 nm, and a protruding half-height at least one-dimensional dimension of 1-500 nm, preferably 1 — Nano-convex bodies between 100 nm, and the distribution density of the nano-convex bodies in the nano-structure region is greater than 1 / μm ² , preferably greater than 10 / um ² ; and the kit further comprises Labeling system for matrix / nanoparticle / molecular labeling substance complex. In the present invention, the ligand / nanoparticle / molecular labeling substance complex refers to a label containing a nanoparticle. An invention (application number PCT / CN2003437, the contents of which are incorporated herein by reference).

 According to the kit, the crane is a weak target signal-the background ratio is less than 0.80, the crane is less than 0.50, and more preferably less than 0.25. In the present invention, the term "weak target signal-background ratio" refers to the ratio c = al / b of the signal strength al of the weak target to the signal strength b of the background. In our other invention (PCT / CN2004 / 000713), we awakened the cage to make the chip weak target signal-background ratio is less than 0.80, «less than 0.50, more crane less than 0.25, which can improve the detection spirit«. Due to the prolonged flow of samples in the reactor of the present invention, which can increase background noise, we have found that this method is particularly meaningful for the chip of the present invention. The contents of this document are incorporated herein by reference.

According to another aspect of the present invention, there is also provided a qualitative analysis method and a quantitative analysis method, which include adding a sample with or without a labeled reaction to a humidified chamber chip according to the present invention, and passing the The labeled target reacts with the probe. The reaction between the target M and the probe is performed at a linear speed of 0.25-2.5 ml / min / mm ² .

 The basic advantages of the chip of the present invention are: it combines the advantages of various chips described in the background art such as a closed chamber chip and a capillary chip, and has both continuous operability and reliability of uniform distribution of the reaction medium, so that The reaction efficiency is increased, and in particular, the sensitivity is improved. Under the condition that a reversible isolation structure is used, the cleaning is convenient and efficient. Under the condition of using nanostructures or / and small weak target signal-background ratio design, its sensitivity is higher. In short, the chip of the present invention is simple to manufacture, has high sensitivity, consumes less reaction medium, is convenient to operate, and shortens the detection time. Examples

 In this embodiment, ¾i ÷ are display slides (hereinafter referred to as i¾ †, dimensions 75 X25X 1.0 mm) and covers ¾j † (dimensions 60 X 24 X 0.15 mm) purchased from ESCO SCIENTIFIC, USA. The substrate in this embodiment is an epoxy-based glass slide (American TeleChem International, Inc.). The weights in this embodiment are HIV antigen and HCV (Beijing People's Medical Center for Dry Diseases Research), and their point suspicions are between 1.0-1.5 mgml.

In this example, sample No. 1 is HCV antibody-positive serum, sample No. 2 is HIV _{1 + 2} antibody-positive Λfree · qing, sample No. 3 is HBs antibody-positive AifiL clear, and sample No. 4 is HBs antigen A-free clear, No. 5 Like The product is a negative control. All samples were determined in advance using the classic single-reactor open chip under the same reaction conditions. Example 1

Preparation of a wettable chamber chip with a closed structure ^ i ^ ffl

 1) Preparation of the bottom element

 The bottom surface element in this embodiment includes the preparation of the bottom surface element for preparing the single-reactor and multi-reactor wet chamber chip of the present invention. The bottom element includes a probe board (probe + substrate) containing only the probe array, and a ten-plate (橼 十 + 片 ¾ ^ isolation structure) containing a reactor isolation structure. In this embodiment, the hanging board containing only the »array is prepared by spot-cutting the substrate in a conventional manner. In this embodiment, the preparation method of the bottom surface element of the single-chip and multi-reaction chip is consistent, so there are many methods for preparing the bottom surface element of the chamber chip.

(1) The bottom surface element of the sealing structure combined with an elastic material

 As shown in Figure 1 f ^, the surface element 1 ¾§ includes the bottom surface of the liquid adding area 2 and the bottom surface of the liquid discharging area 3

5 base material), »4, elastic material isolation structure 7, cavity clock formed by elastic material isolation structure 6.

 The preparation of the surface element 1 is based on the substrate ± ffl elastic material solution (self-drying silicon earning solution, Chengdu Chenguang to! Research Institute). The 8 bottom surfaces of the fixed probe are reserved (the bottom surface length X width is 12 mm X4 Bandages other than the bandit are evenly coated, and after they are dried overnight, an elastic material isolation structure layer (thickness less than 0.1 mm) is formed. Then, use the spotting machine (GM 417 ARRAYER, GENETIC MICROSYSTEMS) to make The form of the 3 points of the ligand is within the ij ± ¾ reserved area to form a 2X3 »array. After coating reaction for 3 hours at room temperature, it was blocked with calf serum, washed and dried for later use. All surface elements are designated Al.

(2) The bottom element of the sealed structure combined with high-sparse LZW materials

The preparation method is the same as that of the ± ¾ (1) household »¾ face element. Gai ^ J ^ cai (high sparse ice organic silicon solution, Chengdu Chenguang Research Institute) layer thickness is less than 0.06. And then coated with ± ¾ 酉 5 »legs, The method is the same as (the bottom surface element obtained by Do

(3) Bottom element with high willow elastic material beach structure

For example, the bottom surface element 1 includes a bottom surface (base area), a punch, an elastic material isolation structure, a high-quality material isolation structure, and a cavity formed by the isolation structure. The first method is to use a highly ice-repellent material ( Silicone high sparse ice coating, Chengdu Chenguang tt Research Institute) will paint a fixed border of 8 bottom surfaces (each bottom length X width 12 mm X 4 mm) with a width of about 1 band and a thickness of about 0.05 mm. Line to form a high-shed structure, and then use the bomb bucket solution after self-drying (self-drying silicon solution, Chengdu Chenguang Research Institute)

After drying, an elastic material isolation structure layer (thickness of about 0.1 mm) is formed. Then, a ¾ 酉 β¾ί§ίM prototype (GM 417ARRAYER, GENETIC MICROSYSTEMS) is clicked into the reserved area in the form of 3 points of 酉 SS, forming 2X3 »Array. After being coated at room temperature for 3 hours, it was blocked with calf serum, cleaned and used for later. All «face elements are denoted as A4.

 (4) Bottom element combined with visible sealing structure

 On the base, using reversible adhesive material (water-insoluble, ethanol-soluble double-sided adhesive with Teflon matrix, Chengdu Chenguang ^ X Research Institute) will reserve 8 areas of fixed 酉 5S (as shown in Figure 1A hemp) , The area is 4 pixels wide) and the other areas are uniformly bonded (layer j ^, drawn at 0.2). The coating method is the same as (1). The surface element is called A5o

(5) Broadband reverse / SI air-slave surface element without sealing structure

 Directly form 8 2X3 «columns on the film base, the coating method is the same as (1). All the crane face elements are denoted as A6. 2) Preparation of the top element

The 11® element in this example includes the preparation of a surface element for preparing the single-reactor and multi-reaction sensible chamber chip of the present invention. Lai components include GU components without and with ± ¾ reactor isolation structure. In the present example, the preparation method of the Iffi element of a single device and a multi-reaction sensible wetting chamber chip, It is consistent, so only the method for preparing the surface element of the multi-reaction calendar chip is listed.

(1) Top element without isolation structure

 The top surface element without the isolation structure in this embodiment includes a top surface (corresponding to a bottom surface), a liquid inlet, a liquid outlet, a liquid inlet structure (a liquid inlet pipe), a liquid outlet structure (a liquid outlet pipe), and a positioning structure.

 The above-mentioned top surface element is prepared by a machining method. This top surface element is a stainless steel plate with dimensions of 100 mm X 40 mm X 2mm (length X width X thickness), which can be reused. The liquid inlet structure is a liquid inlet pipe, and the liquid outlet structure is a liquid outlet pipe, which is easy to communicate with other systems (such as flow

Pipe connection. The surface in contact with the bottom surface element is a flat surface. It has no foreign sealed structure. Each pair of liquid inlet and outlet ports corresponds to the liquid inlet and outlet areas of each reaction cell on the bottom element. The obtained top element is denoted Bl.

(2) Top surface element with isolation structure

 As shown in FIG. 2A and FIG. 2B, the Lai element 8 includes Lai 19 (corresponding to the bottom surface), a wide-band cavity port 10, a wide-band cavity outlet 9, a liquid inlet structure 13, a liquid outlet structure 14, a positioning structure 12, and a reactor. Isolation structure 11.

 The preparation of ± ¾ facet components is first made by a mouth method to produce a stainless steel plate with dimensions of 100 mmX40 mm X 2 mm (length X width X thickness). The liquid inlet structure is the liquid inlet pipe, and the liquid outlet structure is the liquid outlet pipe, which is easy to connect with the pipes of other systems (such as Liu Yunyu of the Office). Then, the bottom of the top surface and the corresponding position of the reactor isolation structure on the top of the element is coated with a high sparse material (organic silicone high i ice coating, Chengdu Chenguang tt Research Institute) coating (thickness less than 0.05 mm), or elastic Material solution (self-drying silicon solution, Chengdu Chenguang Research Institute) coating (thickness less than 0.08 mm). Each pair of liquid inlets and outlets corresponds to the liquid inlet and outlet areas of each reaction cell on the bottom element. In short, the isolation structures on the bottom surface element and the top surface element must correspond to each other. The prepared top element with the isolation structure is designated B2.

3) Top element with reaction medium leakage monitoring structure

This top surface component is a 100 mmX40 mmX2 mm (length X width X thickness) Reused glass plates include a liquid inlet, a liquid outlet, inlet and outlet pipes, a positioning structure, and a reaction medium leakage monitoring structure. The surface that is in contact with the bottom surface element is flat and has no sealing structure. Through the glass plate, it can be observed whether the reaction medium in the reactor has leaked. The beautiful element with the closed reaction medium leakage monitoring structure prepared in this example is referred to as B3.

 This top surface element can also be a 100 mmX 40 mmX 2mm (length X width X thickness), reusable liquid inlet, liquid outlet, inlet and outlet pipe, positioning structure, and reaction medium leakage Stainless steel plate for monitoring port. The leak detection opening is 2 mm away from the boundary of the reaction boundary (this is for sealing by the isolation structure), the width is 2 mm, and the length is equal to the reactor length. The leakage monitoring port can be used to observe whether the reaction medium in the reactor is leaking. Observation can be visual inspection or instrument monitoring. The rain element with the open reaction medium leakage monitoring structure prepared in this example is denoted as B4.

 3) Preparation of Wet Chamber Chip

 In this example, the preparation of the wet chamber chip, the force of the top and bottom elements of the chip (获得 clamp pressure) obtained in ¾ 1 1) and 2) may come from the connection (Table 2).

 Lin, magnetic force and other mechanisms can also be used to prepare the wet chamber chip of this example.

4) Feature inspection of humidified chamber chip

 The wet chamber chip prepared by the preparation method in this example has a cavity bottom width of 2-7 mm and a cavity wall height of 0.05-0.3 m. The cross-section of the broadband chamber reactor is rectangular, and the width ratio of the rectangle is greater than 5 . The time required for the deionized water to fill the broadband chamber by the wetting phenomenon in the horizontal direction is less than 1 second, or even less than 0.5 seconds.

In this implementation, the measurement of the absorption capacity improvement rate of the wet chamber chip is performed by detecting the adsorption capacity of the reactors comparing the open non-flow chip (static suction) and the wet chamber chip (dynamic suction) respectively. Adsorption capacity improvement rate = (humidity chamber chip reactor adsorption capacity / control open non-flow chip adsorption capacity) X 100%] o The measurement of adsorption capacity is performed according to a known method, and it is adsorbed at room temperature for 30 minutes at static absorption. Dynamic aspiration is performed at a flow rate of 1 ml / hour at room temperature. According to the following 4) method of use, the target substance (such as HCV antibody) in the appropriately diluted positive serum is sucked on the chip probe and then rhodamine anti-antibody (using Molecular Probes F-6163 standard) The signal obtained after labeling with IgG) is ¾, which is the anti-absorption capacity. In the humidified chamber chip prepared by the preparation method in this example, the improvement rates of the adsorption capacity were all greater than 200%, some were more than 300%, and more than 400%. 5) Shed for Wet Chamber Chips

 During the experiment, 4 samples (HCV antibody positive serum, HIV antibody positive serum, positive control, and negative control) were added to the above-mentioned humidified chamber chip (Table 2), and each sample was added with 2 reaction cells. The sample was appropriately diluted when loading.

 There are two ways to add samples in this embodiment:

 (1) Batch addition: During the operation, the sample is added to the addition tank and stopped after it reaches the outlet. Due to the capillary effect, the sample automatically fills the entire broadband reaction chamber from the inlet to the outlet, and no air bubbles are observed. Place the chip in the incubator, the reaction temperature is 37 ° C, and the reaction time is 30 minutes. After the reaction is completed, the sample is blotted with paper through the liquid outlet or mechanically withdrawn.

 (2) Continuous sample loading: The sample is heated to 37 ° C during operation, and the sample is added to the reactor at a flow rate of 1 ml / hour using a micro pump. The loading time was 60 minutes and the reaction temperature was room temperature.

 The washing solution can be added in a batch or continuous manner, and the total amount is 100 μ1. After washing, the chip can be turned on and only its probe card can be operated.

 The label was rhodamine-labeled goat anti-human secondary antibody (Jackson ImmunoRresearch Laboratories). In order to reduce the amount of the label as much as possible, this embodiment uses batch-type addition. The amount of each reactor is about 5μ1, the reaction temperature is 37 ° C, and the reaction time is 30 minutes. Wash after labeling reaction. After drying, the laser confocal microscopy was used to detect the results (Afymettix GMS 418 chip scanner). The results obtained after data processing are shown in Table 2.

As a control, the open-type movable chip is a chip prepared by using the same substrate, the same probe, and the same spot preparation method. The deletion of the cake is the same as that of the conventional batch reaction cake. Table 2

 + For positive result

One is negative result Example 2

 —Preparation of a Wet Chamber Chip with a Non-flareable Structure ^ S ^ ffl

 The substrate and two probes (HCV antigen and HIV antigen) in this example are the same as in Example 1.

1) Preparation of the top element

The β element in this embodiment includes the preparation of the Iβ element of the single-reactor and the multi-sensing chamber chip of the present invention. 1M yuan ^ Includes components that do not contain and contain and discharge structure. In this embodiment, the method for preparing the surface elements of the single reactor and the multi-sensing chamber chip is the same. Therefore, the method for preparing the multi-reaction chamber chip of the multi-reaction chamber chip is used. (1) Top surface element without inlet and outlet structure

 As shown in Figure 3, 3M element 8 is a cover with a width of 12.5 (2) a top element with a liquid inlet and a liquid outlet structure

 As shown in FIG. 4B, the surface element 8 includes a surface 19 (corresponding to the bottom surface), a liquid inlet 10, a liquid outlet 9, a liquid inlet structure 13, and a liquid outlet structure 14.

 Preparation of Lai element, made of translucent miscellaneous vinyl, size 750mm X 250 mmX 0.5 mm (length X width X thickness). The liquid inlet structure is a liquid inlet pipe (2mm in height) and the liquid outlet structure is a liquid outlet pipe (2mm in height), which is easy to connect with the pipes of other systems (such as flow machinery). Each pair of liquid inlet and outlet ports corresponds to the liquid inlet and outlet areas of each reaction cell on the bottom element. In short, the structures on the bottom element and the top element must correspond to each other.

2) Preparation method of bottom surface element

 The bottom surface element in this embodiment includes the preparation of the bottom surface element for preparing the single-reactor and multi-reactor wet chamber chip of the present invention. Bottom face elements include bottom face elements that are free of and contain liquid and discharge structures. In this embodiment, the preparation method of the single-reactor and the bottom-surface elements of the multi-chamber chamber chip is consistent. (1) Bottom element with isolation structure for inlet and outlet

 As shown in FIG. 3, the bottom surface element 1 includes a bottom surface 5 (base area), a probe 4, a reactor isolation structure (hydrophobic zone) 11, a reactor liquid inlet structure 13, a reactor liquid outlet structure 14, and a reactor inlet The liquid structure isolation structure 15, the reactor liquid discharge structure isolation structure 16, the reactor liquid inlet pool 17 and the reactor liquid outlet pool 18.

The preparation of the Jl ^ i¾ surface element is to apply high-quality materials (organic silicon high-xian coating, Chengdu Chengguang t) to the health of the area other than the substrate ± / ¾ surface (length X width 12.5 to X 3 mm). Research Institute) Shaped K-coated male fluid and fluid isolation structure coatings (thickness less than 0.05). The method of applying a high-glazing coating insulation structure can refer to our other PCT national application Please (PCT / CN2004 / 000169). Then, a HCV and HIV fusion antigen melting point prototype (GM417ARRAYE, GENETIC MICROSYSTEMS) was spotted in the form of 3 points of each 酉 SS into the ± ^ plane to form a 2X3 橼 10 array. After coating reaction at 37 ° C for 3 hours, it was blocked with calf serum, washed and dried for later use.

 The highly hydrophobic liquid materials used can be "polyacrylic paint" (provided by Chengdu Chenguang Chemical Design Institute, China, static water contact 85 degrees), "organic silicon waterproof coating" (provided by Chengdu Chenguang Chemical Design Institute, China, static water contact) 116 degrees), "Super High Hydrophobic Latex Paint" (provided by Chengdu Chenguang Chemical Design Institute of China, static water contact 123 degrees) and "High Hydrophobic Silicon Oxide Coating" (provided by China Zhoushan Mingri Nano Materials Co., Ltd., static water contact 151 degrees) ), The highly hydrophobic solid materials used are "polytetrafluoroethylene self-adhesive tape" (provided by Chengdu Chenguang Chemical Design Institute, China, static water contact 117 degrees) and "nano textile" (provided by China Zhoushan Mingri Nanomaterials Company, static) Water contact: 155 degrees).

(2) Bottom element without liquid inlet and outlet isolation structure

 As shown in FIG. 4A, the bottom surface element 1 includes a bottom surface 5 (moving area ship, liquid discharge area) and a scale 4, and the scale exists in the form of one or more scales.

 The preparation of the surface element is made by spotting the substrate in accordance with known methods.

3) Preparation of single-sided chip for broadband cavity

 The single-sided chip of the broadband anti-cavity chamber without the element prepared in this example is denoted as A1, and the single-sided chip of the wide-band ventricle with ventilator element is denoted as A2.

 Preparation of Wet Chamber Chips: Prepared by ± ¾: β element and bottom element ¾ ^ glue method. The adhesive used is a binary redox resin (wan ^ ¾ mixture, Chengdu Chenguang Chemical Research Institute). Apply the adhesive to the component containing the substrate according to the OT instruction manual, and then attach another component.

An important but often ignored problem in the application is: If only m reactors are deleted in an experiment, and there are π reactors (n> m) on the chip, how can we provide another n-m reactors for Future experiments ^ ffl. The solution to this problem is a necessary part of the measured high-density device chip. For a single reactor chip, the current reactor system It is a plastic box containing a chip holder (lattice). It is very unfortunate that for multi-reactor chips, there is currently no dedicated iW> structure placed on a reactor chip, which is a single-reactor chip system. For the manufacture of the single-sided chip of the broadband reaction chamber with a protection element of the present invention, please refer to our other

The PCT application (PCT / CN2004 / 000169), the liquid and liquid discharge structures in this embodiment. The liquid and liquid discharge structures are closed by the Aohu element, and when the sample is added.

 The single-sided chip of the broadband cavity formed by bonding the top surface element without the liquid inlet and outlet structures described above to the bottom surface component containing the liquid inlet and outlet structures is referred to as Al 1 and A21. The broadband single-sided chip formed by bonding the above-mentioned top surface element with a liquid inlet or / and liquid outlet structure to the bottom surface component not containing the liquid inlet and liquid outlet structures is denoted as A12 and A22.

4) Preparation of ¾-sided chip for broadband cavity

 The wide-band cavity without the cake element prepared in this example is also referred to as A3, and the single-sided chip of the broadband chamber with AW elements is referred to as A4.

 The preparation of the surface element and the bottom element used is the same as 1) and 2). The difference is that both the top and bottom surfaces will be ft ¾ t, and the broadband cavity] M and the bottom surface are fixed »h will be ¾ 酉 ^ ∞ ¾ point prototype (GM417ARRAYER, GENETIC MICROSYSTEMS) on the corresponding components of the component and the bottom surface In the same form, two points each of ± iHCV and HIV were taken to form a 2X2 rattan square, and the coating mouth was closed and cleaned. The combination of the used element and the bottom element and the formation of the liquid inlet and outlet are the same as the above 3). Fabrication of a double-sided chip for a broadband reaction chamber with a protective element, same as ± ¾3).

 The broadband cavity double-sided chip formed by bonding the top surface element without the liquid inlet and outlet structures described above to the bottom surface component containing the liquid inlet and outlet structures is denoted as A31 and A41. The broadband cavity double-sided chip formed by bonding the top surface element with the liquid inlet and outlet structures described above to the bottom surface component that does not contain the liquid inlet and / or liquid outlet structures is denoted as A32 and A42.

 Some examples are shown in Figure 2-4.

5) Feature inspection of broadband anti-aircraft chamber chip

Three characteristics of the width of the anti-cavity chamber and the adsorption capacity of the wet chamber The detection method is the same as the first example.

 In this embodiment, the chamber chip is prepared, and the broadband reaction chamber] M and the bottom surface are 4 mm X 12.5 mm (width X height), the distance between the bottom and the bottom surface is less than 0.08, and the static water on the surface is ¾j † The worm angle is 44 degrees, and the time required to fill the wide-band cavity of the legs with the deionized 7-wet ¾ »7 plane is less than 1 second, or even less than 0.5 seconds.

 With the humidified chamber chip prepared by the preparation method in this example, the adsorption capacity of the wide-band chamber reactor is improved by more than 200%, some more than 300%, and more than 400%.

6) Wet chamber chip shed

 During the experiment, four samples (HCV antibody positive serum, HIV antibody positive serum, positive control, and negative control) were added to the above-mentioned humidified chamber chip (Table 3), and two reaction cells were added to each sample. The sample was appropriately diluted when loading.

 There are two ways to add samples:

 (1) Batch addition: He I "uses the above-prepared wet chamber chip containing the bottom element of the liquid inlet and outlet structures, and the operation method is the same as that of the first embodiment.

 (2) Continuous sample addition: The above-mentioned wet chamber chip without the bottom-surface components containing the liquid inlet and outlet structures is used. The operation method is the same as that of the first embodiment.

 After the reaction is completed, the sample is blotted with paper through the liquid discharge tank or drawn out with a pipette. The washing solution can be added in batch or continuous mode, and the total amount is 20 / d.

 After washing, you can carry out »self and batch cleaning (by blotting the paper or extracting it with a pipette), and you can also wash it in the Nagisa. Broadband month The single-sided chip can be vacuum-sucked with a vacuum grapefruit sucker to suck off the top surface element, add it after cleaning, and clean after reaction. The marker was rhodamine-labeled goat anti-human secondary antibody (Jackson ImmunoResearch Laboratories). If you want to reduce the amount of markers as much as possible, it is advisable to add in batches, the amount is 3μ1.

Other is the same as in Example 1. The results obtained after data processing are shown in Table 3. table 3

 "+" Is a positive result, "-" is a negative result

 * And ** are the results of 10-fold and 30-fold dilutions of the samples, respectively Example 3

 — Preparation of a Wet Chamber Chip with a Nanostructured Active Region and a Chip Kit ^ ffl

 In this embodiment, a method for preparing a humidified chamber chip containing a nanostructure active region is to first prepare a top surface element and / or a bottom surface element including the nanostructure active region.

The method for preparing the top-side element ^ / and the bottom-side element including the substrate is the same as the method for preparing the top-side element ^ / and the bottom-surface element including the substrate in Examples 1 and 2. Then, according to the method for preparing a nanostructured active carrier in another PCT international patent application (PCT / CN2004 / 000437), the active nanoparticles containing HIV antigen and HCV antigen are respectively fixed on the top surface element and the bottom surface element. Pieces on the base. In this example, the nanoparticles are silicon oxide particles (Straight ¾-20-40nm, Sigma-alderich). The method for identifying the characteristics of the nanostructure active region is the same as the method for identifying the characteristics in another PCT international patent application (PCT / CN / 2004/000437). In this example, the measurement of the nano-structure unit and its distribution, the nano-convex and its height, the minimum size at the half-height, and its distribution density were all measured using a SPA-300HV scanning probe microscope (DFM) and an electron scanning microscope. The nanostructured units on the nanostructure active region in the top surface element ¾ and the bottom surface element prepared in this example (the protruding height is greater than 3 nm, and the protruding half height is at least one-dimensional in the range of 1-500 nm, preferably 1- The distribution of nano-convex bodies between 100 nm is greater than 10 / μηι ² .

 In this embodiment, a method for preparing a humidified chamber chip containing a nano-structured active region by using a top surface element and a bottom surface element containing a nano-structured active region is the same as the method for preparing a humidified chamber chip in Examples 1 and 2.

 The feature identification method of the nano-structured active region-containing luminous chamber chip prepared in this embodiment is the same as the feature identification method of the humidified chip in Examples 1 and 2. Difficulties are as follows: The size of the top and bottom of the broadband anti-air chamber is 4 mmX 12.5 mm (width X height), and the distance from the bottom is 0.08-0.25 mm. The time required for the cavity surface is less than 1 second, or even less than 0.5 seconds; the adsorption capacity of the broadband chamber reactor is improved by «I greater than foot%, and more than 8000%. The increase rate of the adsorption capacity of the open-type throbbing reactor with the nano-structured active zone was 450%, and the rate of the adsorption of the broadband chamber reactor without the nano-structured active zone was 500%.

 The kit of this embodiment includes a humidified chamber chip containing a nanostructured active region of this example and a label. The hexamethylene compounds are Chang shan ii (such as rhodamine ¾ΗSecondary antibody) and 酉 2¾ nano heterozygous / standard substance complex (such as rhodamine heterozygous heterozygote / secondary antibody complex). The preparation method of the complex of heterozygous weeds / molecular markers is the same as the preparation method of 酉 fig / Na ^ Lizi / ^ T¾H in another PCT international patent application (PCT / CN2004 / 000437).

The control chips in this embodiment are an open non-flowing chip containing a nanostructured active region and a wet chamber chip without a nanostructured active region. The control chip is a chip prepared using the same substrate, the same probe, and the same spot preparation method. Preparation method of open-type non-flowing chip containing nano-structure active area and nano-structure activity in another PCT international patent application (PCT / CN2004 / 000437) The method of preparing the open-type non-flowing chip in the zone is the same. The side cake is the same as above, but the reaction is carried out under well-known batch reaction conditions. The wet chamber chip without the nano-structure active region is the same as the method for preparing the wet chamber chip in Examples 1 and 2. The use method is the same as the use method of the wet chamber chip containing the nano-structure active area.

 The method of the chamber chip and the kit containing the nano-structured active region prepared in this embodiment is the same as the method for the surface of the chamber chip in Qian Examples 1 and 2. The results are shown in Table 4. Table 4

Chicken case 4

 Preparation of wide-band chamber gene chip

 Biological samples used for nucleic acid detection, especially human plasma, are mostly complex mixtures of biomolecules. Existing chips cannot directly add sample reactions, and samples must be amplified before labeling. It is very difficult to specifically amplify. Often, while the target molecule is amplified, other nucleic acid molecules are amplified, which increases false positiveness. Because the wet chamber chip of the present invention uses flow loading, the amount of sample added can be increased by thousands of times compared with a general non-flow chip, and the detection sensitivity is correspondingly increased.

The method for manufacturing the wide-band chamber gene chip of the present invention is the same as the method for manufacturing the humidified chamber chip in Examples 1, 2, and 3. «The production method is to make a humid chamber suitable for continuous addition of the reaction medium How to make a chip. However, in this example, the probes used are the four segments (concentrations of 1 mg ml) of the disclosed HCV conserved regions. The characteristics identification of the wide-band chamber gene chip of the present invention is the same as that in Examples 1, 2, and 3, and the characteristics are similar. 4 regions of the HCV conserved region »are listed as: Sequence 1

 261 GCCTTGGT ACTGCCTGAT

AGGGTGCTTG CGAGTGCCCC

 301 GGGAGGTCTC GTAGACCGTG CACCATGAGC ACGAATCCTA AACCTCAAAG AAAAACCAAA

361 CGTAACACCA ACCGCCGTCC ACAGGACGTC AAGTTCCCGG GCGGTGGTCA GATCGTTGGT

 421 GGAGTTTACC TGTTGCCGCG CAGGGGCCCC AGGTTGGGTG TGCGCGCGCT CAGGAAGACT sequence 2

 4651 GGCTTTACCG

GCGACTTTGA CTCAGTGATC

 4681 GACTGTAACA CATGTGTCAC CCAAACAGTC GATTTCAGCT TGGACCCTAC CTTCACCATT

4741 GAGACGACGA CCGTGCCCA AGACGCAGTG TCGCGCTCGC AACGGCGAGG CAGGACTGGT

 4801 AGGGGCAGGA GAGGCATCTA CAGGTTTGTG ACTCCGGGAG AGCGGCCCTC GGGCATGTTC

4861 GATTCCTCGG TCCTGTGTGA GTGCTATGAC GCGGGCTGTG CTTGGTATGA GCT 4913 Sequence 3

 6117 CACG

 6121 CACTATGTGC CTGAGAGCGA CGCCGCAGCG CGTGTCACTC AGATCCTCTC CAGCCTTACC

6181 ATCACCCAGC TGTTGAAGAG GCTCCACCAG TGGATTAACG AGGACTGCTC CACGCCATGC

 6241 TCCGGTTCGT GGCTAAGGGA TGTTTGGGAC TGGATATGCA CGGTGT 6286 sequence 4

 8424 AAGCGGC GTGCTGACGA CTAGCTGCGG TAATACCCTT

 8461 ACATGTTACT TGAAGGCCTC TGCGGCCTGT CGAGCTGCAA AGCTCCAGGA CTGCACGATG

8521 CTCGTGTGCG GAGACGACCT CGTCGTTATC TGTGAAAGCG CGGGAACCCA AGAGGACGCG

 8581 GCGAGCCTAC GAGTCTTCAC GGAGGCCATG ACTAGGTACT CTGCCCCCCC CGGGGACCCG

 8641 CCCCAACCAG AATACGACCT GGAGCTGATA ACATCATGCT CCTCGAATGT GTCGGTCGCG

8701 CACGATGCAT CCGGCAAGAG AGTATACTAC CTCACCCGTG 8740 The positive serum is the positive serum of the above 4 segments. The samples were deproteinized by boiling denaturation, and the deproteinized samples were labeled with rhodamine (TAMRA), and the hybridization solution was used to prepare a concentration equivalent to 10-fold dilution and 20-fold dilution of the original serum. At 42 ° C, a 3 ml sample was continuously added to the reactor for 3 hours using a micro pump (such as an HPLC pump). Then stop solution was added to stop hybridization, and the washing solution was thoroughly washed. Put the dried chip into Afymetrix Scanned by the company's GMS 418 scanner and analyzed by AGUAR II software, the results are shown in Table 5.

 As a control, the open-type non-flowing chip is a chip prepared by using the same substrate, the same probe and the same spot preparation method. The shed was the same as above, but the reaction was carried out under a well-known batch reaction cake. Table 5 Chip test results

 "+" Is a positive result, "-" is a negative result; Example 5

Preparation of a humidified chamber chip containing two or more ordered sub-arrays ^ iOT

 The manufacturing method of the wet chamber chip in this example is the same as the manufacturing method and feature identification of the wet chamber chip in Examples 1, 2, and 3. The manufacturing method of the crane is a manufacturing method of a humidified chamber chip suitable for continuously adding a reaction medium. The feature identification method of the wet chamber chip in this example is the same as the feature identification method of the wet chamber chip in Examples 1, 2, and 3, and the results are similar.

Only in this example, the probe arrays used are three HBV antigen lines and three HCV antigen lines, respectively. Another solution is shown in Fig. 5, where two sub-probe arrays are connected through a thin channel. In the figure, the bottom bin Two sub-arrays of the probe 4 are fixed on the piece 1, and there are a broadband chamber outlet 9, a broadband chamber inlet 10, and a hydrophobic material isolation structure 11.

 The samples deleted in this example are HBs antigen positive Alfil clear, HBs antigen negative serum and HBs antibody positive human serum. All samples were determined in advance using the classic single-reactor open chip under the same reaction conditions.

 During the experiment, three samples were divided into the chips prepared above.

 The method of using the humidified chamber chip in this example may be the same as the method of using the similarly similar humidified chamber chip in Embodiments 1, 2, and 3, respectively. This example uses the method of continuously adding the reaction medium.

 As a control, the open-type non-flowing chip is a chip prepared by using the same substrate, the same probe and the same spot preparation method. The deletion is the same as above, but the reaction is performed under a well-known batch reaction cake.

 In this example, the measurable dilution multiples of the HBs antigen-positive sera from the humidified chamber chip are all greater than 100 times. Compared with the results obtained by the open non-flow chip of the control (less than 10 times), the sensitivity is increased by 10 times or even more than 20 times. . Example 6

Preparation of a low-weak target signal-background ratio chip kit for a chamber chip ^^

 The low-weak target signal-background ratio chip kit in this example includes a humidified chamber chip and a labeling substance. The probe in this example is the same as the probe of Embodiment 1. The fabrication method of the humidified chamber chip in this example is the same as the fabrication method and characteristic identification of the humidified chamber chip in Examples 1, 2, and 3. A preferred manufacturing method is a manufacturing method of a humidified chamber chip suitable for continuously adding a reaction medium. The method for identifying the characteristics of the wet chamber chip in this example is the same as the method for identifying the characteristics of the wet chamber chip in Examples 1, 2, and 3, and the results are similar.

 In this example, the humidified chamber chip is a low-weak target signal-background ratio chip. The preparation of the background signal enhancement chip in this example is the same as the preparation method of our other PCT international patent application (PCT / CN2004 / 000713) background signal enhancement chip. The labeling substance used in this example is rhodamine. In this example, Rhodamine was used to act on a chip that had been blocked with calf serum to form a background signal enhancement chip.

The method of using the humidified chamber chip in this example can be similar to that in Examples 1, 2, and 3, respectively. The method of using the chamber chip is the same. This example uses the method of continuously adding the reaction medium. The same sample as in Example 1 was diluted to 1/20 to 1/3000, and rhodamine was separately added to the chip kit. After 30 minutes of reaction at room temperature, it was added to the chip reactor. Wash 5 times when done. Scan after drying.

 The scanner is a confocal laser scanner (Afymetrix Corporation GMS 418 or Chengdu Optoelectronics Institute Scan-2). The scanning excitation light wavelength is 532nm, the light wavelength is 570nm, the laser light is ¾¾ and the gain is 60/69. ZoCSoft ImageBoos t (for low-weak target signal-background ratio chip kit) and JAGUARII (for reference chip kit) are processed, and then the average value is obtained to obtain the result. Detection using low-weak target signal-background ratio chip kit, weak target signal-background ratio is less than 0.1, and the negative and positive results are consistent with the sample used.

 As a comparison, the open-type non-flowing chip is a low-weak target signal-background ratio chip prepared by using the same substrate, the same probe and the same spot preparation method. The conditions of use are the same as above, but the reaction is carried out under well-known batch reaction conditions.

 The measurable dilution multiples of the HCV antibody-positive sera and HIV antibody-positive sera of the humidified chamber chip in this example are greater than 3000 and 2000 times, respectively, compared with the results obtained by the open non-flow chip of the control (less than 1000 and 700 times, respectively). ), Its sensitivity improvement is obvious.

Claims

Claim

1. An analysis chip comprising one or more multiple broadband chamber reactors formed by a top surface element, a bottom surface element, a reversible or irreversible reactor isolation structure, and optionally other structures, wherein said reactors Including a broadband chamber having a cavity width greater than 600 μm, preferably greater than 1000 μm, a liquid inlet structure, a liquid outlet structure, and the reactor isolation structure, and A) the wideband cavity includes a liquid inlet and a liquid outlet Chamber wall, top and bottom surfaces and probes fixed on the bottom surface and top surface; B) the cavity width, the height of the chamber wall, and the bottom surface and the static water on the top surface The contact angle is selected as itb ^, so that when the bottom surface ^ and the top surface of the fixed probe are parallel to the horizontal plane, the ion-free water added to the liquid inlet moves by itself to fill the broadband chamber. The time is less than 2 seconds, preferably less than 1 second.

 2. The chip according to claim 1, wherein the static water contact angle of the top and bottom surfaces is 40-80, the cross-section of the broadband cavity is rectangular or a derivative with a rectangle as a boundary, and the The ^ / width ratio of the rectangle is greater than 3, preferably greater than 5.

3. The chip according to claim 1 or 2, wherein the cavity width is 2000-7000 μm, and the cavity wall height is 10-600 μm, ¾ 50-400 m ₀

 4. The chip according to claim 3, wherein the height of the cavity wall is 100-300 μm.

5. The chip according to any one of claims 1 to 4, wherein the improvement rate of the adsorption capacity of the device is greater than 200%, the crane is greater than 300%, and the crane is greater than 400%.

6. The chip according to any one of claims 1 to 5, wherein the probe is included in a nanostructure active region on the bottom surface and the top surface, and the nanostructure active region includes a conventional solid-phase carrier and a substrate thereon. A fixed active nanostructure, the active nanostructure comprising a nanostructure unit and a probe fixed on the nanostructure unit, the nanostructure unit includes a protrusion height greater than 3 nm, and at least a one-dimensional size of the protrusion at a half height of 1-500 nm, preferably between 1 and 100 nm, and the nano-convex has a distribution density in the nano-structure region of more than 1 / μηι ² , preferably more than 10 / μ η ² .

 A chip according to any one of claims 1 to 6, wherein said isolation structure includes a reversibly closed isolation structure.

8. The chip according to claim 7, wherein the household-closeable isolation structure is one or more of the following structures: a reversible adhesive structure, an isolation structure, and a physical-chemical isolation structure.

9. The chip of claim 8, wherein the reversible adhesive structure includes a reversible adhesive layer; the isolation structure includes a highly polar structure; and the physical-chemical isolation structure includes a wetting-resistant structure.

 10. The chip according to claim 9, wherein the household height ^ material elastic structure includes a high material coating ^ 20-100 degrees ^ j † or a belt; the household moisture resistance structure includes a static water moving angle greater than 80 degrees, the boat is more than 100 degrees high! !! / Talent coating or tape.

 11. A fiber chip according to any one of claims 7 to 10, wherein the household surface element and the bottom surface element contain a reaction medium leakage monitoring structure.

 12. The chip according to claim 11, wherein the leakage monitoring structure of the household M includes a sealed or open-type leak observation area.

 13. The chip according to claim 1, wherein the isolation structure comprises an irreversible closed isolation structure.

 14. The chip according to claim 13, wherein the irreversible isolation structure comprises irreversible bonding.

 15. The chip according to claim 13 or 14, wherein the thickness of the part of the wide-legged chamber reactor in the β-element and the β-element is less than 0.5, the female is less than or equal to 0.15, and the collision is detected. The rate is greater than 90%.

 16. The chip according to claim 15, wherein the household broadband chamber is a reactor that scans and detects a reaction result with a fluorescence scanner.

 17. A chip according to one of claims 7-16, wherein the liquid-liquid structure and the liquid-liquid structure of the household include an open structure; an isolation structure.

 18. A chip according to claim 17; M, wherein the ¾ square «isolation structure comprises a hydrophobic convex body and a highly hydrophobic convex body and a water-absorbing convex body having a height of less than 1000 μm, preferably less than 500 μm.

 19. The chip according to any one of claims 1 to 18, wherein the probe array has two or more sub-arrays in order, and one sub-array contains one or more ligand probes , And the other sub-array contains one or more matching deletions corresponding to leg scales.

20. According to Quan He waist, find a chip of one of the legs 1 to 19, wherein the probe is from a biological substance, and the biological substance is a question from one or two or more of any combination of the following groups: Antigens, antibodies, ligands, ligand index-enhanced adaptation molecules, peptides and single- or multi-stranded DNA, nucleotides, polynucleotides, sugars, coenzymes, cofactors, antibiotics, classes Solid Alcohol, virus, and cell.

 21. The chip according to one of claims 1-20, wherein the other structures of the household include {¾ ^ elements, and the household elements close at least part of the reactor structure when the sample is not micro-injected; Partially irreversibly removed.

 22. The chip according to claim 21, wherein the household M element and the household M reactor MM include one or more of the following reversible sealing structures connected: a sealing structure, a chemical sealing structure, and a reversible adhesion concept.

 23. The chip according to claim 21 or 22, wherein the protection element is pre-cut to facilitate sealing.

 24. A device comprising a top surface element, a bottom surface element, or a preparation element for said top surface element or a top surface element of a humidified chamber chip as claimed in any preceding claim.

 25. An apparatus according to claim 24, comprising at least one bottom or top surface of a broadband chamber reactor and part or all of a closed structure, and optionally more than one of said probe arrays.

 26. A chip kit comprising one or more humidified chamber chips according to any one of claims 1-23.

 27. The kit according to claim 26, wherein the humidified chamber chip is the humidified chamber chip of claim 2; and the kit further comprises a compound containing a ligand / molecular compound / molecular labeling substance Tagging system.

 28. The micro-U box according to claim 27, which is a kit of weak target signal-background ratios less than 0.80, cranes less than 0.50, and cranes less than 0.25.

 29. A method for quantitative analysis, which includes adding a sample from a country to a humidified chamber chip according to any one of claims 1-23.

 The ¾¾ target reacts with the ship fiber.

 30. The method according to claim 29, wherein the reaction of TO and household?

It is performed under a cake of 0.25-2.5 ml / hr / mm ² .