WO2022252004A1 - 检测芯片及其制备方法和进样方法 - Google Patents

检测芯片及其制备方法和进样方法 Download PDF

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Publication number
WO2022252004A1
WO2022252004A1 PCT/CN2021/097193 CN2021097193W WO2022252004A1 WO 2022252004 A1 WO2022252004 A1 WO 2022252004A1 CN 2021097193 W CN2021097193 W CN 2021097193W WO 2022252004 A1 WO2022252004 A1 WO 2022252004A1
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WIPO (PCT)
Prior art keywords
substrate
reaction
detection chip
liquid inlet
chip according
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PCT/CN2021/097193
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English (en)
French (fr)
Inventor
刘祝凯
丁丁
Original Assignee
京东方科技集团股份有限公司
北京京东方技术开发有限公司
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Application filed by 京东方科技集团股份有限公司, 北京京东方技术开发有限公司 filed Critical 京东方科技集团股份有限公司
Priority to CN202180001343.8A priority Critical patent/CN115989406A/zh
Priority to US17/772,647 priority patent/US20240165619A1/en
Priority to PCT/CN2021/097193 priority patent/WO2022252004A1/zh
Publication of WO2022252004A1 publication Critical patent/WO2022252004A1/zh

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0621—Control of the sequence of chambers filled or emptied
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0642—Filling fluids into wells by specific techniques
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0673—Handling of plugs of fluid surrounded by immiscible fluid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0874—Three dimensional network
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1883—Means for temperature control using thermal insulation

Definitions

  • Embodiments of the present disclosure relate to a detection chip, a preparation method and a sample injection method thereof.
  • Digital Polymerase Chain Reaction PCR Digital Polymerase Chain Reaction, referred to as DPCR
  • DPCR Digital Polymerase Chain Reaction
  • This technology does not depend on the standard curve, is less affected by the amplification efficiency, has good accuracy and reproducibility, can achieve absolute quantitative analysis, and shows great technical advantages in the research fields of nucleic acid detection and identification; Compared with traditional real-time fluorescent quantitative PCR, it is especially suitable for copy number variation, rare mutation detection and typing, NGS verification, single cell expression analysis and other fields.
  • the implementation forms of digital PCR mainly include array type and droplet type.
  • the micro-reaction volume generated by the array type digital PCR detection chip is more uniform, the stability is higher, and the influence between the systems is small. It is beneficial to obtain high-accuracy analysis results.
  • the processing of the microarray is relatively complicated, and the process of injecting the sample solution on the detection chip, that is, the process of the sample solution entering each micro-reaction chamber, is often inefficient, and the sample solution cannot fill the entire chamber smoothly, resulting in The uneven distribution of the sample solution in each micro-reaction chamber directly affects the amplification efficiency and result interpretation, which restricts the application of the array digital PCR detection chip.
  • an embodiment of the present disclosure provides a detection chip, wherein at least one functional area is divided, the functional area includes: a reaction area and a non-reaction area surrounding the reaction area, and the detection chip includes: a relative A first substrate and a second substrate are provided, the first substrate faces the side of the second substrate and is located in the reaction area, and is provided with a plurality of reaction tanks arranged in an array along the first direction and the second direction ;
  • a first circulation channel connected to the two adjacent reaction tanks in the first direction is provided between the two reaction tanks, and the first flow channel extends along the first direction;
  • a second flow groove connected to the two first flow grooves adjacent in the second direction is arranged between the two first flow grooves, and the second flow groove extends along the second direction;
  • the first direction intersects the second direction.
  • the width of the first flow channel is greater than the width of the second flow channel.
  • the depth of the reaction tank is greater than or equal to the depth of the second circulation tank.
  • the first flow channel includes: a first part, a second part and a third part connected in sequence along the first direction, and the first part and the third part respectively connect two adjacent The reaction tank, the second part is connected to the second flow tank;
  • Both the depth of the first portion and the depth of the third portion are greater than or equal to the depth of the second flow groove
  • the depth of the second portion is equal to the depth of the second flow channel.
  • a first liquid inlet and a first liquid outlet penetrating through the second substrate are provided on the second substrate and located in the non-reaction area;
  • a plurality of the reaction tanks and a plurality of the first flow channels arranged alternately in the first direction form a first flow channel, and the two ends of the first flow channel are connected to the first liquid inlet and the first flow channel respectively.
  • the first liquid outlet is connected.
  • the first liquid inlet and the first liquid outlet are respectively located on opposite sides of the reaction zone in the first direction.
  • a line connecting the center of the first liquid inlet and the center of the first liquid outlet extends along the first direction and passes through the center of the reaction area.
  • a first liquid inlet connection groove and a first liquid outlet connection groove corresponding to the first flow channel are further provided on the side of the first substrate facing the second substrate;
  • One end of the first liquid inlet connection groove is connected to one end corresponding to the first flow channel, and the other end of the first liquid inlet connection groove extends to the non-reaction area and is connected to the first liquid inlet ;
  • One end of the first liquid outlet connection groove is connected to the other end corresponding to the first flow channel, and the other end of the first liquid inlet connection groove extends to the non-reaction area and is connected to the first liquid outlet. connected.
  • a second liquid inlet and a second liquid outlet penetrating through the second substrate are formed on the second substrate and located in the non-reaction area;
  • a plurality of the second flow grooves arranged in the second direction form a second flow channel, and two ends of the second flow channel communicate with the second liquid inlet and the second liquid outlet respectively.
  • the second liquid inlet and the second liquid outlet are respectively located on opposite sides of the reaction zone in the second direction.
  • a line connecting the center of the second liquid inlet and the center of the second liquid outlet extends along the second direction and passes through the center of the reaction area.
  • a second liquid inlet connection groove and a second liquid outlet connection groove corresponding to the second flow channel are further provided on the side of the first substrate facing the second substrate;
  • One end of the second liquid inlet connection groove is connected to one end corresponding to the second flow channel, and the other end of the second liquid inlet connection groove extends to the non-reaction area and is connected to the second liquid inlet ;
  • One end of the second liquid outlet connection groove is connected to the other end corresponding to the second flow channel, and the other end of the second liquid inlet connection groove extends to the non-reaction area and is connected to the second liquid outlet. connected.
  • the width range of the first flow channel is: 20um-30um;
  • the width range of the second circulation groove is: 10um-20um.
  • the first substrate includes: a base substrate and a hole-defining layer located on a side of the base substrate facing the second substrate;
  • a first hole structure is provided on the hole-defining layer in a region where the first flow channel is to be formed, and the first flow channel includes the first hole structure;
  • a second hole structure is provided on the hole-defining layer in a region where the second flow channel is to be formed, and the second flow channel includes the second hole structure;
  • a third pore structure is provided on the pore-defining layer in a region where the reaction tank is to be formed, and the reaction tank includes the third pore structure.
  • the hole defining layer is to be formed with the first liquid connection groove.
  • a fourth hole structure is provided in the region of a liquid inlet connection groove, and a fifth hole structure is arranged on the region of the hole limiting layer where the first liquid outlet connection groove is to be formed, and the first liquid inlet connection groove includes the The fourth hole structure, the first liquid outlet connection groove includes the fifth hole structure.
  • the hole-defining layer is to be formed on the first
  • the area of the second liquid inlet connection groove is provided with a sixth hole structure
  • the hole limiting layer is provided with a seventh hole structure in the area where the second liquid outlet connection groove is to be formed
  • the second liquid inlet connection groove includes the The sixth hole structure
  • the second liquid outlet connection groove includes the seventh hole structure.
  • a heating electrode is disposed between the base substrate and the hole-defining layer, and the heating electrode is configured to heat the region where the reaction tank is located.
  • a control electrode is provided between the heating electrode and the base substrate, a first insulating layer is provided between the control electrode and the heating electrode, and the control electrode passes through the first insulating layer.
  • a via hole in an insulating layer is connected to the heating electrode, and the control electrode is configured to apply an electrical signal to the heating electrode.
  • a second insulating layer is disposed between the heating electrode and the hole-defining layer, and a light-shielding layer is disposed between the second insulating layer and the hole-defining layer, and on the light-shielding layer
  • the area where the reaction tank is to be formed is provided with a hollow structure
  • the reaction tank also includes the hollow structure.
  • the side of the base substrate facing the hole-defining layer is provided with a first accommodation groove in the area where the reaction groove is to be formed;
  • the reaction tank also includes the first holding tank.
  • the first flow channel includes: a first part, a second part and a third part connected in sequence along the first direction, and the first part and the third part respectively connect two adjacent The reaction tank, the second part is connected to the second flow tank;
  • the base substrate is provided with a second receiving groove in the area where the first part is to be formed, and the base substrate is provided with a third receiving groove in the area where the third part is to be formed. Both the second accommodation and the third accommodation groove are connected to the corresponding first accommodation groove;
  • the first circulation tank further includes the second receiving tank and the third receiving tank.
  • a light-shielding layer is disposed between the base substrate and the hole-defining layer, and a hollow structure is disposed on the light-shielding layer where the reaction tank is to be formed;
  • the reaction tank also includes the hollow structure.
  • the second substrate includes: a cover plate and a heating electrode located on a side of the cover plate facing the first substrate, and the heating electrode is configured to heat an area where the reaction tank is located.
  • the side of the heating electrode facing away from the cover plate is provided with a first protective layer.
  • a control electrode is provided between the heating electrode and the cover plate, a first insulating layer is provided between the control electrode and the heating electrode, and the control electrode passes through the first The via hole on the insulating layer is connected to the heating electrode, and the control electrode is configured to apply an electric signal to the heating electrode.
  • the material of the hole-defining layer includes: photoresist.
  • the bottom of the reaction tank, the side wall of the reaction tank, the bottom of the first flow tank and/or the side wall of the first flow tank are provided with a hydrophilic layer.
  • the bottom of the second flow channel and/or the sidewall of the second flow channel is provided with a hydrophobic layer.
  • the embodiment of the present disclosure also provides a method for preparing a detection chip as in the first aspect, wherein the detection chip is divided into at least one functional area, and the functional area includes: a reaction area and a In the non-reactive area of the area, the preparation method comprises:
  • one side of the first substrate is provided with a plurality of reaction tanks arranged in an array along the first direction and the second direction, and the two adjacent ones in the first direction
  • a first circulation tank connected to the two reaction tanks is arranged between the reaction tanks, the first circulation tank extends along the first direction, and between two adjacent first flow tanks in the second direction
  • a second flow groove connected to the two first flow grooves is disposed between them, the second flow groove extends along a second direction, and the first direction intersects the second direction;
  • the side of the first substrate provided with the reaction cell, the first circulation cell and the second flow cell is opposite to the second substrate, and the first substrate and the second The substrate is packaged.
  • the embodiment of the present disclosure also provides a method for injecting samples into the detection chip as described in the first aspect, which includes:
  • the oil phase is injected into the second circulation tank to isolate the oil phase from each of the reaction tanks.
  • FIG. 1 is a schematic structural diagram of a detection chip provided by an embodiment of the present disclosure
  • FIG. 2 is a schematic top view of a detection chip provided by an embodiment of the present disclosure
  • Fig. 3 is a schematic top view of the reaction zone in an embodiment of the present disclosure.
  • Fig. 4 is a schematic cross-sectional view of AA' direction in Fig. 1;
  • Fig. 5 is a schematic cross-sectional view of B-B' direction in Fig. 1;
  • Fig. 6 is another schematic cross-sectional view of AA' direction in Fig. 1;
  • Fig. 7 is another kind of cross-sectional schematic view of B-B ' in Fig. 1;
  • Fig. 8 is another schematic cross-sectional view of AA' direction in Fig. 1;
  • Fig. 9 is another schematic cross-sectional view of B-B' direction in Fig. 1;
  • FIG. 10 is a schematic structural diagram of a partial area on the detection chip in an embodiment of the present disclosure.
  • FIG. 11 is a schematic top view of a local area of the base substrate in FIG. 10;
  • FIG. 12 is another schematic structural diagram of a detection chip provided by an embodiment of the present disclosure.
  • FIG. 13 is a flow chart of a method for preparing a detection chip provided by an embodiment of the present disclosure
  • FIG. 14 is a flow chart of a method for injecting samples into a detection chip provided by an embodiment of the present disclosure.
  • Figure 1 is a schematic structural view of a detection chip provided by an embodiment of the present disclosure
  • Figure 2 is a schematic top view of a detection chip provided by an embodiment of the present disclosure
  • Figure 3 is a schematic top view of a reaction zone in an embodiment of the present disclosure
  • FIG. 4 is a schematic cross-sectional view of AA' in FIG. 1
  • FIG. 5 is a schematic cross-sectional view of BB' in FIG. 1 .
  • the detection chip is divided into at least one functional area 1, the functional area 1 includes: a reaction area 101 and a non-reaction area 102 surrounding the reaction area 101, and the detection chip includes: a first substrate 9 oppositely arranged and the second substrate 10 , the first substrate 9 facing the second substrate 10 and located in the reaction area 101 is provided with a plurality of reaction tanks 4 arranged in an array along the first direction X and the second direction Y. Between the two adjacent reaction tanks 4 in the first direction X, a first flow tank 5 connected to the two reaction tanks 4 is provided, and the first flow tank 5 extends along the first direction X; in the second direction Y A second flow groove 6 connected to the two first flow grooves 5 is arranged between the two adjacent first flow grooves 5. The second flow groove 6 extends along the second direction Y, and the first direction X is in line with the second flow groove.
  • the two directions Y intersect.
  • the oil phase for liquid seal (that is, the input oil phase), the oil phase for liquid seal can cut the sample solution in the first flow tank 5 into two parts, so as to realize the oil phase isolation of each reaction tank 4 .
  • the oil phase for the liquid seal may be mineral oil, liquid paraffin, isopropyl palmitate and butyl laurate, perfluoroalkane oil, and the like.
  • the shape of the orthographic projection of the reaction tank 4 on the second substrate 10 may be circular, square, or other regular or irregular shapes, which is not limited in the present disclosure.
  • the pore diameter of the micro-reaction tank 4 includes 40um ⁇ 60um, such as 50um.
  • the detection chip provided by the embodiment of the present disclosure has a simple structure and is convenient to prepare; at the same time, the detection chip has a stable sampling process and high sampling efficiency, and can make the sample solution fully enter the reaction tanks 4, thereby effectively improving the reaction capacity of each reaction tank. 4 Homogeneity of internal sample solution.
  • the first direction X is perpendicular to the second direction Y; during the process of injecting the oil phase for liquid seal, the flow direction of the oil phase is perpendicular to the extension direction of the first flow groove 5, so that the oil flows relatively to the first flow groove.
  • the sample solution in the tank 5 has a better cutting effect.
  • first liquid inlets 6a and first liquid outlets 6b that penetrate the second substrate 10; alternately arranged in the first direction X
  • a plurality of reaction tanks 4 and a plurality of first flow tanks 5 form a first flow channel 2 , and both ends of the first flow channel 2 communicate with a first liquid inlet 6 a and a first liquid outlet 6 b respectively.
  • the sample solution can be injected into the first flow channel 2 through the first liquid inlet 6a; in order to ensure the injection effect of the sample solution as much as possible, the sample solution can be injected into the first liquid inlet 6a at the same time , giving negative pressure to the first liquid outlet 6b (for example, vacuumizing the first liquid outlet 6b); when each reaction tank 4 is filled with the sample solution, stop the injection of the sample solution.
  • the first flow channel 2 is also filled with sample solution.
  • the first liquid inlet 6a and the first liquid outlet 6b are respectively located on opposite sides of the reaction area 101 in the first direction X;
  • the movement in one direction X ensures that the sample solution is quickly injected into the reaction tank 4 located in the first circulation channel 2 .
  • a line connecting the center of the first liquid inlet 6a and the center of the first liquid outlet 6b extends along the first direction X and passes through the center of the reaction region 101 . This setting is beneficial for the uniform input of the sample solution into each first flow channel 2 and the uniform discharge of gas in the first flow channel 2 to ensure that the sample solution is fully injected into each reaction tank 4 .
  • a first liquid inlet connection groove 8a and a first liquid outlet connection groove 8b corresponding to the first flow channel 2 are also provided on the side of the first substrate 9 facing the second substrate 10; One end of the liquid connection groove 8a is connected to one end corresponding to the first flow channel 2, and the other end of the first liquid inlet connection groove 8a extends to the non-reaction area 102 and is connected to the first liquid inlet 6a; the first liquid outlet connection groove 8b One end of the channel 8a is connected to the other end corresponding to the first flow channel 2, and the other end of the first liquid inlet connection groove 8a extends to the non-reaction area 102 and is connected to the first liquid outlet 6b.
  • a second liquid inlet 7a and a second liquid outlet 7b that penetrate the second substrate 10 are formed on the second substrate 10 and located in the non-reaction area 102;
  • a plurality of second circulation grooves 6 form a second circulation channel 3 , and the two ends of the second circulation channel 3 communicate with the second liquid inlet 7 a and the second liquid outlet 7 b respectively.
  • the sample solution can be injected into the second circulation channel 3 through the second liquid inlet 7a; in order to ensure the injection effect of the sample oil phase as much as possible, the oil can be injected into the second liquid inlet 7a.
  • negative pressure is applied to the second liquid outlet 7b (eg, vacuuming is performed on the second liquid outlet 7b); when no bubbles are discharged from the second liquid outlet 7b, the injection of the oil phase is stopped.
  • the second liquid inlet 7 a and the second liquid outlet 7 b are located on opposite sides of the reaction zone 101 in the second direction Y, respectively.
  • This design can make the oil phase entering the second flow channel 3 have a faster flow velocity in the second direction Y, so as to improve the cutting effect on the sample solution in the first flow channel 5 .
  • a line connecting the center of the second liquid inlet 7a and the center of the second liquid outlet 7b extends along the second direction Y and passes through the center of the reaction region 101 .
  • This arrangement is beneficial to the uniform input of the oil phase to each second circulation channel 3 , and the uniform discharge of gas in each second circulation channel 3 , so as to ensure that the oil phase is fully injected into each second connection groove 6 .
  • a second liquid inlet connection groove 9a and a second liquid outlet connection groove 9b corresponding to the second flow channel 3 are provided on the side of the first substrate 9 facing the second substrate 10; One end of the liquid connection groove 9a is connected to one end corresponding to the second flow channel 3, and the other end of the second liquid inlet connection groove 9a extends to the non-reaction area 102 and is connected to the second liquid inlet 7a; the second liquid outlet connection groove 9b One end of the channel 9a is connected to the other end of the corresponding second flow channel 3, and the other end of the second liquid inlet connection groove 9a extends to the non-reaction area 102 and is connected to the second liquid outlet 7b.
  • the first flow channel 2 used to transmit the sample solution and the second flow channel 3 used to transmit the oil phase are respectively provided.
  • the sample injection process it is only necessary to inject the sample into the first flow channel 2 first. solution, and then inject the oil phase into the first circulation channel 2, the whole injection process is relatively simple and convenient to operate.
  • the width of the first flow channel 5 is greater than the width of the second flow channel 6 . That is, the width of the first circulation groove 5 is relatively wide, and the width of the second circulation groove 6 is relatively narrow.
  • the wider first flow channel 5 can effectively increase the speed of injecting the sample solution into the reaction tank 4, which is beneficial to reduce the sampling time; the injection of the oil phase is to cut the sample solution in the first flow channel 5, and the narrower
  • the first flow channel 5 can achieve a faster flow velocity of the oil phase, so that the cutting effect of the sample solution in the first flow channel 5 is better.
  • the width of the first flow channel 5 ranges from 20 um to 30 um; the width of the second flow channel 6 ranges from 10 um to 20 um.
  • the depth of the reaction tank 4 is greater than or equal to the depth of the second circulation tank 6 .
  • the depth of the reaction tank 4 may be greater than the depth of the second circulation tank 6 , and at this time, the reaction tank 4 can accommodate a larger volume of sample solution.
  • the first flow channel 5 includes: a first part 501, a second part 502 and a third part 503 which are sequentially connected along the first direction X, and the first part 501 and the third part 503 respectively connect adjacent two Reaction tank, the second part 502 is connected with the second flow groove 6; the depth of the first part 501 and the depth of the third part 503 are greater than or equal to the depth of the second flow groove 6; the depth of the second part 502 is equal to the second flow groove 6 depths.
  • the depth of the second part 502 connected to the second flow groove 6 in the first flow groove 5 is set to be equal to the depth of the second flow groove 6.
  • the depth of each place in the second flow channel 3 remains consistent, which is beneficial to oil
  • the phase flows quickly in the second flow channel 3, which is beneficial to improve the cutting effect of the oil relative to the sample solution.
  • the depth of the first part 501 and the third part 503 connected to the reaction tank 4 in the first flow tank 5 can be greater than the depth of the second flow tank 6, so that there can be more sample solutions around the reaction tank 4, thereby It is beneficial to inject the sample solution into the reaction tank 4 .
  • the first substrate 9 includes: a base substrate 11 and a hole-defining layer 12 located on the side of the base substrate 11 facing the second substrate 10 .
  • the base substrate 11 can be a glass substrate;
  • the hole-defining layer 12 can form some hole structures, and these hole structures can be used to form the reaction tank 4, the first flow groove 5, the second flow groove 6, and the first liquid inlet connection groove 8a , the first liquid outlet connection groove 8b, the second liquid inlet connection groove 8a and the second liquid outlet connection groove 8b.
  • the various hole structures on the hole-defining layer 12 in the embodiments of the present disclosure can be selectively set as a through-hole structure penetrating through the hole-defining layer 12 or a blind hole structure not penetrating through the hole-defining layer 12 (blind hole structure). can be seen as grooves formed in the hole-defining layer 12).
  • a first hole structure 15 is provided in the area where the first flow channel 5 is to be formed, and the first flow channel 5 includes the first hole structure 15; on the hole-limiting layer 12, the area where the second flow channel 6 is to be formed A second hole structure 16 is provided, and the second circulation cell 6 includes a second hole structure 16; a third hole structure 14 is arranged on the hole-limiting layer 12 in the area where the reaction tank 4 is to be formed, and the reaction tank 4 includes a third hole structure 14 .
  • the first hole structure 15 when the first flow channel 5 only includes the first hole structure 15, the first hole structure 15 can be a through hole structure or a blind hole structure; when the second flow channel 6 only includes the second hole structure 16, the second hole structure 15 can be a through hole structure or a blind hole structure; when the reaction tank 4 only includes the third hole structure 14, the third hole structure 14 can be a through hole structure or a blind hole structure. In some embodiments, the first hole structure 15, the second hole structure 16, and the third hole structure 14 have the same depth.
  • the first liquid inlet connecting groove 8 b when the first liquid inlet connecting groove 8 a and the first liquid outlet connecting groove 8 b are provided on the side of the first substrate 9 facing the second substrate 10 , the first liquid inlet connecting groove 8 b is to be formed on the hole defining layer 12 .
  • the area of the connection groove 8a is provided with a fourth hole structure 17, and the hole defining layer 12 is provided with a fifth hole structure 18 in the area where the first liquid outlet connection groove 8b is to be formed, and the first liquid inlet connection groove 8a includes a fourth hole structure 17.
  • the first liquid outlet connection groove 8b includes a fifth hole structure 18.
  • the second liquid inlet connection groove 9 b when the second liquid inlet connection groove 9 a and the second liquid outlet connection groove 9 b are provided on the side of the first substrate 9 facing the second substrate 10 , the second liquid inlet connection groove 9 b is to be formed on the hole defining layer 12 .
  • the region of the connection groove 9a is provided with a sixth hole structure 19, and the hole-limiting layer 12 is provided with a seventh hole structure 20 in the region where the second liquid outlet connection groove 9b is to be formed, and the second liquid inlet connection groove 9a includes the sixth hole structure 19 .
  • the second liquid outlet connection groove 9 b includes a seventh hole structure 20 .
  • Fig. 6 is another schematic cross-sectional view of AA' in Fig. 1
  • Fig. 7 is another schematic cross-sectional view of B-B' in Fig. 1 .
  • a heating electrode 23 is disposed between the base substrate 11 and the hole-defining layer 12 , and the heating electrode 23 is configured to heat the area where the reaction tank 4 is located.
  • the double-strand structure of the DNA fragment denatures at high temperature to form a single-strand structure.
  • the primer and the single strand are combined according to the principle of base complementary pairing, and base-binding extension is achieved at the optimum temperature for DNA polymerase.
  • the above process is the temperature cycle process of denaturation-annealing-extension. Through multiple temperature cycling processes of denaturation-annealing-extension, DNA fragments can be replicated in large quantities. In order to realize the above-mentioned temperature cycle process, it is usually necessary to use a series of external equipment to heat and cool the detection chip, which makes the equipment bulky, complicated to operate, and high in cost.
  • the overall temperature of the detection chip changes accordingly, so that the temperature of other structures and components in the detection chip except the microcavity for accommodating DNA fragments also changes accordingly, thereby increasing, for example, Risk of damage to components such as electrical circuits.
  • Common dPCR products are often used in conjunction with droplet preparation systems, which makes the cost of the detection chip high and the processing complicated.
  • the embodiments of the present disclosure set the heating electrode 23 in the first substrate 9 to effectively control the temperature of the micro-reaction chamber, and can effectively realize the temperature control of the reaction tank 4 of the detection chip without the need for
  • the temperature cycle can be realized by the driving operation of the droplet, and no external heating equipment is needed. It has high integration, simple operation, low production cost, and can realize effective sample injection.
  • the heating electrode 23 can receive an electric signal, so when a current flows through the heating electrode, heat will be generated, and the heat will be conducted to at least part of the micro-reaction chamber for adjusting the temperature of the micro-reaction chamber.
  • the heating electrode can be made of a conductive material with a relatively high resistivity, so that the heating electrode can generate a large amount of heat while providing a small electrical signal, so as to improve the energy conversion rate.
  • the heating electrode 23 can be made of transparent conductive materials, such as indium tin oxide (ITO), tin oxide, etc., and can also be made of other suitable materials, such as metals, etc. The embodiments of the present disclosure are based on this No limit.
  • the heating electrode 23 may be a planar electrode, for example, uniformly formed on the base substrate 11 by using a conductive material, so that multiple micro-reaction chambers are evenly heated.
  • the embodiments of the present disclosure are not limited thereto, and the heating electrodes 23 may also have a specific figure or pattern, such as a zigzag shape, an arc shape, etc., which may be determined according to the distribution of the multiple reaction tanks 4 .
  • a control electrode 21 is provided between the heating electrode 23 and the base substrate 11, a first insulating layer 22 is provided between the control electrode 21 and the heating electrode 23, and the control electrode 21 passes through the first insulating layer 22.
  • the via hole is connected to the heating electrode 23, and the control electrode 21 is configured to transmit an external electrical signal to the heating electrode.
  • the number of control electrodes 21 may be one or more, which is not limited in the embodiments of the present disclosure. When multiple control electrodes 21 are used to apply electrical signals to the heating electrodes 23, different parts of the heating electrodes 23 can receive the electrical signals at the same time, so that the heating of the heating electrodes 23 is more uniform.
  • the first insulating layer 22 may include a plurality of via holes, each of which exposes a part of the control electrode 21, so that the heating electrode 23 is connected to the plurality of control electrodes through the plurality of via holes. 21 are electrically connected respectively.
  • the number of via holes may also be greater than the number of control electrodes 21, and each control electrode is electrically connected to the heating electrode 23 through one or more via holes.
  • the control electrode 21 can be made of a material with low resistivity, so as to reduce the energy loss on the control electrode 21 .
  • the control electrode 21 can be made of metal material, such as copper or copper alloy, aluminum or aluminum alloy, etc., and can be a single metal layer or a composite metal layer, which is not limited in the embodiments of the present disclosure.
  • the heating electrode 23 is made of indium tin oxide (ITO) or tin oxide
  • the control electrode 21 is made of a metal material. Since ITO is not easily oxidized, it can prevent partial oxidation of the heating electrode exposed to the air, thereby avoiding problems such as uneven heating or increased power consumption caused by the oxidation of the heating electrode 23 .
  • the control electrode is covered by an insulating layer, so even if it is made of a metal material, the problem of oxidation is not easy to occur.
  • the control electrode 21 may further include a contact portion 21a, and the contact portion 21a extends to the edge of the base substrate 11 and is not covered by the first insulating layer 22 .
  • the contact portion 21a is a larger square shape (four contact portions are schematically shown in FIGS. 1 and 2 ), so that it can be conveniently contacted and connected with the probe or electrode in the electrical signal supply device, It has a large contact area and can stably receive electrical signals. In this way, the detection chip can be plug-and-play, easy to operate, and convenient to use.
  • the contact part can be treated by electroplating, thermal spraying or vacuum plating, so as to form protection on the surface of the contact part 21a to prevent oxidation of the contact part without affecting its conductivity.
  • a second insulating layer 24 is disposed between the heating electrode 23 and the hole-defining layer 12, and a light-shielding layer 25 is disposed between the second insulating layer 24 and the hole-defining layer 12.
  • the reaction to be formed A hollow structure 30 is provided in the area where the tank 4 is located; the reaction tank 4 also includes a hollow structure 30 .
  • the reaction tank 4 needs to be optically inspected to obtain a fluorescence image, and a light-shielding layer is set to block other areas except the area where the reaction tank 4 is located, so as to avoid External light interferes with the inside of the reaction tank 4, which is beneficial to improving the accuracy of optical detection.
  • the third hole structure 14 is a through hole structure to ensure communication with the hollow structure 30 .
  • FIG. 8 is another schematic cross-sectional view of AA' in Fig. 1
  • Fig. 9 is another schematic cross-sectional view of BB' in Fig. 1
  • Fig. 10 is a schematic view of a part of the detection chip in an embodiment of the present disclosure
  • FIG. 11 is a schematic top view of a partial area of the base substrate 11 in FIG. 10 .
  • the base substrate 11 faces the side of the hole-defining layer 12 and is provided with a first accommodating groove 27 in the area where the reaction groove 4 is to be formed, and the reaction groove 4 also includes a second A receiving groove 27 .
  • the first accommodating groove 27 is used as a part of the reaction tank 4, which can effectively increase the depth of the reaction tank 4, so that the reaction tank 4 can Inject more sample solution for easier detection.
  • the third hole structure 14 is a through hole structure to ensure communication with the first storage tank 27 .
  • the first flow channel 5 includes: a first part 501, a second part 502 and a third part 503 which are sequentially connected along the first direction X, and the first part 501 and the third part 503 respectively connect adjacent two
  • the reaction tank 4 the second part 502 is connected with the second flow tank 6 (the second part 502 is located on the flow path of the second flow channel 3);
  • the substrate substrate 11 is provided with a second The receiving groove 28, the substrate substrate 11 is provided with a third receiving groove 29 in the area where the third part 503 is to be formed, and the second receiving groove 28 and the third receiving groove 29 are connected to the corresponding first receiving groove 27;
  • the circulation tank 5 also includes a second receiving tank 28 and a third receiving tank 29 .
  • the depths of the first part 501 and the third part 503 are the same as the depth of the reaction tank 4 and greater than the depth of the second communication tank 6 , and the depth of the second part is the same as the depth of the second communication tank 6 .
  • a light-shielding layer 25 is disposed between the base substrate 11 and the hole-defining layer 12, and a hollow structure 30 is disposed on the light-shielding layer 28 where the reaction tank 4 is to be formed; the reaction tank 4 also includes a hollow structure 30 .
  • the material of the light-shielding layer 25 is a black resin material and the base substrate 11 is a glass substrate, since the black resin material is easy to peel off from the surface of the glass substrate, in order to increase the bonding firmness between the two, it can be placed on the base substrate 11.
  • An auxiliary layer is provided between the light-shielding layer, and the material of the auxiliary layer includes inorganic insulating materials, such as silicon oxide, silicon nitride or a laminated structure composed of the two.
  • the second substrate 10 includes: a cover plate 13 and a heating electrode 23 located on a side of the cover plate 13 facing the first substrate 9 , and the heating electrode 23 is configured to heat the area where the reaction tank 4 is located.
  • the cover plate 13 may be a glass cover plate 13 or a hard plastic cover plate 13 .
  • the side of the heating electrode 23 facing away from the cover plate 13 is provided with a first protective layer 26 to avoid direct contact between the heating electrode and the sample solution or oil phase.
  • a control electrode 21 is provided between the heating electrode 23 and the cover plate 13
  • a first insulating layer 22 is provided between the control electrode 21 and the heating electrode 23, and the control electrode 21 passes through the first insulating layer 22.
  • the via hole is connected to the heating electrode 23 , and the control electrode 21 is configured to apply an electric signal to the heating electrode 23 .
  • the material of the hole-defining layer 12 includes: photoresist; at this time, the photoresist can be exposed and developed to form corresponding hole structures .
  • the bottom of the reaction tank 4 , the sidewall of the reaction tank 4 , the bottom of the first circulation tank 5 and/or the sidewall of the first circulation tank 5 are provided with a hydrophilic layer (not shown).
  • a hydrophilic layer is provided at least one of the bottom of the reaction tank 4, the side wall of the reaction tank 4, the bottom of the first flow tank 5 and the side wall of the first flow tank 5, it is beneficial to limit the sample solution to the first flow tank.
  • a hydrophilic layer is provided on the bottom of the reaction tank 4 and/or on the side wall of the reaction tank 4 , which facilitates the entry of the sample solution into the reaction tank 4 .
  • the bottom of the second circulation groove 6 and/or the sidewall of the second circulation groove 6 are provided with a hydrophobic layer (not shown), which is conducive to better adsorption of the oil phase for the liquid seal to the second circulation channel within 3.
  • a second protective layer (not shown) is provided on the side of the pore-defining layer 12 facing away from the substrate 11 to prevent the pore-defining layer 12 from directly contacting the sample solution or oil phase. It should be noted that, when the above-mentioned hydrophilic/hydrophobic layer and the second protective layer are provided in the detection chip at the same time, the hydrophilic/hydrophobic layer is arranged at a corresponding position on the side of the second protective layer facing away from the base substrate 11 .
  • the second protective layer can also be used as a hydrophilic layer and a hydrophobic layer.
  • the material of the second protective layer is silicon oxide, and the untreated silicon oxide film itself has hydrophilicity, and then the surface of the silicon oxide film in the area where the hydrophobic layer needs to be provided is treated (for example, plasma treatment), so that the corresponding The surface energy of the domain is reduced to assume hydrophobicity.
  • FIG. 12 is another schematic structural diagram of a detection chip provided by an embodiment of the present disclosure. As shown in Figure 12, different from the previous embodiments, the number of functional areas 1 in the embodiment of the present disclosure is multiple (four functional areas are illustrated in Figure 12), that is, multiple independent functional areas are set on the detection chip.
  • the reaction area 101 is designed to meet the detection requirements in different application scenarios.
  • the embodiments of the present disclosure also provide a method for preparing a detection chip, which can be used to prepare the detection chip provided in the above embodiments.
  • Fig. 13 is a flowchart of a method for preparing a detection chip provided by an embodiment of the present disclosure. As shown in Fig. 13 , the preparation method includes:
  • Step S101 preparing a first substrate and a second substrate respectively.
  • one side of the first substrate is provided with a plurality of reaction tanks arranged in an array along the first direction and the second direction, and a reaction tank connected to the two reaction tanks is arranged between the two adjacent reaction tanks in the first direction.
  • connected first flow grooves, the first flow grooves extend along the first direction
  • a second flow groove connected to the two first flow grooves is arranged between two adjacent first flow grooves in the second direction, The second flow groove extends along a second direction, and the first direction intersects the second direction.
  • the process of preparing the first substrate is as follows: firstly, a base substrate is provided; then, a hole-defining layer is prepared on the base substrate.
  • the base substrate may be a glass substrate.
  • the process of preparing the pore-defining layer can be as follows: first, spin-coat the photoresist at a speed of 300 rpm for 10 seconds, and bake the photoresist at a temperature of 90° C.
  • the process of preparing the second substrate is as follows: firstly, a cover is provided; then, the first liquid inlet/outlet and the second liquid inlet/outlet are respectively formed on the cover.
  • the cover plate can be a glass cover plate or a hard plastic cover plate; the first liquid inlet/outlet port and the second liquid inlet/outlet port can be formed on the cover plate by laser drilling or etching.
  • the step of preparing the control electrode in the process of preparing the first substrate, the step of preparing the control electrode, the step of preparing the first The step of insulating layer, the step of preparing heating electrode, the step of preparing second insulating layer and the step of preparing light-shielding layer.
  • a step of preparing a second protective layer and a step of preparing a hydrophilic/hydrophobic layer may also be included.
  • control electrode can adopt metal material, and for example control electrode can adopt the lamination structure that molybdenum-aluminum neodymium-molybdenum (Mo-AlNd-Mo) forms; Wherein, the thickness of bottom molybdenum can be The thickness of aluminum neodymium can be The thickness of the upper molybdenum can be The material of the first insulating layer can be silicon oxide (SiO2), and the thickness can be The material of the heating electrode can be indium tin oxide (ITO), and the thickness can be The material of the second insulating layer can be a stacked structure formed of silicon oxide and silicon nitride, wherein the thickness of silicon oxide can be The thickness of silicon nitride (SiNx) can be The material of the light-shielding layer can adopt black resin material.
  • Mo-AlNd-Mo molybdenum-aluminum neodymium-molybdenum
  • the material of the second protective layer can be silicon oxide, and the thickness can be At this time, the second protective layer is reused as a hydrophilic layer and a hydrophobic layer; specifically, the second protective layer (silicon oxide) covers the bottom of the reaction tank, the sidewall of the reaction tank, the bottom of the first flow tank and the first flow tank Part of the side wall of the second flow cell is hydrophilic to reuse as a hydrophilic layer, and the second protective layer (silicon oxide) covers the bottom of the second flow cell and the portion of the side wall of the second flow cell for surface treatment (such as plasma treatment), so that the surface energy of the second protective layer at the corresponding position can be reduced to exhibit hydrophobicity, so that it can be reused as a hydrophobic layer.
  • the hydrophilic/hydrophobic layer may also have a different structure from the second protective layer.
  • the step of preparing the auxiliary layer and the step of preparing the light-shielding layer are also included before the step of preparing the hole-defining layer.
  • the steps of preparing the light-shielding layer the steps of preparing the control electrode, the steps of preparing the first insulating layer, and the steps of preparing the heating electrode, please refer to the previous content, and will not repeat them here.
  • the material of the auxiliary layer can be a stacked structure formed of silicon oxide and silicon nitride, wherein the thickness of silicon oxide can be The thickness of silicon nitride can be The material of the first protective layer can be silicon oxide, and the thickness can be
  • control electrode when the control electrode, the first insulating layer, the first insulating layer, the first protective layer and other structures are arranged on the cover plate, these structures arranged on the cover plate will not interfere with the first liquid inlet/outlet. and the second liquid inlet/outlet are covered to ensure that the first liquid inlet/outlet and the second liquid inlet/outlet can communicate with the corresponding connection grooves on the first substrate.
  • Step S102 setting the side of the first substrate provided with the reaction tank, the first circulation tank and the second circulation tank opposite to the second substrate, and packaging the first substrate and the second substrate.
  • a pressure-sensitive adhesive film can be pasted on the side opposite to the first substrate on the second substrate, and then the first substrate and the second substrate are combined and rolled to apply pressure to complete chip packaging,
  • an embodiment of the present disclosure also provides a method for injecting a sample of a detection chip, which is based on the detection chip provided in the above embodiment.
  • FIG. 14 is a flow chart of a method for injecting samples into a detection chip provided by an embodiment of the present disclosure. As shown in Figure 14, the preparation method comprises:
  • Step S201 injecting a sample solution into the reaction tank through the first flow tank.
  • Step S202 injecting the oil phase for liquid sealing into the second circulation tank, so as to isolate the oil phase of each reaction tank.
  • rubber caps can be used to fasten the first liquid inlet, the first liquid outlet, the second liquid inlet, and the second liquid outlet before the injection starts, so that the first liquid inlet , the first liquid outlet, the second liquid inlet and the second liquid outlet are all in a closed state.
  • two metal needles are pierced into the rubber caps at the first liquid inlet and the first liquid outlet respectively, so that the first liquid inlet and the first liquid outlet are unblocked, and then through the first
  • the metal needle at the liquid inlet presses the pre-mixed sample solution into the first liquid inlet (at the same time, a certain negative pressure can also be applied at the first liquid outlet), and the sample solution flows in the first flow channel, one by one.
  • the reaction tank is filled, and after all the reaction tanks are filled, the metal needles at the first liquid inlet and the first liquid outlet are taken out, so that the first liquid inlet and the first liquid outlet are in a sealed state again. Then, two metal needles are pierced into the rubber caps at the second liquid inlet and the second liquid outlet respectively, so that the second liquid inlet and the second liquid outlet are unblocked, and then through the second liquid inlet.
  • the metal needle at the second liquid inlet presses the oil phase for liquid seal into the second liquid inlet (at the same time, a certain negative pressure can also be applied at the second liquid outlet), the oil phase flows in the second flow channel, and the first flow channel
  • the sample solution in the tank is cut into two parts (the second part in the first circulation tank is the oil phase, and the first part and the third part in the first flow tank are the sample solution), so as to realize the analysis of each reaction.
  • the oil phase is isolated in the tank, and the injection of the oil phase is stopped when no air bubbles are pressed out
  • the heating electrode when the heating electrode is included in the first substrate, it is also possible to provide electrical signals to the heating electrode according to actual needs during the sample injection process and during the PCR reaction after the sample injection is completed to control the reaction.
  • the tank temperature is adjusted.

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Abstract

一种检测芯片，其中，检测芯片划分有至少一个功能区域（1），功能区域（1）包括：反应区域（101）和围绕反应区域（101）的非反应区域（102），检测芯片包括：相对设置的第一基板（9）和第二基板（10），第一基板（9）朝向第二基板（10）的一侧且位于反应区域（101）内设置有沿第一方向（X）和第二方向（Y）呈阵列排布的多个反应槽（4）；在第一方向（X）上相邻的两个反应槽（4）之间设置有与该两个反应槽（4）相连的第一流通槽（5），第一流通槽（5）沿第一方向（X）延伸；在第二方向（Y）上相邻的两个第一流通槽（5）之间设置有与该两个第一流通槽（5）相连的第二流通槽（6），第二流通槽（6）沿第二方向（Y）延伸；第一方向（X）与第二方向（Y）相交。还提供了一种检测芯片的检测方法和进样方法。

Description

检测芯片及其制备方法和进样方法 技术领域
本公开的实施例涉及一种检测芯片及其制备方法和进样方法。
背景技术
数字聚合酶链式反应PCR(Digital Polymerase Chain Reaction，简称DPCR)是近年来迅速发展起来的第三代核酸分子定量分析技术，其原理是将一个样本均匀分配到几万个不同的反应单元，每个单元至少包含一个拷贝的目标DNA模板，随后在每个反应单元中分别进行PCR扩增，扩增结束后对各个反应单元的荧光信号进行统计学分析。该技术不依赖于标准曲线，受扩增效率的影响较小，具有很好的准确度和重现性，可以实现绝对定量分析，在核酸检测、鉴定等研究领域显示出巨大的技术优势；相比于传统的实时荧光定量PCR，特别适用于拷贝数变异、稀有突变检测与分型、NGS验证、单细胞表达分析等领域。
目前数字PCR的实现形式主要有阵列式和液滴式，其中，阵列式的数字PCR检测芯片相比液滴式生成的微反应体积更加均一，稳定性较高且体系之间影响较小，更有利于获得准确度高的分析结果。但同时微阵列的加工相对复杂，而且样品溶液在检测芯片上的进样过程，也就是样本溶液进入各个微反应腔的过程，其效率往往不高，样本溶液无法顺利填充满整个腔室，造成样本溶液在各个微反应腔的分配不均一，直接影响扩增效率和结果判读，制约了阵列式数字PCR检测芯片的应用。
发明内容
第一方面，本公开实施例提供了一种检测芯片，其中，划分有至少一个功能区域，所述功能区域包括：反应区域和围绕所述反应区域 的非反应区域，所述检测芯片包括：相对设置的第一基板和第二基板，所述第一基板朝向所述第二基板的一侧且位于所述反应区域内设置有沿第一方向和第二方向呈阵列排布的多个反应槽；
在第一方向上相邻的两个所述反应槽之间设置有与该两个反应槽相连的第一流通槽，所述第一流通槽沿所述第一方向延伸；
在第二方向上相邻的两个所述第一流通槽之间设置有与该两个第一流通槽相连的第二流通槽，所述第二流通槽沿第二方向延伸；
所述第一方向与所述第二方向相交。
在一些实施例中，所述第一流通槽的宽度大于所述第二流通槽的宽度。
在一些实施例中，所述反应槽的深度大于或等于所述第二流通槽的深度。
在一些实施例中，所述第一流通槽包括：沿第一方向依次相连的第一部分、第二部分和第三部分，所述第一部分和所述第三部分分别连接相邻的两个所述反应槽，所述第二部分与所述第二流通槽相连；
所述第一部分的深度和所述第三部分的深度均大于或等于所述第二流通槽的深度；
所述第二部分的深度等于所述第二流通槽的深度。
在一些实施例中，在所述第二基板上且位于所述非反应区域内设置有贯穿所述第二基板的第一进液口和第一出液口；
在第一方向上交替设置的多个所述反应槽和多个所述第一流通槽构成第一流通通道，所述第一流通通道的两端分别与所述第一进液口和所述第一出液口连通。
在一些实施例中，所述第一进液口和所述第一出液口分别位于所述反应区域在所述第一方向上的相对侧。
在一些实施例中，所述第一进液口的中心与所述第一出液口的中心的连线沿第一方向延伸且经过所述反应区域的中心。
在一些实施例中，在所述第一基板朝向所述第二基板的一侧还设 置有与所述第一流通通道相对应的第一进液连接槽和第一出液连接槽；
所述第一进液连接槽的一端与对应所述第一流通通道的一端相连，所述第一进液连接槽的另一端延伸至所述非反应区域且与所述第一进液口相连；
所述第一出液连接槽的一端与对应所述第一流通通道的另一端相连，所述第一进液连接槽的另一端延伸至所述非反应区域且与所述第一出液口相连。
在一些实施例中，在所述第二基板上且位于所述非反应区域内形成有贯穿所述第二基板的第二进液口和第二出液口；
在第二方向上排布的多个所述第二流通槽构成第二流通通道，所述第二流通通道的两端分别与所述第二进液口和所述第二出液口连通。
在一些实施例中，所述第二进液口和所述第二出液口分别位于所述反应区域在所述第二方向上的相对侧。
在一些实施例中，所述第二进液口的中心与所述第二出液口的中心的连线沿第二方向延伸且经过所述反应区域的中心。
在一些实施例中，在所述第一基板朝向所述第二基板的一侧还设置有与所述第二流通通道相对应的第二进液连接槽和第二出液连接槽；
所述第二进液连接槽的一端与对应所述第二流通通道的一端相连，所述第二进液连接槽的另一端延伸至所述非反应区域且与所述第二进液口相连；
所述第二出液连接槽的一端与对应所述第二流通通道的另一端相连，所述第二进液连接槽的另一端延伸至所述非反应区域且与所述第二出液口相连。
在一些实施例中，所述第一流通槽的宽度范围为：20um～30um；
所述第二流通槽的宽度范围为：10um～20um。
在一些实施例中，所述第一基板包括：衬底基板和位于所述衬底基板朝向所述第二基板一侧的孔限定层；
所述孔限定层上在待形成所述第一流通槽的区域设置有第一孔结构，所述第一流通槽包括所述第一孔结构；
所述孔限定层上在待形成所述第二流通槽的区域设置有第二孔结构，所述第二流通槽包括所述第二孔结构；
所述孔限定层上在待形成所述反应槽的区域设置有第三孔结构，所述反应槽包括所述第三孔结构。
在一些实施例中，当在所述第一基板朝向所述第二基板的一侧设置第一进液连接槽和第一出液连接槽时，所述孔限定层上在待形成所述第一进液连接槽的区域设置有第四孔结构，所述孔限定层上在待形成所述第一出液连接槽的区域设置有第五孔结构，所述第一进液连接槽包括所述第四孔结构，所述第一出液连接槽包括所述第五孔结构。
在一些实施例中，当在所述第一基板朝向所述第二基板的一侧设置第二进液连接槽和第二出液连接槽时，所述孔限定层上在待形成所述第二进液连接槽的区域设置有第六孔结构，所述孔限定层上在待形成所述第二出液连接槽的区域设置有第七孔结构，所述第二进液连接槽包括所述第六孔结构，所述第二出液连接槽包括所述第七孔结构。
在一些实施例中，在所述衬底基板和所述孔限定层之间设置有加热电极，所述加热电极配置为对所述反应槽所在区域进行加热。
在一些实施例中，在所述加热电极与所述衬底基板之间设置有控制电极，所述控制电极与所述加热电极之间设置有第一绝缘层，所述控制电极通过所述第一绝缘层上的过孔与所述加热电极相连，所述控制电极配置为向所述加热电极施加电信号。
在一些实施例中，所述加热电极与所述孔限定层之间设置有第二绝缘层，所述第二绝缘层与所述孔限定层之间设置有遮光层，所述遮光层上在待形成所述反应槽所处区域设置有镂空结构；
所述反应槽还包括所述镂空结构。
在一些实施例中，所述衬底基板朝向孔限定层的一侧且在待形成所述反应槽的区域设置有第一容纳槽；
所述反应槽还包括所述第一容纳槽。
在一些实施例中，所述第一流通槽包括：沿第一方向依次相连的第一部分、第二部分和第三部分，所述第一部分和所述第三部分分别连接相邻的两个所述反应槽，所述第二部分与所述第二流通槽相连；
所述衬底基板上在待形成所述第一部分所处区域设置有第二容纳槽，所述衬底基板上在待形成所述第三部分所处区域设置有第三容纳槽，所述第二容纳和所述第三容纳槽均与对应所述第一容纳槽相连；
所述第一流通槽还包括所述第二容纳槽和所述第三容纳槽。
在一些实施例中，所述衬底基板与所述孔限定层之间设置有遮光层，所述遮光层上在待形成所述反应槽所处区域设置有镂空结构；
所述反应槽还包括所述镂空结构。
在一些实施例中，所述第二基板包括：盖板和位于所述盖板朝向所述第一基板一侧的加热电极，所述加热电极配置为对所述反应槽所在区域进行加热。
在一些实施例中，所述加热电极背向所述盖板的一侧设置有第一保护层。
在一些实施例中，在所述加热电极与所述盖板之间设置有控制电极，所述控制电极与所述加热电极之间设置有第一绝缘层，所述控制电极通过所述第一绝缘层上的过孔与所述加热电极相连，所述控制电极配置为向所述加热电极施加电信号。
在一些实施例中，所述孔限定层的材料包括：光刻胶。
在一些实施例中，所述反应槽的底部、所述反应槽的侧壁、所述第一流通槽的底部和/或所述第一流通槽的侧壁设置有亲水层。
在一些实施例中，所述第二流通槽的底部和/或所述第二流通槽的侧壁设置有疏水层。
在一些实施例中，所述功能区域的数量为多个。
第二方面，本公开实施例还提供了一种如第一方面中检测芯片的制备方法，其中，所述检测芯片划分有至少一个功能区域，所述功能区域包括：反应区域和围绕所述反应区域的非反应区域，所述制备方法包括：
分别制备第一基板和第二基板，所述第一基板的一侧设置有沿第一方向和第二方向呈阵列排布的多个反应槽，在第一方向上相邻的两个所述反应槽之间设置有与该两个反应槽相连的第一流通槽，所述第一流通槽沿所述第一方向延伸，在第二方向上相邻的两个所述第一流通槽之间设置有与该两个第一流通槽相连的第二流通槽，所述第二流通槽沿第二方向延伸，所述第一方向与所述第二方向相交；
将所述第一基板设置有所述反应槽、所述第一流通槽和所述第二流通槽的一侧与所述第二基板相对设置，并将所述第一基板与所述第二基板进行封装。
第三方面，本公开实施例还提供了一种如第一方面中所述检测芯片的进样方法，其中，包括：
通过所述第一流通槽向所述反应槽注入样本溶液；
向所述第二流通槽中注入油相，以对所述各反应槽进行油相隔离。
附图说明
图1为本公开实施例提供的检测芯片的一种结构示意图；
图2为本公开实施例提供的检测芯片的一种俯视示意图；
图3为本公开实施例中反应区的一种俯视示意；
图4为图1中A-A'向的一种截面示意图；
图5为图1中B-B’向的一种截面示意图；
图6为图1中A-A'向的另一种截面示意图；
图7为图1中B-B’向的另一种截面示意图；
图8为图1中A-A'向的又一种截面示意图；
图9为图1中B-B’向的又一种截面示意图；
图10为本公开实施例中检测芯片上部分区域的一种结构示意图；
图11为图10中衬底基板的局部区域的一种俯视示意图；
图12为本公开实施例提供的检测芯片的另一种结构示意图；
图13为本公开实施例提供的一种检测芯片的制备方法的流程图；
图14为本公开实施例提供的一种检测芯片的进样方法的流程图。
具体实施方式
为使本领域的技术人员更好地理解本公开的技术方案，下面结合附图对本公开提供的一种检测芯片及其制备方法和进样方法进行详细描述。
为使本公开实施例的目的、技术方案和优点更加清楚，下面将结合本公开实施例的附图，对本公开实施例的技术方案进行清楚、完整地描述。显然，所描述的实施例是本公开的一部分实施例，而不是全部的实施例。基于所描述的本公开的实施例，本领域普通技术人员在无需创造性劳动的前提下所获得的所有其他实施例，都属于本公开保护的范围。
除非另作定义，此处使用的技术术语或者科学术语应当为本公开所属领域内具有一般技能的人士所理解的通常意义。本公开中使用的“第一”、“第二”以及类似的词语并不表示任何顺序、数量或者重要性，而只是用来区分不同的组成部分。同样，“包括”或者“包含”等类似的词语意指出现该词前面的元件或者物件涵盖出现在该词后面列举的元件或者物件及其等同，而不排除其他元件或者物件。“连接”或者“相连”等类似的词语并非限定于物理的或者机械的连接，而是可以包括电性的连接，不管是直接的还是间接的。“上”、“下”、“左”、“右”等仅用于表示相对位置关系，当被描述对象的绝对位置改变后，则该相对位置关系也可能相应地改变。
图1为本公开实施例提供的检测芯片的一种结构示意图，图2为 本公开实施例提供的检测芯片的一种俯视示意图，图3为本公开实施例中反应区的一种俯视示意，图4为图1中A-A'向的一种截面示意图，图5为图1中B-B’向的一种截面示意图。如图1至图5所示，该检测芯片划分有至少一个功能区域1，功能区域1包括：反应区域101和围绕反应区域101的非反应区域102，检测芯片包括：相对设置的第一基板9和第二基板10，第一基板9朝向第二基板10的一侧且位于反应区域101内设置有沿第一方向X和第二方向Y呈阵列排布的多个反应槽4。在第一方向X上相邻的两个反应槽4之间设置有与该两个反应槽4相连的第一流通槽5，第一流通槽5沿第一方向X延伸；在第二方向Y上相邻的两个第一流通槽5之间设置有与该两个第一流通槽5相连的第二流通槽6，第二流通槽6沿第二方向Y延伸，第一方向X与第二方向Y相交。
在进行进样时，先通过第一流通槽5向反应槽4注入样本溶液，并使得样本溶液能够充分进入到各反应槽4内(即输入水相)；然后向第二流通槽6中注入液封用油相(即输入油相)，液封用油相可将第一流通槽5内的样本溶液切割为两部分，从而实现对各反应槽4进行油相隔离。其中，液封用油相可以为矿物油、液体石蜡、棕榈酸异丙酯后月桂酸丁酯、全氟烷类油等。
需要说明的是，在本公开实施例中，反应槽4在第二基板10上的正投影的形状可以为圆形、方形、其他规则或不规则的形状，本公开对此不作限定。在一些实施例中，微反应槽4的孔径包括40um～60um，例如50um。
本公开实施例提供的检测芯片结构简单、方便制备；同时，该检测芯片的进样过程稳定、进样效率高，能使得样本溶液充分进入至各反应槽4内，从而能有效提升各反应槽4内样本溶液的均一性。
在一些实施例中，第一方向X与第二方向Y垂直；在注入液封用油相过程中，油相流动方向与第一流通槽5的延伸方向相垂直，以使得油相对第一流通槽5内的样本溶液具有较佳的切割效果。
在一些实施例中，在第二基板10上且位于非反应区域102内设置 有贯穿第二基板10的第一进液口6a和第一出液口6b；在第一方向X上交替设置的多个反应槽4和多个第一流通槽5构成第一流通通道2，第一流通通道2的两端分别与第一进液口6a和第一出液口6b连通。在进样过程中，可通过第一进液口6a向第一流通通道2输入注入样本溶液；为尽可能的保证样本溶液的注入效果，可在向第一进液口6a注入样本溶液的同时，对第一出液口6b给予负压(例如，对第一出液口6b进行抽真空处理)；在各反应槽4内均注满样本溶液时，停止样本溶液的注入。一般而言，此时第一流通通道2内也注满有样本溶液。
在一些实施例中，第一进液口6a和第一出液口6b分别位于反应区域101在第一方向X上的相对侧；该设计可使得进入第一流通通道2的样本溶液优先沿第一方向X进行运动，以保证将样本溶液快速注入至位于第一流通通道2内的反应槽4。进一步地，第一进液口6a的中心与第一出液口6b的中心的连线沿第一方向X延伸且经过反应区域101的中心。该设置有利于样本溶液均匀的输入至各第一流通通道2，有利于第一流通通道2内气体的均匀排出，以保证样本溶液注入充分注入至各反应槽4内。
在一些实施例中，在第一基板9朝向第二基板10的一侧还设置有与第一流通通道2相对应的第一进液连接槽8a和第一出液连接槽8b；第一进液连接槽8a的一端与对应第一流通通道2的一端相连，第一进液连接槽8a的另一端延伸至非反应区域102且与第一进液口6a相连；第一出液连接槽8b的一端与对应第一流通通道2的另一端相连，第一进液连接槽8a的另一端延伸至非反应区域102且与第一出液口6b相连。
在一些实施例中，在第二基板10上且位于非反应区域102内形成有贯穿第二基板10的第二进液口7a和第二出液口7b；在第二方向Y上排布的多个第二流通槽6构成第二流通通道3，第二流通通道3的两端分别与第二进液口7a和第二出液口7b连通。在完成样本溶液的注入后，可通过第二进液口7a向第二流通通道3输入注入样本溶液；为尽可能的保证样油相的注入效果，可在向第二进液口7a注入油相的 同时，对第二出液口7b给予负压(例如，对第二出液口7b进行抽真空处理)；在第二出液口7b无气泡排出时，停止油相注入。
在一些实施例中，第二进液口7a和第二出液口7b分别位于反应区域101在第二方向Y上的相对侧。该设计可使得进入第二流通通道3的油相在第二方向Y上具有较快的流动速度，以提升对第一流通槽5内的样本溶液的切割效果。进一步地，第二进液口7a的中心与第二出液口7b的中心的连线沿第二方向Y延伸且经过反应区域101的中心。该设置有利于油相均匀的输入至各第二流通通道3，有利于各第二流通通道3内气体的均匀排出，以保证油相注入充分注入至各第二连接槽6。
在一些实施例中，在第一基板9朝向第二基板10的一侧还设置有与第二流通通道3相对应的第二进液连接槽9a和第二出液连接槽9b；第二进液连接槽9a的一端与对应第二流通通道3的一端相连，第二进液连接槽9a的另一端延伸至非反应区域102且与第二进液口7a相连；第二出液连接槽9b的一端与对应第二流通通道3的另一端相连，第二进液连接槽9a的另一端延伸至非反应区域102且与第二出液口7b相连。
在本公开实施例中，用于传输样本溶液的第一流通通道2与用于传输油相的第二流通通道3分别设置，在进样过程中，仅需先向第一流通通道2注入样本溶液，再向第一流通通道2注入油相，整个进样过程相对简单、方便操作。
在一些实施例中，第一流通槽5的宽度大于第二流通槽6的宽度。即，第一流通槽5的宽度相对较宽，而第二流通槽6的宽度相对较窄。较宽的第一流通槽5能够有效提升将样本溶液注入至反应槽4的速度，有利于减少进样时间；油相的注入是为了对第一流通槽5内样本溶液进行切割，较窄的第一流通槽5能够实现油相具有更快的流动速度，使得对第一流通槽5内样本溶液的切割效果更佳。在一些实施例中，第一流通槽5的宽度范围为：20um～30um；第二流通槽6的宽度范围为：10um～20um。
在一些实施例中，反应槽4的深度大于或等于第二流通槽6的深度。在本公开实施例中，反应槽4的深度可以大于第二流通槽6的深度，此时可反应槽4内可以容纳更多体积的样本溶液。
在一些实施例中，第一流通槽5包括：沿第一方向X依次相连的第一部分501、第二部分502和第三部分503，第一部分501和第三部分503分别连接相邻的两个反应槽，第二部分502与第二流通槽6相连；第一部分501的深度和第三部分503的深度均大于或等于第二流通槽6的深度；第二部分502的深度等于第二流通槽6的深度。将第一流通槽5内与第二流通槽6相连的第二部分502的深度设置为与第二流通槽6的深度相等，此时第二流通通道3内各处深度保持一致，有利于油相在第二流通通道3内快速流动，有利于提升油相对样本溶液的切割效果。与此同时，第一流通槽5内与反应槽4相连的第一部分501和第三部分503的深度可以大于第二流通槽6的深度，使得反应槽4周围可以存在更多的样本溶液，有利于将样本溶液注入至反应槽4内。
参见图4和图5所示，在一些实施例中，第一基板9包括：衬底基板11和位于衬底基板11朝向第二基板10一侧的孔限定层12。其中，衬底基板11可采用玻璃基板；孔限定层12可以形成一些孔结构，这些孔结构可用于形成反应槽4、第一流通槽5、第二流通槽6、第一进液连接槽8a、第一出液连接槽8b、第二进液连接槽8a以及第二出液连接槽8b。
需要说明的是，本公开实施例中孔限定层12上的各种孔结构可以选择性的设置为贯穿孔限定层12的通孔结构或未贯穿孔限定层12的盲孔结构(盲孔结构可看作为是形成于孔限定层12上的槽)。
孔限定层12上在待形成第一流通槽5的区域设置有第一孔结构15，第一流通槽5包括第一孔结构15；孔限定层12上在待形成第二流通槽6的区域设置有第二孔结构16，第二流通槽6包括第二孔结构16；孔限定层12上在待形成反应槽4的区域设置有第三孔结构14，反应槽4包括第三孔结构14。
在一些实施例中，当第一流通槽5仅包括第一孔结构15时，第一孔结构15可以为通孔结构也可以为盲孔结构；当第二流通槽6仅包括第二孔结构16时，第二孔结构15可以为通孔结构也可以为盲孔结构；当反应槽4仅包括第三孔结构14时，第三孔结构14可以为通孔结构也可以为盲孔结构。在一些实施例中，第一孔结构15、第二孔结构16和第三孔结构14的深度相同。
在一些实施例中，当在第一基板9朝向第二基板10的一侧设置第一进液连接槽8a和第一出液连接槽8b时，孔限定层12上在待形成第一进液连接槽8a的区域设置有第四孔结构17，孔限定层12上在待形成第一出液连接槽8b的区域设置有第五孔结构18，第一进液连接槽8a包括第四孔结构17，第一出液连接槽8b包括第五孔结构18。
在一些实施例中，当在第一基板9朝向第二基板10的一侧设置第二进液连接槽9a和第二出液连接槽9b时，孔限定层12上在待形成第二进液连接槽9a的区域设置有第六孔结构19，孔限定层12上在待形成第二出液连接槽9b的区域设置有第七孔结构20，第二进液连接槽9a包括第六孔结构19，第二出液连接槽9b包括第七孔结构20。
图6为图1中A-A'向的另一种截面示意图，图7为图1中B-B’向的另一种截面示意图。如图6和图7所示，在一些实施例中，在衬底基板11和孔限定层12之间设置有加热电极23，加热电极23配置为对反应槽4所在区域进行加热。
在进行PCR反应时，DNA片段的双链结构在高温时变性形成单链结构，在低温时引物与单链按照碱基互补配对原则实现结合，在DNA聚合酶最适宜温度实现碱基结合延伸，上述过程即为变性-退火-延伸的温度循环过程。通过变性-退火-延伸的多个温度循环过程，DNA片段可实现大量复制。为了实现上述温度循环过程，通常需要采用一系列的外部设备对检测芯片进行加热和冷却，使得设备体积庞大，操作复杂，且成本较高。并且，对检测芯片进行加热和冷却的过程中，检测芯片的整体温度随之变化，使得检测芯片中除了容纳DNA片段的微腔以外的其他结构及部件的温度也随之变化，从而增加了例如电 路等部件的损坏风险。通常的dPCR产品多采用与液滴制备系统相配合，使得检测芯片的成本高昂且加工复杂。
为克服上述技术问题，本公开实施例通过在第一基板9内设置加热电极23，以可对微反应腔的温度进行有效控制，可以有效实现对检测芯片的反应槽4的温度控制，无需对液滴进行驱动操作即可实现温度循环，也无需外部加热设备，其集成度高，操作简单，生产成本低，并且可以实现有效进样。
加热电极23可以接收电信号，由此当有电流流过加热电极时会产生热量，该热量被传导至至少部分微反应腔中，以用于调节微反应腔的温度。其中，加热电极可采用电阻率较大的导电材料制备，从而使该加热电极在提供较小的电信号下产生较大的热量，以提高能量转化率。在一些实施例中，加热电极23可以采用透明导电材料制备，例如采用氧化铟锡(ITO)、氧化锡等制备,也可以采用其他适用的材料制备，例如金属等，本公开的实施例对此不作限制。
在本公开实施例中，加热电极23可以为面状电极，例如采用导电材料均匀形成在衬底基板11上，以使多个微反应腔受热均匀。当然，本公开的实施例不限于此，加热电极23也可以具有特定图形或图案，例如为折线形、圆弧形等，这可以根据多个反应槽4的分布方式而定。
在一些实施例中，在加热电极23与衬底基板11之间设置有控制电极21，控制电极21与加热电极23之间设置有第一绝缘层22，控制电极21通过第一绝缘层22上的过孔与加热电极23相连，控制电极21配置为将外部电信号传递至加热电极。
控制电极21的数量可以为一个或多个，本公开的实施例对此不作限制。当采用多个控制电极21对加热电极23施加电信号时，可以使加热电极23的不同部位同时接收该电信号，从而使得加热电极23的发热更加均匀。例如，当控制电极21为多个时，第一绝缘层22可以包括多个过孔,每个过孔都暴露控制电极21的一部分，从而使加热电极23通过多个过孔与多个控制电极21分别电连接。例如，多个控制电极21和多个过孔—一对应。又例如，多个过孔的数量也可以大于多 个控制电极21的数量，每个控制电极通过一个或多个过孔与加热电极23电连接。
控制电极21可以采用电阻率较小的材料，从而降低控制电极21上的能量损耗。控制电极21可以采用金属材料制备，金属材料例如可以为铜或铜合金、铝或铝合金等，可以为单一金属层或复合金属层，本公开的实施例对此不作限制。
在一些实施例中，在本公开的一些实施例中，加热电极23采用氧化铟锡(ITO)或氧化锡制备，控制电极21采用金属材料制备。由于ITO不易氧化，因此可以防止加热电极暴露于空气中的部分氧化，进而避免加热电极23氧化所导致的加热不均匀或功耗增大等问题。控制电极被绝缘层覆盖，因此即使采用金属材料制备，也不易出现氧化的问题。
为了便于使控制电极21与外部的电信号供给设备电连接以接收电信号，控制电极21还可以包括接触部分21a，接触部分21a延伸至衬底基板11的边缘且不被第一绝缘层22覆盖。例如，该接触部分21a为尺寸较大的方块形(图1和图2中示例性性画出了4个接触部分)，从而可以方便地与电信号供给设备中的探针或电极接触连接，其接触面积大，能够稳定地接收电信号。通过这种方式，可以使检测芯片实现即插即用，操作简单，使用方便。例如，当控制电极采用金属材料制备时，可以对接触部分进行电镀、热喷镀或真空镀等处理，从而在接触部分21a的表面形成保护，以防止接触部分氧化，且不影响其导电性能。
在一些实施例中，加热电极23与孔限定层12之间设置有第二绝缘层24，第二绝缘层24与孔限定层12之间设置有遮光层25，遮光层25上在待形成反应槽4所处区域设置有镂空结构30；反应槽4还包括镂空结构30。一般地，在反应槽4内完成PRC反应后，还需对反应槽4进行光学检测以获得焚光图像，通过设置遮光层以对除反应槽4所在区域之外的其他区域进行遮挡，以避免外部光线对反应槽4内产生干扰，有利于提高光学检测的精准度。
需要说明的是，当反应槽4包括有镂空结构时，则第三孔结构14为通孔结构，以保证与镂空结构30的连通。
图8为图1中A-A'向的又一种截面示意图，图9为图1中B-B’向的又一种截面示意图，图10为本公开实施例中检测芯片上部分区域的一种结构示意图，图11为图10中衬底基板11的局部区域的一种俯视示意图。如图8至图11所示，在一些实施例中，衬底基板11朝向孔限定层12的一侧且在待形成反应槽4的区域设置有第一容纳槽27，反应槽4还包括第一容纳槽27。在本公开实施例中，通过在衬底基板11上设置第一容纳槽27，第一容纳槽27作为反应槽4的一部分，可有效增大反应槽4的深度，以使得反应槽4中能注入更多样本溶液，更便于进行检测。需要说明的是，当反应槽4包括第一容纳槽27时，第三孔结构14为通孔结构，以保证与第一容纳槽27的连通。
在一些实施例中，第一流通槽5包括：沿第一方向X依次相连的第一部分501、第二部分502和第三部分503，第一部分501和第三部分503分别连接相邻的两个反应槽4，第二部分502与第二流通槽6相连(第二部分502位于第二流通通道3的流动路径上)；衬底基板11上在待形成第一部分501所处区域设置有第二容纳槽28，衬底基板11上在待形成第三部分503所处区域设置有第三容纳槽29，第二容纳槽28和第三容纳槽29均与对应第一容纳槽27相连；第一流通槽5还包括第二容纳槽28和第三容纳槽29。
在一些实施例中，第一部分501、第三部分503的深度与反应槽4的深度相同且大于第二连通槽6的深度，第二部分的深度与第二连通槽6的深度相同。
在一些实施例中，衬底基板11与孔限定层12之间设置有遮光层25，遮光层28上在待形成反应槽4所处区域设置有镂空结构30；反应槽4还包括镂空结构30。当遮光层25材料采用黑色树脂材料、衬底基板11为玻璃基板时，由于黑色树脂材料容易从玻璃衬底的表面剥离，为增加二者之间的结合牢固度，因此可在衬底基板11与遮光层之间设置辅助层，辅助层的材料包括无机绝缘材料，例如氧化硅、氮化 硅或二者构成的层叠结构。
在一些实施例中，第二基板10包括：盖板13和位于盖板13朝向第一基板9一侧的加热电极23，加热电极23配置为对反应槽4所在区域进行加热。其中，盖板13可以为玻璃盖板13或硬质塑料盖板13。
在一些实施例中，加热电极23背向盖板13的一侧设置有第一保护层26，以避免加热电极与样本溶液或油相直接接触。
在一些实施例中，在加热电极23与盖板13之间设置有控制电极21，控制电极21与加热电极23之间设置有第一绝缘层22，控制电极21通过第一绝缘层22上的过孔与加热电极23相连，控制电极21配置为向加热电极23施加电信号。
参见图4至图9所示，在一些实施例中，孔限定层12的材料包括：光刻胶；此时，可通过对光刻胶进行曝光、显影的工艺，以形成相应的各孔结构。
在一些实施例中，反应槽4的底部、反应槽4的侧壁、第一流通槽5的底部和/或第一流通槽5的侧壁设置有亲水层(未示出)。通过在反应槽4的底部、反应槽4的侧壁、第一流通槽5的底部和第一流通槽5的侧壁中至少之一位置设置亲水层，有利于将样本溶液限定在第一流通通道2内。其中，在反应槽4的底部和/或反应槽4的侧壁设置亲水层，有利于样本溶液进入至反应槽4内。
在一些实施例中，第二流通槽6的底部和/或第二流通槽6的侧壁设置有疏水层(未示出)，有利于液封用油相更好的吸附于第二流通通道3内。
在一些实施例中在孔限定层12背向衬底基板11的一侧还设置有第二保护层(未示出)，以避免孔限定层12与样本溶液或油相直接接触。需要说明的是，检测芯片内同时设置有上述亲/疏水层以及第二保护层时，亲/疏水层设于第二保护层背向衬底基板11的一侧的相应位置。
当然第二保护层也可以复用作亲水层和疏水层，此时在设置有第二保护层的情况下无需再额外设置亲水层或疏水层。例如，第二保护 层的材料采用氧化硅，未经过处理的氧化硅薄膜本身具有亲水性，然后对需要设置疏水层的区域的氧化硅薄膜表面进行处理(例如等离子体处理)，以使得相应区域的表面能减小以呈现疏水性。
图12为本公开实施例提供的检测芯片的另一种结构示意图。如图12所示，与前面实施例中不同，本公开实施例中功能区域1的数量为多个(图12中示例性画出了4个功能区)，即检测芯片上设置有多个独立的反应区域101，以满足不用应用场景下的检测需求。
基于同一发明构思，本公开实施例还提供的一种检测芯片的制备方法，该制备方法可用于制备上述实施例所提供的检测芯片。
图13为本公开实施例提供的一种检测芯片的制备方法的流程图，如图13所示，该制备方法包括：
步骤S101、分别制备第一基板和第二基板。
其中，第一基板的一侧设置有沿第一方向和第二方向呈阵列排布的多个反应槽，在第一方向上相邻的两个反应槽之间设置有与该两个反应槽相连的第一流通槽，第一流通槽沿第一方向延伸，在第二方向上相邻的两个第一流通槽之间设置有与该两个第一流通槽相连的第二流通槽，第二流通槽沿第二方向延伸，第一方向与第二方向相交。
以制备图4和图5中所示第一基板和第二基板为例。制备第一基板的过程如下：首先提供衬底基板；然后在衬底基板基板上制备孔限定层。其中，衬底基板可以为玻璃基板。制备孔限定层的过程可以如下：首先以300转/分钟的速度旋涂10秒光刻胶，并在90℃温度下对光刻胶烘2分钟；然后重复旋涂一次光刻胶并进行上述过程以得到光刻胶层；接着通过掩膜板对光刻胶层进行曝光；再接着利用显影液对曝光后的光刻胶层显影45秒，在230℃的温度下，将显影后的光刻胶层固化30分钟，以得到孔限定层。制备第二基板的过程如下：首先提供一盖板；然后，在盖板上分别形成第一进/出液口和第二进/出液口。其中，盖板可以玻璃盖板，也可以为硬质塑料盖板；可通过激光打孔或刻蚀的方式在盖板上形成第一进/出液口和第二进/出液口。
需要说明的是，若第一基板和第二基板采用图6和图7中所示， 在制备第一基板过程中，在制备孔限定层的步骤之前还包括制备控制电极的步骤、制备第一绝缘层的步骤、制备加热电极的步骤、制备第二绝缘层的步骤和制备遮光层的步骤。可选地，在制备孔限定层之后还可以包括制备第二保护层的步骤以及制备亲/疏水层的步骤。
控制电极的材料可以采用金属材料，例如控制电极可采用钼-铝钕-钼(Mo-AlNd-Mo)形成的叠层结构；其中，下层钼的厚度可以为
Figure PCTCN2021097193-appb-000001
铝钕的厚度可以为
Figure PCTCN2021097193-appb-000002
上层钼的厚度可以为
Figure PCTCN2021097193-appb-000003
第一绝缘层的材料可以为氧化硅(SiO2)，厚度可以为
Figure PCTCN2021097193-appb-000004
加热电极的材料可以为氧化铟锡(ITO)，厚度可以为
Figure PCTCN2021097193-appb-000005
第二绝缘层的材料可以为氧化硅与氮化硅形成的层叠结构，其中氧化硅的厚度可以
Figure PCTCN2021097193-appb-000006
氮化硅(SiNx)的厚度可以为
Figure PCTCN2021097193-appb-000007
遮光层的材料可采用黑色树脂材料。第二保护层的材料可以为氧化硅，厚度可以为
Figure PCTCN2021097193-appb-000008
此时第二保护层复用作亲水层和疏水层；具体地，第二保护层(氧化硅)覆盖反应槽的底部、反应槽的侧壁、第一流通槽的底部和第一流通槽的侧壁的部分呈亲水性以复用作亲水层，对第二保护层(氧化硅)覆盖第二流通槽的底部和第二流通槽的侧壁的部分进行表面处理(例如等离子体处理)，以使得相应位置处的第二保护层的表面能减小呈现疏水性以复用作疏水层。当然，亲/疏水层也可以是与第二保护层不同的结构。
需要说明的是，若第一基板和第二基板采用图8和图9中所示，在制备第一基板过程中，在制备孔限定层的步骤之前还包括制备辅助层步骤和制备遮光层的步骤；在制备第二基板的过程中，在盖板上分别形成第一进/出液口和第二进/出液口的步骤之后还包括：制备控制电极的步骤、制备第一绝缘层的步骤、制备加热电极的步骤和制备第一保护层的步骤。对于制备遮光层的步骤、制备控制电极的步骤、制备第一绝缘层的步骤、制备加热电极的步骤可参见前面内容，此处不再赘述。
辅助层的材料可以为氧化硅与氮化硅形成的层叠结构，其中氧化硅的厚度可以
Figure PCTCN2021097193-appb-000009
氮化硅的厚度可以为
Figure PCTCN2021097193-appb-000010
第一保护层的材 料可以为氧化硅，厚度可以为
Figure PCTCN2021097193-appb-000011
需要说明的是，当盖板上设置有控制电极、第一绝缘层、第一绝缘层、第一保护层等结构时，设置在盖板上的这些结构不会对第一进/出液口和第二进/出液口产生覆盖，以保证第一进/出液口和第二进/出液口能够与第一基板上对应的连接槽连通。
步骤S102、将第一基板设置有反应槽、第一流通槽和第二流通槽的一侧与第二基板相对设置，并将第一基板与第二基板进行封装。
在步骤S102中，可以在第二基板上用于与第一基板相对的一侧贴附压敏胶膜，然后将第一基板与第二基板进行对合并滚动施压，以完成芯片封装、
基于同一发明构思，本公开实施例还提供的一种检测芯片的进样方法，该进样方法基于上述实施例所提供的检测芯片。
图14为本公开实施例提供的一种检测芯片的进样方法的流程图。如图14所示，该制备方法包括：
步骤S201、通过第一流通槽向反应槽注入样本溶液。
步骤S202、向第二流通槽中注入液封用油相，以对各反应槽进行油相隔离。
在一些实施例中，在进样开始之前，可利用橡胶帽扣紧在第一进液口、第一出液口、第二进液口和第二出液口内，以使得第一进液口、第一出液口、第二进液口和第二出液口内均处于封闭状态。在进样时，通过两个金属针头分别扎入至第一进液口和第一出液口处的橡胶帽，以使得第一进液口和第一出液口解除封闭，然后通过第一进液口处的金属针头向第一进液口内压入预先混合好的样本溶液(同时，也可以在第一出液口处施加一定负压)，样本溶液在第一流通通道内流动，逐个填充反应槽，待全部反应槽填充完成后将第一进液口和第一出液口处的金属针头取出，以使得第一进液口和第一出液口再次处于密封状态。接着，通过两个金属针头分别扎入至第二进液口和第二出液口处的橡胶帽，以使得第二进液口和第二出液口解除封闭，然后通过第二进液口处的金属针头向第二进液口内压入液封用油相(同时，也可 以在第二出液口处施加一定负压)，油相在第二流通通道内流动，并将第一流通槽内的样本溶液切割为两部分(第一流通槽内第二部分所容纳的为油相，第一流通槽内第第一部分和第三部分所容纳的为样本溶液)，从而实现对各反应槽进行油相隔离，待第二出液口无气泡压出时停止油相的注入，进样完成。
在一些实施例中，当第一基板内包括有热加电极时，还可在进样过程中以及进样完成后进行PCR反应的过程中，根据实际需要相加热电极提供电信号，以对反应槽的温度进行调节。
可以理解的是，以上实施方式仅仅是为了说明本公开的原理而采用的示例性实施方式，然而本公开并不局限于此。对于本领域内的普通技术人员而言，在不脱离本公开的精神和实质的情况下，可以做出各种变型和改进，这些变型和改进也视为本公开的保护范围。

Claims (31)

  1. 一种检测芯片，其中，划分有至少一个功能区域，所述功能区域包括：反应区域和围绕所述反应区域的非反应区域，所述检测芯片包括：相对设置的第一基板和第二基板，所述第一基板朝向所述第二基板的一侧且位于所述反应区域内设置有沿第一方向和第二方向呈阵列排布的多个反应槽；
    在第一方向上相邻的两个所述反应槽之间设置有与该两个反应槽相连的第一流通槽，所述第一流通槽沿所述第一方向延伸；
    在第二方向上相邻的两个所述第一流通槽之间设置有与该两个第一流通槽相连的第二流通槽，所述第二流通槽沿第二方向延伸；
    所述第一方向与所述第二方向相交。
  2. 根据权利要求1所述的检测芯片，其中，所述第一流通槽的宽度大于所述第二流通槽的宽度。
  3. 根据权利要求1或2所述的检测芯片，其中，所述反应槽的深度大于或等于所述第二流通槽的深度。
  4. 根据权利要求1至3中任一所述的检测芯片，其中，所述第一流通槽包括：沿第一方向依次相连的第一部分、第二部分和第三部分，所述第一部分和所述第三部分分别连接相邻的两个所述反应槽，所述第二部分与所述第二流通槽相连；
    所述第一部分的深度和所述第三部分的深度均大于或等于所述第二流通槽的深度；
    所述第二部分的深度等于所述第二流通槽的深度。
  5. 根据权利要求1至4中任一所述的检测芯片，其中，在所述第二基板上且位于所述非反应区域内设置有贯穿所述第二基板的第一进液口和第一出液口；
    在第一方向上交替设置的多个所述反应槽和多个所述第一流通槽构成第一流通通道，所述第一流通通道的两端分别与所述第一进液口和所述第一出液口连通。
  6. 根据权利要求5所述的检测芯片，其中，所述第一进液口和所述第一出液口分别位于所述反应区域在所述第一方向上的相对侧。
  7. 根据权利要求6所述的检测芯片，其中，所述第一进液口的中心与所述第一出液口的中心的连线沿第一方向延伸且经过所述反应区域的中心。
  8. 根据权利要求5至7中任一所述的检测芯片，其中，在所述第一基板朝向所述第二基板的一侧还设置有与所述第一流通通道相对应的第一进液连接槽和第一出液连接槽；
    所述第一进液连接槽的一端与对应所述第一流通通道的一端相连，所述第一进液连接槽的另一端延伸至所述非反应区域且与所述第一进液口相连；
    所述第一出液连接槽的一端与对应所述第一流通通道的另一端相连，所述第一进液连接槽的另一端延伸至所述非反应区域且与所述第一出液口相连。
  9. 根据权利要求1至8中任一所述的检测芯片，其中，在所述第二基板上且位于所述非反应区域内形成有贯穿所述第二基板的第二进液口和第二出液口；
    在第二方向上排布的多个所述第二流通槽构成第二流通通道，所 述第二流通通道的两端分别与所述第二进液口和所述第二出液口连通。
  10. 根据权利要求9所述的检测芯片，其中，所述第二进液口和所述第二出液口分别位于所述反应区域在所述第二方向上的相对侧。
  11. 根据权利要求10所述的检测芯片，其中，所述第二进液口的中心与所述第二出液口的中心的连线沿第二方向延伸且经过所述反应区域的中心。
  12. 根据权利要求9至11中任一所述的检测芯片，其中，在所述第一基板朝向所述第二基板的一侧还设置有与所述第二流通通道相对应的第二进液连接槽和第二出液连接槽；
    所述第二进液连接槽的一端与对应所述第二流通通道的一端相连，所述第二进液连接槽的另一端延伸至所述非反应区域且与所述第二进液口相连；
    所述第二出液连接槽的一端与对应所述第二流通通道的另一端相连，所述第二进液连接槽的另一端延伸至所述非反应区域且与所述第二出液口相连。
  13. 根据权利要求1至12中任一所述的检测芯片，其中，所述第一流通槽的宽度范围为：20um～30um；
    所述第二流通槽的宽度范围为：10um～20um。
  14. 根据权利要求1至13中任一所述的检测芯片，其中，所述第一基板包括：衬底基板和位于所述衬底基板朝向所述第二基板一侧的孔限定层；
    所述孔限定层上在待形成所述第一流通槽的区域设置有第一孔结构，所述第一流通槽包括所述第一孔结构；
    所述孔限定层上在待形成所述第二流通槽的区域设置有第二孔结构，所述第二流通槽包括所述第二孔结构；
    所述孔限定层上在待形成所述反应槽的区域设置有第三孔结构，所述反应槽包括所述第三孔结构。
  15. 根据权利要求14所述的检测芯片，其中，当在所述第一基板朝向所述第二基板的一侧设置第一进液连接槽和第一出液连接槽时，所述孔限定层上在待形成所述第一进液连接槽的区域设置有第四孔结构，所述孔限定层上在待形成所述第一出液连接槽的区域设置有第五孔结构，所述第一进液连接槽包括所述第四孔结构，所述第一出液连接槽包括所述第五孔结构。
  16. 根据权利要求14所述的检测芯片，其中，当在所述第一基板朝向所述第二基板的一侧设置第二进液连接槽和第二出液连接槽时，所述孔限定层上在待形成所述第二进液连接槽的区域设置有第六孔结构，所述孔限定层上在待形成所述第二出液连接槽的区域设置有第七孔结构，所述第二进液连接槽包括所述第六孔结构，所述第二出液连接槽包括所述第七孔结构。
  17. 根据权利要求14至16中任一所述的检测芯片，其中，在所述衬底基板和所述孔限定层之间设置有加热电极，所述加热电极配置为对所述反应槽所在区域进行加热。
  18. 根据权利要求17所述的检测芯片，其中，在所述加热电极与所述衬底基板之间设置有控制电极，所述控制电极与所述加热电极之间设置有第一绝缘层，所述控制电极通过所述第一绝缘层上的过孔与 所述加热电极相连，所述控制电极配置为向所述加热电极施加电信号。
  19. 根据权利要求17或18所述的检测芯片，其中，所述加热电极与所述孔限定层之间设置有第二绝缘层，所述第二绝缘层与所述孔限定层之间设置有遮光层，所述遮光层上在待形成所述反应槽所处区域设置有镂空结构；
    所述反应槽还包括所述镂空结构。
  20. 根据权利要求14至16中任一所述的检测芯片，其中，所述衬底基板朝向孔限定层的一侧且在待形成所述反应槽的区域设置有第一容纳槽；
    所述反应槽还包括所述第一容纳槽。
  21. 根据权利要求20所述的检测芯片，其中，所述第一流通槽包括：沿第一方向依次相连的第一部分、第二部分和第三部分，所述第一部分和所述第三部分分别连接相邻的两个所述反应槽，所述第二部分与所述第二流通槽相连；
    所述衬底基板上在待形成所述第一部分所处区域设置有第二容纳槽，所述衬底基板上在待形成所述第三部分所处区域设置有第三容纳槽，所述第二容纳和所述第三容纳槽均与对应所述第一容纳槽相连；
    所述第一流通槽还包括所述第二容纳槽和所述第三容纳槽。
  22. 根据权利要求21所述的检测芯片，其中，所述衬底基板与所述孔限定层之间设置有遮光层，所述遮光层上在待形成所述反应槽所处区域设置有镂空结构；
    所述反应槽还包括所述镂空结构。
  23. 根据权利要求20至22中任一所述的检测芯片，其中，所述第二基板包括：盖板和位于所述盖板朝向所述第一基板一侧的加热电极，所述加热电极配置为对所述反应槽所在区域进行加热。
  24. 根据权利要求23所述的检测芯片，其中，所述加热电极背向所述盖板的一侧设置有第一保护层。
  25. 根据权利要求23或24所述的检测芯片，其中，在所述加热电极与所述盖板之间设置有控制电极，所述控制电极与所述加热电极之间设置有第一绝缘层，所述控制电极通过所述第一绝缘层上的过孔与所述加热电极相连，所述控制电极配置为向所述加热电极施加电信号。
  26. 根据权利要求14至25中任一所述的检测芯片，其中，所述孔限定层的材料包括：光刻胶。
  27. 根据权利要求1至26中任一所述的检测芯片，其中，所述反应槽的底部、所述反应槽的侧壁、所述第一流通槽的底部和/或所述第一流通槽的侧壁设置有亲水层。
  28. 根据权利要求1至27中任一所述的检测芯片，其中，所述第二流通槽的底部和/或所述第二流通槽的侧壁设置有疏水层。
  29. 根据权利要求1至28中任一所述的检测芯片，其中，所述功能区域的数量为多个。
  30. 一种如权利要求1至29中任一所述检测芯片的制备方法，其 中，所述检测芯片划分有至少一个功能区域，所述功能区域包括：反应区域和围绕所述反应区域的非反应区域，所述制备方法包括：
    分别制备第一基板和第二基板，所述第一基板的一侧设置有沿第一方向和第二方向呈阵列排布的多个反应槽，在第一方向上相邻的两个所述反应槽之间设置有与该两个反应槽相连的第一流通槽，所述第一流通槽沿所述第一方向延伸，在第二方向上相邻的两个所述第一流通槽之间设置有与该两个第一流通槽相连的第二流通槽，所述第二流通槽沿第二方向延伸，所述第一方向与所述第二方向相交；
    将所述第一基板设置有所述反应槽、所述第一流通槽和所述第二流通槽的一侧与所述第二基板相对设置，并将所述第一基板与所述第二基板进行封装。
  31. 一种如权利要求1至29中任一所述检测芯片的进样方法，其中，包括：
    通过所述第一流通槽向所述反应槽注入样本溶液；
    向所述第二流通槽中注入油相，以对所述各反应槽进行油相隔离。
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