CN1249437C - Method and apparatus for bio-molecular chip minute quantity sample application and reaction - Google Patents

Abstract

The invention relates to a method and a device for directly performing micro-sample addition and immediate reaction on a biomolecular chip. The method comprises: under the action of external pressure, the microarray is in close contact with a smooth chip surface to form a sealed cavity. The bioligand molecules that need to be immobilized enter the cavity through the microchannel to react with the surface of the chip, and the reacted ligand molecules are then discharged through the microchannel. The device includes: the elastic material has grooves, the grooves are arranged in an array of micro-arrays, and the micro-channels matched with it; there are liquid inlets and outlets on both sides of the micro-array or micro-channel substrate, and on the micro-channels Covered diaphragm of elastic material. Since the method of the present invention carries out the preparation of the biochip and the immediate detection reaction in the same device, and the area where the ligand biomolecules are immobilized on the chip is strictly limited, the surface after the immobilization and reaction is washed with a buffer to make the surface The immobilization and reaction of biomolecules are uniform and consistent, which effectively improves the quality of detection.

Description

Method and device for micro-sampling and reaction of biomolecular chip

technical field

The invention relates to a method for preparing a biomolecular chip and a device thereof, in particular to a method for directly performing micro-sample addition and immediate reaction on a biomolecular chip and a device for realizing the method.

Background technique

Biomolecular chips are an integrated and parallel biological detection technology developed in recent years, which can integrate a variety of ligands on a tiny geometric scale, so that multiple indicators of trace samples can be detected at the same time. Due to the high price of biomolecular samples, it is required to use as little as possible, so the biomolecular reagents and samples used in biomolecular chips are required to be miniaturized, which also requires the miniaturization of chip loading and reaction devices. At present, a spotting instrument is mainly used for adding samples to biochips. According to the different spotting methods, it can be divided into two types, one is the contact type, first use the sampling needle to dip the ligand to be used, and then place the ligand on the chip by touching the surface of the chip; the other is the spray printing type, first Use a hollow sampling needle to absorb a small amount of ligand to be spotted, and then add the ligand to the surface of the chip by a method similar to an inkjet printer. The common disadvantage of these two methods is that the sample volume is uneven, and the areal density distribution of ligand molecules in a single point is also uneven, which will seriously affect the quality of the detection results. At present, most of the biochip reactions adopt the overall reaction method, that is, the whole chip is immersed in the sample solution to be tested for reaction. This method requires a large amount of sample solution to be tested, a long reaction time, and low sensitivity.

Another chip loading and reaction technology is microfluidic transport and microcavity reactors. At present, the chip of the commonly used microchannel technology is integrated, that is, the chip and the microchannel are made on the same material, such as Document 1: Dielectrophoretic cell separation and gene expression profiling on microelectronic chip arrays. July 15, 2002 Huang Y, Joo S, Duhon M, Heller M, Wallace B, Xu X Anal Chem 2002 Jul 15;74(14):3362-71. The microfluidic channel used in this method is disposable, and the microfluidic channel is complicated to manufacture and high in cost, which limits its application. The simultaneous biomolecule immobilization and detection reactions are carried out twice in two devices, so the preparation process is cumbersome.

Contents of the invention

The purpose of the present invention is in order to overcome the shortcoming of above-mentioned prior art; In order to greatly reduce the manufacture cost of biochip and simplify the technology of biomolecule immobilization and detection reaction, thereby provide a kind of biochip making, and on this biochip Methods and devices for instant direct immobilization and detection reactions of biomolecules.

The purpose of the present invention is achieved like this:

The method for preparing a biochip provided by the invention includes performing the following steps in sequence:

(1) The substrate material of the chip is closely contacted with the microarray template, and the microflow channel is pressed by external force;

(2) Transport the ligand molecule to the selected area on the surface of the chip substrate through the microtube of the microfluidic channel; wait until the ligand molecule is fixed on the chip substrate;

(3) Rinse the chip substrate immobilized with biomolecules prepared in step (2) with a buffer solution, and transport the buffer solution to different areas on the chip surface through the micro-channel; wash off the components that are not fixed on the chip surface base molecule;

(4) by covering one deck elastic material diaphragm again on the elastic diaphragm 13 of band groove 14, and two layers of membrane peripheries are bonded together, have formed microchannel 5 by groove 14, make fixedly equipped with The regions of the ligand molecules are connected in series; or the regions with the ligand molecules are connected in series through the switch formed by squeezing the nodes on the elastic membrane;

(5) Transport the biological sample to be detected to each unit on the surface of the chip prepared in step (4) through the microfluidic channel, and immediately perform the detection reaction;

(6) Remove the chip prepared in step (5) carrying the detection reaction result, and use a chip detector to detect the reaction result.

The concentration of the ligand molecules used in the step (2) is 0.001-1 mg/ml; the flow rate of the ligand molecules in the microchannel is 0.1-100 microliters/minute.

In the step (5), the flow rate of the biological sample through the microchannel is 1-100 microliters/minute.

The chip base material includes: silicon, glass, metal, plastic and other materials or composite materials of the above, preferably silicon.

This method combines microfluidics with microarrays. Microarray refers to micro grooves made on elastic solid materials, the number of grooves in the microarray depends on the detection index, ranging from one to hundreds or more. Microchannels are pipes with tiny inner diameters connected to microgrooves.

Under the action of external pressure, the microarray can be in close contact with the smooth chip surface to form sealed cavities one by one. The bioligand molecules that need to be immobilized can enter the cavity through the microchannel to react with the surface of the chip, and the reacted ligand molecules are then discharged through the microchannel. This enables micro-loading of samples in different areas on the chip surface. Then, the biological sample to be detected also enters each cavity through the microfluidic channel, reacts with the immobilized bioligand molecules on the surface of the chip, and then is discharged through the outlet. Biochips are used to simultaneously detect multiple biomolecules in biological samples to be tested. In the method, a flow control part is designed to make the same biological sample to be tested sequentially react with various bioligand molecules pre-immobilized on the chip, effectively reducing the amount of samples used. Any number of grooves can be connected in series through the flow control section. The immobilization area of ligand molecules on the chip is strictly limited, and the surface after immobilization and reaction is washed with buffer solution, which can make the immobilization and reaction of biomolecules on the surface uniform, and effectively improve the quality of detection. The chip reaction is limited in a small area, and in the flow state, the mass transfer rate of biomolecules is accelerated, the reaction time is effectively shortened, and the sensitivity is improved. After the biomolecule immobilized and reacted chip is separated from the microarray template, a chip detector is used to detect the reaction result. The microarray template separated from the chip material can be reused for multiple immobilization and reaction of biomolecules on the chip.

The special device for the preparation method of the biochip provided by the present invention is different according to the way of forming the micro flow channel, and the device includes: two types.

Describe the first one at first, this device comprises: an elastic material sheet 3 engraved with the first groove 2 on the surface, its first groove 2 is arranged in an array, and the two ends of the first groove 2 are respectively opened with first Through-hole 10; Wherein the through-hole of each groove comprises two micro flow passages one into one out; Also engrave second groove 2 ' on a surface of a piece of rigid solid material block 4, the second groove 2 ' The two ends are respectively provided with second through holes 11, and the grooved side of the elastic material sheet 3 is relatively fixed with the side of the rigid solid material block 4 without grooves; the through hole on the rigid solid material block 4 and the elastic material The through holes on the sheet 3 are staggered and communicated with each other, that is, a through hole of the first groove 2 is used as a solution outlet to communicate with a through hole of the second groove 2' as a solution inlet, and the outlet of the second groove 2' It communicates with the inlet of the next first groove 2; the two sides of the solid material block 4 are respectively provided with an inlet 7 for the liquid to be tested and an outlet 8 for the liquid to be tested, and a switch is installed on the inlet; the chip 1 is in close contact with the elastic material sheet 3 In the through hole of the solid material block 4, a tube 12 with a similar diameter is inserted, and the micro flow channel is formed by connecting the tube 12 with the through hole on the microarray template.

Under the action of external pressure, the grooves on the microarray template are in close contact with the smooth chip surface to form sealed cavities one by one. The ligand molecules that need to be immobilized can be driven by the pump and enter the cavity through the microchannel to react with the surface of the chip, and the reacted sample is then discharged through the microchannel. This enables micro-loading of samples on different areas on the chip. The chip after adding the sample does not need to be removed from the device, and the detection reaction can be carried out directly. Remove the tiny tubes from the microarray, use an elastic membrane to close the groove 2', and connect the grooves 2 in series, leaving an inlet and an outlet. The biological samples to be detected enter each cavity sequentially through the inlet, react with the immobilized ligand molecules on the surface of the chip, and then discharge through the outlet. After the reaction, the chip can be removed from the device, and the reaction result of ligand and receptor on the chip can be detected by a detector.

It also includes an elastic membrane, and an inlet and an outlet are respectively opened on both sides of the solid material block, or an inlet is opened on one side of the solid material block, and an outlet is opened on one side of the elastic material block; One switch; the chip after adding the sample does not need to be removed from the device, and the detection reaction can be carried out directly. Remove the tiny tubes from the microarray, use an elastic membrane to cover the solid material block, close the first groove 2, connect the grooves on the elastic material sheet in series, and open a hole at the elastic membrane opposite to the solution inlet , as the detection solution inlet, and the hole opened on the side of the solid material block as the detection solution outlet. The biological sample to be detected enters each cavity sequentially through the solution inlet, reacts with the fixed biological sample on the surface of the chip, and then is discharged through the solution outlet. After the reaction, the chip can be removed from the device, and the result can be detected by the detector.

The second structure of the device for directly preparing a biochip and immediately performing a detection reaction of the present invention includes: a microfluidic channel 5, and a microarray template matched therewith; wherein the microarray template includes: one surface is engraved with at least 2 The elastic material sheet 3 of the first groove 2, the elastic material sheet 3 has the face of the first groove 2 facing up, the two ends of each first groove 2 respectively open the first through hole 10, the other of the elastic material sheet 3 One side is in close contact with the surface of the chip 1; the microfluidic channel includes: an elastic material diaphragm 13, on which an elastic material diaphragm 13 is formed with a concave groove 14, and a port of the concave groove 14 is corresponding to the first microarray on the microarray. The position of the first through hole 10 of a groove 2 is set, and the other port communicates with the outside world; the first through hole 10 of each groove corresponds to a groove 14; and the corresponding microarray on the elastic material membrane 13 The film at the through hole of the elastic material diaphragm 13 is opened to form a hole, and a layer of elastic material film is covered on the elastic material diaphragm 13 with groove 14 again, and the peripheries of the two layers of films are bonded together to form a groove 14 The micro-channels 5 are connected to each other, and at the node positions, the first switch 15 and the second switch 17 are formed by extruding with external force.

Also comprises a rigid solid material block 4, this solid material block 4 is fixed on the surface of the first groove 2 of the elastic material diaphragm 3, the first through hole opened at the two ends of the first groove 2 of the elastic material diaphragm 3 10 runs through the rigid solid material block 4, and the elastic material film 16 without grooves is bonded to the upper surface of the solid material block 4 of the microarray.

Under the action of external pressure, the grooves on the microarray can be in close contact with the smooth chip surface to form sealed cavities one by one. When adding samples, the channel between the micro-channels is closed by external force extrusion, that is, the second switch is closed. Each groove has independent flow passages in and out. Driven by the pump, different ligand protein solutions enter the cavity and react with the surface of the chip, thereby immobilizing on the surface. Then wash with a buffer to remove proteins that are not immobilized on the surface. In this way, the purpose of adding micro-sample in different regions on the chip is realized. When the detection reaction is performed, the second switch between the micro-channels is turned on, and the first switch on the micro-channel is turned off, so that multiple cavities are connected in series, leaving only one inlet and one outlet. The protein solution to be tested enters each cavity sequentially along the inlet and reacts with the ligand protein that has been fixed on the surface; after the reaction, it is washed with buffer solution, and the silicon chip is removed from the device for detection.

The elastic material blocks, elastic membranes for sealing, elastic material membranes or elastic material membranes include silica gel, rubber or plastic materials.

The solid material block includes materials such as silica gel, rubber, plastic, metal, glass, etc., and its thickness is 1 mm to 10 mm.

The grooves are strip-shaped grooves, the area of the strip-shaped grooves is from 0.01mm ² to 1mm ² ; the depth is from 10μm-1mm; the number of the strip-shaped grooves is at least 2, for example, it can be from 2 to 1000.

The inner diameter of the through hole can be from 10 μm to 1 mm.

The inner diameter of the micro-channel can be from 10 μm to 1 mm.

The inner diameter of the groove can be from 10 μm to 1 mm.

The advantages of the present invention are:

Since the method for directly preparing a biomolecular chip and immediately performing a detection reaction of the present invention can carry out the preparation of the biochip and the immediate detection reaction in the same device, and the area where the ligand biomolecules are immobilized on the chip is strictly limited, After the fixation and reaction, the surface is washed with buffer solution, which can make the fixation and reaction of biomolecules on the surface uniform and consistent, and effectively improve the quality of detection. The chip reaction is limited in a small area, and in the flow state, the mass transfer rate of biomolecules is accelerated, the reaction time is effectively shortened, and the sensitivity is improved. The microarray template separated from the manufactured chip can be reused for immobilization and reaction of biomolecules on the chip.

Description of drawings

Fig. 1 a is the schematic plan view of the microarray template in the first device of the present invention

Fig. 1 b is a side view of the microarray template in the first device of the present invention

Fig. 2 is the structural diagram when the first device of the present invention is used to directly prepare a biomolecule chip

(The through holes at both ends of the groove of the microarray template and the through holes at both ends of the groove of the rigid solid material block are staggered and correspondingly communicated to form a microchannel coordination diagram)

Fig. 3 is the structure diagram of the device when the first device of the present invention is used for immediate detection reaction

Fig. 4 a is the microarray (elastic material sheet) template plane schematic diagram in the second device of the present invention

Figure 4b is a side view of the microarray template of Figure 4a

Fig. 5 is the structural representation of the second device of the present invention

Fig. 6 is a structural schematic diagram of another embodiment of the second device of the present invention

Fig. 7 is the top view of the microfluidic channel in the second device of the present invention

The illustrations are as follows:

1-chip 2-first groove 3-elastic material block or diaphragm

4-solid material block 5-micro-channel 6-liquid inlet and outlet

7-The inlet of the liquid to be tested 8-The outlet of the liquid to be tested 9-The elastic membrane for sealing

10-the first through hole 11-the second through hole (not shown in the figure) 2'-the second groove

12-pipe 13-diaphragm of elastic material 14-groove

15-first switch 16-film of elastic material (not shown in the figure) 17-second switch

Detailed ways

The present invention is described in detail below in conjunction with accompanying drawing and embodiment

Example 1

Make the first device, and carry out the method of immobilization and detection of twelve kinds of proteins on it. The device includes twelve grooves. The area of the groove is 1mm ² , the depth is 0.1mm, and the inner diameter of the microchannel is 0.5mm. The elastic material is rubber, the solid material is plexiglass, the tube is polytetrafluoroethylene, and the elastic material membrane for closure is silicone. The chip material is silicon.

Make a microarray template according to Figure 1-2; use rubber with a thickness of 1mm as the elastic material block 3, and there are twelve first grooves 2 arranged in an array on it, and the area of the first grooves 2 is 1mm ² , The depth is 0.1 mm; a first through hole 10 with an inner diameter of 0.5 mm is opened at both ends of the first groove 2 as a micro-channel 5 (as shown in FIG. 1 ). Use plexiglass with a thickness of 5mm as the solid material block 4, and twelve second grooves 2' are arranged in an array on the plexiglass block 4. The second groove 2' has an area of 1mm ² and a depth of 0.1mm A second through hole 11 with an inner diameter of 0.5 mm is opened at both ends of the second groove 2'; the through hole of each groove includes two micro-channels 5, one in and one out; the rubber sheet 3 is grooved The surface and the surface of the plexiglass block 4 without grooves are relatively fixed together; the second through hole 11 on the plexiglass block 4 communicates with the first through hole 10 on the rubber sheet 3, and the first through hole 10 on the rubber sheet 3 surface The first through hole 10 at both ends of a groove 2 is staggered corresponding to the second through hole 11 at both ends of the second groove 2' of the organic glass block 4, that is, one of the second through holes 11 of the second groove 2' is used as a solution The outlet communicates with the first through hole 10 of the first groove 2 as the solution inlet, and the outlet of the second groove 2' communicates with the inlet of the first groove; the two sides of the plexiglass block 4 are respectively provided with 2 holes, one of which The hole is used as the inlet 7 of the liquid to be tested during detection, and a switch is installed on the inlet 7 of the liquid to be tested, and the other hole is used as the outlet 8 of the liquid to be tested; Microtube 12 is installed in the two through holes 11, and microfluidic channel 5 is to be connected with the second through hole 11 on the plexiglass block 4 by microtube 12 and forms (as shown in Figure 2). The tube 12 used for the micro-channel 5 of this embodiment is polytetrafluoroethylene, and the elastic material membrane 9 used for sealing is silica gel. Chip 1 is made of silicon.

Using the method of the present invention, carry out the immobilization and detection of twelve kinds of proteins in the device of the above embodiment, the steps are as follows:

(1) under the effect of external pressure (as shown in Figure 2) during work, silicon chip 1 is in close contact with microarray, forms 12 independent sealed cavities between silicon chip 1 and the first groove 2 like this, each One cavity has two micro-channels 5 in and one out;

(2) Under the impetus of a micro plunger pump, for example, 10 microliters of 12 kinds of protein solutions, or hepatitis B surface antigen, hepatitis B e antigen, hepatitis B core antigen, hepatitis B surface antibody, and hepatitis B e antibody solution (concentration is 0.1mg/ ml) are transported to different regions on the silicon chip through micro-channels respectively, and the flow rate is controlled at 1 microliter/minute, after the ligand molecules are immobilized on the chip 1;

(3) Then, transport the phosphate buffer solution to the surface of the chip 1 prepared in the above step (2) through the micro-channel, and wash away the biomolecules that are not fixed on the surface of the silicon chip;

(4) Remove the tube 12 used for the microchannel 5, and cover the microchannel 5 with a closed silica gel membrane 9, so that the regions fixed with ligand molecules are connected in series;

(5) By injecting 100 microliters of the serum of the patient to be tested into the inlet 7 of the liquid to be tested, after injecting the reaction through a microchannel at a flow rate of 10 microliters/min, it flows out through the outlet 8 of the liquid to be tested, and then rinses with phosphate buffer (As shown in Figure 3);

(6) Remove the chip 1 and use a detector to detect the reaction result.

Enter the cavity through the micro-channel and react with the surface of the silicon wafer, thereby immobilizing the protein on the surface of the silicon wafer. Then use the buffer to clean the surface of the silicon chip, the cavity and the micro-channel, and discharge the protein that is not immobilized on the surface of the silicon chip.

Example 2

A device of the invention was prepared having 100 individual sealed cavities.

The elastic material block 3 in this embodiment is silica gel, and 100 first grooves 2 are engraved on it. The area of the first groove 2 is 0.1mm ² or 0.01mm ² ; the depth is 0.1mm; the first through hole 10 with an inner diameter of 0.2mm is opened at both ends of the first groove 2, and a piece of solid material 4 is aluminum, on which Consists of engraved with 100 second grooves 2'. The second groove 2' has an area of 0.1mm ² and a depth of 0.1mm; both ends of the second groove 2' have second through holes 11 with an inner diameter of 0.2mm. The elastic material block 3 has a grooved surface and the solid material 4 does not have a grooved surface and is relatively fixed, wherein the first through hole 10 and the second through hole 11 are misplaced and communicated to form a micro flow channel 5, and the inner diameter of the micro flow channel 5 is 0.2 mm. The upper opening of the flow channel 5 is inserted with a stainless steel pipe 12, and the upper opening of the stainless steel pipe 12 is a liquid inlet 6, and all the other structures are the same as in Embodiment 1.

Also comprise a silica gel membrane 9 that is used for sealing when detecting, this silica gel membrane 9 covers on the second groove 2 ' of organic glass 4, the glass material of gold plating is as chip 1, and all the other structures are with embodiment 1.

Using the device of this embodiment, carry out the immobilization and detection of 1000 kinds of genes, the steps are as follows:

(1) When preparing the gene chip, under the action of external pressure, the silicon chip is in close contact with the microarray template, so that 1000 independent sealed cavities are formed between the silicon chip and the groove, and each cavity has a Into one out of two micro-channels;

(2) Driven by a micro plunger pump, 10 microliters (concentration of 0.1 μg/ml) of DNA molecule solutions of 1000 different sequences were transported to different regions on the silicon wafer through microchannels, and the flow rate was controlled at 1 microliter. L/min, after the DNA molecules are immobilized;

(3) Then, rinse with phosphate buffer to wash away molecules that are not fixed on the surface of the silicon wafer;

(4) Remove the tube 12 used for the micro-channel 5, cover the micro-channel 5 with a closed silica gel membrane 9, connect 1000 grooves in series, and connect the regions fixed with DNA molecules in series;

(5) By injecting 100 microliters of denatured DNA samples to be tested into the inlet 7 of the liquid to be tested, injecting at a flow rate of 10 microliters/minute, and flowing through each of the chips prepared in step (4) through a microchannel. After the regional reaction, it flows out from outlet 8 of the liquid to be tested; it is washed with phosphate buffer;

(7) Take off the silicon wafer, and use a detector to detect the reaction result.

When performing detection: the above-mentioned chip after adding the sample does not need to be removed from the device, and the detection reaction can be directly carried out. Take off the tiny tube 12 from the microarray template, use a piece of silica gel film 9 to seal the second groove 2' on the plexiglass 4, connect 100 grooves on the elastic material sheet 3 in series, and place it opposite to the solution inlet A hole is opened at the elastic membrane as the liquid inlet 7 to be tested, and the hole opened on the side of the solid material block is used as the liquid outlet 8 to be tested. The biological sample to be detected enters each cavity through the liquid inlet 7 to be tested, and is connected with the chip. 1 The biological sample fixed on the surface is reacted and then discharged through the outlet 8 of the liquid to be tested. The reacted chip 1 can be removed from the device, and the result can be detected by a detector.

Example 3

The first device was made, as shown in Figures 1-3; the method of the present invention was used to immobilize and detect 100 kinds of proteins.

The device includes 100 grooves. The area of the groove is 0.06mm ² , the depth is 0.1mm, and the inner diameter of the microchannel is 0.2mm. The elastic material is silicone, the solid material is aluminum, the tube is stainless steel, and the elastic material membrane for closure is silicone. The chip material is gold-plated glass, and the other structures are the same as in Embodiment 1.

Under the action of external pressure, the silicon chip is in close contact with the microarray, so that 100 independent sealed cavities are formed between the silicon chip and the groove. Each cavity has two microchannels, one in and one out. Driven by the micro plunger pump, 100 kinds of protein solutions enter the cavity through the micro flow channel to contact and react with the surface of the silicon wafer, thereby immobilizing the protein on the surface of the silicon wafer. Then use the buffer to clean the surface of the silicon chip, the cavity and the micro-channel, and discharge the protein that is not immobilized on the surface of the silicon chip. Remove the stainless steel tube, use a silicone film to seal the grooves on the plexiglass, and connect 100 grooves in series. Driven by the micro plunger pump, the solution to be tested enters the cavity from the inlet on the side of the plexiglass block and reacts with the protein immobilized on the surface of the silicon wafer. The solution to be tested flows through 100 cavities in sequence, and is finally discharged from the outlet, and then washed with buffer. The reacted silicon chip is removed from the microarray for detection.

Example 4

Make the second device (as shown in Figures 4, 5, and 7), including: microfluidic channel 5 and a microarray template matching it; wherein the microarray template includes: a rubber sheet with an area of 20mm * 20mm as an elastic material Block 3, an elastic material block 3 with 100 first grooves 2 engraved on its surface, each strip-shaped first groove 2 has a depth of 0.01 mm and a cross-sectional area of 0.1 mm ² . 100 are divided into 10 rows and arranged regularly on the rubber sheet 1, and there are 10 first grooves 2 in a row. The surface of the elastic material block 3 with the first groove 2 faces upward, and the two ends of each strip-shaped first groove 2 are respectively opened with a first through hole 10, and the inner diameter of the first through hole 10 is 0.1 mm, and there are 200 in total. The other side of the rubber block 3 is in close contact with the surface of the chip 1; the microfluidic channel is made on the silica gel membrane 13, and the silica gel membrane 13 is formed with a groove 14 with a width of 0.1 mm and a depth of 0.1 mm. One port of the groove 14 is It is set corresponding to the position of the first through hole 10 of the first groove 2 on the microarray, and the other port 6 of the groove 14 extends to the edge of the silica gel membrane 13 to communicate with the outside world; the first through hole 10 of each groove corresponds to a groove 14; and the film at the corresponding microarray silica gel membrane 13 through hole on the silica gel membrane 13 is opened into a hole, and then a layer of plastic film 16 is covered on the silica gel membrane 13 with groove 14, and the silica gel membrane 13 Adhere to the periphery of the two-layer film of the plastic film 16, the micro-flow channel 5 is formed by the groove 14, and there are grooves with a width of 0.1 mm and a depth of 0.1 mm between the micro-flow channels 5 to make them communicate with each other. position, the first switch 15 and the second switch 17 are formed by extruding with external force.

Example 5

Also comprise on the basis of embodiment 4 as rigid solid fast 4 on a plexiglass block 4, this solid material block 4 is the plexiglass block of 25mm * 25mm; One area is the rubber sheet 3 of 20mm * 20mm, this rubber sheet 3 Bonded on the plexiglass block 4 3. The rubber sheet 1 is engraved with 100 strip-shaped first grooves 2 with a depth of 0.01mm, and the cross-sectional area of each strip-shaped first groove 2 is 0.1mm ² . 100 are divided into 10 rows and arranged regularly on the rubber sheet 3, and there are 10 first grooves 2 in a row. The two ends of the 100 first grooves 2 on the elastic material sheet 3 of the substrate are respectively opened with first through holes 10 , and the inner diameter of the first through holes 10 is 0.1 mm, 200 in total. The micro-channel is made on the silica gel membrane 13, and the silica gel membrane 13 is provided with a groove 14 with a width of 0.1 mm and a depth of 0.1 mm (as shown in FIGS. 4, 6, and 7).

Under the action of external pressure, the silicon chip is in close contact with the microarray, forming 100 cavities. When adding samples, close the second switch II between the micro-channels, and each groove has an independent flow channel for entering and exiting. Driven by the plunger pump, different protein solutions enter the cavity and react with the surface of the silicon wafer, thereby immobilizing on the surface. Then wash with a buffer to remove proteins that are not immobilized on the surface. Turn on the second switch 17 between the micro-channels, and close the first switch 15 on the micro-channels, so that 100 cavities are connected in series, leaving only one inlet 6 and one outlet. The protein solution to be tested enters each cavity sequentially along the inlet 6 to react with the protein immobilized on the surface. After the reaction, wash with buffer. The wafer is removed from the device for inspection.

Claims (11)

1. a device that is used for biomolecule chip micro sample-adding and reaction comprises: fluid channel (5); It is characterized in that: also comprise the microarray template that is complementary with its fluid channel (5); This microarray template comprises: be carved with the elastic material sheet (3) of first groove (2) on surface, its first groove (2) is arranged by the array formula, and first groove (2) two ends have first through hole (10); Also be carved with groove (2 ') on one surface of a rigid solid material block (4), the two ends of groove (2 ') have second through hole (11), and the groove one side that is carved with of elastic material sheet (3) is not with groove one side relative fixed together with rigid solid material block (4); Through hole on this rigid solid material block (4) and through hole on the elastic material sheet (3) stagger one corresponding and communicate, solid pieces of material (4) two sides have a testing liquid solution inlet port to be measured (7) and a testing liquid outlet (8), and a switch is installed in the import; Chip (1) closely contact on elastic material sheet (3), the pipe that the plug-in mounting diameter is close in the through hole of solid pieces of material (4) (12), fluid channel is with the formation that is connected of the through hole on the microarray template by pipe (12); The internal diameter of described first through hole (10) or second through hole (11) from 10 μ m to 1mm.

2. by the described device that is used for biomolecule chip micro sample-adding and reaction of claim 1; It is characterized in that: the elastic membrane (9) that also comprises sealing first groove (2); This elastic membrane (9) is covered on the solid pieces of material of taking off pipe (12) (4), open a hole in the elastic material block relative (3) side-walls with solution inlet port, as detecting liquid testing liquid import (7), export (8) with the hole that solid pieces of material (4) side is opened as testing liquid, perhaps have a hole as testing liquid outlet (8), testing liquid import (7) in the solid pieces of material relative (4) two sides with solution inlet port.

3. by claim 1 or the 2 described devices that are used for biomolecule chip micro sample-adding and reaction; It is characterized in that: described elastic material block (3), sealing comprise silica gel, rubber or plastic material with elastic membrane (9), resilient material diaphragm (13) or elastic material membrane (16); Described solid pieces of material (4) comprises silica gel, rubber, plastics, metal or glass material, and its thickness 1mm is to 10mm.

4. by the described device that is used for biomolecule chip micro sample-adding and reaction of claim 1; It is characterized in that: described first groove (2) or second groove (2 ') are strip groove, and strip groove is 2-1000; Its strip groove area is from 0.1mm ²-1mm ²The degree of depth is from 10 μ m-1mm.

5. device that is used for biomolecule chip micro sample-adding and reaction, comprising: fluid channel (5) is characterized in that: also comprise the microarray template that is complementary with it; Wherein the microarray template comprises: be carved with on the surface at least 2 first grooves (2) and elastic material sheet (3), elastic material sheet (3) has facing up of first groove (2), first through hole (10) is opened at the two ends of each first groove (2) respectively, and the another side of elastic material sheet (3) closely contacts with chip (1) surface; Fluid channel comprises a resilient material diaphragm (13), on resilient material diaphragm (13), be shaped on chase (14), one end of its chase (14) is that the position of first through hole (10) of first groove (2) of corresponding described microarray is provided with another port and extraneous UNICOM; The corresponding chase (14) of first through hole (10) of each groove; And the film of the flexible sheet (13) of band chase being gone up flexible sheet (13) through hole of corresponding microarray band chase is got through pore-forming, on the resilient material diaphragm (13) of band chase, cover one deck elastic material membrane more again, and the two membranes periphery is bonded together, formed fluid channel (5) by chase (14), fluid channel interconnects between (5), at node location, the method for pushing by external force forms first switch (15), second switch (17); The internal diameter of described first through hole (10) or second through hole (11) from 10 μ m to 1mm; The internal diameter of described chase (14) from 10 μ m to 1mm.

6. by the described device that is used for biomolecule chip micro sample-adding and reaction of claim 5; It is characterized in that: also comprise a solid pieces of material (4), this solid pieces of material (4) is fixed on the face of resilient material diaphragm (3) with first groove (2), first through hole (10) that open at the two ends of first groove (2) of elastic material sheet (3) runs through solid pieces of material (4), and resilient material diaphragm (16) is not bonded together with solid pieces of material (4) upper surface of the elastic material membrane and the microarray of chase.

7. by claim 5 or the 6 described devices that are used for biomolecule chip micro sample-adding and reaction; It is characterized in that: described elastic material sheet (3), elastic membrane (9), resilient material diaphragm (13) or flexible sheet comprise silica gel, rubber or plastic material; Described solid pieces of material (4) comprises silica gel, rubber, plastics, metal or glass material, and its thickness 1mm is to 10mm.

8. by claim 5 or the 6 described devices that are used for biomolecule chip micro sample-adding and reaction; It is characterized in that: described first groove (2) or second groove (2 ') are strip groove, and strip groove is 2-1000: its strip groove area is from 0.1mm ²-1mm ²The degree of depth is from 10 μ m-1mm.

9. the method that application rights requires 1,2, the 5 or 6 described devices that are used for biomolecule chip micro sample-adding and reaction to carry out application of sample and reaction comprises, order is carried out as follows:

(1) base material with chip closely contacts with the microarray template, and compresses fluid channel by external force;

(2) the fine pipe of aglucon molecule by fluid channel is transported to the lip-deep selection area of chip base; By the time after the aglucon molecule is fixed on the chip base;

(3) chip base that is fixed with biomolecule that step (2) is prepared is washed with damping fluid, damping fluid is transported in the zones of different of chip surface by fluid channel; Wash the aglucon molecule that is not fixed on the chip surface;

(4) by on the flexible sheet (13) of band chase (14), covering one deck resilient material diaphragm more again, and the two membranes periphery is bonded together, has formed fluid channel (5), the zone that is fixed with the aglucon molecule is together in series by chase (14); Perhaps the switch that forms by the node on the extruding flexible sheet is together in series the zone that is fixed with the aglucon molecule;

(5) biological sample to be detected is transported in each unit on the chip surface that step (4) prepares by fluid channel again, carries out detection reaction immediately;

(6) take off step (5) preparation and the chip that is loaded with the detection reaction result that obtains, use chip detector to detect its reaction result.

10. the method for carrying out application of sample and reaction by the described device that is used for biomolecule chip micro sample-adding and reaction of claim 9, it is characterized in that: described chip base material comprises: the silicon chip of silicon chip or gold-plated film; Described aglucon molecule is protein or DNA, and its aglucon molecular conecentration is 0.001-1mg/ml.

11., it is characterized in that the flow velocity of aglucon molecule in fluid channel is the 0.1-100 mul/min in the step (2) by the method that the described device that is used for biomolecule chip micro sample-adding and reaction of claim 1 carries out application of sample and reaction.

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