Silicon-based flow path micropore type digital nucleic acid detection chip, detection system comprising same and application of silicon-based flow path micropore type digital nucleic acid detection chip

Abstract

The invention belongs to the technical field of nucleic acid detection, and particularly relates to a silicon-based flow path micropore type digital nucleic acid detection chip, a detection system containing the same and application of the silicon-based flow path micropore type digital nucleic acid detection chip. According to the microporous digital nucleic acid detection chip, a microporous array structure connected by a micro-channel is formed on the surface of a silicon-based wafer, and the microporous array structure is bonded with an anode of a glass wafer, so that the microporous digital nucleic acid detection chip with a closed channel structure is obtained. Silicon is used as a substrate material of the microporous digital PCR chip, so that sample evaporation and aerosol pollution can be avoided, the detection sensitivity is guaranteed, and the microporous digital PCR chip has the potential of batch processing and application. According to the integrated nucleic acid digital detection system comprising the detection chip, a lead-acid storage battery module is used as a standby power supply system, and it is guaranteed that the detection system conducts nucleic acid detection in a non-laboratory scene.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid detection, and in particular relates to a silicon-based flow path microporous digital nucleic acid detection chip, a detection system containing the same, and applications thereof. Background Art

[0002] Nucleic acid amplification test (NAAT) is a target gene in vitro replication and detection technology. It can directly extract a small amount of target genes from the inside of cell tissues for in vitro replication and subsequent detection without culturing cells. Compared with cell culture, NAAT has obvious advantages in detection accuracy and detection time. As typical representatives of NAAT, variable temperature amplification technology (PCR) and isothermal amplification technology (LAMP, RPA, RCA, etc.) are now widely used in many fields such as infectious diseases, oncology, genetics, blood screening, and microbiology. Among them, PCR technology has become the gold standard for nucleic acid detection. With the advancement of globalization, new infectious diseases are gradually transforming from common epidemics to pandemics. Therefore, deploying nucleic acid instruments to clinical environments closer to the front line, while improving detection speed and efficiency, can better curb the large-scale spread of infectious diseases. In addition, early screening and timely diagnosis of genetic diseases and viruses can achieve "early detection and early treatment" and reduce the mortality rate of patients. As the third-generation nucleic acid amplification detection technology, digital NAAT based on microfluidics technology can realize the early detection and absolute quantification of trace target genes. Therefore, this method is of great significance for disease prevention and virus control.

[0003] As a new generation of precise nucleic acid molecule detection technology, digital NAAT, at its core, uses microfluidics technology to monodisperse target nucleic acids into thousands of independent micro-reaction chambers, maximize the proportion of target nucleic acids in a single micro-reaction chamber, and then achieve high-sensitivity detection of trace target nucleic acids, which can effectively improve the detection rate of patients with low viral loads (<200 copies / mL) in the early stages of infection. Droplet digital NAAT technology mainly produces monodisperse droplets of nucleic acid molecules wrapped in oily liquids through microfluidic channels, inkjet printing, centrifugation, etc. The number and size of droplets are adjustable, and the degree of automation is high. Therefore, it is widely used in commercial digital NAAT instruments. However, the thermal stability of microdroplets at high temperatures is poor, which makes them prone to rupture and fusion during the thermal cycle required for nucleic acid amplification, resulting in loss of droplet quantity, making it difficult to meet the needs of high-sensitivity detection.

[0004] In contrast, micropore digital NAAT technology mainly uses micromachining technology to create thousands of micropores on chips with silicon or polymer as the base material, which is easier to process in batches and can reduce the cost of detection. If the micropores are completely independent, an independent micropore digital NAAT chip is formed: first, a liquid containing a nucleic acid sample is spread on the surface of the chip using a pipette, and the sample is allowed to enter the micropores through a scraper or capillary force. The chip is then pressed and packaged to remove excess residual liquid. The average cost of commercial chips can be reduced to $100 per chip. However, this open independent micropore is prone to aerosol contamination during the monodisperse process of nucleic acids. Therefore, if the micropores are connected by microchannels to form an integrated flow path micropore digital NAAT chip, the sample liquid can enter the micropores through the flow channel in the chip to achieve monodisperse nucleic acids, which can effectively avoid aerosol contamination. In recent years, the research group of Professor Mou Ying of Zhejiang University, the research group of Professor Li Gang of Chongqing University, and the research group of Researcher Mao Hongju of Shanghai Institute of Microsystem and Information Technology have proposed and prepared a self-priming and liquid-separating integrated flow path microporous digital NAAT chip: First, the integrated flow path microporous digital NAAT chip is prepared on the flexible polymer material polydimethylsiloxane (PDMS) substrate using soft lithography; second, the chip is vacuumed to form a pressure difference inside and outside the chip; finally, the porous and permeable characteristics of the PDMS material are used to achieve the transport of liquid samples in the microchannel and the monodispersity of nucleic acid samples through negative pressure. This method does not rely on the drive of the pump and the control of the valve, which simplifies the chip structure. However, under the high temperature state in the thermal cycle, the liquid sample will evaporate in the form of water vapor through the porous structure of PDMS, which not only affects the test results, but also causes aerosol contamination. In addition, soft lithography technology requires multiple molds, and the manufacturing process is complicated, which is not conducive to mass production. Summary of the invention

[0005] In view of the above technical problems, the present invention uses silicon-based materials to produce an integrated flow path microporous digital NAAT chip. This method can not only realize batch processing and reduce production and testing costs, but also avoid contamination risks. It can be used as a new idea for the preparation of digital NAAT chips.

[0006] The specific technical solutions provided by the present invention are as follows:

[0007] In a first aspect, the present invention provides a silicon-based flow path microporous digital nucleic acid detection chip, which is a microporous array structure connected by microchannels formed on the surface of a silicon-based wafer, and anodically bonded to a glass wafer to obtain a microporous digital nucleic acid detection chip with a closed flow channel structure.

[0008] Preferably, the microporous digital nucleic acid detection chip is prepared according to the following steps:

[0009] Growing a SiO2 layer on the surface of the silicon wafer;

[0010] Depositing a monolayer of hexamethyldisilazane on the surface of the SiO2 layer to enhance the adhesion of the photoresist on the SiO2 surface;

[0011] A positive photoresist is coated on the surface of the SiO2 layer pretreated with a hexamethyldisilazane monomolecular layer;

[0012] Using photolithography technology, a photoresist pattern designed with access holes, flow channels and micro-hole arrays is transferred from a mask to a photoresist;

[0013] Using the positive photoresist as a mask, etching the SiO2 layer, transferring the photoresist pattern from the positive photoresist to the SiO2 layer, and removing and cleaning;

[0014] Align photolithography on the front side of the SiO2 surface to form an access hole structure;

[0015] Using etching technology, the access hole structure is transferred from the photoresist to the silicon wafer, and the silicon wafer is penetrated to remove the photoresist and clean;

[0016] Using the SiO2 layer as a mask, etching the flow channel and the micro-hole array on the surface of the silicon wafer to remove the SiO2 layer;

[0017] A silicon wafer with access holes, flow channels and a micropore array structure is anodically bonded to a glass wafer and then diced to form a digital nucleic acid detection chip with a closed flow channel structure.

[0018] Further preferably, the hexamethyldisilazane monolayer is deposited on the surface of the SiO2 layer by vapor deposition for 5 to 10 minutes.

[0019] Further preferably, the thickness of the positive photoresist applied when forming the photoresist pattern is 1 to 4 μm;

[0020] The thickness of the positive photoresist applied during front alignment photolithography is 4 to 12 μm.

[0021] Preferably, the microporous digital nucleic acid detection chip is prepared according to the following steps:

[0022] Coating a positive photoresist on the pretreated silicon wafer surface;

[0023] Photolithography to transfer a photoresist pattern designed with access holes, flow channels and microwell arrays from a mask to a photoresist;

[0024] Deep silicon etching to transfer the photoresist pattern from the positive photoresist to the silicon wafer surface, and then remove the photoresist and clean it;

[0025] A silicon wafer with a photoresist pattern is anodically bonded to a glass wafer to form a digital nucleic acid detection chip array with a closed flow channel structure;

[0026] Aligning photolithography on the surface of the silicon wafer away from the glass wafer to form an access hole structure on the photoresist layer;

[0027] Deep silicon etching to transfer the access hole structure from the photoresist to the silicon wafer to form a through-hole structure, and then remove the resist and clean it;

[0028] Slice to form independent digital nucleic acid detection chips.

[0029] Further preferably, when forming the photoresist pattern, the coating thickness of the positive photoresist is 1 to 4 μm;

[0030] The thickness of the positive photoresist coated on the silicon wafer surface away from the glass wafer during alignment photolithography is 4-12 μm.

[0031] In a second aspect, the present invention provides an integrated digital nucleic acid detection system containing the microporous digital nucleic acid detection chip, which further comprises:

[0032] A temperature control system, used to control the temperature of the microporous digital nucleic acid detection chip;

[0033] A fluorescence signal acquisition and analysis system, used for collecting and analyzing the fluorescence signals of the detection results of the microporous digital nucleic acid detection chip; and

[0034] The backup power supply system is used to provide backup power for the temperature control system and the fluorescence signal acquisition and analysis system.

[0035] Preferably, the temperature control system comprises:

[0036] A thermoelectric cooler (TEC), used for heating or cooling the digital nucleic acid detection chip;

[0037] Pt100 platinum resistance temperature sensor, used to measure the temperature of the digital nucleic acid detection chip in real time;

[0038] A closed-loop feedback temperature control system, which is used to receive the output signal of the Pt100 platinum resistance temperature sensor and adjust the power and polarity of the thermoelectric semiconductor refrigerator according to the real-time temperature of the chip collected; the closed-loop feedback temperature control system has a current limiting protection function for the thermoelectric semiconductor refrigerator, which can avoid the life reduction or damage of the TEC caused by long-term operation at the maximum current; and

[0039] The PC end is used to control the thermoelectric semiconductor refrigerator, the Pt100 platinum resistance temperature sensor and the closed-loop feedback temperature control system, and to collect and analyze the fluorescence signal of the detection results of the microporous digital nucleic acid detection chip.

[0040] Preferably, the fluorescence signal acquisition and analysis system comprises:

[0041] LED light, used as the excitation light source for the fluorescence signal collection system;

[0042] A filter module, used for exciting the light signal of the fluorescence signal collection system and filtering the fluorescence signal to avoid interference from background light;

[0043] A CMOS camera, connected to the filter module with a macro lens and a magnifying lens, for collecting fluorescent photos of the microporous digital nucleic acid detection chip; and

[0044] The analysis system is used to analyze the detection results of the microporous digital nucleic acid detection chip.

[0045] Preferably, the backup power supply system is a lead-acid battery module, which is used to provide backup power for nucleic acid detection of the integrated digital nucleic acid detection system in non-laboratory scenarios.

[0046] The third aspect of the present invention provides an application of the microporous digital nucleic acid detection chip or the integrated digital nucleic acid detection system in nucleic acid amplification detection.

[0047] Compared with the prior art, the beneficial effects of the present invention are:

[0048] 1. Silicon is a non-porous material with a dense structure, which can effectively prevent liquid evaporation. In addition, silicon, as the base material of integrated circuit chips, has a mature processing technology and good thermal properties and chemical stability. Therefore, the present invention uses silicon as the base material of the microporous digital PCR chip, which can not only avoid sample evaporation and aerosol contamination, ensure detection sensitivity, but also has the potential for batch processing and application. In addition, the high thermal conductivity of silicon-based chips can reduce thermal delays in the nucleic acid amplification process and shorten detection time.

[0049] 2. The method for preparing silicon-based flow path microporous digital PCR chip provided by the present invention can not only realize batch processing and reduce production and detection costs, but also avoid contamination risks, and can be used as a new idea for the preparation of digital NAAT chips.

[0050] 3. The closed-loop feedback temperature control system in the integrated digital nucleic acid detection system provided by the present invention can adjust the power and polarity of the TEC in real time according to the real-time temperature of the chip to ensure efficient and stable amplification of nucleic acids. The present invention increases the current limiting protection function on the premise of improving the TEC heating and cooling rate to avoid the life reduction or damage of the TEC caused by long-term operation at the maximum current.

[0051] 4. The integrated digital nucleic acid detection system provided by the present invention is also equipped with a backup power supply system based on a lead-acid battery module, which can be powered independently and has the function of outdoor instant detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is a process flow chart of the preparation of the integrated microporous digital nucleic acid detection chip provided in Example 1 of the present invention;

[0053] Figure 2 is a flow chart of the preparation process of the integrated microporous digital nucleic acid detection chip provided in Example 2 of the present invention;

[0054] Figure 3 The figure is a physical image of the prepared chip (a) and a side cross-sectional SEM image of the chip (b);

[0055] Figure 4 It is the connection diagram of the temperature control system;

[0056] Figure 5 It is a connection diagram of the fluorescence signal acquisition and analysis system.

[0057] Figure 6 This is a schematic diagram of the principle prototype of a portable integrated digital nucleic acid detection system. DETAILED DESCRIPTION

[0058] The present invention is further described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0059] The present invention provides a silicon-based flow path microporous digital nucleic acid detection chip, which is a microporous digital nucleic acid detection chip with a closed flow path structure obtained by forming a microporous array structure connected by microchannels on the surface of a silicon-based wafer and anodically bonding it to a glass wafer.

[0060] The present invention adopts Nanolithography Toolbox to design the channel and micropore structure of the silicon-based integrated nucleic acid detection chip, and then uses micro-nano processing technology to process the chip.

[0061] The silicon-based flow path microporous digital nucleic acid detection chip provided by the present invention can be manufactured in two ways:

[0062] Method 1:

[0063] Growing a SiO2 layer on the surface of the silicon wafer;

[0064] Depositing a monolayer of hexamethyldisilazane on the surface of the SiO2 layer to enhance the adhesion of the photoresist on the SiO2 surface, the deposition time is 5 to 10 minutes;

[0065] On the surface of the SiO2 layer pretreated with a hexamethyldisilazane monolayer, a positive photoresist is coated with a thickness of 1 to 4 μm;

[0066] Using photolithography technology, a photoresist pattern designed with access holes, flow channels and micro-hole arrays is transferred from a mask to a photoresist;

[0067] Using positive photoresist as a mask, etching the SiO2 layer, transferring the photoresist pattern from the positive photoresist to the SiO2 layer, and then removing and cleaning;

[0068] Performing front alignment photolithography on the SiO2 surface to form an access hole structure, wherein the thickness of the positive photoresist applied during the front alignment photolithography is 4 to 12 μm;

[0069] Using etching technology, the access hole structure is transferred from the photoresist to the silicon wafer, and the silicon wafer is penetrated to remove the resist and clean it;

[0070] Using the SiO2 layer as a mask, the flow channel and micro-hole array are etched on the surface of the silicon wafer to remove the SiO2 layer;

[0071] A silicon wafer with access holes, flow channels and a micropore array structure is anodically bonded to a glass wafer and then diced to form a digital nucleic acid detection chip with a closed flow channel structure.

[0072] Method 2:

[0073] Apply positive photoresist on the pretreated silicon wafer surface with a thickness of 1 to 4 μm;

[0074] Photolithography to transfer the photoresist pattern designed with access holes, flow channels and micro-well arrays from the mask to the photoresist;

[0075] Deep silicon etching to transfer the photoresist pattern from the positive photoresist to the silicon wafer surface, stripping and cleaning;

[0076] A silicon wafer with a photoresist pattern is anodically bonded to a glass wafer to form a digital nucleic acid detection chip array with a closed flow channel structure;

[0077] Align photolithography on the surface of the silicon wafer facing away from the glass wafer to transfer the access hole structure from the mask to the photoresist. The thickness of the positive photoresist applied during the back alignment photolithography is 4 to 12 μm.

[0078] Deep silicon etching to transfer the access hole structure from the photoresist to the silicon wafer to form a through-hole structure, and then remove the resist and clean it;

[0079] Slice to form independent digital nucleic acid detection chips.

[0080] In addition, based on the silicon-based flow path microporous digital nucleic acid detection chip, the present invention also provides an integrated digital nucleic acid detection system including the detection chip, and the integrated digital nucleic acid detection system also includes:

[0081] A temperature control system, used to control the temperature of a microporous digital nucleic acid detection chip;

[0082] Fluorescence signal acquisition and analysis system, used for collecting fluorescence signals and analyzing the detection results of microporous digital nucleic acid detection chips;

[0083] The backup power supply system is used to provide backup power for the temperature control system and the fluorescence signal acquisition system.

[0084] Since commercial digital NAAT instruments are large in size and lack integration, their liquid circuit system, temperature control system, and fluorescence signal acquisition and analysis system are all independent instruments, resulting in complicated operation and low detection efficiency (2 to 3 hours). Moreover, their systems are all powered by 220V high-voltage AC, which cannot achieve rapid and highly sensitive nucleic acid detection in non-laboratory environments. Therefore, the closed-loop feedback temperature control system used in the integrated digital nucleic acid detection system of the present invention can adjust the power and polarity of the TEC in real time according to the real-time temperature of the chip to ensure efficient and stable amplification of nucleic acids. Under the premise of improving the heating and cooling rate of the TEC, the integrated digital nucleic acid detection system adds a current limiting protection function to avoid the reduction or damage of the life of the TEC due to long-term operation at the maximum current.

[0085] The following describes the invention in conjunction with specific embodiments.

[0086] Example 1

[0087] Preparation of Silicon-based Flow Path Micropore Digital Nucleic Acid Detection Chip

[0088] Structural design and process flow Figure 1 As shown, the details are as follows:

[0089] (1) Cleaning the silicon wafer: First, use hydrofluoric acid to clean a 4-inch silicon wafer with a thickness of 300 μm for about 2 minutes to remove the uneven native oxide layer on the surface of the silicon wafer; then rinse with deionized water for about 3 minutes to remove the residual acid; finally, use nitrogen to blow dry.

[0090] (2) Thermal oxidation and surface treatment: The silicon wafer is placed in an oxidation furnace, and a SiO2 layer with a thickness of about 500 nm is grown on the silicon wafer by wet oxygen and dry oxygen oxidation at a temperature of 850°C. The silicon wafer is placed in a vapor deposition furnace, and a monolayer of hexamethyldisilazane (HMDS) is deposited at a temperature of 150°C for about 5 minutes to enhance the adhesion of the photoresist on the surface of the SiO2 layer.

[0091] (3) Photolithography: Transfer the chip structure (access holes, flow channels and micropore array) from the mask to the photoresist, which has a thickness of about 1 μm.

[0092] (4) SiO2 etching: Using the positive photoresist as a mask, use hydrofluoric acid or plasma to etch the SiO2 layer to transfer the chip structure from the positive photoresist to the SiO2 layer.

[0093] (5) Debonding: First, use acetone to remove the photoresist; then use anhydrous ethanol and deionized water to rinse and remove the residual acetone on the surface of the silicon wafer; finally, blow dry with nitrogen.

[0094] (6) Alignment photolithography: Alignment photolithography is performed on the front side of the SiO2 surface to form an access hole structure. The thickness of the positive photoresist applied during the front alignment photolithography is 4 μm.

[0095] (7) Through-hole etching: Using photoresist as a mask and etching technology, the access hole structure is transferred from the photoresist to the silicon wafer, and the silicon material at the access hole structure is etched through by a deep silicon etching process.

[0096] (8) Debonding: First, use acetone to remove the photoresist; then use anhydrous ethanol and deionized water to rinse and remove the residual acetone on the surface of the silicon wafer; finally, blow dry with nitrogen.

[0097] (9) Structural etching: Using SiO2 as a mask layer, a deep silicon etching process is used to form flow channels and micropore arrays on the surface of the silicon wafer. The etching depth is determined according to actual requirements.

[0098] (10) SiO2 etching: Use hydrofluoric acid to remove the remaining SiO2 layer.

[0099] (11) Bonding + Slicing: A silicon wafer containing access holes, flow channels and micropore array structures is anodically bonded to a glass wafer to form a digital nucleic acid detection chipset with a closed flow channel micropore array. The wafer is sliced ​​according to the designed scribe grooves to obtain an independent digital PCR chip.

[0100] Chip physical diagram Figure 3 (a) shown.

[0101] Example 2

[0102] Preparation of Silicon-based Flow Path Micropore Digital Nucleic Acid Detection Chip

[0103] The only difference from Example 1 is that the deposition time of the hexamethyldisilazane (HMDS) monolayer is 10 min.

[0104] Example 3

[0105] Preparation of Silicon-based Flow Path Micropore Digital Nucleic Acid Detection Chip

[0106] The only difference from Example 1 is that the deposition time of the hexamethyldisilazane (HMDS) monolayer is 8 minutes.

[0107] Example 4

[0108] Preparation of Silicon-based Flow Path Micropore Digital Nucleic Acid Detection Chip

[0109] The only difference from Example 1 is that when the photoresist pattern designed with access holes, flow channels and micro-hole arrays is transferred from the mask to the photoresist, the thickness of the photoresist is about 4 μm.

[0110] Example 5

[0111] Preparation of Silicon-based Flow Path Micropore Digital Nucleic Acid Detection Chip

[0112] The only difference from Example 1 is that when the access hole structure is formed by front-side alignment photolithography on the SiO2 surface, the thickness of the positive photoresist coated by the front-side alignment photolithography is 12 μm.

[0113] Example 6

[0114] Preparation of Silicon-based Flow Path Micropore Digital Nucleic Acid Detection Chip

[0115] The only difference from Example 1 is that when the access hole structure is formed on the SiO2 surface by front-side alignment photolithography, the thickness of the positive photoresist coated by the front-side alignment photolithography is 8 μm.

[0116] Example 7

[0117] Preparation of Silicon-based Flow Path Micropore Digital Nucleic Acid Detection Chip

[0118] Structural design and process flow Figure 2 As shown, the details are as follows:

[0119] (1) Cleaning the silicon wafer: First, use hydrofluoric acid to clean the 4-inch silicon wafer to remove the native oxide layer on the surface of the silicon wafer; then rinse with deionized water to remove residual acid; finally, use nitrogen to blow dry.

[0120] (2) Photolithography: A positive photoresist with a thickness of 1 μm is applied to the pre-treated silicon wafer surface; photolithography is used to transfer the chip structure (entry and exit holes, flow channels, and micro-hole arrays) from the mask to the photoresist.

[0121] (3) Deep silicon etching: Transferring the chip structure from the positive photoresist to the silicon wafer surface.

[0122] (4) De-gumming and cleaning: removing the photoresist from the surface of the silicon wafer.

[0123] (5) Anodic bonding: Anodic bonding of a silicon wafer to a glass wafer to form a digital nucleic acid detection chip array with a closed flow channel structure.

[0124] (6) Back-alignment photolithography: transferring the access hole structure from the mask to the photoresist, and applying a 4 μm thick positive photoresist on the silicon wafer surface facing away from the glass wafer during the alignment photolithography.

[0125] (7) Deep silicon etching: Transfer the inlet and outlet hole structures of the microchannel from the photoresist to the silicon wafer surface.

[0126] (8) De-bonding and cleaning + dicing: Remove the photoresist on the surface of the silicon wafer, and after dicing, it becomes an independent nucleic acid detection chip.

[0127] Example 8

[0128] Preparation of Silicon-based Flow Path Micropore Digital Nucleic Acid Detection Chip

[0129] The only difference from Example 7 is that the thickness of the positive photoresist coated on the pre-treated silicon wafer surface is 4 μm.

[0130] Example 9

[0131] Preparation of Silicon-based Flow Path Micropore Digital Nucleic Acid Detection Chip

[0132] The only difference from Example 7 is that when transferring the access hole structure from the mask to the photoresist, the thickness of the positive photoresist coated during the alignment photolithography on the surface of the silicon wafer facing away from the glass wafer is 12 μm.

[0133] Example 10

[0134] Preparation of Silicon-based Flow Path Micropore Digital Nucleic Acid Detection Chip

[0135] The only difference from Example 7 is that when transferring the access hole structure from the mask to the photoresist, the thickness of the positive photoresist coated during the back side alignment lithography is 8 μm.

[0136] The detection chips with the expected effects of the present invention were prepared using the solutions provided in Examples 1 to 10, and the performance of the detection chips prepared in each example was basically similar.

[0137] The present invention takes the detection chip prepared in Example 1 as an example, and observes its structure under an electron microscope.

[0138] The results are as follows Figure 3As shown in FIG. 2 , it can be seen from the figure that the structure depth of the prepared chip is 50 μm, which meets the design requirements of Example 1 of the present invention. It should be noted that the structure depth is only an exemplary description of the present invention, and is not a special limitation on the structure depth of the detection chip provided by the present invention. Those skilled in the art can reasonably etch the required depth according to the design ideas and requirements of the present invention during specific implementation.

[0139] Embodiment 11

[0140] An integrated digital nucleic acid detection system including a silicon-based flow path microporous digital nucleic acid detection chip also includes: a temperature control system for controlling the temperature of the microporous digital nucleic acid detection chip; a fluorescence signal acquisition system for acquiring fluorescence signals from the detection results of the microporous digital nucleic acid detection chip; and a backup power supply system for providing power to the temperature control system and the fluorescence signal acquisition system.

[0141] In this system, the present invention selects high-power TEC and combines the proportional-integral-differential (PID) control algorithm to build a temperature control system based on closed-loop temperature control feedback. The hardware part of the system is mainly composed of a thermoelectric semiconductor cooler (TEC), a Pt100 platinum resistance temperature sensor, a closed-loop feedback temperature control system and a PC terminal, specifically a driving power supply, TEC, a TEC controller, a Pt100 platinum resistance temperature sensor, a temperature control circuit and a personal computer. Figure 4 shown.

[0142] Among them, the thermoelectric semiconductor refrigerator is used to heat and cool the digital nucleic acid detection chip; the Pt100 platinum resistance temperature sensor is used to measure the temperature of the digital nucleic acid detection chip in real time; the closed-loop feedback temperature control system is used to receive the output signal of the Pt100 platinum resistance temperature sensor, and adjust the power and polarity of the thermoelectric semiconductor refrigerator according to the collected real-time temperature of the chip; the closed-loop feedback temperature control system has a current limiting protection function, which can avoid the life reduction or damage of TEC due to long-term operation at the maximum current; the PC end is used to control the thermoelectric semiconductor refrigerator, the Pt100 platinum resistance temperature sensor and the closed-loop feedback temperature control system, and collect and analyze the fluorescence signal of the detection results of the microporous digital nucleic acid detection chip. The specific working principle of the temperature control system is as follows:

[0143] The real-time temperature of TEC is measured in real time by Pt100 platinum resistance temperature sensor, and the measurement circuit is driven by constant current source. Pt100 temperature sensor first collects the actual temperature of TEC surface, and then feeds back the temperature to TEC controller in the form of resistance value; when the temperature changes, the resistance value of Pt100 changes accordingly, and the voltage signal at both ends also changes. The voltage signal is amplified and filtered, and fed back to the control system inside the program control chip (i.e. PIC chip). By comparing with the set temperature, the change of TEC polarity and power in the closed-loop temperature control feedback system is implemented, so that TEC executes heating, cooling or constant temperature instructions.

[0144] In addition, the fluorescent signal acquisition system provided by the embodiment of the present invention mainly includes a fluorescent filter module, LED, CMOS camera and analysis system. The LED lamp is used as the excitation light source of the fluorescent signal acquisition system; the filter module is used to excite the optical signal of the fluorescent signal acquisition system and filter the fluorescent signal to avoid interference from background light; the CMOS camera is used to collect fluorescent photos of the microporous digital nucleic acid detection chip; the analysis system is used to analyze the detection results of the microporous digital nucleic acid detection chip. Figure 5 shown.

[0145] The backup power supply system of the integrated digital nucleic acid detection system provided in the embodiment of the present invention is an independent power supply system, which can adopt a lead-acid battery module. The independent backup power supply system can provide power for nucleic acid detection of the integrated digital nucleic acid detection system in non-laboratory scenarios, thereby achieving the purpose of outdoor instant detection.

[0146] Principle prototype of the integrated digital nucleic acid detection system provided by the embodiment of the present invention ( Figure 6 )as follows:

[0147] Temperature control module: The lower surface of TEC is fixed to the pure copper radiator through thermal conductive silicone. The radiator is connected to an external 12V cooling fan to discharge the heat generated by TEC at high temperature in a timely manner during the thermal cycle. A brass dPCR chip tray is fixed to the upper surface of TEC through thermal conductive silicone. This tray can not only use the high thermal conductivity of pure copper material to transfer heat to the liquid inside the chip in a timely manner during the thermal cycle, but also facilitate the fixation of the chip position, which is conducive to the acquisition of later fluorescence images. The resistive temperature sensor is fixed to the tray using thermal conductive silicone. The heat dissipation system is fixed to a one-dimensional transmission displacement stage by screws, which is convenient for the early placement of the chip and the focus during the later photography process.

[0148] Fluorescence signal acquisition and analysis module: embed the filter module into the package shell and connect it to the LED and CMOS camera. There is a macro lens between the CMOS camera and the filter module (add a magnification lens as needed), and the macro lens can be a fixed focus or zoom lens. This connection method is to maximize the chip image in the camera's CMOS sensor.

[0149] Control module: This system uses a Windows-based host computer software (digital detection system) to achieve mechanical transmission control, temperature control, fluorescence signal acquisition control and result analysis.

[0150] The above embodiments are only preferred implementation cases of the present invention, and are not intended to limit the implementation scope of the present invention. All technical solutions within the scope of the present invention belong to the protection scope of the present invention.

Claims

1. A silicon-based flow path microporous digital nucleic acid detection chip, characterized in that: The microporous array structure connected by microchannels is formed on the surface of a silicon-based wafer, and is anodically bonded to a glass wafer to obtain a microporous digital nucleic acid detection chip with a closed channel structure.

2. The microporous digital nucleic acid detection chip according to claim 1, characterized in that: It is prepared according to the following steps: Growing a SiO2 layer on the surface of the silicon wafer; After depositing a monolayer of hexamethyldisilazane on the surface of the SiO2 layer, a positive photoresist is applied and photolithography is performed to form a photoresist pattern having access holes, flow channels and micro-hole arrays; Using the positive photoresist as a mask, etching the SiO2 layer to transfer the photoresist pattern from the positive photoresist to the SiO2 layer, and removing and cleaning; Align the photolithography on the front side of the SiO2 surface to form the access hole structure; Transferring the access hole structure from the photoresist to the silicon wafer by etching technology, and penetrating the silicon wafer to remove the photoresist and clean it; Using the SiO2 layer as a mask, etching the flow channel and the micro-hole array on the surface of the silicon wafer to remove the SiO2 layer; A silicon wafer with access holes, flow channels and a micropore array structure is anodically bonded to a glass wafer and then diced to form a digital nucleic acid detection chip with a closed flow channel structure.

3. The microporous digital nucleic acid detection chip according to claim 2, characterized in that: The hexamethyldisilazane monolayer is deposited on the surface of the SiO2 layer by vapor deposition for 5 to 10 minutes.

4. The microporous digital nucleic acid detection chip according to claim 2, characterized in that: The thickness of the positive photoresist applied when forming the photoresist pattern is 1 to 4 μm; The thickness of the positive photoresist applied during front alignment photolithography is 4 to 12 μm.

5. The microporous digital nucleic acid detection chip according to claim 1, characterized in that: It is prepared according to the following steps: A positive photoresist is coated on the surface of the pre-treated silicon wafer and photolithography is performed to form a photoresist pattern having access holes, flow channels and micro-hole arrays; Deep silicon etching to transfer the photoresist pattern from the positive photoresist to the silicon wafer surface, and then remove the photoresist and clean it; A silicon wafer with a photoresist pattern is anodically bonded to a glass wafer to form a digital nucleic acid detection chip array with a closed flow channel structure; Aligning photolithography on the surface of the silicon wafer away from the glass wafer to form an access hole structure on the photoresist layer; Deep silicon etching to transfer the access hole structure from the photoresist to the silicon wafer to form a through-hole structure, and then remove the photoresist and clean it; Slice to form independent digital nucleic acid detection chips.

6. The microporous digital nucleic acid detection chip according to claim 5, characterized in that: When forming a photoresist pattern, the coating thickness of the positive photoresist is 1 to 4 μm; The thickness of the positive photoresist coated on the silicon wafer surface away from the glass wafer during alignment photolithography is 4-12 μm.

7. An integrated digital nucleic acid detection system comprising the microporous digital nucleic acid detection chip according to any one of claims 1 to 6, characterized in that: It also includes: A temperature control system, used to control the temperature of the microporous digital nucleic acid detection chip; A fluorescence signal acquisition and analysis system, used for collecting and analyzing the fluorescence signals of the detection results of the microporous digital nucleic acid detection chip; and The power supply system is used to provide power to the temperature control system and the fluorescence signal acquisition system.

8. The integrated digital nucleic acid detection system according to claim 7, characterized in that: The temperature control system comprises: A thermoelectric semiconductor refrigerator, used to heat or cool the microporous digital nucleic acid detection chip; Pt100 platinum resistance temperature sensor, used for real-time measurement of the temperature of the microporous digital nucleic acid detection chip; A closed-loop feedback temperature control system, used to receive the output signal of the Pt100 platinum resistance temperature sensor and adjust the power and polarity of the thermoelectric semiconductor refrigerator according to the collected real-time temperature of the chip; and The PC end is used to control the thermoelectric semiconductor cooler, the Pt100 platinum resistance temperature sensor and the closed-loop feedback temperature control system.

9. The integrated digital nucleic acid detection system according to claim 7, characterized in that: The power supply system is a lead-acid battery module, which is used to provide power for nucleic acid detection by the integrated digital nucleic acid detection system in non-laboratory scenarios.

10. Use of the microporous digital nucleic acid detection chip according to claim 1 or the integrated digital nucleic acid detection system according to claim 7 in nucleic acid amplification detection.

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