A single cell temperature detection sensor and its manufacturing method and detection method

Abstract

Provided are a single cell temperature detection sensor and a manufacturing method and a detection method thereof. The sensor comprises a glass substrate (1) and a PDMS sheet (2) bonded to each other. A Pt working electrode block (3) and its lead wire (4) for conducting electricity and temperature sensing are sputtered on the bonding surface of the glass substrate (1). A flow channel (7) is provided on the bonding surface of the PDMS sheet (2). After bonding, a flow channel is formed on the glass substrate (1). Sample inlet holes (5) and sample outlet holes (6) are provided at both ends of the flow channel (7). An opening dam for single cells to flow in and be retained is provided in the flow channel (7). The dam is located above the Pt working electrode block (3) and its opening faces the direction of sample injection. A height gap is reserved between the two for sample fluid and air to flow through during detection. The height gap is smaller than the outer diameter of the single cell to be detected. The present invention can detect tiny temperature fluctuations during the life activities of single cells and can realize online continuous monitoring of temperature indicators.

Description

Technical Field

[0001] The present invention relates to the technical field of cell detection, and in particular to a single cell temperature detection sensor and a manufacturing method thereof, and a corresponding single cell temperature detection method. Background Art

[0002] In the field of biology, temperature is one of the important indicators that are indispensable for studying the life process of organisms in the field of life sciences. In life science research, on the one hand, various physiological and chemical reactions are constantly taking place in living cells, and the external manifestation of energy changes closely related to these reactions is the change in temperature. On the other hand, when stimulated by external factors, cells will quickly adjust their metabolic activities to cope with environmental changes, which will also be accompanied by obvious temperature changes. Therefore, the real-time detection of the temperature of a single cell is of great significance both for the study of cell physiological functions and heterogeneity, and from the perspective of cell adaptability and stress response to the external environment.

[0003] Traditional cell temperature measurement methods mainly use fluorescent materials and nanoprobes to measure cell temperature. However, the linearity between fluorescent materials and cell temperature is not high enough, which affects the accuracy of cell temperature. The nanoprobe method will destroy the cell structure and is not suitable for detecting temperature changes during cell division and proliferation. The temperature changes of single cells themselves are small and easily affected by the temperature of the extracellular environment. Therefore, the single cell temperature detection method needs high precision and fast response speed to accurately detect the temperature fluctuations of cells. At present, there is no mature and effective detection method.

[0004] With the continuous improvement and development of microfluidics and micromachining technology, the advantages of micro total analysis system (μ-TAS) in the fields of biological research and clinical experimental diagnosis at the cellular level are becoming increasingly significant. Microfluidic chips have shown obvious advantages in cell analysis due to the advantages of functional unit size comparable to cell size, high precision, and fast and convenient detection. This technology can integrate all current cell analysis steps and processes (such as cell manipulation, cell capture / screening, cell culture, and online real-time dynamic monitoring and analysis, etc.) on a microchip to achieve the integration of analysis operations, which can reduce damage and contamination to cell samples during operation, and is very suitable for rapid and highly sensitive detection of characteristic parameters of a small number of cells. The single-cell temperature detection sensor of the present invention is such a microfluidic sensor, also called a microfluidic chip.

[0005] At present, a series of new microfluidic chips and devices have been developed for single-cell detection, which can be mainly divided into the following categories: (1) Using emulsion droplet units to encapsulate single cells and provide independent space for cell detection. Then use microstructures to manipulate droplet fusion to achieve single-cell lysis and nucleic acid amplification, such as patent document CN109988821A. (2) Using microcavity structure units to capture single cells, and then control the opening and closing of specific channels through microvalve structures, pass lysis solution to lyse cells and drive lysis products into microreaction chambers for amplification, such as patent document CN106065391A. (3) Using the principle of dielectrophoresis, electrodes are integrated in microchips to capture single cells, and then high voltage is applied to lyse cells, such as patent document CN107267382A. The main methods for cell gene detection in microfluidic chips are: (1) Add specific Taqman probes to nucleic acid amplification reagents and analyze gene expression based on fluorescence signals. (2) Recover nucleic acid amplification products and use second-generation sequencing technology to detect target nucleic acids. (3) In situ nucleic acid hybridization technology can be used to analyze genes without lysing cells.

[0006] The above microfluidic devices used for single-cell gene detection still have the following problems: 1) The chip that can realize integrated single-cell detection has a complex structure and requires external sampling or manipulation equipment. 2) The chip with a simple structure only realizes single-cell capture and lysis, and does not integrate all detection steps.

[0007] Polydimethylsiloxane, also known as PDMS, is a high molecular weight organic polymer with optical transparency, porous internal microstructure, and good biocompatibility. The applications of PDMS include microfluidic systems, caulking agents, lubricants, and contact lenses in bio-MEMS. Dimethylsiloxane in liquid state is a viscous liquid called silicone oil, which is a mixture of organic siloxanes with chain structures of different polymerization degrees, and its end groups and side groups are all hydrocarbon groups (such as methyl, ethyl, phenyl, etc.). Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a single cell temperature detection sensor that integrates the functions of single cell capture, lysis, and temperature detection, thereby solving the problems of complex chip structure and cumbersome steps in the prior art. The present invention also provides a method for making such a single cell temperature detection sensor, and a single cell gene detection method using such a single cell temperature detection sensor.

[0009] In order to solve the above technical problems, the technical solution adopted by the single cell temperature detection sensor of the present invention is:

[0010] A single cell temperature detection sensor comprises a glass substrate (1) and a PDMS sheet (2) bonded to each other, a Pt electrode block (3) and a lead wire (4) for conducting and temperature sensing being sputtered on the bonding surface of the glass substrate (1), the bonding surface of the PDMS sheet (2) being a grooved structure surface, a flow channel (7) being provided on the bonding surface of the PDMS sheet (2), and a flow channel for a single cell solution to flow through during detection being formed on the glass substrate (1) after bonding to the glass substrate (1), the flow channel (7) Through holes are respectively formed at both ends of the PDMS sheet (2) to form a sample inlet (5) and a sample outlet (6); the characteristic is that an opening dam is provided in the flow channel (7) for the single cell to flow in and be retained, and the opening of the opening dam faces the sample injection direction; the opening dam is located above the Pt electrode block (3) on the glass substrate (1), and a height gap is retained between the opening dam and the glass substrate (1) for the sample fluid and air to flow through during detection, and the height gap is smaller than the outer diameter of the single cell to be detected.

[0011] The following is a further solution of the single cell temperature detection sensor of the present invention:

[0012] The open dam is a C-shaped dam (8) with an opening in the form of a C-shaped circular ring, the opening width of the C-shaped dam (8) being greater than the inner radius of the C-shaped dam (8); or, the open dam is a square dam with an opening on one side in the form of a square ring, the opening width of the square dam being greater than half the inner side length of the opening side of the C-shaped dam (8).

[0013] The height of the opening dam is 50um, and the height gap between the opening dam and the glass substrate (1) is 5um; the inner diameter of the C-shaped dam (8) is 150um, or the inner side length of the square dam is 150um.

[0014] The flow channel (7) comprises a sample inlet section (7A), a sample outlet section (7B) and two symmetrically distributed detection sections (7C); the detection section (7C) flow channel (7) is surrounded by a rectangle; the lower flow channel (7) of the rectangle is connected to the sample inlet hole (5) via the sample inlet section (7A) flow channel (7); the upper flow channel (7) of the rectangle is connected to the sample outlet hole (6) via the sample outlet section (7B) flow channel (7); the opening dams are respectively arranged in the two side flow channels (7) of the rectangle.

[0015] The Pt electrode block (3) comprises a Ti layer with a thickness of 500a which is first sputtered on the glass substrate (1) as an adhesion layer, and a Pt layer with a thickness of 1500a which is sputtered to form a Ti / Pt working layer with a thickness of 500 / 1500a, and a Si3N4 layer (12) with a thickness of 5000a is deposited on the Ti / Pt working layer as an insulating layer.

[0016] The Pt electrode block (3) is in the shape of a Pt wire that meanders back and forth. The two ends of the Pt electrode block (3) are respectively connected to its two side lead wires (4), and the two side lead wires (4) are arranged in a herringbone shape. The Pt electrode block (3) is located at the top of the herringbone shape, and the bottom ends of the two side lead wires (4) are located at the side edge of the glass substrate (1).

[0017] In order to solve the above technical problems, the technical solution adopted by the method for manufacturing the single-cell temperature detection sensor of the present invention is:

[0018] A method for manufacturing a single-cell temperature detection sensor includes the manufacturing of a PDMS sheet (2) and a glass substrate (1) which can be performed in either order, and bonding the two. The manufacturing of the PDMS sheet (2) first includes the preparation of a silicon substrate (9) as a mold and the manufacturing of the PDMS sheet (2). The manufacturing of the PDMS sheet (2) specifically includes the following steps: Step 1, pretreatment of the silicon substrate (9): 98% H2SO4, 30% H2O2, mixed in a ratio of 7:3 to prepare a piranha solution, the silicon substrate (9) is continuously immersed in the 120°C piranha solution for 30 minutes, then the silicon substrate (9) is taken out and repeatedly washed with deionized water, and the silicon substrate (9) is placed in a spin dryer to dry the surface moisture for later use; step 2, coating: using a coating machine to evenly coat the LC100A photoresist (10) on the surface of the silicon substrate (9); step 3, photolithography: after exposure for 15 seconds, develop to obtain the required pattern; step 4, etching: using deep reactive ion etching to etch the silicon substrate (9) downward 5um depth; Step 5, degumming: use piranha solution to remove LC100A photoresist (10); Step 6, glue coating: SU8-3050 photoresist (11) is applied on the cleaned silicon substrate (9) with a thickness of 50um, and left to stand horizontally for 2 hours; Step 7, photolithography: after exposure for 10 seconds, the required silicon substrate (9) is obtained after development and hard baking; Step 8: PDMS that has been evacuated is poured on the silicon substrate (9), left to stand for half an hour, and then placed in a 90°C oven for baking for 1 hour; Step 9, demoulding: cut into individual PDMS sheets (2) with a cutting knife.

[0019] The production of the glass substrate (1) specifically includes the following steps: step 10, sputtering: sputtering a Ti / Pt layer with a thickness of 500 / 1500a on the entire glass substrate; step 11, coating: using a coating machine to evenly coat the LC100A photoresist (10) on the metal surface; step 12, photolithography: developing after exposing for 15s to obtain the required pattern; step 13, etching: using deep reactive ion etching to etch 2000a downward on the glass substrate to remove excess metal and only retain the Pt electrode block (3) and the electrode lead; step 14, degumming: using acetone to degumming to remove the photoresist on the electrode surface; step 15, PECVD: using A Si3N4 layer (12) with a thickness of 5000a is deposited by vapor deposition as an insulating layer; Step 16, coating: using a coating machine to evenly coat the LC100A photoresist (10) on the surface of the Si3N4 layer (12); Step 17, photolithography: after exposure for 15s, development is performed to obtain a required pattern; Step 18, etching: using deep reactive ion etching to etch downward 5000a to remove excess Si3N4 layer (12), that is, remove the Si3N4 layer (12) on the Pt layer of the lead wire (4); Step 19, degumming: using dry degumming to remove excess LC100A photoresist (10) to obtain the glass substrate (1) for standby use.

[0020] Step 20, bonding: the treated PDMS sheet (2) and the glass substrate are cleaned together, taken out, and quickly aligned and bonded under a microscope in a manner that the structural surfaces meet, and then placed on a hot plate at 105° C. and heated for 2 h to ensure that the two are firmly bonded.

[0021] In order to solve the above technical problems, the technical solution adopted by the single cell gene detection method of the present invention is:

[0022] A single cell temperature detection method, characterized by using a high-precision constant temperature water tank (13), an electrochemical workstation (14), a computer (15), and two identical sensors as described above, one of which is used as a sample injection detection sensor and the other is used as a reference sensor, specifically comprising the following steps:

[0023] Step (1), using the good and stable linear relationship between the resistance and temperature of the Pt electrode block (3), the linear relationship can be expressed as: R=XT+Y, adjusting the temperature T of the water contained in the constant temperature water tank (13), respectively T1, T2, T3...Tn, using the electrochemical workstation (14) to measure the resistance R1, R2, R3, Rn of the corresponding temperature in turn, and obtaining the X and Y values ​​in the linear relationship between the resistance and temperature of the temperature sensor Pt electrode block (3).

[0024] Step (2), using negative pressure injection mode to introduce the cell solution to be tested into the injection detection sensor, inserting a gun tip filled with culture medium at its injection hole (5) to provide a continuous supply of nutrients for the cells in the chip, placing it in a cell culture incubator and culturing it until the cells grow by adhering to the wall, placing the sensor in a constant temperature water tank (13), and placing a cell-free reference sensor in the same environment for comparative detection, and connecting the Pt electrode blocks (3) of the two sensors to an electrochemical workstation (14) and a computer (1) through their lead wires (4). 5), respectively detecting the voltage value and constant current value of the Pt electrode block (3) of the sample injection detection sensor and the reference sensor through the electrochemical workstation (14), calculating the corresponding resistance value of the Pt electrode block (3), and then calculating the temperature value of the Pt electrode block (3) of the sample injection detection sensor and the reference sensor according to the linear relationship between the resistance and temperature of the Pt electrode block (3) and its X, Y values, and the difference between the two and the fluctuation of the cell temperature over a period of time is the cell temperature value and the fluctuation of the cell temperature over a period of time.

[0025] A method for detecting temperature changes caused by certain life phenomena occurring after a single cell is stimulated, characterized in that a high-precision constant temperature water tank (13), an electrochemical workstation (14), a computer (15), and two identical sensors as described above are used, one of which is used as a sample detection sensor and the other is used as a reference sensor, and specifically comprises the following steps:

[0026] Step (1), using the good and stable linear relationship between the resistance and temperature of the Pt material, the linear relationship can be expressed as: R=XT+Y, adjusting the temperature T of the water in the constant temperature water tank (13), respectively T1, T2, T3...Tn, using the electrochemical workstation (14) to measure the resistance R1, R2, R3, Rn of the corresponding temperature in turn, and obtaining the X and Y values ​​in the linear relationship between the resistance and temperature of the Pt electrode block (3).

[0027] Step (2), maintaining the temperature of the constant temperature water tank (13) at 37°C, using the negative pressure injection mode to introduce the cell solution to be tested into the two sensors respectively, inserting the gun tips filled with culture medium at the injection holes (5) respectively, providing a continuous supply of nutrients for the cells in the chip, placing the cells in a cell culture incubator with a humidified box environment of 37°C and 5% CO2 until the cells grow attached to the wall, injecting PBS to rinse the sensor for detection, and then injecting drugs for stimulating single cells, and then sealing the injection holes (5) and the output holes (6) of the two sensors and placing them in the water of the constant temperature water tank (13), and separating the Pt electrode blocks (3) of the two sensors. The two sensors are connected to an electrochemical workstation (14) and a computer (15) respectively via their leads; after the temperature of the sensor is stabilized, the voltage value and the constant current value of the two sensors detected by the electrochemical workstation (14) are used to calculate the resistance value of the corresponding Pt electrode block (3); then, based on the linear relationship between the resistance and temperature of the Pt electrode block (3) and its X and Y values, the temperature values ​​of the Pt electrode blocks (3) of the detection sample injection detection sensor and the reference sensor are calculated; the difference between the two and their fluctuation over a period of time are the temperature change value of the single cell under drug stimulation and the temperature fluctuation of the single cell under drug stimulation over a period of time.

[0028] Further scheme: used to detect the temperature changes during apoptosis of Hela cells stimulated by the drug Cisplatin.

[0029] The single-cell temperature measurement microfluidic sensor of the present invention comprises two main functional modules: a single-cell capture and in-situ culture module, and a high-precision temperature measurement sensing module. The temperature measurement sensing module comprises a Pt electrode block (3) made of Pt and an isolation layer of Si3N4; the single-cell capture and culture module is composed of a microfluidic channel made of PDMS. Using micro-nano processing technology, a Pt1000-based microelectrode system is prepared using glass as a substrate, and a single-cell capture and culture chip is prepared using a patterned silicon wafer as a mold. The two modules are aligned and bonded to form a complete microfluidic sensor. The chip can detect tiny temperature fluctuations during the life activities of single cells (growth, proliferation, drug stimulation, etc.), and can realize online continuous monitoring of temperature indicators.

[0030] The single-cell temperature measurement microfluidic sensor of the present invention is a single-cell temperature high-precision detection chip prepared based on the MEMS process. It uses the physical property that the linearity of the metal material Pt temperature-resistance is greater than 0.999. It can also continuously monitor the temperature changes of single cells in real time online without affecting the normal physiological functions of cells. It can continuously monitor the temperature changes of cells in the process of exercising normal physiological functions in real time online. It is mainly composed of two parts of devices bonded together, namely the electrode layer on the glass sheet and the PDMS layer with specific channels. The former uses photolithography, sputtering, and wet degumming processes, while the latter uses photolithography, deep reactive ion etching (DRIE), and SU8 glue photolithography processes. Although both are photolithography processes, there are obvious differences in the processing of the two. In order to facilitate the observation of cell morphology under a microscope, the electrode part is sputtered on the glass sheet, and a layer of Si3N4 is made on the Pt electrode to prevent the electrode from conducting electricity after contacting water. After the above is completed, the glass sheet is diced. After the required structure is photoetched on the silicon wafer, the pattern structure on the silicon wafer mold is transferred to the PDMS through the epoxy resin inverting process. At this time, the required microfluidic chip channel will appear on the PDMS film, and the cured PDMS film is cut and punched with a cutting knife. Finally, the treated PDMS chip and the scribed electrode chip are plasma cleaned, and after cleaning, they are quickly aligned and bonded under a microscope, and then placed on a 105℃ hot plate and heated for 2 hours to ensure that the two are firmly bonded.

[0031] Since the diameter of the cell is greater than the channel height under the C-type dam structure, the cell can be trapped in the C-type dam structure, and the basal layer in the structure is covered with Pt electrodes, so the microfluidic chip can accurately measure the temperature changes during the growth and proliferation of adherent cells. The chip with sampled cells is placed in a constant temperature water tank, and the constant current method of the electrochemical workstation is used to measure the changes in cell temperature. Through this measure of maintaining the stable ambient temperature and the high-performance Pt microelectrode array device, the thermal stability of the system is achieved, and the temperature changes of cells in normal physiological activities (metabolism, division, apoptosis, etc.) and under drug stimulation are detected in real time. In addition, the present invention can be mass-produced, has low cost, high detection sensitivity, and has important practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a three-dimensional schematic diagram of the appearance of the single-cell temperature detection sensor of the present invention.

[0033] Figure 2 It is a three-dimensional schematic diagram of the separation state of the PDMS sheet and the glass substrate of the single-cell temperature detection sensor of the present invention.

[0034] Figure 3 This is a three-dimensional schematic diagram of the PDMS sheet.

[0035] Figure 4It is a three-dimensional schematic diagram of a glass substrate.

[0036] Figure 5 This is a top view of a C-type dam.

[0037] Figure 6 This is a top view of the square dam.

[0038] Figure 7 Schematic diagram of the changes in cross-sectional shape during each step of the PDMS sheet production process.

[0039] Figure 8 Schematic diagram of the changes in cross-sectional shape during each step in the glass substrate manufacturing process.

[0040] Fig. 9 Schematic diagram of the single-cell temperature detection system.

[0041] Fig.10 for Figure 4 Enlarged schematic diagram of part A in the middle.

[0042] Figure 7 , Figure 8 It is schematic and does not correspond to the actual cross-sectional shape of the PDMS sheet or glass substrate. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below with reference to the accompanying drawings.

[0044] The single cell temperature detection sensor of the present invention is as follows: Figure 1 As shown, it includes a glass substrate 1 and a PDMS sheet 2 bonded to each other, and a Pt electrode block 3 and its lead wire 4 for conduction and temperature sensing are sputtered on the bonding surface of the glass substrate 1. Figure 2 As shown, the bonding surface of the PDMS sheet 2 is a grooved structure surface, and a flow channel 7 is provided on the bonding surface of the PDMS sheet 2. After bonding with the glass substrate 1, a flow channel for the single cell solution to flow through during detection is formed on the glass substrate 1. Through holes are respectively provided at both ends of the flow channel 7 to penetrate the PDMS sheet 2, forming a sample inlet 5 and a sample outlet 6. Figure 3 As shown, an opening dam is provided in the flow channel 7 for the single cell to flow in and be retained, and the opening of the opening dam faces the injection direction. The opening dam is located above the Pt electrode block 3 on the glass substrate 1, and a height gap is reserved between the opening dam and the glass substrate 1 for the sample fluid and air to flow through during detection, and the height gap is smaller than the outer diameter of the single cell to be detected.

[0045] like Figure 3 , Figure 5As shown, the opening dam is a C-shaped ring-shaped C-shaped dam 8 with an opening, and the opening width of the C-shaped dam 8 is greater than the inner radius of the C-shaped dam 8; the height of the opening dam is 50um. Because most cells have a diameter of 10-20um, the height gap between the opening dam and the glass substrate 1 is set to 5um. The inner diameter of the C-shaped dam 8 is 150um. Alternatively, Figure 6 As shown, the opening dam is a square dam with one side open and a square ring in the shape of a U. The opening width of the square dam is greater than half of the inner side length of the opening side of the C-shaped dam 8, and the inner side length of the square dam is 150um. Because most single cells have a diameter of 10-20um, the single cells to be tested cannot pass through the gap and are confined in the C-shaped ring. Therefore, the special C-shaped structure can fix the cells on the microelectrode. At the same time, due to the existence of a 5um height gap between the opening dam and the glass substrate 1, it does not affect the circulation of air and liquid, so it does not affect the survival of the single cells to be tested. The same principle applies to the use of a square dam. Figure 3 As shown, the width of the flow channel 7 should be at least slightly larger than the outer diameter of the opening dam, and the flow channel 7 may be provided with an arc-shaped notch 16 at the location of the opening dam to facilitate the continued forward flow of the cell solution to be tested.

[0046] like Figure 3 As shown, the flow channel 7 includes a sample inlet section 7A, a sample outlet section 7B and two symmetrically distributed detection sections 7C. The detection section 7C flow channel 7 is surrounded by a rectangle. The lower flow channel 7 of the rectangle is connected to the sample inlet hole 5 through the sample inlet section 7A flow channel 7, and the upper flow channel 7 of the rectangle is connected to the sample outlet hole 6 through the sample outlet section 7B flow channel 7. Opening dams are respectively arranged in the two side flow channels 7 of the rectangle.

[0047] like Figure 4 As shown, the Pt electrode block 3 includes a Ti layer with a thickness of 500a sputtered on the glass substrate 1 as an adhesion layer, and a Pt layer with a thickness of 1500a sputtered to form a Ti / Pt working layer with a thickness of 500 / 1500a, and a Si3N4 layer 12 with a thickness of 5000a is deposited on the Ti / Pt working layer as an insulating layer. Fig.10 As shown in FIG. 1 , the Pt electrode block 3 is in the shape of a Pt wire that meanders back and forth. Figure 4 As shown, the two ends of the Pt electrode block 3 are respectively connected to its two side lead wires 4, and the two side lead wires 4 are arranged in a herringbone shape. The Pt electrode block 3 is located at the top of the herringbone, and the bottom ends of the two side lead wires 4 are located at the side edge of the glass substrate 1.

[0048] The method for manufacturing a single-cell temperature detection sensor of the present invention includes the manufacturing of a PDMS sheet 2 and a glass substrate 1, which can be performed in no particular order, and bonding the two. The manufacturing of the PDMS sheet 2 first includes the preparation of a silicon substrate 9 as a mold thereof and the manufacturing of the PDMS sheet 2.

[0049] like Figure 7 As shown, the preparation of the PDMS sheet 2 specifically includes the following steps:

[0050] Step 1, pretreatment of silicon substrate 9: 98% H2SO4 and 30% H2O2 are mixed in a ratio of 7:3 to prepare a piranha solution, and the silicon substrate 9 is continuously immersed in the 120°C piranha solution for 30 minutes, then the silicon substrate 9 is taken out and repeatedly washed with deionized water, and the silicon substrate 9 is placed in a spin dryer to dry the surface moisture for later use; see Figure 7-1 .

[0051] Step 2, coating: Use a coating machine to evenly coat the LC100A photoresist 10 on the surface of the silicon substrate 9; Figure 7-2 .

[0052] Step 3, photolithography: After exposure for 15 seconds, develop to obtain the required pattern; see Figure 7-3 .

[0053] Step 4, etching: using deep reactive ion etching, the silicon substrate 9 is etched down to a depth of 5 μm; see Figure 7-4 .

[0054] Step 5, stripping: Use piranha solution to remove LC100A photoresist 10; see Figure 7-5 .

[0055] Step 6, glue coating: Spin SU8-3050 photoresist 11 on the cleaned silicon substrate 9 with a thickness of 50um and leave it horizontally for 2 hours; Figure 7-6 .

[0056] Step 7, photolithography: After exposure for 10 seconds, the desired silicon substrate 9 is obtained after development and hard baking; see Figure 7-7 .

[0057] Step 8: Pour the vacuumed PDMS on the silicon substrate 9, see Figure 7-8 , after standing for half an hour, put it in a 90℃ oven for 1 hour.

[0058] Step 9, demoulding: Use a cutting knife to cut into individual PDMS sheets 2, see Figure 7-9 .

[0059] like Figure 8 As shown, the production of the glass substrate 1 specifically includes the following steps:

[0060] Step 10, sputtering: sputtering a Ti / Pt layer with a thickness of 500 / 1500a on the entire glass substrate; see Figure 8-2 .

[0061] Step 11, glue coating: Use a glue coating machine to evenly coat the LC100A photoresist 10 on the metal surface; see Figure 8-3 .

[0062] Step 12, photolithography: After exposure for 15 seconds, develop to obtain the required pattern; see Figure 8-4 .

[0063] Step 13, etching: Use deep reactive ion etching to etch downward 2000a on the glass substrate to remove excess metal, leaving only the Pt electrode block (3) and the electrode lead; see Figure 8-5 .

[0064] Step 14: Remove the photoresist from the electrode surface using acetone. Figure 8-6 .

[0065] Step 15, PECVD: Deposit a Si3N4 layer 12 with a thickness of 5000a by vapor deposition as an insulating layer; see Figure 8-7 .

[0066] Step 16, glue coating: Use a glue coating machine to evenly coat the LC100A photoresist 10 on the surface of the Si3N4 layer 12; see Figure 8-8 .

[0067] Step 17, photolithography: After exposure for 15 seconds, develop to obtain the required pattern; see Figure 8-9 .

[0068] Step 18, etching: Use deep reactive ion etching to etch down 5000a to remove the excess Si3N4 layer 12, that is, remove the Si3N4 layer 12 on the Pt layer of the lead wire 4; see Figure 8-10 .

[0069] Step 19: Remove the excess LC100A photoresist 10 by dry stripping to obtain the glass substrate 1 for later use. Figure 8-1 1.

[0070] Step 20, bonding: clean the treated PDMS sheet 2 and the glass substrate together, take them out, quickly align and bond them under a microscope in a way that the structural surfaces meet, and then place them on a hot plate at 105° C. and heat them for 2 hours to ensure that the two are firmly bonded.

[0071] The single cell temperature detection method of the present invention is as follows: Fig. 9 As shown, a high-precision constant temperature water tank 13, an electrochemical workstation 14, a computer 15, and two identical sensors as any of the above are used, one of which is used as a sample detection sensor and the other is used as a reference sensor. The water temperature fluctuation range in the high-precision constant temperature water tank 13 is ±0.002°C. The detection specifically includes the following steps:

[0072] Step (1), using the good and stable linear relationship between the resistance and temperature of the Pt electrode block 3, when the chip temperature changes, the resistance will change accordingly, and its linear relationship can be expressed as: R=XT+Y, adjust the temperature T of the water in the constant temperature water tank 13, respectively T1, T2, T3...Tn, use the electrochemical workstation 14 to measure the resistance R1, R2, R3, Rn of the corresponding temperature in turn, and obtain the X and Y values ​​in the linear relationship between the resistance and temperature of the temperature sensor Pt electrode block 3. The water temperature T1, T2, T3...Tn in the constant temperature water tank can be set to 20℃, 25℃, 30℃, 35℃, 40℃ respectively, and the resistance of the corresponding temperature can be measured in turn using the electrochemical workstation, and then the X and Y values ​​in the linear relationship between the resistance and temperature of the temperature sensor Pt electrode block 3 can be obtained. For example, R=4.13757T+2239.66932.

[0073] Step (2), using negative pressure injection mode to introduce the cell solution to be tested into the injection detection sensor, inserting a gun tip filled with culture fluid at its injection hole 5 to provide a continuous supply of nutrients for the cells in the chip, placing the sensor in a cell culture incubator and culturing until the cells grow attached to the wall, placing the sensor in a constant temperature water tank 13, and placing a cell-free reference sensor in the same environment for comparative detection, the Pt electrode blocks 3 of the two sensors are respectively connected to the electrochemical workstation 14 and the computer 15 through their lead wires 4, and the voltage value and constant current value of the Pt electrode block 3 of the injection detection sensor and the reference sensor are respectively detected by the electrochemical workstation 14, and the corresponding resistance value of the Pt electrode block 3 is calculated, and then the temperature value of the Pt electrode block 3 of the injection detection sensor and the reference sensor is calculated according to the linear relationship between the resistance and temperature of the temperature sensor and its X, Y values, and the difference between the two and the fluctuation of the cell temperature over a period of time is the cell temperature value and the fluctuation of the cell temperature over a period of time.

[0074] The present invention can also detect the temperature change caused by certain life phenomena after a single cell is stimulated, using a high-precision constant temperature water tank 13, an electrochemical workstation 14, a computer 15, and two identical sensors such as any of the above, one of which is used as a sample detection sensor and the other as a reference sensor, specifically including the following steps:

[0075] Step (1), using the good and stable linear relationship between the resistance and temperature of the Pt material, the linear relationship can be expressed as: R=XT+Y, adjusting the temperature T of the water contained in the constant temperature water tank 13 to T1, T2, T3...Tn, respectively, using the electrochemical workstation 14 to measure the resistance R1, R2, R3, Rn of the corresponding temperature in turn, and obtaining the X and Y values ​​in the linear relationship between the resistance and temperature of the temperature sensor.

[0076] Step (2), maintaining the temperature of the constant temperature water tank 13 at 37°C, using the negative pressure injection mode to introduce the cell solution to be tested into the two sensors respectively, inserting the gun tips filled with culture medium at the injection holes 5 respectively, providing a continuous supply of nutrients for the cells in the chip, placing the chip in a cell culture incubator with a humidified box environment of 37°C and 5% CO2 until the cells grow attached to the wall, injecting PBS into the sensor for testing to rinse, and then injecting drugs used to stimulate single cells, and then sealing the injection holes 5 and the outlet holes 6 of the two sensors and placing them in the water of the constant temperature water tank 13, and the Pt electrode blocks 3 of the two sensors are respectively The leads are connected to an electrochemical workstation 14 and a computer 15 respectively; after the temperature of the sensor is stabilized, the voltage value and the constant current value of the two sensors detected by the electrochemical workstation 14 are used to calculate the corresponding resistance value of the Pt electrode block 3, and then the temperature value of the Pt electrode block 3 of the detection sample detection sensor and the reference sensor is calculated based on the linear relationship between the resistance and temperature of the temperature sensor and its X, Y values. The difference between the two and its fluctuation over a period of time are the temperature change value of the single cell under drug stimulation and the temperature fluctuation of the single cell under drug stimulation over a period of time.

[0077] For example, the present invention can be used to detect the temperature change of Hela cells during apoptosis under the stimulation of the drug Cisplatin: set the temperature of the thermostatic water tank to 37°C. Inject the Hela cells into the sterilized sensor, and culture the cells in an incubator (37°C, 5% CO2 humid environment) for 24 hours. Observe the cells. If they have grown attached to the wall, prepare a sensor with only Hela cells and culture medium for comparison, and inject PBS into the other sensor for washing, and then inject the drug Cisplatin, which can kill Hela cells. Stimulate the Hela cells with drugs, measure and compare the two chips in the same environment, and eliminate the influence of external environmental interference factors on the temperature change of the cells. Seal the inlet / outlet ports of the two chips and put them into the thermostatic water tank. After the chip temperature stabilizes, the constant current method of the electrochemical workstation is used to measure the voltage fluctuation, and the resistance is calculated based on the constant current value. The temperature fluctuation is then calculated based on the known calibration results. Since Cisplatin will cause Hela cells to gradually die, the temperature changes of Hela cells during apoptosis under the stimulation of Cisplatin can be measured.

Claims

1. A single cell temperature detection sensor, comprising a glass substrate (1) and a PDMS sheet (2) bonded to each other, a Pt electrode block (3) and its lead wire (4) for conduction and temperature sensing being sputtered on the bonding surface of the glass substrate (1), the bonding surface of the PDMS sheet (2) being a grooved structure surface, a flow channel (7) being provided on the bonding surface of the PDMS sheet (2), and a flow channel for a single cell solution to flow through during detection is formed on the glass substrate (1) after bonding with the glass substrate (1), through holes penetrating the PDMS sheet (2) are provided at both ends of the flow channel (7) to form a sample inlet (5) and a sample outlet (6); characterized in that: The flow channel (7) is provided with an opening dam for the single cell to flow in and be retained, and the opening of the opening dam faces the injection direction; the opening dam is located above the Pt electrode block (3) on the glass substrate (1), and a height gap is reserved between the opening dam and the glass substrate (1) for the sample fluid and air to flow through during detection, and the height gap is smaller than the outer diameter of the single cell to be detected; the opening dam is a C-shaped dam (8) with an opening in the shape of a C-shaped circular ring, and the opening width of the C-shaped dam (8) is greater than the inner radius of the C-shaped dam (8); or the opening dam is a square dam with an opening on one side in the shape of a square ring, and the opening width of the square dam is greater than half of the inner side length of the opening side of the C-shaped dam (8); the flow channel (7) includes an injection port. The invention relates to a glass substrate (1) and a glass substrate having a plurality of flow channels (7A), a sample outlet section (7B), and two symmetrically distributed detection sections (7C); the flow channel (7) of the detection section (7C) is surrounded by a rectangle; the lower flow channel (7) of the rectangle is connected to the sample injection hole (5) via the flow channel (7) of the sample injection section (7A); the upper flow channel (7) of the rectangle is connected to the sample outlet hole (6) via the flow channel (7) of the sample outlet section (7B); the opening dams are respectively arranged in the two side flow channels (7) of the rectangle; the Pt electrode block (3) is in the shape of a Pt wire that meanders back and forth; the two ends of the Pt electrode block (3) are respectively connected to its two side lead wires (4); the two side lead wires (4) are arranged in a herringbone shape; the Pt electrode block (3) is located at the top of the herringbone shape; and the bottom ends of the two side lead wires (4) are located at the side edge of the glass substrate (1).

2. The single cell temperature detection sensor according to claim 1, characterized in that: The height of the opening dam is 50um, and the height gap between the opening dam and the glass substrate (1) is 5um; the inner diameter of the C-shaped dam (8) is 150um, or the inner side length of the square dam is 150um.

3. The single cell temperature detection sensor according to claim 1, characterized in that: The Pt electrode block (3) comprises a Ti layer with a thickness of 500a which is first sputtered on the glass substrate (1) as an adhesion layer, and a Pt layer with a thickness of 1500a which is sputtered to form a Ti / Pt working layer with a thickness of 500 / 1500a, and a Si3N4 layer (12) with a thickness of 5000a is deposited on the Ti / Pt working layer as an insulating layer.

4. The method for manufacturing a single-cell temperature detection sensor as claimed in claim 1, comprising the manufacturing of a PDMS sheet (2) and a glass substrate (1), which can be carried out in no particular order, and bonding the two. The manufacturing of the PDMS sheet (2) first includes the preparation of a silicon substrate (9) as a mold thereof and the manufacturing of the PDMS sheet (2), characterized in that: The preparation of the PDMS sheet (2) specifically includes the following steps: Step 1, pretreatment of the silicon substrate (9): 98% H2SO4 and 30% H2O2 are mixed in a ratio of 7:3 to prepare a piranha solution, and the silicon substrate (9) is continuously immersed in the 120°C piranha solution for 30 minutes, then the silicon substrate (9) is taken out and repeatedly washed with deionized water, and the silicon substrate (9) is placed in a spin dryer to dry the surface moisture for standby use; Step 2, glue coating: LC100A photoresist (10) is uniformly coated on the surface of the silicon substrate (9) using a glue coating machine; Step 3, photolithography: after exposure for 15 seconds, development is performed to obtain a desired pattern; Step 4, etching: using a deep reactive ion etching method, the silicon substrate (9) is etched downward 5um depth; Step 5, degumming: use piranha solution to remove LC100A photoresist (10); Step 6, glue coating: SU8-3050 photoresist (11) is applied on the cleaned silicon substrate (9) with a thickness of 50um, and left to stand horizontally for 2 hours; Step 7, photolithography: after exposure for 10 seconds, the required silicon substrate (9) is obtained after development and hard baking; Step 8: PDMS that has been evacuated is poured on the silicon substrate (9), left to stand for half an hour, and then placed in a 90°C oven for baking for 1 hour; Step 9, demoulding: cut into individual PDMS sheets (2) with a cutting knife; The production of the glass substrate (1) specifically includes the following steps: step 10, sputtering: sputtering a Ti / Pt layer with a thickness of 500 / 1500a on the entire glass substrate; step 11, coating: using a coating machine to evenly coat the LC100A photoresist (10) on the metal surface; step 12, photolithography: developing after exposing for 15s to obtain the required pattern; step 13, etching: using deep reactive ion etching to etch 2000a downward on the glass substrate to remove excess metal and only retain the Pt electrode block (3) and the electrode lead; step 14, degumming: using acetone to degumming to remove the photoresist on the electrode surface; step 15, PECVD: using A Si3N4 layer (12) with a thickness of 5000a is deposited by vapor deposition as an insulating layer; step 16, coating: using a coating machine to evenly coat the LC100A photoresist (10) on the surface of the Si3N4 layer (12); step 17, photolithography: developing after exposure for 15s to obtain a desired pattern; step 18, etching: using deep reactive ion etching to etch downward 5000a to remove excess Si3N4 layer (12), that is, remove the Si3N4 layer (12) on the Pt layer of the lead wire (4); step 19, degumming: using dry degumming to remove excess LC100A photoresist (10) to obtain the glass substrate (1) for standby use; Step 20, bonding: clean the treated PDMS sheet (2) and the glass substrate (1) together, take them out, quickly align and bond them under a microscope in a way that the structural surfaces meet, and then place them on a hot plate at 105° C. and heat for 2 hours to ensure that the two are firmly bonded.

5. A single cell temperature detection method, characterized in that: Using a high-precision constant temperature water tank (13), an electrochemical workstation (14), a computer (15), and two identical sensors as described in any one of claims 1 to 3, one of which is used as a sample injection detection sensor and the other as a reference sensor, the following steps are specifically included: Step (1), using the good and stable linear relationship between the resistance and temperature of the Pt electrode block (3), the linear relationship is expressed as: R=XT+Y, adjusting the temperature T of the water in the constant temperature water tank (13), respectively T1, T2, T3...Tn, using the electrochemical workstation (14) to measure the resistance R1, R2, R3, Rn of the corresponding temperature in turn, and obtaining the X and Y values ​​in the linear relationship between the resistance and temperature of the Pt electrode block (3); Step (2), using negative pressure injection mode to introduce the cell solution to be tested into the injection detection sensor, inserting a gun tip filled with culture fluid at the injection hole (5) thereof to provide a continuous supply of nutrients for the cells in the chip, placing the sensor in a cell culture box and culturing until the cells grow by adhering to the wall, placing the sensor in a constant temperature water tank (13), and placing a cell-free reference sensor in the same environment for comparative detection, the Pt electrode blocks (3) of the two sensors are respectively connected to an electrochemical workstation (14) and a computer (15) via their lead wires (4), the voltage value and constant current value of the Pt electrode block (3) of the injection detection sensor and the reference sensor are respectively detected by the electrochemical workstation (14), and the corresponding resistance value of the Pt electrode block (3) is calculated, and then the temperature value of the Pt electrode block (3) of the injection detection sensor and the reference sensor is calculated according to the linear relationship between the resistance and temperature of the temperature detection sensor and its X, Y values, and the difference between the two and the fluctuation of the cell temperature over a period of time is the cell temperature value and the fluctuation of the cell temperature over a period of time.

6. A method for detecting temperature changes caused by certain life phenomena in a single cell after being stimulated, characterized in that: Using a high-precision constant temperature water tank (13), an electrochemical workstation (14), a computer (15), and two identical sensors as described in any one of claims 1 to 3, one of which is used as a sample injection detection sensor and the other as a reference sensor, the following steps are specifically included: Step (1), using the good and stable linear relationship between the resistance and temperature of the Pt material, the linear relationship is expressed as: R=XT+Y, adjusting the temperature T of the water in the constant temperature water tank (13) to T1, T2, T3...Tn, respectively, using the electrochemical workstation (14) to measure the resistance R1, R2, R3, Rn of the corresponding temperature in turn, and obtaining the X and Y values ​​in the linear relationship between the resistance and temperature of the temperature detection sensor; Step (2), maintaining the temperature of the constant temperature water tank (13) at 37°C, using the negative pressure injection mode to introduce the cell solution to be tested into the two sensors respectively, inserting the gun tips filled with culture medium at the injection holes (5) respectively, providing a continuous supply of nutrients for the cells in the chip, placing the cells in a cell culture incubator with a humidified box environment of 37°C and 5% CO2 until the cells grow attached to the wall, injecting PBS to rinse the sensor for detection, and then injecting drugs used to stimulate single cells, and then sealing the injection holes (5) and the output holes (6) of the two sensors and placing them in the water of the constant temperature water tank (13), and the Pt electrode blocks (3) of the two sensors are respectively The leads are connected to an electrochemical workstation (14) and a computer (15) respectively; after the temperature of the sensor is stabilized, the voltage value and the constant current value of the two sensors detected by the electrochemical workstation (14) are used to calculate the resistance value of the corresponding Pt electrode block (3); then, based on the linear relationship between the resistance and temperature of the temperature detection sensor and its X and Y values, the temperature values ​​of the Pt electrode blocks (3) of the detection injection detection sensor and the reference sensor are calculated; the difference between the two and the fluctuation over a period of time are the temperature change value of the single cell under drug stimulation and the temperature fluctuation of the single cell under drug stimulation over a period of time.

7. The detection method according to claim 6, characterized in that Used to detect the changes in temperature during apoptosis of Hela cells stimulated by the drug Cisplatin.

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