CN114544719B - PH sensing electrode, preparation method thereof and electrochemical sensor - Google Patents

Abstract

The invention provides a pH sensing electrode, a preparation method thereof and an electrochemical detector, wherein the pH sensing electrode comprises a glass capillary tube and an electrochemical probe, one end of the glass capillary tube is a tip, openings are formed in both ends of the glass capillary tube, electrolyte is arranged in the glass capillary tube, and the diameter of the opening of the tip is smaller than that of the opening of the other end of the glass capillary tube; the electrochemical probe is arranged on one side of the glass capillary tube with a tip, and is BSA hydrogel which has a cross-linked network structure. Therefore, the pH sensing electrode has good protein pollution resistance, high space-time resolution sensing capability on the concentration of hydrogen ions, excellent specificity, stability and reversibility, and can accurately and sensitively realize pH sensing in the brain of a living body, and the living body level has good stability.

Description

pH sensing electrode and preparation method thereof and electrochemical sensor

Technical Field

The present invention relates to the field of in-situ electroanalytical chemistry in living bodies, and in particular to a pH sensing electrode and a preparation method thereof, and an electrochemical sensor.

Background technique

The brain is the most sophisticated and complex organ in the human body. It participates in life activities such as sensation, cognition, movement, language and emotion. Revealing the chemical nature of brain neurological phenomena is one of the main goals and important frontiers of modern science. Research on brain function also helps to understand the nature of complex physiological processes such as human cognition and emotion, as well as the formation and development of neurological diseases. The transmission of brain nerve signals and metabolic processes cannot be separated from the participation of chemical substances. Therefore, conducting brain neuroanalytical chemistry research on many neurochemical substances in the brain, such as neurotransmitters, modulators, energy metabolites, free radicals, and ions, is of great significance for exploring and understanding the molecular mechanisms of neurophysiology and pathology.

Acid-base balance and pH regulation are essential for normal tissue metabolism and physiological function, and the pH of brain tissue changes in many disease states. In the past few decades, some breakthroughs have been made in the detection technology of pH changes in the brain, such as nuclear magnetic resonance, fluorescence, surface-enhanced Raman scattering, and electrochemical methods. However, due to the lack of accurate in situ analytical technology, the relationship between local pH fluctuations and brain diseases has not been widely studied. At present, the most commonly used method for in situ measurement of pH in vivo is based on electrochemical determination of traditional microelectrodes. Although it has high spatial resolution, simplicity and high sensitivity, it still faces problems such as selectivity and anti-pollution in the actual detection process. Therefore, it is very necessary to develop a simple, efficient, anti-pollution and highly sensitive in vivo in situ pH sensing method.

In situ electrochemical methods have great advantages as a living in situ sensing method with high spatiotemporal resolution. The classic electrochemical analysis method is to use ultramicroelectrodes implanted in situ for the detection of electrically active neurotransmitters or neuromodulators, but there are certain challenges for some non-electrochemically active substances. In recent years, micro-nanopore technology has been vigorously developed, and sensing technology based on ion current rectification has realized the detection of chemical substances at the single cell and living body level. However, the analysis method based on the regulation of ion transport in this confined micron pore, especially at the living body level, is limited by protein contamination on the sensing interface.

Summary of the invention

The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, one purpose of the present invention is to provide a pH sensing electrode and a preparation method thereof and an electrochemical sensor, wherein the pH sensing electrode has good anti-protein contamination performance, the sensing interface of the pH sensing electrode is an in-situ polymerized BSA hydrogel, and the detection of the hydrogen ion concentration level can be achieved without undergoing a redox reaction process, and the sensing ability has high temporal and spatial resolution, and has excellent specificity, stability, and reversibility, and can accurately and sensitively realize pH sensing in the living brain, and has good stability at the living level.

In one aspect of the present invention, a pH sensing electrode is proposed, comprising a glass capillary, one end of the glass capillary is a tip, both ends of the glass capillary are provided with openings, an electrolyte is provided in the glass capillary, and the opening diameter of the tip is smaller than the opening diameter of the other end of the glass capillary; an electrochemical probe, the electrochemical probe is arranged on a side of the glass capillary having the tip, the electrochemical probe is a BSA hydrogel, and the BSA hydrogel has a cross-linked network structure.

According to some embodiments of the present invention, the pore size of the cross-linked network structure is 10 to 30 nanometers.

According to some embodiments of the present invention, the BSA hydrogel comprises bovine serum albumin, glutaraldehyde and water.

According to some embodiments of the present invention, in the BSA hydrogel, the mass ratio of the bovine serum albumin, the glutaraldehyde and the water is (5-20):(5-10):100.

According to some embodiments of the present invention, the length of the electrochemical probe is 10 to 100 micrometers.

According to some embodiments of the present invention, the electrochemical probe is bonded to the glass capillary via a chemical bond.

According to some embodiments of the present invention, the diameter of the tip opening is 3 to 5 microns.

In another aspect of the present invention, a method for preparing the above-mentioned pH sensing electrode is proposed, including providing a glass capillary with two ends open, one end of the glass capillary being a tip, and the opening diameter of the tip being smaller than the opening diameter of the other end of the glass capillary; immersing the tip of the glass capillary into BSA hydrogel, so that the BSA hydrogel fills the side of the glass capillary having the tip, polymerizes to form an electrochemical probe; and adding an electrolyte into the glass capillary to obtain the pH sensing electrode. Thus, the pH sensing electrode prepared by this method has good anti-protein contamination performance and high temporal and spatial resolution sensing capability for hydrogen ion concentration, has excellent specificity, stability, and reversibility, can more sensitively realize real-time monitoring of pH changes in the brain, and has good stability during in vivo level detection.

In another aspect of the present invention, an electrochemical detector is proposed, including the aforementioned pH sensing electrode, whereby the electrochemical detector has all the characteristics and advantages of the aforementioned pH sensing electrode, which will not be elaborated here. In general, it has at least the advantages of good resistance to protein contamination and good stability.

According to some embodiments of the present invention, the electrolyte is a NaCl solution.

According to some embodiments of the present invention, the working electrode and the reference electrode are Ag/AgCl electrodes.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

FIG1 shows a schematic structural diagram of a pH sensing electrode according to an embodiment of the present invention;

FIG2 shows a schematic flow chart of a method for preparing a pH sensing electrode according to an embodiment of the present invention;

FIG3a shows a schematic diagram of the tip structure of the pH sensing electrode prepared in Example 1 of the present invention;

FIG3 b shows a SEM image of the BSA hydrogel prepolymer solution of the pH sensing electrode prepared in Example 1 of the present invention;

FIG4a shows a cyclic voltammetry test diagram of the pH sensing electrode prepared in Example 1 of the present invention;

FIG4 b shows a cyclic voltammetry test diagram of the pH sensing electrode prepared in Example 1 of the present invention;

FIG5a shows a graph showing the anti-protein contamination performance test of the pH sensing electrode prepared in Example 1 of the present invention;

FIG5 b shows a test diagram of the anti-protein contamination performance of the pH sensing electrode prepared in Example 1 of the present invention;

FIG6a shows a graph showing the anti-protein contamination performance test of the pH sensing electrode prepared in Example 1 of the present invention;

FIG6 b shows a graph showing the anti-protein contamination performance test of the pH sensing electrode prepared in Example 1 of the present invention;

FIG7a shows a sensing performance test diagram of the pH sensing electrode prepared in Example 1 of the present invention;

FIG7 b shows a sensing performance test diagram of the pH sensing electrode prepared in Example 1 of the present invention;

FIG7c shows a sensing performance test diagram of the pH sensing electrode prepared in Example 1 of the present invention;

FIG8 shows a repeatability test diagram of the pH sensing electrode prepared in Example 1 of the present invention;

FIG9 shows a reversibility test diagram of the pH sensing electrode prepared in Example 1 of the present invention;

FIG10 shows a test diagram of the anti-interference performance of the pH sensing electrode prepared in Example 1 of the present invention;

FIG11 shows a graph of the in vivo performance test of the pH sensing electrode prepared in Example 1 of the present invention;

FIG. 12 shows a graph of the in vivo performance test of the pH sensing electrode prepared in Example 1 of the present invention.

Reference numerals:

100: pH sensing electrode; 110: tip; 120: electrolyte; 130: electrochemical probe.

Detailed ways

The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in this area or the product instructions are used. The reagents or instruments used without specifying the manufacturer are all conventional products that can be obtained commercially.

In one aspect of the present invention, a pH sensing electrode is proposed. Referring to FIG1 , the pH sensing electrode 100 includes a glass capillary and an electrochemical probe, wherein one end of the glass capillary is a tip 110, both ends of the glass capillary are provided with openings, an electrolyte 120 is provided in the glass capillary, and the opening diameter of the tip 110 is smaller than the opening diameter of the other end of the glass capillary; the electrochemical probe 130 is provided on the side of the glass capillary having the tip 110, and the electrochemical probe 130 is a BSA hydrogel, and the BSA hydrogel has a cross-linked network structure. Therefore, the pH sensing electrode 100 has good anti-protein contamination performance, as well as a high temporal and spatial resolution sensing capability for hydrogen ion concentration, has excellent specificity, stability, and reversibility, can accurately and sensitively realize pH sensing in the living brain, and has good stability at the living level.

The following is a brief description of the principle by which the present invention can achieve the above beneficial effects:

As mentioned above, the currently commonly used ion transmission sensors usually modify the inner surface of the glass tube with functionalized regulatory molecules to achieve the analysis and detection of specific substances. The biggest disadvantage of this detection method is that the sensing interface is easily contaminated by proteins. The pH sensing electrode proposed by the present invention has an electrochemical probe at the extreme end of the glass capillary, and the electrochemical probe is a BSA hydrogel with a cross-linked network structure. The in-situ polymerized BSA hydrogel constitutes the sensing interface of the pH sensing electrode, so that the pH sensing electrode can detect the hydrogen ion concentration level without undergoing a redox reaction process. The detection principle is that by applying a continuous voltage step, the hydrogen ions in the solution enter the BSA hydrogel filled in the tube under the action of the electric field force and the electroosmotic flow. The entry of hydrogen ions triggers the protonation of the amino acid residues on the bovine serum albumin, so that the charge of the BSA hydrogel changes with the change of the pH of the external solution. Specifically: as the pH of the external solution decreases, the charge of the BSA hydrogel filled in the tube decreases, and the ion current becomes smaller; as the pH of the external solution increases, the charge of the BSA hydrogel filled in the tube increases, and the ion current becomes larger. Thus, the inventor can use this electrode to achieve high sensitivity and high time resolution real-time detection of hydrogen ion levels in the range of pH 5.8-8.0, and has good stability at the in vivo level (such as detection of pH level changes in mouse brain). At the same time, the tight cross-linked network of BSA hydrogel can prevent protein from passing through the electrochemical probe to enter the sensing interface to produce false positive signals, preventing protein contamination. The aperture of the cross-linked network structure can prevent protein but will not form obstacles to ions. In the actual pH sensing process, by applying a high-frequency square wave potential to regulate the ion transmission behavior, the update speed of the glass capillary tip tube mouth interface is improved, and the anti-protein performance of the electrode is further improved. Therefore, the pH sensing electrode based on the limited ion transmission regulation of the present invention can realize accurate and sensitive monitoring of pH level changes in the brain, and this method lays the foundation for the diagnosis and treatment of diseases such as epilepsy, ischemia, and Alzheimer's disease, and has a wide range of applications.

According to some embodiments of the present invention, when the pH sensing electrode 100 is working, one end of the working electrode can be inserted into the electrolyte solution 120 in the glass capillary, and one end of the reference electrode can be inserted into the sample solution to be tested, and the other end of the working electrode is connected to the other end of the reference electrode through a power supply; the power supply is turned on to form an ion current path between the working electrode and the reference electrode, and the applied voltage value and current value are recorded to detect the pH value of the sample to be tested.

According to some embodiments of the present invention, the diameter of the opening of the glass capillary tip 110 is 3-5 micrometers, so that the electrochemical probe 130 has good robustness and can be used for in-situ detection and analysis in vivo.

According to some specific embodiments of the present invention, the BSA hydrogel includes bovine serum albumin, glutaraldehyde and water. Specifically, the mass ratio of bovine serum albumin, glutaraldehyde and water in the BSA hydrogel is (5-20): (5-10): 100, and the BSA hydrogel finally formed has better anti-protein pollution performance. The inventors found that if the content of bovine serum albumin in the BSA hydrogel is too low, the cross-linking density will be too low, which will reduce the anti-protein pollution effect of the pH sensing electrode to a certain extent; if the content of bovine serum albumin is too high, the chemical cross-linking speed will be faster after adding glutaraldehyde, which will increase the difficulty of repeated preparation of the pH sensing electrode and reduce the process reproducibility; if the content of glutaraldehyde is too low, the cross-linking density of bovine serum albumin is too low, which will reduce the anti-protein pollution effect of the pH sensing electrode to a certain extent; if the content of glutaraldehyde is too high, the chemical cross-linking speed of bovine serum albumin is too fast, which will also increase the difficulty of preparing the pH sensing electrode and reduce the process reproducibility.

According to some embodiments of the present invention, the preparation method of BSA hydrogel is not particularly limited. For example, in the present application, a bovine serum albumin solution and a glutaraldehyde aqueous solution can be mixed to obtain a BSA hydrogel. According to some specific embodiments of the present invention, the concentrations of the bovine serum albumin solution and the glutaraldehyde solution are not particularly limited, as long as the mass ratio of bovine serum albumin, glutaraldehyde and water in the final formed BSA satisfies (5-20): (5-10): 100. For example, the concentration of bovine serum albumin in the bovine serum albumin solution is 50-200 mg/mL. According to other embodiments of the present invention, based on the total mass of the glutaraldehyde aqueous solution, the content of glutaraldehyde in the glutaraldehyde aqueous solution is 5% to 10%.

According to some embodiments of the present invention, the pore size of the cross-linked network structure in the BSA hydrogel is 10 to 30 nanometers, which can prevent proteins from entering the pH sensing electrode and prevent proteins from contaminating the pH sensing electrode. The inventors found that if the pore size of the cross-linked network structure is too small, the process requirements are high and the process reproducibility is poor; if the pore size of the cross-linked network structure is too large, the ability of the pH sensing electrode to resist protein contamination will be reduced.

According to some embodiments of the present invention, the length of the electrochemical probe 130 is not particularly limited, and those skilled in the art can design it according to the needs of detection. Specifically, in the present invention, the length of the electrochemical probe 130 can be 10 to 100 microns. The inventors found that if the length of the electrochemical probe 130 is too short, the preparation process requirements are high and the process reproducibility is reduced; if the length of the electrochemical probe 130 is too long, the production cost will be increased to a certain extent, resulting in unnecessary waste.

According to some embodiments of the present invention, due to the presence of carboxyl groups on the surface of the glass, glutaraldehyde can cross-link the amino groups on the surface of the protein and the carboxyl groups on the surface of the glass, so that the electrochemical probe 130 and the glass capillary are bonded through chemical bonds, that is, the electrochemical probe 130 and the inner surface of the glass capillary are tightly combined without gaps. At this time, ions can only enter the electrolyte 120 through the pores of the electrochemical probe 130, and cannot enter through the gap between the electrochemical probe 130 and the glass, thereby improving the detection accuracy to a certain extent.

In another aspect of the present invention, a method for preparing the aforementioned pH sensing electrode is proposed, comprising: (1) providing a glass capillary with two ends open, one end of the glass capillary being a tip, and the opening diameter of the tip being smaller than the opening diameter of the other end of the glass capillary; (2) immersing the tip of the glass capillary into a BSA hydrogel, so that the BSA hydrogel fills the side of the glass capillary having the tip, polymerizes, and forms an electrochemical probe; (3) adding an electrolyte into the glass capillary to obtain the pH sensing electrode. Thus, the pH sensing electrode prepared by the method has a simple process and good reproducibility. The prepared pH sensing electrode has good resistance to protein contamination and high spatiotemporal resolution sensing capability for hydrogen ion concentration, has excellent specificity, stability, and reversibility, can accurately and sensitively realize pH sensing in the living brain, and has good stability at the living level.

Below, according to an embodiment of the present invention, each step of the method is described in detail. Referring to FIG. 2 , the method may include:

S100: Preparation of glass capillaries with sharp tips

In this step, a glass capillary with two ends opened is provided, one end of the glass capillary is a tip, and the opening diameter of the tip is smaller than the opening diameter of the other end of the glass capillary. Specifically, a glass capillary with a tip can be prepared by drawing a borosilicate glass with an outer diameter of 1.5 mm and an inner diameter of 0.86 mm, and the drawing conditions are as follows: first cycle: temperature of 430°C, focusing range of 4, speed of 28, delay of 200, and tension of 0; second cycle: temperature of 325°C, focusing range of 4, speed of 30, delay of 200, and tension of 0, thereby obtaining a glass capillary with a tip at one end, and the inner diameter of the opening at the tip is 3 to 5 microns.

S200: Electrochemical probe formation by capillary action

In this step, the tip of the glass capillary is immersed in the BSA hydrogel, so that the BSA hydrogel fills the side of the glass capillary with the tip, and polymerizes to form an electrochemical probe. According to some embodiments of the present invention, the immersion time of the glass capillary in the BSA hydrogel is not particularly limited, and those skilled in the art can select it according to the length of the electrochemical probe finally formed. For example, the tip of the glass capillary with a tip opening diameter of 3 to 5 microns is immersed in the BSA hydrogel prepolymer solution, and the solution is immersed in the tip of the tube by capillary action. After 3 minutes of polymerization, a BSA hydrogel-filled anti-protein contamination pH sensing electrode is obtained.

S300: Add electrolyte into glass capillary

In this step, an electrolyte is added into the glass capillary obtained in step (2) to obtain a pH sensing electrode.

In another aspect of the present invention, an electrochemical detector is proposed, which includes the aforementioned pH sensing electrode; an electrochemical workstation; a working electrode, wherein one end of the working electrode contacts the electrolyte in the glass capillary of the pH sensing electrode; and a reference electrode, wherein one end of the reference electrode contacts the sample to be tested, and the other end of the working electrode is connected to the other end of the reference electrode through the electrochemical workstation. Thus, real-time, pollution-resistant, and highly sensitive detection of many substances (such as hydrogen ions) can be achieved, and the electrode has good robustness and anti-protein pollution performance, and can be used for real-time analysis and detection in complex environments such as physiology and pathology.

According to some embodiments of the present invention, when the electrochemical detector is used for in vivo detection, the electrolyte in the pH sensing electrode may be a NaCl solution. According to some embodiments of the present invention, the working electrode and the reference electrode are Ag/AgCl electrodes.

Example 1

(1) A borosilicate glass tube with an outer diameter of 1.50 mm, an inner diameter of 0.86 mm, and a length of 10 cm is drawn using a P-2000 _CO2 laser drawing instrument using the following procedure to obtain a glass nanotube with a pointed end and an average tip opening size of 3 μm:

(First cycle) Temperature = 430°C, Focus Range = 4, Speed = 28, Delay = 200, Tension = 0

(Second cycle) Temperature = 325°C, Focus Range = 4, Speed = 30, Delay = 200, Tension = 0

(2) The tip of the drawn glass capillary was immersed in a BSA hydrogel prepolymer solution containing 100 μl of a mixed solution of 200 mg/ml BSA and 20 μl of 50% glutaraldehyde. The solution was allowed to penetrate into the tip of the capillary by capillary action. After polymerization for 3 minutes, a BSA hydrogel-filled pH sensing electrode was obtained. Referring to Figures 3a and 3b, Figure 3a is an optical microscope image of the tip of the pH sensing electrode, and Figure 3b is a SEM image of a cross-linked network structure. It can be seen that the BSA hydrogel presents a relatively small porous cross-linked network structure.

The obtained pH sensor electrode was subjected to performance test:

(1) Cyclic voltammetry test

Referring to Figures 4a and 4b, the working electrode is placed in the glass capillary of the pH sensing electrode and in contact with the electrolyte in the glass capillary of the pH sensing electrode. A Chenhua 660e electrochemical workstation is used, the potential window is set to -1V~1V, and the scan rate is 50mV/s. Figure 4a is a cyclic voltammetry test diagram of the pH sensing electrode and the glass capillary. It can be seen that the pH sensing electrode exhibits nonlinear ion current rectification, while the glass capillary without BSA hydrogel shows a straight line, indicating the presence of BSA hydrogel in the glass capillary of the pH sensing electrode. Figure 4b is a cyclic voltammetry test diagram under different pH conditions, indicating that the pH sensing electrode has a pH response within the physiological pH range.

(2) Anti-protein contamination performance test

Referring to Figures 5a and 5b, the glass capillary containing the electrolyte and the pH sensing electrode were immersed in a 40 mg/ml FITC-BSA solution for 12 hours. Referring to Figure 5a, the first line is a laser confocal image of the pH sensing electrode after immersion for 12 hours. Figures a, b and c in Figure 5a are laser confocal images of the glass capillary after immersion for 12 hours. Figures d, e and f in Figure 5a are laser confocal images of the glass capillary after immersion for 12 hours, wherein Figures a and d are test images under dark field conditions, Figures b and e are test images under bright field conditions, and Figures c and f are test images superimposed with bright field and dark field. It can be seen from the figures that there is no fluorescence at the tip of the pH sensing electrode, indicating that the BSA hydrogel can prevent the infiltration of protein and has better anti-protein contamination performance. The tip of the glass capillary has fluorescence (Figure e also has fluorescence, but it is not obvious under bright field conditions), indicating that it has been contaminated by protein. FIG. 5 b shows that during the amperometric test, the addition of 10 mg/ml BSA does not interfere with the signal of the pH sensing electrode.

(3) Anti-pollution performance test of different concentrations of protein and different types of protein

Referring to Figures 6a and 6b, 10 mg/ml, 20 mg/ml, and 40 mg/ml BSA protein were added to the samples to be tested, respectively. It can be seen from the figure that after adding different concentrations of the same protein, the pollution generated is far less than 20% of the response compared to the 0.8 pH drop, indicating that the pH sensing electrode has good anti-protein pollution ability for proteins of different concentrations; referring to Figure 6b, 10 mg/ml BSA (bovine serum albumin), Trypsin (bovine trypsin), Fibrinogen (fibrinogen), Hb (hemoglobin), HRP (horseradish peroxidase) and Serum (serum) were added to the samples to be tested, respectively. It can be seen that the pollution generated by different types of proteins is far less than 20% of the response, indicating that the pH sensing electrode can not only resist the pollution of BSA protein, but also has good anti-protein pollution ability for other types of proteins.

(4) pH sensing performance test

Referring to FIG7a, the time resolution of the pH sensing electrode is tested within the physiological pH range. It can be seen from the figure that the pH sensing electrode has a high time resolution within the physiological pH range, so that when it is applied to a living body, it can quickly detect the change of pH in the living body caused by external environmental stimulation; referring to FIG7b, FIG7b is the relationship between different pH and ion current size obtained according to FIG7a. It can be seen from FIG7b that within the physiological pH range, there is a linear relationship between pH and ion current, and the pH sensing electrode can be used to detect the change of pH level within the physiological pH range. Referring to FIG7c, one sample to be tested is artificial cerebrospinal fluid, and the other sample to be tested is artificial cerebrospinal fluid containing BSA. After the test is completed, the pH sensing electrode is calibrated. It can be seen from the figure that in the presence of 10mg/ml BSA, there is a small deviation in the linear calibration, indicating that the pH sensing electrode has good stability.

(5) Repeatability verification

8 , 100 mM NaCl solution was perfused into the glass capillary of the pH sensing electrode, and the solution to be tested outside the tube was artificial cerebrospinal fluid, and its pH 8.0-5.8 was tested three times. It can be seen from the figure that the pH sensing electrode has good repeatability and stability.

(6) Reversibility verification

Referring to Figure 9, a 100mM NaCl solution is perfused into the glass capillary of the pH sensing electrode, and the test solutions outside the tube are 100mM NaCl and 50mM PBS solutions of pH 7.4 and pH 5.8. Cyclic voltammetry test is performed, the voltage is set to -1V~1V, the scan rate is 50mV/s, and the external test solutions are replaced with the above solutions of pH 7.4 and pH 5.8, respectively, and the cycle is repeated 8 times. It can be seen from the figure that the pH sensing electrode has excellent reversibility.

(7) Anti-interference ability test

Referring to Figure 10, the pH sensing electrode was subjected to an amperometric current test, with an applied voltage of -1 V, and 10 μM DA, 10 μM E, 200 μM AA, 50 μM DOPAC, 30 μM 5-HT, 10 μM NE, 1 mM KCl, 1 mM NaCl, 1 mM MgCl ₂ , 1 mM CaCl ₂ and 0.8 pH drop were added. It can be seen from the figure that, except for pH response, the pH sensing electrode has no current response to other substances, proving that the pH sensing electrode has good specificity for pH response.

(8) In vivo verification experiment

Referring to Figure 11, the square wave potential method was used for testing, and the step potential application parameters were as shown in the above experimental example. The microperfusion hose and the pH sensing electrode were co-implanted into the cerebral cortex, and the microperfusion hose contained artificial cerebrospinal fluid with a pH of 3. As can be seen from the figure, the pH sensing electrode can monitor the changes in pH level after continuous acid stimulation within 1 hour. Referring to Figure 12, the pH sensing electrode was taken out from the living body and calibrated. There was little difference in the calibration before and after the experiment, indicating that the pH sensing electrode has excellent anti-pollution performance and stability in in-situ detection of the living body.

In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims (11)

1. A pH sensing electrode, comprising: A glass capillary, wherein one end of the glass capillary is a tip, both ends of the glass capillary are provided with openings, an electrolyte is provided in the glass capillary, and the opening diameter of the tip is smaller than the opening diameter of the other end of the glass capillary; The electrochemical probe is arranged on the side of the glass capillary having the tip, and the electrochemical probe is a BSA hydrogel having a cross-linked network structure. 2 . The pH sensing electrode according to claim 1 , wherein the pore size of the cross-linked network structure is 10 to 30 nanometers. 3. The pH sensing electrode according to claim 1, wherein the BSA hydrogel comprises bovine serum albumin, glutaraldehyde and water. 4 . The pH sensing electrode according to claim 3 , wherein in the BSA hydrogel, the mass ratio of the bovine serum albumin, the glutaraldehyde and the water is (5-20):(5-10):100. 5 . The pH sensing electrode according to claim 1 , wherein the length of the electrochemical probe is 10 to 100 microns. 6 . The pH sensing electrode according to claim 5 , wherein the electrochemical probe is bonded to the glass capillary via a chemical bond. 7 . The pH sensing electrode according to claim 6 , wherein the diameter of the tip opening is 3 to 5 μm. 8. A method for preparing the pH sensing electrode according to claims 1 to 7, characterized in that it comprises: (1) providing a glass capillary tube with openings at both ends, wherein one end of the glass capillary tube is a tip, and the opening diameter of the tip is smaller than the opening diameter of the other end of the glass capillary tube; (2) immersing the tip of the glass capillary into BSA hydrogel, so that the BSA hydrogel fills the side of the glass capillary having the tip, and polymerizes to form an electrochemical probe; (3) Adding electrolyte into the glass capillary to obtain the pH sensing electrode. 9. An electrochemical detector, comprising: A pH sensing electrode, wherein the pH sensing electrode is the pH sensing electrode according to any one of claims 1 to 7 or is prepared by the method according to claim 8; Electrochemical Workstation; A working electrode, one end of which is in contact with the electrolyte in the glass capillary of the pH sensing electrode; A reference electrode, one end of which is in contact with the sample to be tested, and the other end of the working electrode is connected to the other end of the reference electrode through an electrochemical workstation. 10 . The electrochemical detector according to claim 9 , wherein the electrolyte is a NaCl solution. 11 . The electrochemical detector according to claim 9 , wherein the working electrode and the reference electrode are Ag/AgCl electrodes.