CN114544719B - PH sensing electrode, preparation method thereof and electrochemical sensor - Google Patents
PH sensing electrode, preparation method thereof and electrochemical sensor Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/302—Electrodes, e.g. test electrodes; Half-cells pH sensitive, e.g. quinhydron, antimony or hydrogen electrodes
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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
Technical Field
The invention relates to the field of in-situ electroanalytical chemistry of living bodies, in particular to a pH sensing electrode, a preparation method thereof and an electrochemical sensor.
Background
The brain is the most precise and complex organ in the human body, participates in life activities such as feeling, cognition, movement, language and emotion, and the chemical nature of revealing the neurological phenomenon in the brain is one of the main targets and important fronts of modern science. Research on brain function is also helpful for understanding the nature of complex physiological processes such as human cognition, emotion and the like, and the formation and development rules of nervous system diseases. The transmission of the cranial nerve signals and the metabolic processes are independent of the participation of chemical substances. Therefore, the development of the brain nerve analysis chemistry research aiming at a plurality of neurochemical substances such as neurotransmitters, tempering substances, energy metabolism substances, free radicals, ions and the like in the brain has extremely important significance for exploring and understanding the molecular mechanisms of neurophysiologic and pathology.
Acid-base balance and pH adjustment are critical to normal tissue metabolism and physiology, and the pH of brain tissue can change in many disease states. Over the past decades, detection techniques for changes in pH in the brain have made some breakthroughs, such as nuclear magnetic resonance, fluorescence, surface enhanced Raman scattering, and electrochemical methods. However, due to the lack of accurate in situ analysis techniques, the relationship between local pH fluctuations and brain diseases has not been widely studied. The most commonly used method for in-situ measurement of pH value of living body at present is based on electrochemical measurement of traditional microelectrode, and has high spatial resolution, simplicity and high sensitivity, but the problems of selectivity, pollution resistance and the like still face in the actual detection process. It is therefore highly desirable to develop a simple, efficient, contamination resistant, high sensitivity in vivo in situ pH sensing method.
The in situ electrochemical method has great advantages as a living body in situ sensing method with high space-time resolution. Classical electrochemical analysis methods are for detection of electroactive neurotransmitters or neuromodulators using in situ implantation of ultra-microelectrodes, but for some non-electrochemically active substances there are certain challenges. In recent years, micro-nano pore technology has been greatly developed, and single-cell and living body layered chemical substance detection is realized based on ionic current rectification sensing technology. However, the analysis method based on ion transmission regulation in the restricted-domain micro-pore is limited by the pollution of sensing interface proteins especially for living body layers.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the related art to some extent. Therefore, an object of the present invention is to provide a pH sensing electrode, a method for preparing the same, and an electrochemical sensor, wherein the pH sensing electrode has a good protein pollution resistance, a sensing interface of the pH sensing electrode is an in-situ polymerized BSA hydrogel, the detection of the concentration level of hydrogen ions can be achieved without a redox reaction process, the sensing capability of high space-time resolution is provided, the stability and the reversibility are excellent, the in-vivo brain pH sensing can be accurately and sensitively achieved, and the in-vivo level has a good stability.
In one aspect of the invention, a pH sensing electrode is provided, comprising a glass capillary, wherein one end of the glass capillary is a tip, both ends of the glass capillary are provided with openings, electrolyte is arranged in the glass capillary, and the diameter of the opening of the tip is smaller than that of the other end of the glass capillary; the electrochemical probe is arranged on one side of the glass capillary tube, which is provided with the tip, and is BSA hydrogel, and the BSA hydrogel is provided with a cross-linked network structure.
According to some embodiments of the invention, the cross-linked network has a pore size of 10 to 30 nanometers.
According to some embodiments of the invention, the BSA hydrogel comprises bovine serum albumin, glutaraldehyde and water.
According to some embodiments of the 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 invention, the electrochemical probe has a length of 10 to 100 microns.
According to some embodiments of the invention, the electrochemical probe is bound to the glass capillary by a chemical bond.
According to some embodiments of the invention, the tip opening has a diameter of 3-5 microns.
In another aspect of the present invention, a method for preparing the pH sensing electrode is provided, including providing a glass capillary tube with two ends open, 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; immersing the tip of the glass capillary into a BSA hydrogel, causing the BSA hydrogel to fill a side of the glass capillary having the tip, and polymerizing to form an electrochemical probe; electrolyte is added into the glass capillary tube to obtain the pH sensing electrode. Therefore, the pH sensing electrode prepared by the method has good protein pollution resistance, high space-time resolution sensing capability on the concentration of hydrogen ions, excellent specificity, stability and reversibility, can realize the real-time monitoring of the pH change level in the brain more sensitively, and has good stability when in-vivo level detection.
In yet another aspect of the present invention, an electrochemical detector is provided, which includes the aforementioned pH sensing electrode, and thus, the electrochemical detector has all the features and advantages of the aforementioned pH sensing electrode, which are not described herein, and generally has at least the advantages of good protein contamination resistance, good stability, and the like.
According to some embodiments of the invention, the electrolyte is a NaCl solution.
According to some embodiments of the invention, the working electrode and the reference electrode are Ag/AgCl electrodes.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 shows a schematic diagram of the structure of a pH sensing electrode according to an embodiment of the present invention;
FIG. 2 is a schematic flow chart of a method for preparing a pH sensing electrode according to one embodiment of the invention;
FIG. 3a is a schematic view showing the tip structure of the pH sensing electrode prepared in example 1 of the present invention;
FIG. 3b shows an SEM image of a pH sense electrode BSA hydrogel prepolymer prepared according to example 1 of the present invention;
FIG. 4a shows a cyclic voltammetry test pattern of a pH sensing electrode prepared in example 1 of the present invention;
FIG. 4b shows a cyclic voltammetry test pattern of a pH sensing electrode prepared in example 1 of the present invention;
FIG. 5a is a graph showing the anti-protein contamination performance test of the pH sensor electrode prepared in example 1 of the present invention;
FIG. 5b shows a graph for testing the anti-protein contamination performance of the pH sensor electrode prepared in example 1 of the present invention;
FIG. 6a is a graph showing the anti-protein contamination performance test of the pH sensor electrode prepared in example 1 of the present invention;
FIG. 6b shows a graph for testing the anti-protein contamination performance of the pH sensor electrode prepared in example 1 of the present invention;
FIG. 7a is a graph showing the sensing performance test of the pH sensing electrode prepared in example 1 of the present invention;
FIG. 7b shows a graph of the sensing performance of the pH sensing electrode prepared in example 1 of the present invention;
FIG. 7c shows a graph of the sensing performance of the pH sensing electrode prepared in example 1 of the present invention;
FIG. 8 shows a graph of reproducibility test of the pH sensor electrode prepared in example 1 of the present invention;
FIG. 9 shows a reversibility test chart of the pH sensing electrode prepared in example 1 of the present invention;
FIG. 10 is a graph showing the anti-interference performance test of the pH sensor electrode prepared in example 1 of the present invention;
FIG. 11 shows a graph of in vivo performance testing of the pH sensing electrode prepared in example 1 of the present invention;
FIG. 12 shows a graph of in vivo performance testing of the pH sensing electrode prepared in example 1 of the present invention.
Reference numerals:
100: a pH sensing electrode; 110: a tip; 120: an electrolyte; 130: an electrochemical probe.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In one aspect of the present invention, a pH sensing electrode is provided, referring to fig. 1, the pH sensing electrode 100 includes a glass capillary and an electrochemical probe, wherein one end of the glass capillary is a tip 110, two ends of the glass capillary are provided with openings, an electrolyte 120 is provided in the glass capillary, and the diameter of the opening of the tip 110 is smaller than that of the other end of the glass capillary; the electrochemical probe 130 is provided at a side of the glass capillary having the tip 110, and the electrochemical probe 130 is a BSA hydrogel having a cross-linked network structure. Therefore, the pH sensing electrode 100 has good protein pollution resistance, high space-time resolution sensing capability on hydrogen ion concentration, 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.
The principle of the present invention that the above advantageous effects can be achieved is briefly described below:
As described above, the currently commonly used ion transmission type sensor usually realizes the analysis and detection of specific substances by modifying functional regulation molecules on the inner surface of a glass tube, and the greatest disadvantage of the detection method is that a sensing interface is easy to be polluted by protein, the pH sensing electrode provided by the invention is characterized in that an electrochemical probe is arranged at the extreme position of a glass capillary, the electrochemical probe is BSA hydrogel with a crosslinked network structure, the in-situ polymerized BSA hydrogel forms the sensing interface of the pH sensing electrode, the pH sensing electrode can realize the detection of the concentration level of hydrogen ions without a redox reaction process, the detection principle is that the hydrogen ions in solution enter the BSA hydrogel filled in the tube under the action of electric field force and electroosmotic flow by applying continuous voltage steps, the entering of the hydrogen ions causes the protonation of amino acid residues on bovine serum albumin, and the charge quantity of the BSA hydrogel changes along with the change of the pH of an external solution, and the specific principle is that: as the pH of the external solution decreases, the charge amount of the BSA hydrogel filled in the tube decreases and the ionic current becomes smaller; as the pH of the external solution increases, the charge amount of the BSA hydrogel filled in the tube increases and the ionic current increases. Thus, the inventor can realize high-sensitivity and high-time-resolution real-time detection of the hydrogen ion level in the pH range of 5.8-8.0 by using the electrode, and has good stability in living body level (such as detection of pH level change in mouse brain). Meanwhile, the BSA hydrogel compact crosslinked network can prevent proteins from penetrating through the electrochemical probe and entering the sensing interface to generate false positive signals, so that protein pollution is prevented. The pore diameter of the cross-linked network structure can prevent protein but can not form barriers to ions, and in the actual pH sensing process, the ion transmission behavior is regulated and controlled by applying high-frequency square wave potential, so that the updating speed of the orifice interface of the tip end of the glass capillary is improved, and the protein resistance of the electrode is further improved. Therefore, the pH sensing electrode based on finite field ion transmission regulation and control can realize accurate and sensitive monitoring of the pH level change in the brain, and the method lays a foundation for the disease diagnosis and treatment of epilepsy, ischemia, alzheimer disease and other diseases and has wide application prospect.
According to some embodiments of the present invention, the pH sensing electrode 100 may be operated by inserting one end of the working electrode into the electrolyte solution 120 in the glass capillary, inserting one end of the reference electrode into the sample solution to be measured, and connecting the other end of the working electrode with the other end of the reference electrode through a power supply; and switching on a power supply to form an ion current path between the working electrode and the reference electrode, and recording the applied voltage value and the current value so as to detect the pH value of the sample to be detected.
According to some embodiments of the present invention, the diameter of the glass capillary tip 110 opening is 3-5 microns, whereby the electrochemical probe 130 is more robust and can be used for in situ detection analysis of living subjects.
According to some specific embodiments of the present invention, the BSA hydrogel comprises bovine serum albumin, glutaraldehyde and water, and specifically, the mass ratio of bovine serum albumin, glutaraldehyde and water in the BSA hydrogel is (5-20): (5-10): 100, the finally formed BSA hydrogel has better protein pollution resistance. The inventors found that if the bovine serum albumin content in the BSA hydrogel is too low, the crosslinking density is too low, so that the protein pollution resistance effect of the pH sensing electrode is reduced to a certain extent; if the content of bovine serum albumin is too high, the chemical crosslinking speed is higher after glutaraldehyde is added, the difficulty of repeatedly preparing the pH sensing electrode is increased, and the process reproducibility is reduced; if the glutaraldehyde content is too low, the crosslinking density of the bovine serum albumin is too low, so that the protein pollution resistance effect of the pH sensing electrode is reduced to a certain extent; if the glutaraldehyde content is too high, the chemical crosslinking speed of the bovine serum albumin is too high, and the difficulty in preparing the pH sensing electrode is increased, so that the process reproducibility is reduced.
According to some embodiments of the present application, the preparation method of the BSA hydrogel is not particularly limited, and for example, in the present application, a bovine serum albumin solution and an aqueous glutaraldehyde solution may be mixed to obtain the BSA hydrogel. According to some embodiments of the present application, the concentration of the bovine serum albumin solution and the glutaraldehyde solution is not particularly limited as long as the mass ratio of bovine serum albumin, glutaraldehyde and water in the finally formed BSA satisfies (5 to 20): (5-10): 100. For example, the concentration of bovine serum albumin in the bovine serum albumin solution is 50 to 200mg/mL. According to other embodiments of the present application, the glutaraldehyde content of the glutaraldehyde aqueous solution is 5% to 10% based on the total mass of the glutaraldehyde aqueous solution.
According to some embodiments of the invention, the pore size of the cross-linked network structure in the BSA hydrogel is 10-30 nanometers, so that proteins can be prevented from entering the pH sensing electrode, and pollution of the pH sensing electrode by the proteins is prevented. The inventor finds that if the pore diameter of the crosslinked network structure is too small, the requirement on the process is high, and the process reproducibility is poor; if the pore size of the crosslinked network is too large, the resistance of the pH sensing electrode to protein contamination is reduced.
According to some embodiments of the present invention, the length of the electrochemical probe 130 is not particularly limited, and one skilled in the art can design itself according to the detection needs, and in particular, the length of the electrochemical probe 130 may be 10 to 100 micrometers according to the present invention. The inventors found that if the length of the electrochemical probe 130 is too short, the requirements on the manufacturing process are high, and the process reproducibility is reduced; if the length of the electrochemical probe 130 is excessively long, the production cost is increased to some extent, resulting in unnecessary waste.
According to some embodiments of the present invention, since carboxyl groups exist on the surface of the glass, glutaraldehyde can crosslink amino groups on the surface of the protein and carboxyl groups on the surface of the glass, so that the electrochemical probe 130 and the glass capillary are combined through chemical bonds, that is, the electrochemical probe 130 and the inner surface of the glass capillary are tightly combined, and no gap exists, at this time, ions can only enter the electrolyte 120 through the pore canal of the electrochemical probe 130, but not enter through the gap between the electrochemical probe 130 and the glass, and the detection accuracy is improved to a certain extent.
In another aspect of the present invention, a method for preparing the aforementioned pH sensing electrode is provided, comprising (1) providing a glass capillary tube having two ends open, one end of the glass capillary tube being a tip, the opening diameter of the tip being smaller than the opening diameter of the other end of the glass capillary tube; (2) Immersing the tip of the glass capillary into a BSA hydrogel, causing the BSA hydrogel to fill a side of the glass capillary having the tip, and polymerizing to form an electrochemical probe; (3) Electrolyte is added into the glass capillary tube to obtain the pH sensing electrode. Therefore, the pH sensing electrode prepared by the method has simple process and good reproducibility, has good protein pollution resistance, has excellent specificity, stability and reversibility on the high space-time resolution sensing capability of the concentration of hydrogen ions, can accurately and sensitively realize the pH sensing in the brain of a living body, and has good stability on the level of the living body.
In the following, various steps of the method are described in detail, referring to fig. 2, according to an embodiment of the present invention, the method may include:
s100: preparation of glass capillary with tip
In this step, a glass capillary tube with two ends open is provided, 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. Specifically, a glass capillary having a tip can be prepared by drawing borosilicate glass having an outer diameter of 1.5mm and an inner diameter of 0.86mm under the following conditions: first cycle: the temperature is 430 ℃, the focusing range is 4, the speed is 28, the delay is 200, and the pulling force is 0; second cycle: the temperature is 325 ℃, the focusing range is 4, the speed is 30, the delay is 200, and the pulling force is 0, so that the glass capillary with one end as a tip end can be obtained, and the inner diameter of an opening at the tip end part is 3-5 microns.
S200: electrochemical probe formation by capillary action
In this step, the tips of the glass capillaries were immersed in the BSA hydrogel, so that the BSA hydrogel filled the side of the glass capillaries having the tips, and polymerized to form the electrochemical probes. According to some embodiments of the present invention, the immersion time of the glass capillary in the BSA hydrogel is not particularly limited, and one skilled in the art may select according to the length of the finally formed electrochemical probe, for example, immersing a glass capillary tip having a tip opening diameter of 3 to 5 μm into a BSA hydrogel pre-polymerization solution, immersing the solution into the tip by capillary action, and polymerizing for 3 minutes to obtain the BSA hydrogel-filled anti-protein contamination pH sensing electrode.
S300: electrolyte is added into the glass capillary tube
In the step, electrolyte is added into the glass capillary tube obtained in the step (2), so that the pH sensing electrode can be obtained.
In yet another aspect of the invention, an electrochemical detector is presented, comprising the aforementioned pH sensing electrode; an electrochemical workstation; the working electrode, wherein one end of the working electrode contacts with electrolyte in the glass capillary of the pH sensing electrode; and the other end of the working electrode is connected with the other end of the reference electrode through an electrochemical workstation. Therefore, the real-time, anti-pollution and high-sensitivity detection of various substances (such as hydrogen ions) can be realized, and the electrode has better robustness and protein pollution resistance and can be used for real-time analysis and detection in complex environments such as physiology, pathology and the like.
According to some embodiments of the invention, when the electrochemical detector is used for detection of a level of a living body, the electrolyte within the pH sensing electrode may be a NaCl solution. According to some embodiments of the invention, the working electrode and the reference electrode are Ag/AgCl electrodes.
Example 1
(1) Borosilicate glass tube with an outer diameter of 1.50mm, an inner diameter of 0.86mm and a length of 10cm was drawn by a P-2000CO 2 laser drawing machine by the following procedure to obtain glass nanotubes with a tip end and an average size of 3 μm in the opening of the tip:
(first cycle) temperature=430 ℃, focus range=4, speed=28, delay=200, pull force=0
(Second cycle) temperature=325 ℃, focus range=4, speed=30, delay=200, pull force=0
(2) Immersing the tip of the drawn glass capillary into a BSA hydrogel prepolymerization solution, wherein the prepolymerization solution contains 100 microliters of a mixed solution of 200mg/ml BSA and 20 microliters of 50% glutaraldehyde, immersing the solution into the tip of the tube by utilizing capillary action, and polymerizing for 3 minutes to obtain the BSA hydrogel-filled pH sensing electrode, wherein referring to fig. 3a and 3b, fig. 3a is an optical microscope image of the tip of the pH sensing electrode, and fig. 3b is an SEM image of a crosslinked network structure, and the BSA hydrogel can be seen to show a smaller porous crosslinked network structure.
And performing performance test on the obtained pH sensing electrode:
(1) Cyclic voltammetry test
Referring to fig. 4a and 4b, the working electrode is placed in the glass capillary of the pH sensing electrode and is contacted with electrolyte in the glass capillary of the pH sensing electrode, a Chenhua 660e electrochemical workstation is used, and the potential window is set to be-1V and the sweeping speed is 50mV/s. FIG. 4a is a cyclic voltammetry graph of a pH sensing electrode and a glass capillary, showing that the pH sensing electrode exhibits non-linear ionic 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. FIG. 4b is a cyclic voltammetry graph at different pH conditions, illustrating the pH response of the pH sensing electrode over a physiological pH range.
(2) Protein contamination resistance test
Referring to fig. 5a and 5b, a glass capillary containing an electrolyte and a pH sensing electrode were immersed in a 40mg/ml FITC-BSA solution for 12h, and referring to fig. 5a, a laser confocal image after the first behavioural pH sensing electrode was immersed for 12h, a, b and c in fig. 5a were laser confocal images after the glass capillary was immersed for 12h, d, e and f in fig. 5a were laser confocal images after the glass capillary was immersed for 12h, wherein a and d were test images under dark field conditions, b and e were test images under bright field conditions, c and f were test images superimposed with dark field conditions, in which it can be seen that the tip of the pH sensing electrode was not fluorescent, indicating that BSA hydrogel can prevent protein immersion, had better anti-protein contamination properties, and the tip of the glass capillary was fluorescent (e was also fluorescent, but not apparent under bright field conditions), indicating that protein was contaminated. FIG. 5b shows that the addition of 10mg/ml BSA did not interfere with the signal from the pH sensing electrode during the amperometric test.
(3) Anti-fouling Performance test on proteins of different concentrations and different classes
Referring to fig. 6a and 6b, 10mg/ml, 20mg/ml and 40mg/ml BSA proteins are respectively added into a sample to be detected, and it can be seen that after the same protein with different concentrations is added, compared with the pH drop of 0.8, the generated pollution is far less than 20% of the response, which indicates that the pH sensing electrode has better protein pollution resistance to the proteins with different concentrations; referring to FIG. 6b, 10mg/ml BSA (bovine Serum albumin), trypsin (bovine Trypsin), fibrinogen (fibrinogen), hb (hemoglobin), HRP (horseradish peroxidase) and Serum (Serum) are respectively added into a sample to be tested, and it can be seen that the pollution generated by different kinds of proteins is far less than 20% of the response, which indicates that the pH sensing electrode can not only resist the pollution of BSA protein, but also has better protein pollution resistance to other kinds of proteins.
(4) PH sensing Performance test
Referring to fig. 7a, the time resolution of the pH sensing electrode is tested in the physiological pH range, and it can be seen that the pH sensing electrode has high time resolution in the physiological pH range, so that when it is applied to a living body, the change of pH in the living body due to the external environmental stimulus can be rapidly detected; referring to fig. 7b, fig. 7b shows the relationship between pH and ionic current according to fig. 7a, and it can be seen from fig. 7b that in the physiological pH range, there is a linear relationship between pH and ionic current, and the pH sensing electrode can be used for detecting pH level change in the physiological pH range. Referring to FIG. 7c, one sample to be tested is artificial cerebrospinal fluid, the other sample to be tested is artificial cerebrospinal fluid containing BSA, and the pH sensing electrode is calibrated after the test is completed, so that the linear calibration has smaller deviation under the existence of 10mg/ml BSA, which indicates that the pH sensing electrode has good stability.
(5) Repeatability verification
Referring to fig. 8, 100mM NaCl solution is poured into a glass capillary of the pH sensing electrode, the solution to be measured outside the tube is artificial cerebrospinal fluid, and the pH of the solution to be measured is 8.0-5.8, and the repeated test is performed three times.
(6) Reversibility verification
Referring to FIG. 9, 100mM NaCl solution was poured into the glass capillary of the pH sensing electrode, and the solution to be measured outside the tube was 100mM NaCl and 50mM PBS solution at pH 7.4 and pH 5.8. The cyclic voltammetry test is carried out, the voltage is set to be-1V, the sweeping speed is 50mV/s, the external solution to be tested is respectively replaced by the solution with the pH of 7.4 and the pH of 5.8, and the cyclic voltammetry test is circulated for 8 times, so that the pH sensing electrode has excellent reversibility.
(7) Tamper resistance test
Referring to fig. 10, amperometric current tests were performed on the pH sensing electrode at an applied voltage of-1V, with 10μM DA,10μM E,200μM AA,50μM DOPAC,30μM 5-HT,10μM NE,1mM KCl,1mM NaCl,1mM MgCl2,1mM CaCl2 and a 0.8pH drop added, respectively. From the graph, except the pH response, the pH sensing electrode has no current response to other substances, and the pH sensing electrode has good specificity to the pH response.
(8) Live experience verification test
Referring to fig. 11, the test was performed using a square wave potential method, and the applied parameters of the step potential are shown in the above experimental examples. The micro-irrigation hose and the pH sensing electrode are implanted into the cerebral cortex together, and the interior of the micro-irrigation hose is artificial cerebrospinal fluid with pH=3. As can be seen from the figure, the pH sensing electrode can monitor the change in pH level by continuous administration of acid stimulation during 1 h. Referring to fig. 12, the pH sensing electrode was calibrated after in vivo removal, and the calibration was not significantly different before and after the experiment, indicating that the pH sensing electrode has excellent anti-contamination performance and stability for in-situ detection of living body.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (11)
1. A pH sensing electrode, comprising:
the device comprises a glass capillary, wherein one end of the glass capillary is a tip, openings are formed in two ends of the glass capillary, electrolyte is arranged in the glass capillary, and the diameter of the opening of the tip is smaller than that of the other end of the glass capillary;
The electrochemical probe is arranged on one side of the glass capillary tube, which is provided with the tip, and is BSA hydrogel, and the BSA hydrogel is provided with a cross-linked network structure.
2. The pH sensing electrode of claim 1, wherein the cross-linked network has a pore size of 10 to 30 nanometers.
3. The pH sensing electrode of claim 1, wherein the BSA hydrogel comprises bovine serum albumin, glutaraldehyde and water.
4. A pH sensing electrode according to claim 3, wherein in the BSA hydrogel, the mass ratio of bovine serum albumin, glutaraldehyde and water is (5-20): (5-10): 100.
5. The pH sensing electrode of any one of claims 1-4, wherein the electrochemical probe has a length of 10-100 microns.
6. The pH sensing electrode of claim 5, wherein the electrochemical probe is chemically bonded to the glass capillary.
7. The pH sensing electrode of claim 6, wherein the tip opening has a diameter of 3-5 microns.
8. A method of making the pH sensing electrode of claims 1-7, comprising:
(1) Providing a glass capillary tube with two open ends, wherein one end of the glass capillary tube is a tip, and the diameter of the opening of the tip is smaller than that of the other end of the glass capillary tube;
(2) Immersing the tip of the glass capillary into a BSA hydrogel, causing the BSA hydrogel to fill a side of the glass capillary having the tip, and polymerizing to form an electrochemical probe;
(3) Electrolyte is added into the glass capillary tube to obtain the pH sensing electrode.
9. An electrochemical detector, comprising:
A pH sensing electrode according to any one of claims 1-7 or prepared by the method of claim 8;
An electrochemical workstation;
one end of the working electrode is contacted with electrolyte in the pH sensing electrode glass capillary;
one end of the reference electrode is contacted with the sample to be detected, and the other end of the working electrode is connected with the other end of the reference electrode through an electrochemical workstation.
10. The electrochemical detector of claim 9, wherein the electrolyte is a NaCl solution.
11. The electrochemical detector of claim 9, wherein the working electrode and the reference electrode are Ag/AgCl electrodes.
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