Extracellular calcium ion light-addressable potential sensor and calcium ion concentration imaging method

Abstract

The invention discloses a cell extracellular calcium ion light-addressable potential sensor and a calcium ion concentration imaging method, wherein the sensor comprises a sapphire silicon chip substrate and a solid-state sensitive membrane. A calcium ion sensitive membrane solution is prepared, and is spin-coated on a sapphire silicon chip by a coating machine, and a layer of solid-state ion sensitive membrane is formed after drying. A light-addressable potential sensor system is composed of a 520 nm laser, a constant potentiostat, a lock-in amplifier, a reference electrode and a counter electrode. After adding a calcium ion solution, a relationship curve between a photocurrent and a bias voltage is measured, the photocurrent value at the working point is analyzed, a standard curve is established and the calcium ion concentration of the solution to be tested is measured. The photocurrent value is converted into a color by point-by-point scanning, and the cell contour and the extracellular calcium ion concentration image are drawn. The invention utilizes the specificity of calcium ion carriers to solution calcium ions, and realizes quantitative detection of the extracellular calcium ion concentration by analyzing the change of the photocurrent value, which is low in cost, convenient in operation and short in detection time.

Description

Technical Field

[0001] The present invention relates to a cell extracellular calcium ion detection and analysis technology, and in particular to a cell extracellular calcium ion light addressing potential sensor and a calcium ion concentration imaging method. Background Art

[0002] Calcium ions are one of the most common signal factors in the human body. As an intracellular messenger, it is responsible for transmitting information to regulate cell activities and plays an important role in the birth, death and life of cells, including a wide range of physiological activities such as neurotransmitter and hormone release, gene transcription activation and muscle contraction. There are many diseases, especially heart-related diseases in the human body, and their etiology analysis and treatment methods need to start from the microscopic level. Diseases such as atrial fibrillation, hypertension, epilepsy and a series of nervous system problems are closely related to the dysfunction of human calcium channels. At present, the main methods for detecting extracellular calcium ions at home and abroad are fluorescent calcium imaging and microelectrode array (MEA). Fluorescent calcium imaging can achieve short-term instant observation, but it cannot be monitored continuously for a long time due to the attenuation of the fluorescent signal; MEA can monitor the changes of extracellular field potential signals in real time for a long time, but the specificity is poor. In addition, these two methods are highly professional and complicated to operate. The silicon layer of the sapphire silicon chip is only 1 μm thick and a chromium / gold composite layer is directly magnetron sputtered on the front of the silicon layer as an ohmic contact, which greatly improves the sensing performance. In addition to having comparable or even superior sensing performance to traditional PVC sensitive membranes, the solid-state sensitive membrane made of silicone rubber also exhibits better stability and longer service life. The imaging method realizes a qualitative change from one-dimensional data to two-dimensional images. In addition to ensuring the effectiveness of the sensing information, its richness and amount of information are also greatly improved. The cell extracellular calcium ion concentration detection sensor and imaging method have the characteristics of stable signal, real-time response, good specificity, low detection limit, and two-dimensional imaging, which facilitates the establishment of an analytical platform for cell extracellular calcium ion concentration detection and imaging, and is expected to become a new tool in the field of cell extracellular calcium ion analysis and be promoted. Summary of the invention

[0003] The object of the present invention is to provide a cell extracellular calcium ion light addressable potential sensor and a calcium ion concentration imaging method in view of the deficiencies of the prior art.

[0004] The objective of the present invention is achieved through the following technical solutions: on the one hand, the present invention provides a cell extracellular calcium ion light-addressable potential sensor, which includes a sapphire silicon chip substrate, a solid-state sensitive membrane and a calcium ion gradient solution; the prepared calcium ion sensitive membrane solution is taken and spin-coated on the sapphire silicon chip by a coating machine, and a layer of solid-state sensitive membrane is formed after drying; a sensor laser light path of the light-addressable potential sensor irradiating the solid-state sensitive membrane is constructed; after the calcium ion gradient solution is added to the light-addressable potential sensor system, a relationship curve between a photogenerated current and a bias voltage is measured, the photogenerated current value at the working point is analyzed, a standard curve is established, and the calcium ion concentration of the solution to be tested is measured.

[0005] Furthermore, the sapphire silicon chip substrate is: the sapphire silicon chip uses a sapphire wafer as a supporting substrate; first, a silicon layer is epitaxially grown on the sapphire substrate. A silicon dioxide layer is oxidized and grown on the silicon layer through a thermal oxidation process; a selected area of ​​the silicon dioxide layer is etched to remove the silicon dioxide in the area and expose the bottom silicon layer; then a chromium layer and a gold layer are deposited in the etched area using magnetron sputtering technology; the entire wafer after processing is diced and divided into a number of chips for standby use.

[0006] Furthermore, the solid-state sensitive membrane is: 300 mg of silicone rubber is weighed in a centrifuge tube; 1.4 mL of tetrahydrofuran is added to the centrifuge tube, manually shaken and then ultrasonicated, and then shaken and shaken again until completely dissolved; the dissolved silicone rubber solution is centrifuged; 1 mL of supernatant is drawn, and 3.6 mg of calcium ion carrier and 1.45 mg of ion additive are added to the supernatant, and the ion additive is sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate, NaTFPB); after being mixed evenly, an ultrasonic water bath is conducted to obtain a completely dissolved ion-sensitive membrane mixed solution; before spin coating, a layer of polyimide tape is applied to the gold layer area of ​​the sapphire silicon chip substrate; the spin coating process is controlled by a glue spreader, the sapphire silicon chip substrate is adsorbed on a turntable through a vacuum pump, the ion-sensitive membrane mixed solution is dripped on the surface of the sapphire silicon chip substrate, and spin coating is conducted; after the spin coating is completed, the chip is dried at room temperature overnight, and the polyimide tape is torn off the next day after ensuring that the sensitive film is completely dry, and the solid-state sensitive membrane is prepared.

[0007] Furthermore, the calcium ion gradient solution is: -1 A series of isocratic gradient calcium ion solutions are prepared by diluting a 10 M calcium chloride solution. -1 M, 10 -2 M, 10 -3 M, 10 -4 M, 10 -5 M, 10-6 M, 10 -7 M, 10 -8 M, 10 -9 M.

[0008] Furthermore, the light-addressable potentiometric sensor system is composed of a 520 nm laser, a potentiostat, a lock-in amplifier, a reference electrode, and a counter electrode.

[0009] Furthermore, the sensor laser optical path realizes laser beam contraction and makes the laser emit vertically upward. The optical path of the sensor laser is designed as follows:

[0010] (1) Use a fiber collimator to connect the single-mode fiber FC / PC connector of the laser diode; the laser emitted from the fiber is first collimated by the collimator to become a parallel beam;

[0011] (2) The collimated parallel light beam is further reduced using two plano-convex lenses. After being converged by the first plano-convex lens L1, the parallel light beam is adjusted to parallel light by the second plano-convex lens L2 and finally emitted.

[0012] (3) Four reflectors are added between the first plano-convex lens L1 and the second plano-convex lens L2 to realize the construction of a three-dimensional system, so that the laser beam after beam reduction can be emitted vertically upward and irradiated onto the chip surface to generate a light spot.

[0013] On the other hand, the present invention also provides a calcium ion concentration imaging method of a cell extracellular calcium ion light-addressable potential sensor, which uses a three-axis translation stage to move the sensor to gradually scan the spatial area where the cell is located at a certain rate, converts the obtained photocurrent value into color, and draws the cell contour and extracellular calcium ion concentration image; the scanning method adopts "S-type" scanning, that is, first performing an uninterrupted scan in the X direction from the beginning to the end, then taking a step in the Y direction, and continuing to perform the next column of continuous scanning in the X direction from the end to the beginning.

[0014] Furthermore, the specific steps of imaging method data processing are:

[0015] Each column of data obtained by the stage scanning in the X direction is called valid data, denoted as X. In theory, each column of X only needs to be merged into a two-dimensional array to draw a two-dimensional image. Therefore, the data collected during the Y-direction stepping needs to be discarded, which is called invalid data. First, accurately determine the valid data of the first column of X, namely X1, and then merge Y1, X2 and Y2 in sequence to obtain a new sequence C2. C2 is reversed and compared with X1. The comparison process is based on the principle that the values ​​of adjacent pixels are closest. The comparison process is as follows:

[0016] (1) If the number of valid data points is m, take the first m values ​​in C2, subtract each value from the adjacent value in X1, and calculate the sum of the m differences, S1;

[0017] (2) Take the m values ​​from the 2nd to the m+1th digit in C2, and calculate the difference and the sum of the differences S2 in the same way as step 1;

[0018] (3) Repeat the above steps until the sum of the differences between the values ​​from the mth to the last digit in C2 and X1 is obtained;

[0019] (4) Compare the values ​​of S1 to SE, and select the m-bit data corresponding to the smallest S as the valid data X2;

[0020] Then the comparison is repeated in a cycle. In the process of merging Yn-1, Xn and Yn to obtain the new sequence Cn, if n is an odd number, Cn does not need to be arranged in reverse order and can be directly compared with Xn-1. After determining all Xs, Xs are arranged in sequence, and a two-dimensional pseudo-color map can be drawn based on the merged two-dimensional array. All data processing processes are automatically completed in MATLAB software.

[0021] The beneficial effects of the present invention are as follows: the present invention utilizes the specificity of calcium ion carriers to solution calcium ions, realizes quantitative detection of extracellular calcium ion concentration by analyzing the change of photogenerated current value, and realizes drawing of cell contour and extracellular calcium ion concentration image by combining with a three-axis translation stage. The detection method of the present invention is low in cost, convenient in operation, and short in detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a typical structure and equivalent circuit diagram of the optically addressed potential sensor system of the method of the present invention.

[0023] Figure 2 It is a schematic diagram of the constant current mode and constant voltage mode of the light-addressable potentiometric sensor according to the method of the present invention.

[0024] Figure 3 The present invention is a schematic diagram of the process flow of sapphire silicon chip processing and light-addressable potential sensor chip preparation.

[0025] Figure 4 This is a graph showing the response of the light-addressable potential sensor of the present invention to calcium ions.

[0026] Figure 5 It is the three-dimensional laser light path design diagram of the present invention.

[0027] Figure 6 It is a schematic diagram of the S-type scanning mode of the imaging method of the present invention.

[0028] Figure 7 It is a block diagram of scanning data processing of the imaging method of the present invention.

[0029] Figure 8 The imaging method of the present invention scans the original image and the calibrated image.

[0030] Fig. 9 This is a comparison chart of imaging effects with different numbers of pixels of the present invention. DETAILED DESCRIPTION

[0031] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] The invention provides a method for detecting and imaging extracellular calcium ion concentration of cells using a light-addressable potential sensor based on a sapphire silicon chip and a solid-state sensitive membrane. The method comprises the following substances: a sapphire silicon chip substrate, a solid-state sensitive membrane and a calcium ion gradient solution.

[0033] The sapphire silicon chip substrate is: the sapphire silicon chip uses a sapphire wafer as a supporting substrate. First, a silicon layer (Si, N-type) is epitaxially grown on the sapphire substrate. A silicon dioxide layer is oxidatively grown on the silicon layer through a thermal oxidation process. The selected area of ​​the silicon dioxide layer is etched to remove the silicon dioxide in the area and expose the bottom silicon layer. Then, a chromium layer and a gold layer are deposited in the etched area using magnetron sputtering technology. After the processing is completed, the entire wafer is diced and divided into several chips for standby;

[0034] Specifically, the solid-state sensitive membrane is: 300 mg of silicone rubber is weighed in a centrifuge tube. 1.4 mL of tetrahydrofuran is added to the centrifuge tube, and the tube is shaken manually and then ultrasonicated, and then shaken again until completely dissolved. The dissolved silicone rubber solution is centrifuged at a certain speed for a period of time. 1 mL of supernatant is taken, and 3.6 mg of calcium ion carrier and 1.45 mg of ion additive are added to the supernatant. After mixing evenly, an ultrasonic water bath is performed to obtain a completely dissolved ion-sensitive membrane mixed solution. A layer of polyimide tape is attached to the gold layer area of ​​the SOS chip before spin coating. The spin coating process is controlled by a glue spreader, and the SOS chip is adsorbed on the turntable by a vacuum pump, and the solid-state sensitive membrane solution is dripped on the surface of the chip for spin coating. After the spin coating is completed, the chip is dried at room temperature overnight. The polyimide tape is torn off the next day after ensuring that the sensitive film is completely dry, and the solid-state sensitive membrane is prepared.

[0035] Specifically, the calcium ion gradient solution is: -1 A series of isocratic gradient calcium ion solutions are prepared by diluting a 10 M calcium chloride solution. -1 M, 10 -2 M, 10 -3 M, 10 -4 M, 10 -5 M, 10 -6 M, 10-7 M, 10 -8 M, 10 -9 M.

[0036] Specifically, the imaging method is: using a three-axis translation stage to move the sensor to scan point by point, converting the obtained photocurrent value into color, and drawing the cell contour and extracellular calcium ion concentration image.

[0037] The above-mentioned method for detecting extracellular calcium ions in cells based on a light-addressable potential sensor of a sapphire silicon chip and a solid-state sensitive membrane is based on the principle of Figure 1 and Figure 2 As shown, specifically:

[0038] Take 60 μL of the prepared calcium ion sensitive membrane solution and spin coat it on a sapphire silicon chip through a coating machine. After drying, a solid ion sensitive membrane is formed. The peripheral detection system consists of a 520 nm laser, a constant potential instrument, a lock-in amplifier, a reference electrode and a counter electrode. After adding the calcium ion solution, the relationship curve between the photocurrent and the bias voltage is measured, the photocurrent value at the working point is analyzed, the standard curve is established and the calcium ion concentration of the solution to be tested is determined. The sensor is moved by a three-axis translation stage for point-by-point scanning, and the obtained photocurrent value is converted into color to draw the cell contour and the image of the extracellular calcium ion concentration.

[0039] Example 1: Preparation of sapphire silicon chip base: The sapphire silicon chip uses a 460 μm thick sapphire wafer (composed of 99.995% high-purity aluminum oxide) as a supporting substrate. The principle diagram of its preparation process is as follows: Figure 3 As shown. First, a 1 μm thick silicon layer (Si, N-type, resistivity 0.3±0.08 Ohm·cm) was epitaxially grown on a sapphire substrate. A 100 nm thick silicon dioxide layer was oxidatively grown on the silicon layer by a thermal oxidation process. A 2 mm wide area of ​​the silicon dioxide layer was selected for etching to remove the silicon dioxide in the area and expose the silicon layer at the bottom. Then, a 20 nm thick chromium layer and an 80 nm thick gold layer were deposited in the etched area using magnetron sputtering technology. After processing, the entire wafer was diced and divided into several 1 cm×1 cm chips for standby use.

[0040] Example 2: Preparation of solid-state sensitive membrane: 300 mg of silicone rubber was weighed in a 2 mL centrifuge tube. 1.4 mL of tetrahydrofuran was added to the centrifuge tube, and the solution was shaken manually for 1 min, ultrasonicated for 15 min, and then shaken again until completely dissolved. The dissolved silicone rubber solution was centrifuged at 9.5 k rpm for 20 min. 1 mL of supernatant was taken, and 3.6 mg of calcium ion carrier and 1.45 mg of ion additive were added to the supernatant. After mixing evenly, an ultrasonic water bath was conducted for 30 min to obtain a completely dissolved ion-sensitive membrane mixed solution. A layer of polyimide tape was attached to the gold layer area of ​​the SOS chip before spin coating. The spin coating process was controlled by a glue spreader, and the SOS chip was adsorbed on the turntable by a vacuum pump. 60 µL of ISM solution was dropped on the surface of the chip and spin coated for 30 s. After the spin coating was completed, the chip was dried at room temperature overnight. The polyimide tape was torn off the next day after ensuring that the sensitive membrane was completely dry, and the solid-state sensitive membrane was prepared. The schematic diagram of the preparation process of solid-state sensitive membrane is as follows Figure 3 shown.

[0041] Example 3: Preparation of a 10 -1 M CaCl2 solution and 0.1 M CH3COOLi solution were used to investigate the performance of sensor chips based on sapphire silicon chips and solid-state sensitive membranes. First, 3 mL of 10 -1 Then, 2.7 mL of the solution was aspirated and an equal amount of 0.1 M CH3COOLi background solution was added. The solution was mixed evenly by slowly blowing with a pipette. At this time, the calcium ion concentration was diluted to 10 -2 M. After that, each concentration was tested by dilution method, and each experiment was repeated three times. Figure 4 As shown. The sensor is -7 -10 -2 It shows good linearity in the range of M. The linear relationship is Y = 21.77 X + 151.8 (R 2 =0.9870), where X represents calcium ion concentration, Y represents bias voltage, and the linearity is 0.9870.

[0042] Example 4: The optical path design steps of the optically addressable potentiometric sensor are as follows: (1) Use a fiber collimator to connect the single-mode fiber FC / PC connector of the laser diode. The laser emitted from the optical fiber is first collimated by the collimator to be a parallel beam. (2) The collimated parallel beam is further reduced using two plano-convex lenses. After the parallel beam is converged by lens L1, it is adjusted to parallel light again by lens L2 and finally emitted. (3) Four reflectors are added between lens L1 and lens L2 to realize the construction of a three-dimensional system, such as Figure 5 The three-dimensional laser optical path enables the laser beam after beam contraction to be emitted vertically upward and illuminate the chip surface to generate a light spot.

[0043] Embodiment 5: Imaging "S-type" scanning mode: The scanning mode adopts "S-type" scanning, that is, firstly, a row of uninterrupted scanning in the X direction is performed from the beginning to the end, and then a step is taken in the Y direction, and the next row of continuous scanning in the X direction is continued from the end to the beginning, such as Figure 6 As shown in the figure. The stage has a short turning delay only after each column of X scanning is completed and when it is ready to step in the Y direction. It will not move or stop point by point during the scanning process, which reduces time loss. When performing actual image detection, the scanning area is set to 0.5 mm×0.5 mm. The sampling frequency of the detection system is fixed at 40 Hz, so the displacement speed in the X direction determines the number of sampling points. If the speed is set to 0.2 mm / s, 100 data points can be obtained after completing a column of scanning, and the interval between each point is 5 data points. The interval between each point of the sampling data in the Y direction is determined by the single step distance. If the single step distance is also set to 5 samples, the pixel size of the scanned image is 5 pixels of the scanned image.

[0044] Embodiment 6: Imaging data analysis method: First, accurately determine the valid data of the first column X, i.e., X1, and then sequentially merge Y1, X2, and Y2 to obtain a new number column C2, reverse C2, and compare it with X1. The comparison process is based on the principle that the values ​​of adjacent pixel points are closest, and the comparison process is as follows: (1) If the number of valid data points is m, take the first m digits of C2, and subtract each value from the adjacent position value in X1 in turn, and calculate the sum of m differences S1; (2) Take the m values ​​from the 2nd to the m+1th digit in C2, and calculate the difference and the sum of the differences S2 in the same way as step 1; (3) Repeat the above steps until the sum of the differences SE between the mth to the last digit in C2 and X1 is obtained; (4) Compare the values ​​of S1 to SE, and select the m-bit data corresponding to the smallest S as the valid data X2. After that, the comparison is repeated in a cycle. In the process of merging Yn-1, Xn and Yn to obtain a new sequence Cn, if n is an odd number, Cn does not need to be arranged in reverse order and can be directly compared with Xn-1. After determining all X, X is arranged in sequence, and a two-dimensional pseudo-color image can be drawn based on the merged two-dimensional array. All data processing processes are automatically completed in MATLAB software. The imaging method scan data processing block diagram is shown in the figure. Figure 7 shown.

[0045] Example 7: Under the above imaging scanning and data analysis method, taking HL-1 cell sphere as an example, a 1 mm×1 mm area is scanned point by point, and the pseudo-color image drawn contains a total of 100×100 pixels. The images before and after calibration are as follows Figure 8As shown, the data after automatic calibration ensures to the greatest extent that each column of adjacent data is adjacent sites in the actual sensor and does not contain invalid data at the inflection point. Therefore, the edge contour of the cell ball is clearer and the image quality is improved.

[0046] Example 8: To explore the effects of different scanning rates and total number of pixels on image quality, HL-1 cell spheres were added to the detection chamber. After they were stably attached to the sensor surface, the same 0.5 mm × 0.5 mm area was selected and four scanning modes were set to scan with different parameters. The corresponding images are as follows: Fig. 9 As shown in the figure. In the four repeated scans of the same area with different parameters, as the total number of pixels increases, the image resolution gradually improves, the image burrs gradually decrease, and the edge of the cell ball becomes clearer. In applications that pursue fast imaging, a 50×50 pixel scanning method can be selected, and the imaging time of a single image is about 70 s. In applications that pursue imaging quality, a 100×100 pixel scanning method can be selected to obtain a higher resolution image, but the imaging time will also be extended to about 265 s accordingly. These images are obtained by returning the translation stage to the starting point after each scan and repeating the scan. The location and contour of the cell ball in the four images have not changed significantly, proving that the translation stage has good repeatability, and the slower scanning speed and the adhesion of the cell culture medium make the form of the mobile sensor chip not cause the shaking of the solution in the detection chamber and the displacement of the cell ball. Therefore, this system design and scanning method can be used in the actual detection of cell balls.

[0047] It should be stated that the content and specific implementation methods of the present invention are intended to demonstrate the practical application of the technical solution provided by the present invention and should not be interpreted as limiting the scope of protection of the present invention. Any modifications and changes made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A method for imaging calcium ion concentration using an extracellular calcium ion light-addressable potential sensor, characterized in that: The sensor comprises a sapphire silicon chip substrate, a solid-state sensitive membrane and a calcium ion gradient solution; the prepared calcium ion sensitive membrane solution is taken and spin-coated on the sapphire silicon chip by a coating machine, and a layer of solid-state sensitive membrane is formed after drying; a sensor laser optical path of a light-addressable potential sensor is constructed to irradiate the solid-state sensitive membrane; after the calcium ion gradient solution is added to the light-addressable potential sensor system, a relationship curve between a photogenerated current and a bias voltage is measured, the photogenerated current value at the working point is analyzed, a standard curve is established and the calcium ion concentration of the solution to be measured is measured; The sensor laser optical path realizes laser beam contraction and makes the laser emit vertically upward. The optical path of the sensor laser is designed as follows: (1) Use a fiber collimator to connect the single-mode fiber FC / PC connector of the laser diode; the laser emitted from the fiber is first collimated by the collimator to become a parallel beam; (2) The collimated parallel light beam is further reduced using two plano-convex lenses. After being converged by the first plano-convex lens L1, the parallel light beam is adjusted to parallel light by the second plano-convex lens L2 and finally emitted. (3) Four reflectors are added between the first plano-convex lens L1 and the second plano-convex lens L2 to realize the construction of a three-dimensional system, so that the laser beam after beam reduction can be emitted vertically upward and irradiated onto the chip surface to generate a light spot; The imaging method uses a three-axis translation stage to move the sensor to scan the spatial area where the cells are located at a certain rate step by step, convert the obtained photocurrent value into color, and draw the cell contour and extracellular calcium ion concentration image; the scanning method adopts "S-type" scanning, that is, first perform an uninterrupted scan in the X direction from the beginning to the end, then take a step in the Y direction, and continue to perform the next column of continuous scanning in the X direction from the end to the beginning; the displacement speed in the X direction determines the number of sampling points, and the interval between each point of the Y-direction sampling data is determined by the single step distance, which corresponds to the pixel point size of the scanned image; The specific steps of imaging method data processing are: Each column of data obtained by the stage scanning in the X direction is called valid data, denoted as X. In theory, each column of X only needs to be merged into a two-dimensional array to draw a two-dimensional image. Therefore, the data collected during the Y-direction stepping needs to be discarded, which is called invalid data. First, accurately determine the valid data of the first column X, namely X1, and then merge Y1, X2 and Y2 in sequence to obtain a new number sequence C2. C2 is reversed and compared with X1. The comparison process is based on the principle that the values ​​of adjacent pixel points are closest to each other, so as to ensure that each column of adjacent data is the adjacent position in the actual sensor and does not contain invalid data at the inflection point. The comparison process is as follows: (1) If the number of valid data points is m, take the first m values ​​in C2, subtract each value from the adjacent value in X1, and calculate the sum of the m differences, S1; (2) Take the m values ​​from the 2nd to the m+1th digit in C2, and calculate the difference and the sum of the differences S2 in the same way as step 1; (3) Repeat the above steps until the sum of the differences between the values ​​from the mth to the last digit in C2 and X1 is obtained; (4) Compare the values ​​of S1 to SE, and select the m-bit data corresponding to the smallest S as the valid data X2; Then the comparison is repeated in a cycle. In the process of merging Yn-1, Xn and Yn to obtain the new sequence Cn, if n is an odd number, Cn does not need to be arranged in reverse order and can be directly compared with Xn-1. After determining all Xs, Xs are arranged in sequence, and a two-dimensional pseudo-color map can be drawn based on the merged two-dimensional array. All data processing processes are automatically completed in MATLAB software.

2. The method for imaging calcium ion concentration using the extracellular calcium ion light-addressable potential sensor according to claim 1, characterized in that: The sapphire silicon chip substrate is as follows: the sapphire silicon chip uses a sapphire wafer as a supporting substrate; first, a silicon layer is epitaxially grown on the sapphire substrate; a silicon dioxide layer is oxidized and grown on the silicon layer through a thermal oxidation process; a selected area of ​​the silicon dioxide layer is etched to remove the silicon dioxide in the area and expose the bottom silicon layer; then a chromium layer and a gold layer are deposited in the etched area using magnetron sputtering technology; the entire wafer after processing is diced and divided into a number of chips for standby use.

3. The calcium ion concentration imaging method of the extracellular calcium ion light addressable potential sensor according to claim 2, characterized in that: The solid-state sensitive membrane is as follows: 300 mg of silicone rubber is weighed in a centrifuge tube; 1.4 mL of tetrahydrofuran is added to the centrifuge tube, and the tube is shaken manually and then ultrasonicated, and then shaken again until it is completely dissolved; the dissolved silicone rubber solution is centrifuged; 1 mL of supernatant is drawn, and 3.6 mg of calcium ion carrier and 1.45 mg of ion additive are added to the supernatant; after being evenly mixed, an ultrasonic water bath is conducted to obtain a completely dissolved ion-sensitive membrane mixed solution; before spin coating, a layer of polyimide tape is attached to the gold layer area of ​​the sapphire silicon chip substrate; the spin coating process is controlled by a glue spreader, the sapphire silicon chip substrate is adsorbed on a turntable by a vacuum pump, the ion-sensitive membrane mixed solution is dripped on the surface of the sapphire silicon chip substrate, and spin coating is conducted; after the spin coating is completed, the chip is dried overnight at room temperature, and the polyimide tape is torn off the next day after ensuring that the sensitive membrane is completely dry, and the solid-state sensitive membrane is prepared.

4. The calcium ion concentration imaging method of the extracellular calcium ion light addressable potential sensor according to claim 1, characterized in that: The calcium ion gradient solution is: -1 A series of isocratic gradient calcium ion solutions are prepared by diluting a 10 M calcium chloride solution. -1 M, 10 -2 M, 10 -3 M, 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M.

5. The calcium ion concentration imaging method of the extracellular calcium ion light addressable potential sensor according to claim 1, characterized in that: The light-addressable potentiometric sensor system consists of a 520 nm laser, a potentiostat, a lock-in amplifier, a reference electrode, and a counter electrode.

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