A MEMS sensor chip for cell temperature measurement and its preparation method

Abstract

The present invention discloses a MEMS sensor chip for cell temperature measurement and a preparation method thereof. The sensor chip includes a substrate, on which a polyimide film, a Cr adhesion layer, a Pt resistance layer, and a silicon nitride encapsulation layer are sequentially provided. The sensor chip is prepared using MEMS technology and subjected to surface silanization treatment. Cells adhere and grow on the nano-modified surface of the sensor. The change in cell temperature causes a change in the resistance value of the sensor. Through the resistance-temperature conversion relationship of the sensor, the resistance signal is converted into a change in the sensor temperature, realizing in-situ detection of cell temperature. The sensor has excellent biocompatibility, facilitating cell adhesion and growth and enabling detection of weak thermal signals of cells, which is of great importance for obtaining cell information, drug screening, and clinical medicine.

Description

Technical Field

[0001] The present invention relates to a MEMS sensor chip for cell temperature measurement and a preparation method thereof, belonging to the technical fields of cell informatics, cell temperature measurement, and Pt resistance temperature sensors. Background Art

[0002] The activities of cells are accompanied by the transfer and conversion of energy. The change in energy causes the change of cell temperature. Monitoring and analyzing the cell temperature signal helps to understand the life activities of cells. In addition, being stimulated by external signals such as drugs will cause the change of cell temperature. Collecting and analyzing the cell stress heat signal is of great significance for drug screening, clinical medicine and other fields. Cell temperature measurement can be divided into invasive temperature sensors and non-invasive cell temperature sensors. Invasive cell temperature measurement includes fluorescent polymers, fluorescent proteins, quantum dots, nanodiamonds, etc. Its operation is complex, and the way of invading cells may affect the normal metabolism of cells, which is not conducive to obtaining cell information. Non-invasive contact temperature measurement is a temperature measurement method in which cells adhere to grow on the surface of the sensor, and the cells transfer the heat signal to the temperature sensor through the contact interface, mainly including thermocouples and resistance temperature sensors. Among these existing non-invasive contact cell temperature sensors, there are problems such as large thermal resistance and heat capacity of the sensors, unreasonable structural designs of some sensors, or being not conducive to the adherent growth of cells, etc. The sensors cannot effectively obtain the weak heat signal of cells and cannot quickly and accurately detect the cell temperature signal during cell temperature measurement. Summary of the Invention

[0003] Object of the Invention: Aiming at the problems existing in the prior art, the first object of the present invention is to provide a MEMS sensor chip for cell temperature measurement, and the second object of the present invention is to provide a preparation method of a MEMS sensor chip for cell temperature measurement. The MEMS sensor chip of the present invention has small thermal resistance and heat capacity, a nano-layer on the surface that is conducive to the adherent growth of cells, stable performance, and can monitor the temperature signal of cells for a long time.

[0004] Technical Solution: The MEMS sensor chip for cell temperature measurement of the present invention, the MEMS sensor chip for cell temperature measurement includes a substrate (1), and a polyimide film (2), a Cr adhesion layer (3), a Pt resistance layer (4), and a silicon nitride encapsulation layer (5) are sequentially provided on the substrate (1).

[0005] Among them, the thickness of the Cr adhesion layer (3) is 5 - 10 nm, the thickness of the Pt resistance layer (4) is 80 - 110 nm, and the thickness of the silicon nitride encapsulation layer (5) is 150 - 200 nm.

[0006] The preparation method of a MEMS sensor chip for cell temperature measurement according to the present invention comprises the following steps:

[0007] (1) Pretreat the substrate to obtain a pretreated substrate;

[0008] (2) Spin-coat the polyimide solution on the pretreated substrate and cure it in a nitrogen oven to obtain a substrate with a polyimide film;

[0009] (3) Spin-coat AZ5214 photoresist on the polyimide film of the substrate in step (2), perform photolithography and development through a positive photoresist reversal process, and transfer the sensor pattern on the mask plate to the polyimide film of the substrate;

[0010] (4) Use an electron beam evaporation process to sequentially prepare a Cr adhesion layer and a Pt resistance layer on the upper part of the pattern of the polyimide film;

[0011] (5) Immerse the chip obtained in step (4) in an acetone solution, perform ultrasonic treatment and immersion, and remove the excess photoresist and excess metal through a lift-off process to obtain the Pt resistance of the sensor chip;

[0012] (6) Shield the lead electrodes of the Pt resistance, and use ICPCVD technology to prepare a silicon nitride encapsulation layer in the detection area of the Pt resistance to obtain a sensor chip;

[0013] (7) Perform plasma treatment on the surface of the sensor chip in an oxygen atmosphere, then immerse it in an absolute ethanol solution containing 3-(2-aminoethylamino)propyltrimethoxysilane, let it stand, wash it with pure water and dry it with nitrogen to obtain a MEMS sensor chip for cell temperature measurement.

[0014] Among them, in step (1), the substrate is a glass sheet.

[0015] Among them, in step (1), the pretreatment is to ultrasonically clean the substrate successively with acetone and isopropyl alcohol, wash the substrate with pure water, dry it with nitrogen, and perform plasma treatment in an oxygen atmosphere.

[0016] Among them, the time for ultrasonic cleaning is 3 - 5 min.

[0017] Among them, when performing plasma treatment, the equipment power is 150 - 300 W and the treatment time is 3 - 5 min.

[0018] Among them, in step (2), when spin-coating the polyimide solution, the spin-coating is performed at a speed of 500 - 600 rpm for 3 - 5 s, and then at a speed of 3000 - 3500 rpm for 30 - 35 s.

[0019] Among them, in step (2), during curing, it is kept at 120 - 125 °C for 1 h, 200 - 210 °C for 2 h, and 250 - 260 °C for 2.5 h for curing respectively.

[0020] Among them, in step (3), when spin - coating the photoresist, it rotates at 500 - 600 rpm for 4 - 5 seconds first, and then rotates at 3500 - 4000 rmp for 25 - 30 seconds.

[0021] Among them, in step (5), the ultrasonic time is 5 - 10 min, and the continued soaking time is 24 - 48 h.

[0022] Among them, in step (6), the obtained sensor chip can be divided into multiple sensor chips by a dicing machine.

[0023] Among them, in step (7), the power for plasma treatment on the surface of the sensor chip in an oxygen atmosphere is 150 - 250 W, and the time is 3 - 5 min.

[0024] Among them, in step (7), the concentration of 3-(2 - aminoethylamino)propyltrimethoxysilane in the anhydrous ethanol solution containing 3-(2 - aminoethylamino)propyltrimethoxysilane is 1 - 2%.

[0025] Among them, in step (7), the standing time is 8 - 12 h.

[0026] The silanization principle of the present invention: As Figure 1 shown, after plasma treatment, a large number of hydroxyl groups are carried on the surface of the silicon nitride encapsulation layer within a certain time. Immerse it in the anhydrous ethanol solution of 3-(2 - aminoethylamino)propyltrimethoxysilane, and the Si - OH bonds on the surface of the silicon nitride encapsulation layer hydrolyze and condense with the Si - OH bonds of 3-(2 - aminoethylamino)propyltrimethoxysilane to form Si - O - Si bonds, thereby forming a single - molecule self - assembly layer with a thickness of only a few to dozens of nanometers on the sensor surface. The other end of 3-(2 - aminoethylamino)propyltrimethoxysilane contains an amino functional group, and the amino group carries a positive charge, which can interact with the negative charge on the cell through charge adsorption, contributing to the adherent growth of the cell.

[0027] The present invention uses polyimide as the substrate thermal barrier layer, which can reduce the heat dissipation of the sensor when detecting the thermal signal of the cell, contributing to the perception of the cell temperature. The thickness of the platinum resistor is at the nanometer level, which can effectively reduce the heat capacity of the sensor resistance, contributing to the detection of weak thermal signals by the sensor. Compared with other sensors, using a nanometer - level silicon nitride thin film as the insulating layer (encapsulation layer) is beneficial to the conduction of cell heat in the direction of the sensor resistance. The present invention uses the silanization treatment technology to make the surface of the silicon nitride insulating layer carry amino groups, and the amino groups are beneficial to the adsorption of cells on the surface layer of the sensor.

[0028] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0029] The present invention uses MEMS technology to prepare a surface-silanized sensor, which is beneficial for cells to adhere and grow on the nano-modified surface of the sensor; in addition, the heat transfer layer with a nanoscale thickness is beneficial for the sensor to perceive the thermal signals of cells. When using the sensor prepared by the present invention, the change in cell temperature causes a change in the resistance value of the sensor, and the resistance signal is converted into a change in the sensor temperature through the resistance-temperature conversion relationship of the sensor, enabling in-situ detection of cell temperature. The sensor of the present invention has excellent biocompatibility, facilitates cell adhesion and growth, and can detect weak thermal signals of cells, which is of great value for obtaining cell information, drug screening, and clinical medicine. Brief description of the drawings

[0030] Figure 1 It is a schematic diagram of the silanization principle of the present invention;

[0031] Figure 2 It is a schematic diagram of the MEMS sensor chip structure for measuring cell temperature of the present invention;

[0032] Figure 3 It is a composition analysis diagram of the surface of the sensor chip by SEM-EDS of the present invention;

[0033] Figure 4 It is a calibration and stability test diagram of the sensor chip of the present invention;

[0034] Figure 5 It is a diagram of the sensor chip of the present invention for detecting HEK-293T cells. Detailed implementation manners

[0035] The technical solutions of the present invention will be further described below in conjunction with the drawings.

[0036] Example 1 MEMS sensor chip for measuring cell temperature

[0037] As Figure 2 shown, Figure 2Schematic structural diagram of the MEMS sensor chip for measuring cell temperature in the present invention; the sensor chip of the present invention includes a glass substrate 1 of model B270, a polyimide film 2, a Cr adhesion layer 3 with a thickness of 10 nm in sequence and a Pt resistance layer 4 with a thickness of 100 nm, and a silicon nitride encapsulation layer 5 with a thickness of 200 nm. The Pt resistance temperature measurement area is 120 μm × 150 μm, and both the line width and the line pitch are 3 μm. Such line width and line pitch can ensure that there is always a part of the cell volume growing on the Pt resistance in the detection area. The thickness of the platinum resistance layer 4 is 100 nm. Such thickness is several times the electron mean free path of platinum, which can reduce the heat capacity and thermal resistance of the resistance sensor as much as possible while ensuring a relatively high temperature coefficient of resistance of the platinum resistance, making it suitable for detecting weak thermal signals such as cells.

[0038] Example 2 Preparation of the MEMS sensor chip for measuring cell temperature

[0039] (1) Use acetone and isopropanol to ultrasonically clean a 4-inch B270 glass sheet with a thickness of 0.5 mm for 5 minutes each in sequence. Then wash the glass sheet with pure water and dry it with nitrogen. Plasma-treat the washed glass sheet in an oxygen atmosphere. The power of the plasma equipment is selected as 250 W, and the plasma treatment time is 5 minutes to obtain a pretreated glass substrate.

[0040] (2) Adsorb the glass substrate on a spin coater, and pour polyimide liquid covering the chip area on the glass substrate. Spin coat at 500 rpm for 5 s and then at 3000 rpm for 30 s to make the polyimide evenly cover the glass surface. Then put the glass substrate coated with polyimide liquid into a nitrogen oven, set the nitrogen oven temperature to 120 °C, 200 °C and 250 °C in sequence, and the holding times are 1 h, 2 h and 2.5 h respectively to cure the polyimide and obtain a glass substrate with a polyimide film.

[0041] (3) Place the glass substrate with the polyimide film on a spin coater, and drop AZ5214 photoresist on the glass substrate. Spin coat at 600 rpm for 5 s and 4000 rmp for 30 s respectively for spin coating operation, and then bake on a 95 °C hot plate for 90 s. Through the positive photoresist inversion process, place the sensor mask and the glass substrate at the corresponding positions in the lithography machine, expose for 4 s, take out the glass substrate and bake at 110 °C for 90 s. Put the glass substrate back into the lithography machine, perform overexposure on the glass substrate for 45 s. After completing this operation, develop the glass substrate with JZ3038 developer for 45 s, rinse with pure water and dry with nitrogen to complete the patterning of the sensor pattern on the polyimide film of the glass substrate.

[0042] (4) Prepare a chromium (Cr) adhesion layer and a platinum (Pt) resistance layer on the upper part of the sensor pattern in sequence by electron beam evaporation process: Place the patterned glass substrate into an electron beam evaporation equipment (Ei-5z, ULVAC, JAPAN), and deposit a 10-nm-thick Cr layer as the adhesion layer and a 100-nm-thick Pt material resistance layer in sequence under a vacuum of 5×10 -4 Pa.

[0043] (5) Based on the Lift-off process, place the glass substrate with the metal layer into an acetone solution and perform ultrasonic treatment for 10 min, and then soak it for 48 h to strip the excess photoresist and metal on the glass surface, obtaining the Pt resistance of the sensor.

[0044] (6) Use a polyimide tape with a width of 3 mm to cover the lead area of the platinum resistance sensor on the glass, block the lead electrodes of the Pt resistance, and place it into an ICPCVD (Inductively Coupled Plasma Chemical Vapor Deposition Equipment). Use the ICPCVD technology to prepare a 200-nm-thick silicon nitride encapsulation layer at 75°C. After completion, use a dicing machine to cut the four-inch glass into sensor detection chips of 12 mm×14 mm, complete the preparation of the sensor MEMS process, and obtain the sensor chip.

[0045] (7) Place the sensor chip into a plasma cleaner, select a power of 200 W, a time of 3 min, and an oxygen atmosphere gas for plasma treatment. The plasma treatment is used to increase the hydroxyl groups on the sensor surface. At the same time, prepare an absolute ethanol solution containing 3-(2-aminoethylamino)propyltrimethoxysilane with a concentration of 1%. Place the plasma-treated sensor chip into the prepared absolute ethanol solution of 3-(2-aminoethylamino)propyltrimethoxysilane, let it stand for 12 h, take out the sensor chip, wash it with pure water and dry it with nitrogen, completing the preparation of the MEMS sensor chip for cell temperature measurement.

[0046] Use an energy spectrometer to analyze the element types and contents on the surface of the sensor chip before and after treatment with 3-(2-aminoethylamino)propyltrimethoxysilane. The results are as Figure 3 shown, Figure 3 This is the composition analysis diagram of the surface of the sensor chip by the energy spectrometer of the present invention; among them, a) is the element type diagram of the sensor detection area before treatment with 3-(2-aminoethylamino)propyltrimethoxysilane, and b) is the element type diagram of the sensor detection area after treatment with 3-(2-aminoethylamino)propyltrimethoxysilane. From Figure 3It can be seen that before the treatment with 3-(2-aminoethylamino)propyltrimethoxysilane in a), the mass ratios of N, O, and Si elements are 11.83%, 23.14%, and 10.70% respectively. After the treatment with 3-(2-aminoethylamino)propyltrimethoxysilane in b), the mass ratios of N, O, and Si elements are 14.51%, 27.48%, and 20.63%. The main elements of 3-(2-aminoethylamino)propyltrimethoxysilane increase after the treatment, indicating that the surface of silicon nitride has been successfully silanized.

[0047] Calibration and Stability Test of the Sensor Chip in Example 3

[0048] The calibration test of the sensor chip is as follows: The MEMS sensor chip for cell temperature measurement prepared in Example 1 is connected in a four-wire system to a processing system composed of an ADS1263 data acquisition card and an STM32f103c8t6 single-chip microcomputer. The detection area of the sensor chip is immersed in a constant temperature water bath with an accuracy of 0.1 °C. In the temperature range of 34 - 40 °C, the resistance value of the sensor chip is measured and recorded every one degree, and the calibration between the resistance and the temperature is carried out through the resistance-temperature curve of the sensor chip.

[0049] The stability test of the sensor chip is as follows: The entire sensor chip and the detection circuit are placed in a carbon dioxide cell incubator. Two sensor chips are in a group. After the system is stable (for 1 hour), the temperature difference between the two sensor chips is recorded, and the experiment is repeated three times within three days to observe the stability change of the sensor chip.

[0050] The results are as shown in Figure 4. Figure 4 This is the calibration and stability test diagram of the sensor chip of the present invention. Among them, a) is the calibration curve diagram of the sensor chip of the present invention, and b) is the stability test result diagram of the sensor chip of the present invention. From Figure 4 As can be seen from a) in it, there is a good linearity (R2 = 0.99948) between the calibration measurement values of the sensor chip and the fitting curve. According to the fitting curve, the resistance temperature coefficient of the sensor chip can be calculated to be 2100 ppm / °C. From Figure 4 As can be seen from b) in it, within the measurement time of three days, the temperature difference fluctuation of the sensor chip is mostly within ±0.015 °C, which indicates that the entire system can work stably in the humidity, temperature, and atmosphere environment for normal cell growth. This sensor chip has excellent stability and can be used for long-term monitoring of cell temperature.

[0051] Example 4 Detection of HEK-293T Cells by the MEMS Sensor Chip for Cell Temperature Measurement of the Present Invention

[0052] Take the MEMS sensor chip for cell temperature measurement prepared in Example 1. Two sensor chips are in a group. Add high-glucose DMEM medium (Gibco) containing cells to one sensor chip, while there are no cells on the other sensor chip. Place the above two sensor chips in a cell culture incubator and culture for about 12 hours. Then take out the sensor chips and add DMEM medium to both sensor chips. Subsequently, put the sensor chips and the detection system back into the incubator and start recording the change in the temperature difference between the two sensor chips. After more than ten hours, take out the sensor chips and use a pipette to blow the cells on the sensor chips to make them fall off and replace the DMEM medium with pure water. At this time, there are no cells on both sensor chips. Put the sensor and the detection system back into the incubator and record for more than ten hours as a reference group. The fluctuation of the temperature difference of the sensor chip caused by culturing cells on one sensor chip is considered to be the temperature change caused by the cells.

[0053] The results are shown in Figure 5. Figure 5 It is a diagram of the sensor of the present invention detecting HEK-293T cells. Among them, a) is a diagram of the growth status of cells on the surface of the sensor chip, and b) is a diagram of the temperature change of the sensor chip when detecting HEK-293T. From Figure 5 a) in it, it can be seen that after the cells adhere and grow for 12 hours, the morphology of the cells indicates that the cells can grow normally in large numbers on the surface of the sensor chip. From Figure 5 b) in it, it can be seen that during the detection period, the temperature change of the sensor chip caused by the cells is about 0.1 °C to 0.05 °C. After the cells are removed, the change of the sensor chip returns to 0 °C. This experiment shows that the prepared cell temperature sensor chip can realize the detection of cell temperature.

Claims

1. A preparation method of a MEMS sensor chip for cell temperature measurement, characterized in that, It includes the following steps: (1) Pretreat the substrate to obtain a pretreated substrate; (2) Spin-coat the polyimide solution on the pretreated substrate and cure it in a nitrogen oven to obtain a substrate with a polyimide film; (3) Spin-coat AZ5214 photoresist on the polyimide film of the substrate in step (2), perform photolithography through a positive photoresist inversion process, develop, and transfer the sensor pattern on the mask plate to the polyimide film of the substrate; (4) Use an electron beam evaporation process to sequentially prepare a Cr adhesion layer and a Pt resistance layer on the upper part of the pattern of the polyimide film; (5) Immerse the chip obtained in step (4) in an acetone solution, ultrasonicate, soak, and remove the excess photoresist and excess metal through a lift-off process to obtain the Pt resistance of the sensor chip; (6) Shield the lead electrodes of the Pt resistance, and use ICPCVD technology to prepare a silicon nitride encapsulation layer in the detection area of the Pt resistance to obtain a sensor chip; (7) Perform plasma treatment on the surface of the sensor chip in an oxygen atmosphere, then immerse it in an anhydrous ethanol solution containing 3-(2-aminoethylamino)propyltrimethoxysilane, let it stand, wash it with pure water and dry it with nitrogen to obtain a MEMS sensor chip for cell temperature measurement; The MEMS sensor chip for cell temperature measurement includes a substrate (1), and a polyimide thin film (2), a Cr adhesion layer (3), a Pt resistance layer (4), and a silicon nitride encapsulation layer (5) are sequentially provided on the substrate (1); the thickness of the Cr adhesion layer (3) is 5-10 nm, the thickness of the Pt resistance layer (4) is 80-110 nm, and the thickness of the silicon nitride encapsulation layer (5) is 150-200 nm.

2. The preparation method of the MEMS sensor chip for cell temperature measurement according to claim 1, characterized in that In step (1), the substrate is a glass sheet, and the pretreatment is to ultrasonically clean the substrate with acetone and isopropyl alcohol in sequence, wash the substrate with pure water, dry it with nitrogen, and perform plasma treatment in an oxygen atmosphere.

3. The preparation method of the MEMS sensor chip for cell temperature measurement according to claim 1, characterized in that, The time for ultrasonic cleaning is 3-5 min, the power of the equipment during plasma treatment is 150-300 W, and the treatment time is 3-5 min.

4. The preparation method of the MEMS sensor chip for cell temperature measurement according to claim 1, characterized in that, In step (2), when spin-coating the polyimide solution, spin the glue at 500-600 rpm for 3-5 s respectively, and then spin the glue at 3000-3500 rpm for 30-35 s. When curing, cure it at 120-125 °C for 1 h, 200-210 °C for 2 h, and 250-260 °C for 2.5 h respectively.

5. The preparation method of the MEMS sensor chip for cell temperature measurement according to claim 1, characterized in that, In step (3), when spin-coating the photoresist, rotate at 500-600 rpm for 4-5 seconds first, and then rotate at 3500-4000 rmp for 25-30 seconds.

6. The preparation method of the MEMS sensor chip for cell temperature measurement according to claim 1, characterized in that, In step (5), the time for ultrasonication is 5-10 min, and the time for continuous soaking is 24-48 h.

7. The preparation method of the MEMS sensor chip for cell temperature measurement according to claim 1, wherein, In step (6), the obtained sensor chip can be divided into multiple sensor chips by a dicing machine.

8. The preparation method of the MEMS sensor chip for cell temperature measurement according to claim 1, characterized in that, In step (7), the power of the plasma treatment on the surface of the sensor chip in an oxygen atmosphere is 150 - 250 W, the time is 3 - 5 min, and the concentration of 3-(2-aminoethylamino)propyltrimethoxysilane in the absolute ethanol solution containing 3-(2-aminoethylamino)propyltrimethoxysilane is 1 - 2%.

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