CN107865637B - Living body assay H2S electrode, preparation method and in vivo detection H2S device - Google Patents
Living body assay H2S electrode, preparation method and in vivo detection H2S device Download PDFInfo
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Abstract
The invention discloses a biopsy H2S electrode, preparation method and in vivo detection H2And S. The electrode includes: a carbon fiber matrix; nano-gold particles formed on a surface of the carbon fiber substrate; to be provided withAnd H2S is a permeable membrane, said H2And S is formed on the outer surfaces of the carbon fiber matrix and the nano gold particles through a membrane. Thus, the electrode can avoid HS‑Contacting with carbon fiber matrix to specifically respond to H2And (4) an S signal. The carbon fiber matrix has smaller size and better biocompatibility and stability, and has the advantages of small brain damage, high space-time resolution and the like when being used for in-situ analysis of living bodies.
Description
Technical Field
The invention relates to the field of analytical chemistry, in particular to in-situ detection of H in a living body2S electrode, preparation method and in vivo detection H2And S.
Background
Hydrogen sulfide (H)2S) is an important endogenous gas signaling molecule in the body. In the mammalian body, H2S plays a plurality of physiological functions of resisting oxidative stress, regulating blood pressure, relaxing blood vessels and the like. H2S exists mainly in two forms in the brain: 1/3 with gas H2S form, 2/3 in HS-The morphology exists. Gas H2S and HS-Form dynamic balance in vivo, and maintain H in vivo2And S balancing. Commonly used assay H2The S method comprises a fluorescence probe method, an electrochemiluminescence method, a colorimetric method, a sulfur ion selective electrode method and the like.
However, the current in vivo hydrogen sulfide detection technology still needs to be improved.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art.
The present invention has been completed based on the following knowledge and findings of the inventors:
at present H2The S detection method is mostly only suitable for in vitro detection (cell culture, brain slice, etc.), and the target substance to be detected is H2S and HS-Mixture, not of pure H2An S signal molecule. Thus, current H2S detection method reflects H in brain easily, truly and in real time in physiological and pathological processes2And S change information.
In view of the above, in a first aspect of the invention, the invention proposes a method for activating a living organismBody detection H2And S electrode. The electrode can specifically respond to H2The microelectrode of S can be directly implanted into a specific part in the brain, and H in the brain can be analyzed by using in-situ electrochemical analysis technology of living bodies2And S, carrying out in-situ and real-time dynamic signal acquisition. H is carried out by using the electrode2The in-vivo detection of S has the advantages of high selectivity, small brain injury, high space-time resolution and the like.
In a first aspect of the invention, the invention proposes a method for in vivo detection of H2And S electrode. According to an embodiment of the invention, the electrode comprises: a carbon fiber matrix; nano-gold particles formed on a surface of the carbon fiber substrate; and H2S is a permeable membrane, said H2And S is formed on the outer surfaces of the carbon fiber matrix and the nano gold particles through a membrane. Due to H2The S-permeable membrane is negatively charged so that the electrode can avoid HS-Contacting with carbon fiber matrix to specifically respond to H2And (4) an S signal. The carbon fiber matrix has smaller size, better biocompatibility and stability, and has the advantages of small brain damage, high space-time resolution and the like when being used for in-situ analysis of living bodies. In general, the electrode is used for in vivo detection H2S, has the advantages of high selectivity, small brain injury, high spatial and temporal resolution and the like.
According to the embodiment of the invention, the carbon fiber substrate is formed by carbon fibers with amino groups modified on the surfaces. Therefore, the bonding strength of the nano gold particles and the carbon fiber matrix can be enhanced.
According to the embodiment of the invention, the surfaces of the carbon fiber substrate and the gold nanoparticles are modified with HS-。HS-The ions can play a role in eliminating the interference of oxygen in the system to the electrode, thereby improving the H resistance of the electrode2And (4) selectivity of S.
According to an embodiment of the invention, said H2The S permeable membrane is formed by perfluorosulfonic acid-polytetrafluoroethylene copolymer. H formed of the above materials2The S permeation film is negatively charged, so that HS can be effectively blocked-Passage of molecules, thereby allowing the electrode to be directed only to H2S signal proceedingAnd (6) responding.
In a second aspect of the invention, the invention proposes a method for in vivo detection of H2And S. According to an embodiment of the invention, the apparatus comprises: a detection electrode, said detection electrode being the electrode described above; the detection unit is connected with the detection electrode; and the computing unit is connected with the detection unit. Since the device employs the electrodes described above, the device has the electrodes described above for the in vivo examination H2All features and advantages of S will not be described herein. Generally speaking, the device has at least one of the advantages of high selectivity, small brain injury, high space-time resolution and the like.
According to an embodiment of the invention, the apparatus further comprises: and the reference electrode and the counter electrode are respectively and independently connected with the detection unit. Thus, the detection H of the device can be further improved2And (4) the accuracy of S.
In a third aspect of the invention, the invention proposes a method of preparing an electrode as described above. According to an embodiment of the invention, the method comprises: forming nano gold particles on the surface of the carbon fiber substrate; forming H on the outer surfaces of the carbon fiber matrix and the nano-gold particles2S permeates the film to form the electrode. The method is simple to operate, can quickly prepare the electrode, and is beneficial to reducing the production cost.
According to an embodiment of the present invention, before the gold nanoparticles are formed, the carbon fiber substrate is aminated in advance to form amino groups on the surface of the carbon fiber substrate. Therefore, the efficiency and the effect of forming the nano-gold particles can be improved, and the bonding strength of the nano-gold particles and the carbon fiber matrix can be enhanced.
According to an embodiment of the invention, the H is clad2Performing HS on the carbon fiber matrix and the gold nanoparticles in advance before S permeates the membrane-And (6) chemical treatment. Thus, the prepared electrode can be further reduced in H2The response potential of S.
According to an embodiment of the invention, the H is clad2And before S permeates the membrane, carrying out activation treatment on the carbon fiber matrix and the nano gold particles in advance. Thus, the performance of the electrode prepared by the method can be further improved.
In a fourth aspect of the invention, the invention proposes a method of preparing an electrode as described previously. According to an embodiment of the invention, the method comprises: (1) sequentially cleaning and electrochemically activating a carbon fiber substrate, wherein the electrochemically activating treatment comprises the following steps: polarizing the carbon fiber matrix for 30s at a potential of 2V, then polarizing the carbon fiber matrix for 10s at a potential of-1V, and finally performing cyclic voltammetry scanning on the carbon fiber matrix at a scanning speed of 0.05V/s within a range of 0-1V. (2) And (2) applying a 1.1V potential polarization to the carbon fiber substrate treated in the step (1) in 0.1mol/L ammonium carbamate solution for 60 minutes so as to form amino groups on the surface of the carbon fiber substrate. (3) And (3) soaking the carbon fiber substrate treated in the step (2) in a nanogold solution for not less than 8 hours, and then placing the soaked carbon fiber substrate in a mixed solution containing 2.4mmol/L of hydroxylamine hydrochloride and 1 wt% of chloroauric acid for 6 minutes so as to form nanogold particles on the surface of the carbon fiber substrate. (4) And performing cyclic voltammetry scanning on the carbon fiber substrate with the gold nanoparticles formed on the surface in a 0.5mol/L sulfuric acid solution at a scanning speed of 0.05V/s within a range of-0.2 to 1.0V. (5) And (3) soaking the carbon fiber substrate treated in the step (4) in 5mmol/L NaHS solution for 2 minutes, and washing the soaked carbon fiber substrate and the nano gold particles by adopting secondary distilled water. (6) Coating perfluorosulfonic acid-polytetrafluoroethylene copolymer on the surfaces of the carbon fiber substrate treated in the step (5) and the gold nanoparticles to form H2S permeates the membrane to obtain said electrode. The method is simple to operate, can quickly prepare the electrode, and is beneficial to reducing the production cost.
Drawings
FIG. 1 shows a method for in vivo H detection according to one embodiment of the present invention2Electricity of SA schematic view of a pole structure;
FIG. 2 shows a method for in vivo H detection according to one embodiment of the present invention2S is a schematic structural diagram of the device;
FIG. 3 shows a method for in vivo H detection according to another embodiment of the present invention2S is a schematic structural diagram of the device;
FIG. 4 shows a scanning electron microscope image of an electrode prepared in example 1 of the present invention;
FIG. 5 shows in vitro H of the electrode prepared in example 1 of the present invention2S, responding to a test result;
FIG. 6 shows the results of an electrode selectivity test of the electrode prepared in example 1 of the present invention;
FIG. 7 shows in vitro H of the electrode prepared in example 1 of the present invention2S concentration test results; and
FIG. 8 shows a living body H of the electrode prepared in example 1 of the present invention2And S, detecting a result.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
In a first aspect of the invention, the invention proposes a method for in vivo detection of H2And S electrode. According to an embodiment of the present invention, referring to fig. 1, the electrode includes: carbon fiber substrate 100, gold nanoparticles 200, and H2S permeates the membrane 300. Wherein nano gold particles 200 are formed on the surface of the carbon fiber substrate 100, H2The S-permeable film 300 is formed on the outer surfaces of the carbon fiber substrate 100 and the nano-gold particles 200, that is, H2The S permeable membrane 300 covers the outer surfaces of the carbon fiber substrate 100 and the gold nanoparticles 200.
The specific structure of the above-described electrode will be explained in detail below with reference to the embodiments of the present invention.
According to an embodiment of the present invention, carbon fibers are used to form the carbon fiber matrix 100. Therefore, the connection between the electrode and an external circuit and the output of a sensing signal can be realized by utilizing the conductivity of the carbon fiber. The carbon fiber itself has good biocompatibility and stability, so that the carbon fiber matrix 100 according to the embodiment of the present invention does not damage an organism when used for a biopsy, and can stably exist in a complex environment of the organism. And the carbon fiber has a thinner diameter, which is beneficial to miniaturizing the electrode and implanting the electrode into a body through a smaller wound for in vivo detection. In addition, the carbon fiber has good mechanical properties, so that the carbon fiber matrix 100 can maintain the integrity of the electrode matrix in a complex living environment. The diameter of the carbon fiber matrix 100 according to the embodiment of the present invention is not particularly limited, and those skilled in the art can select carbon fibers with appropriate size as the carbon fiber matrix 100 according to the embodiment of the present invention according to practical circumstances. According to an embodiment of the present invention, the diameter of the carbon fiber may be several micrometers to several tens of micrometers, for example, may be 5 μm to 50 μm. The diameter of the carbon fiber is too thin, so that the electrode is easily broken in the process of in vivo detection, and the diameter of the carbon fiber is too thick, so that the wound is not easy to miniaturize, and the electrical performance of the electrode is also influenced.
According to the embodiment of the invention, the nano gold particles 200 are modified on the surface of the carbon fiber substrate 100, so that the effective surface area of the electrode can be increased, the conductivity of the electrode can be improved, and the nano gold particles 200 have higher electron density, dielectric property and certain catalytic performance, so that the carbon fiber substrate 100 modified with the nano gold particles 200 can obtain better electrochemical performance, and the performance of the electrode can be further improved. In addition, the gold nanoparticles 200 have good biocompatibility, so that the electrode modified with the gold nanoparticles 200 does not cause damage to the living body in the process of in vivo detection.
According to the embodiment of the present invention, in order to improve the bonding force between the gold nanoparticles 200 and the carbon fiber substrate 100, the carbon fiber substrate 100 may be formed by using carbon fibers modified with amino groups on the surface. Thus, the gold nanoparticles 200 may be formed on the surface of the carbon fiber substrate 100 through the amino group. Therefore, the bonding strength of the nano gold particles and the carbon fiber matrix can be enhanced.
According to the embodiment of the present invention, in order to further improve the performance of the electrode, according to the embodiment of the present invention, the surfaces of the carbon fiber substrate 100 and the gold nanoparticles 200 may be further modified with HS-。HS-The ions can play a role in preventing oxygen in the system from interfering detection, and the selectivity of the electrode is improved. Thereby, the electrode can be further improved for the in vivo detection H2And (5) detecting the effect of S.
According to an embodiment of the present invention, H2S-permeable Membrane 300 for blocking HS-The passage of molecules. Thus, detection of H can be avoided2S, HS-The molecules interfere with the detection results. According to a particular embodiment of the invention, H2The S-permeable membrane 300 may be formed of a perfluorosulfonic acid-polytetrafluoroethylene copolymer. H formed of the above materials2The S permeable membrane has negative charges and can effectively block HS-Passage of molecules, thereby allowing the electrode to be directed only to H2The S signal responds. Thus, the electrode can avoid HS-Interference of signal, thereby specifically responding to H2And (4) an S signal.
Since the carbon fiber substrate 100 has electrical conductivity, the electrodes are connected to an external circuit, i.e., the electrochemical signal can be induced and transmitted. However, carbon fibers have a relatively thin diameter, and thus are not easy to handle during transportation and use and easily cause damage to the electrodes. Therefore, the electrode having the structure described above may be packaged in a capillary glass tube, an electrode connection portion being reserved at one end of the capillary glass tube for external circuit connection, and a portion of the electrode being exposed at the other end, so that the detection is completed. Therefore, the electrode can be protected from being damaged by external force in the operations of transportation, circuit connection and the like, and the surface of the electrode can be kept clean.
In a second aspect of the invention, the invention proposes a method for in vivo detection of H2And S. According to an embodiment of the invention, with reference to fig. 2, the apparatus comprises: detection electrode 1000, detection unit 2000, and calculation unit 3000. Specifically, the detecting electrode 1000 is the one described aboveThe electrode, detecting unit 2000 is connected to the detecting electrode 1000 for controlling the detecting electrode 1000 to perform detection using an electrochemical analysis technique, and transmits an electric signal detected by the detecting electrode 1000 to the calculating unit 3000 connected thereto. The calculation unit 3000 may be used to process the signals transmitted by the detection unit 2000 in order to finally realize H2And (5) detecting S. Since the device employs the electrodes described above, the device has the electrodes described above for the in vivo examination H2All features and advantages of S will not be described herein. Generally, the device has the advantages of high selectivity, small brain injury, high spatial-temporal resolution and the like.
According to an embodiment of the present invention, referring to fig. 3, the device may further include a reference electrode 4000 and a counter electrode 5000. The reference electrode 4000 and the counter electrode 5000 are independently connected to the detection unit 2000. The detection electrode 1000 (also called working electrode) forms an electrochemical circuit with the reference electrode 4000 and the counter electrode 5000, thereby further improving the detection H of the device2And (4) the accuracy of S.
In a third aspect of the invention, the invention proposes a method of preparing an electrode as described above. According to an embodiment of the invention, the method comprises:
setting nano gold particles:
according to an embodiment of the present invention, in this step, nanogold particles are formed on the surface of the carbon fiber substrate. The specific method for disposing the gold nanoparticles according to the embodiment of the present invention is not particularly limited, and may be selected by those skilled in the art according to practical circumstances. For example, gold nanoparticles with a certain particle size can be prepared in advance, and then the gold nanoparticles are modified on the surface of the carbon fiber substrate by utilizing the excellent adsorption performance of the gold nanoparticles; alternatively, the nano-gold particles can be grown in situ on the surface of the carbon fiber substrate by using a method for synthesizing the nano-gold particles. For example, according to the embodiment of the present invention, the carbon fiber substrate can be soaked in a mixed solution containing 2.4mmol/L hydroxylamine hydrochloride and 1 wt% chloroauric acid, and after standing for several minutes, nano-gold particles can be formed on the surface of the carbon fiber substrate.
In the present invention, "gold nanoparticles are formed on the surface of the carbon fiber substrate", and the like are to be understood in a broad sense, that is, gold nanoparticles cover at least a part of the surface of the carbon fiber substrate. As will be understood by those skilled in the art, since the sizes of the carbon fiber substrate and the gold nanoparticles are both in the micrometer and nanometer level, if the gold nanoparticles are controlled to completely cover the entire surface of the carbon fiber substrate and only cover the surface of the carbon fiber substrate (no gold nanoparticles are fixed on the surface of the carbon fiber substrate by being adsorbed on the surfaces of other gold nanoparticles), the difficulty of disposing the gold nanoparticles will be undoubtedly greatly increased, and the performance of the finally obtained electrode will not be significantly improved. Therefore, it can be understood by those skilled in the art that, in this step, it is not strictly controlled that the entire surface of the carbon fiber matrix is covered with the nano-gold particles as long as the nano-gold particles are formed on the surface of the carbon fiber matrix. It can be understood by those skilled in the art that, in this step, the larger the area of the carbon fiber matrix covered by the gold nanoparticles, the smaller the particle size of the formed gold nanoparticles is, which is beneficial to improve the performance of the electrode. Therefore, in order to cover the surface of the carbon fiber substrate with as many nanogold particles as possible and control the particle size of the nanogold particles, the carbon fiber substrate may be first soaked in a nanogold solution overnight before being placed in a mixed solution of chloroauric acid and hydroxylamine hydrochloride according to an embodiment of the present invention. Therefore, a part of nano-gold particles can be firstly arranged on the surface of the carbon fiber substrate, so that more nano-gold particles can be formed on the surface of the carbon fiber substrate.
According to the embodiment of the present invention, in order to further improve the effect of providing the gold nanoparticles, the carbon fiber substrate may be aminated in advance before the gold nanoparticles are formed, so that amino groups are formed on the surface of the carbon fiber substrate. Thus, the gold nanoparticles can be bonded to the surface of the carbon fiber substrate via the amino group. Compared with the mode of direct adsorption on the surface of the carbon fiber substrate (the binding force is Van der Waals force and electrostatic adsorption), the mode of binding the amino on the surface of the carbon fiber substrate has stronger binding force, so that the efficiency and the effect of forming the nano gold particles can be further improved. The specific method for amination treatment of the carbon fiber substrate is not particularly limited as long as the carbon fiber substrate can be modified with an amino group on the surface thereof without affecting the stability and conductivity of the carbon fiber substrate itself. For example, according to an embodiment of the present invention, an amino group may be electrochemically modified on the surface of the carbon fiber substrate. Specifically, the carbon fiber substrate may be placed in a solution containing an amino group, for example, in an ammonium carbamate solution, and then a voltage of a fixed potential is applied to the carbon fiber substrate, so that the amino group is modified to the surface of the carbon fiber substrate by an electrochemical reaction.
According to the embodiment of the invention, in order to further improve the detection of H by using the electrode prepared by the method2S, the method may further comprise: performing HS on carbon fiber matrix and nano gold particles-And (6) chemical treatment. The inventor finds that the modified HS is obtained through intensive research and a large number of experiments-Can be remarkably improved for H2And (4) selectivity of S. Therefore, when the surfaces of the carbon fiber matrix and the nano gold particles are modified with HS-When the oxygen content is reduced to H, the oxygen content can be obviously reduced2And S interference. According to a specific embodiment of the present invention, the HS-The chemical treatment can be realized by the following steps: the carbon fiber substrate provided with the gold nanoparticles is soaked in 5mmol/L NaHS solution for 2 minutes, and then washed with secondary distilled water. Thus, HS can be easily modified on the surfaces of the carbon fiber substrate and the gold nanoparticles-。
Form H2S, a permeable membrane:
according to an embodiment of the present invention, in this step, H is provided on the outer surfaces of the carbon fiber matrix and the nano-gold particles2S permeates the membrane. With respect to H2The function and composition of the S permeable membrane have been described in detail above and will not be described herein. According to a specific embodiment of the present invention, in this step, the carbon fiber matrix provided with the gold nanoparticles may be immersed in a solution containing perfluorosulfonic acid-polytetrafluoroethylene co-polymerAnd (3) adding the solution of the polymer or dipping the solution containing the perfluorosulfonic acid-polytetrafluoroethylene copolymer, taking out the solution, and drying to remove the solvent. In this step, in order to make H2S the permeation film fully covers the outer surfaces of the carbon fiber matrix and the nano gold particles, and dipping or immersing operation can be carried out for multiple times.
According to an embodiment of the present invention, in order to further improve the performance of the electrode obtained by the method, the method may further include:
pretreating a carbon fiber matrix:
according to an embodiment of the present invention, the carbon fiber substrate may be pretreated in advance before the gold nanoparticles are disposed. The pretreatment may include a cleaning treatment and an electrochemical activation treatment. Thus, the preparation effect of the subsequent steps can be further improved. According to a specific embodiment of the present invention, the cleaning process may include ultrasonic cleaning of the carbon fibers with acetone, ethanol, and secondary distilled water in sequence. Specifically, the carbon fiber substrate may be sequentially placed in acetone, ethanol and redistilled water at a frequency of 50kHz to be subjected to ultrasonic treatment, and the time of each ultrasonic treatment may be 3 minutes. Thus, impurities such as organic substances and particles adhering to the surface of the carbon fiber substrate can be cleaned. And (3) rinsing the carbon fiber matrix cleaned by ultrasonic cleaning with secondary distilled water, and performing electrochemical activation treatment. According to particular embodiments of the present invention, the electrochemical activation treatment may include: polarizing the carbon fiber matrix for 30s at a potential of 2V, then polarizing the carbon fiber matrix for 10s at a potential of-1V, and finally performing cyclic voltammetry scanning on the carbon fiber matrix at a scanning speed of 0.05V/s within a range of 0-1V until a stable cyclic voltammetry curve is obtained.
Activating the carbon fiber matrix with the formed nano gold particles:
according to an embodiment of the invention, in the coating H2Before S permeates the membrane, the carbon fiber substrate on which the gold nanoparticles are formed may be activated in advance. According to the embodiment of the invention, sodium can be formed on the surface in 0.5mol/L sulfuric acid solution at the scanning speed of 0.05V/s and in the range of-0.2-1.0VAnd (3) carrying out cyclic voltammetry scanning on the carbon fiber matrix of the migold particles until a stable cyclic voltammetry curve is obtained, and then completing activation.
In a fourth aspect of the invention, the invention proposes a method of preparing an electrode as described previously. According to an embodiment of the invention, the method comprises:
(1) and sequentially cleaning and electrochemically activating the carbon fiber matrix. According to an embodiment of the present invention, the cleaning process may have the same features as the cleaning step in the method for preparing an electrode described above, and thus, the description thereof is omitted. The electrochemical activation treatment comprises: polarizing the carbon fiber matrix for 30s at a potential of 2V, then polarizing the carbon fiber matrix for 10s at a potential of-1V, and finally performing multiple cyclic voltammetry scans on the carbon fiber matrix at a scanning speed of 0.05V/s within a range of 0-1V. And after the obtained cyclic voltammetry scanning curve is stable, the electrochemical activation treatment can be completed.
(2) And (2) applying a 1.1V potential polarization to the carbon fiber substrate treated in the step (1) in 0.1mol/L ammonium carbamate solution for 60 minutes so as to form amino groups on the surface of the carbon fiber substrate. Therefore, the efficiency and the effect of subsequently arranging the nano gold particles can be improved.
(3) And (3) soaking the carbon fiber substrate treated in the step (2) in the nanogold solution for not less than 8 hours (for example, soaking overnight), and then placing the soaked carbon fiber substrate in a mixed solution containing 2.4mmol/L of hydroxylamine hydrochloride and 1 wt% of chloroauric acid for 6 minutes so as to form nanogold particles on the surface of the carbon fiber substrate. Thus, the efficiency and effect of disposing the gold nanoparticles can be improved.
(4) And (3) in 0.5mol/L sulfuric acid solution, carrying out cyclic voltammetry scanning on the carbon fiber substrate with the gold nanoparticles formed on the surface for multiple times at a scanning speed of 0.05V/s and within a range of-0.2-1.0V until the cyclic voltammetry curve to be obtained is stable.
(5) Soaking the carbon fiber substrate treated in the step (4) in 5mmol/L NaHS solution for 2 minutes, washing with secondary distilled waterA soaked carbon fiber matrix and gold nanoparticles. Therefore, HS can be modified from the surfaces of the nano gold particles and the carbon fiber matrix-. For modifying HS-The advantages of the ions have been described in detail above and will not be described further herein.
(6) Coating perfluorosulfonic acid-polytetrafluoroethylene copolymer on the surfaces of the carbon fiber substrate and the nano gold particles treated in the step (5) so as to form H2S permeates the film, thereby obtaining an electrode according to an embodiment of the present invention.
The method is simple to operate, can quickly prepare the electrode, and is beneficial to reducing the production cost.
The present invention is illustrated below by way of specific examples, which are intended to be illustrative only and not to limit the scope of the present invention in any way, and unless otherwise specified, conditions or steps not specifically recited are generally conventional and reagents and materials used therein may be commercially available.
Example 1: living body assay H2Preparation of S electrode
Firstly, ultrasonically cleaning a carbon fiber matrix in acetone, ethanol and secondary water in sequence. The ultrasonic frequency was 50kHz and the ultrasonic time was 3 minutes. Washing the electrode with secondary water, and adding 0.5mmol/L H2SO4Electrochemical activation in solution: applying a potential of 2V to the electrodes, and polarizing for 30 s; applying a potential of-1V to the electrodes, and polarizing for 10 s; and carrying out multiple cyclic voltammetry scans in a potential range of 0-1V at a scan speed of 0.05V/s until a stable cyclic voltammogram is obtained.
The carbon fiber substrate prepared previously was placed in a 0.1mol/L ammonium carbamate solution, and a potential of 1.1V was applied for polarization for 60 minutes. Obtaining the carbon fiber matrix modified by amino. And soaking the amino modified carbon fiber substrate in the nano-gold solution overnight. After being taken out, the mixture is washed by secondary water. Then, the mixture was immersed in a mixture containing 2.4mmol/L of hydroxylamine hydrochloride and 1 wt% of chloroauric acid for 6 minutes. Thus obtaining the carbon fiber substrate with the surface modified with the nano-gold particles. Subsequently, the surface is modified withThe carbon fiber matrix of the nano gold particles is 0.5mol/L H2SO4Activation is carried out in solution, and a plurality of cyclic voltammetry scans are carried out in a potential range of-0.2-1.0V at a scanning speed of 0.05V/s until a stable cyclic voltammogram is obtained.
Soaking the activated carbon fiber matrix in 5mmol/L NaHS solution for 2 minutes, taking out the carbon fiber matrix, and washing the carbon fiber matrix with secondary water to clean the carbon fiber matrix and the surface of the nano-gold particles to modify HS-。
A layer of perfluorosulfonic acid-polytetrafluoroethylene copolymer film is modified on the front or the surface of the carbon fiber substrate by adopting a dipping mode. The scanning electron micrograph of the obtained electrode is shown in fig. 4. The diameter of the electrode is about 5 microns.
Performance testing
The following electrode Performance test and Living body H2S response tests all used the electrode obtained in example 1 as a working electrode, Ag/AgCl as a reference electrode, and a platinum wire as a counter electrode. The electrochemical workstation adopts CHI1030C workstation manufactured by Shanghai Chenghua instruments Co., Ltd to realize in vivo electrochemical analysis under a three-electrode system.
In vitro H2S response test
And (3) placing the three electrodes in 5mL of artificial cerebrospinal fluid solution, and performing cyclic voltammetry scanning at a scanning speed of 0.05V/s within a potential range of-0.3-0.6V. Subsequently, the solution was concentrated in 5mL of a solution containing 100. mu. mol/L H2S artificial cerebrospinal fluid, the cyclic voltammetric scan described above was repeated. The cyclic voltammetry scan results are referenced in fig. 5. The electrode contains H2In the solution of S, a characteristic redox peak appears, indicating that for H2S response is good.
Electrode selectivity test
Following Living body examination H2S, detecting common interferents to test the electrodes for H2And (4) selectivity of S. Specifically, the response condition of the electrode to the interferent is analyzed by adopting the three-electrode system and utilizing a constant potential technology. Specifically, a voltage of-0.05V was applied to the working electrode, and a current-time curve test was performed, with reference to fig. 6 for the test results. After the background current stabilized (about 100 s), the two phases were sequencedAdding Ascorbic Acid (AA), Dopamine (DA), dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT), Uric Acid (UA), Glutathione (GSH), cysteine (L-Cys) and Glucose (Glucose) at a time interval of 50 s. Although the current intensity fluctuates slightly after the addition of the interfering substances, the current intensity does not rise significantly, indicating that the electrode does not respond to these common interfering substances. After the background current is stabilized, H is added2S solution (about 550S), the current intensity increased significantly and reached equilibrium in a shorter time, after which the current intensity remained unchanged and the current/time curve plateaued. Repeating the above addition of H2The S solution was run twice and the electrode showed good correspondence to hydrogen sulfide. And then sequentially adding Ascorbic Acid (AA), Dopamine (DA), dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT), Uric Acid (UA), Glutathione (GSH), cysteine (L-Cys) and Glucose (Glucose), wherein the current does not obviously change. The electrode has good selectivity to the hydrogen sulfide, and the selectivity of the electrode is not affected before and after the hydrogen sulfide signal is detected.
In vitro H2S concentration test
The test conditions were tested with the electrode selectivity, and the test results obtained are shown in FIG. 7. Except that H was added to the electrolyte after the background current had stabilized (at about 100 s), at 50s intervals2S solution, H in the electrolyte is adjusted in sequence2The S concentration is 3, 6, 9, 12, 15, 18, 21, 24, 27 and 30 mu mol/L. As can be seen from fig. 7, the current intensity on the surface of the electrode is significantly increased after the addition of hydrogen sulfide, and can be maintained for a period of time after the addition of hydrogen sulfide, so that the current curve shows a plateau after each adjustment of the hydrogen sulfide concentration. After the concentration of the hydrogen sulfide is adjusted for many times, the concentration of the hydrogen sulfide in the electrolyte changes in a gradient manner, and the current of the electrode also increases in a gradient manner along with the concentration of the hydrogen sulfide, which shows that the electrode responds sensitively to the change of the concentration of the hydrogen sulfide.
Living body H2S detection
Placing the working electrode in the brain of a living mouse, selectively testing the working electrode under the same test condition, and applying the working electrode to the cerebral ischemia process of the mouseH in mouse cerebral ischemia process2The change of S content was monitored.
Rats were anesthetized with 10% chloral hydrate injection (345mg/kg, i.p.), placed in a stereotaxic apparatus, and maintained in a thermostatted pad to maintain normal body temperature. An incision is made in the middle of the parietal cranium and the tissue is pulled open to expose bregma and bregma. Drilling a hole with a diameter of about 1mm (AP 2.5mm, L4.36 mm from bregma) in the right brain, and fixing with a stereotaxic instrument H2S working electrode, slowly inserted into CA1 brain region (AP 2.5mm, L4.36 mm from bregma, V3 mm from dura). Detection of H in rat brain using current-time method2The S content.
The mouse cerebral ischemia model adopts a 2-VO whole cerebral ischemia model. In this model, ligation of the bilateral common carotid arteries in rats resulted in total cerebral ischemia in rats. A longitudinal incision was made in the ventral cervical correction of the rat and the bilateral common carotid arteries were isolated from the accompanying vagus nerve after exposure. The separated bilateral common carotid arteries are respectively ligated by using 3-0 silk threads, and a permanent 2-VO whole-brain ischemia model can be formed.
The detection results are shown in fig. 8. The result shows that the electrode can generate obvious current response to the mouse cerebral ischemia process in the living body detection environment. Therefore, the H in the mouse brain can be monitored in real time2The concentration of S varies.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean 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 invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer 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, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (11)
1. H for in vivo detection2S, characterized in that it comprises:
a carbon fiber matrix;
nano-gold particles formed on a surface of the carbon fiber substrate; and
H2s is a permeable membrane, said H2S is formed on the outer surfaces of the carbon fiber matrix and the nano gold particles through a membrane, and H is2S is negatively charged through the membrane, said H2The S permeable membrane is formed by perfluorosulfonic acid-polytetrafluoroethylene copolymer.
2. The electrode of claim 1, wherein the carbon fiber substrate is formed of carbon fibers modified with amino groups on the surface.
3. The electrode according to claim 2, wherein the gold nanoparticles are formed on the surface of the carbon fiber substrate through the amino groups.
4. The electrode of claim 1, wherein the carbon fiber matrix and the gold nanoparticles are surface modified with HS-。
5. H for in vivo detection2S, characterized in that it comprises:
a detection electrode according to any one of claims 1 to 4;
the detection unit is connected with the detection electrode; and
and the computing unit is connected with the detection unit.
6. The apparatus of claim 5, further comprising: and the reference electrode and the counter electrode are respectively and independently connected with the detection unit.
7. A method of making the electrode of any one of claims 1 to 4, comprising:
forming nano gold particles on the surface of the carbon fiber substrate;
forming H on the outer surfaces of the carbon fiber matrix and the nano-gold particles2S permeates the film to form the electrode.
8. The method according to claim 7, wherein the carbon fiber substrate is aminated in advance before the gold nanoparticles are formed, so that amino groups are formed on the surface of the carbon fiber substrate.
9. The method of claim 7, wherein the H is clad2Performing HS on the carbon fiber matrix and the gold nanoparticles in advance before S permeates the membrane-And (6) chemical treatment.
10. The method of claim 9, wherein the H is clad2And before S permeates the membrane, carrying out activation treatment on the carbon fiber matrix and the nano gold particles in advance.
11. A method of making the electrode of any one of claims 1 to 4, comprising:
(1) sequentially cleaning and electrochemically activating a carbon fiber substrate, wherein the electrochemically activating treatment comprises the following steps: polarizing the carbon fiber matrix for 30s at a potential of 2V, then polarizing the carbon fiber matrix for 10s at a potential of-1V, and finally performing cyclic voltammetry scanning on the carbon fiber matrix at a scanning speed of 0.05V/s within a range of 0-1V;
(2) applying 1.1V potential polarization to the carbon fiber substrate treated in the step (1) in 0.1mol/L ammonium carbamate solution for 60 minutes to form amino on the surface of the carbon fiber substrate;
(3) soaking the carbon fiber substrate treated in the step (2) in a nano gold solution for not less than 8 hours, and then placing the soaked carbon fiber substrate in a mixed solution containing 2.4mmol/L of hydroxylamine hydrochloride and 1 wt% of chloroauric acid for 6 minutes so as to enable gold nanoparticles on the surface of the carbon fiber substrate to grow and cover on the carbon fiber substrate;
(4) performing cyclic voltammetry scanning on the carbon fiber substrate with the gold nanoparticles formed on the surface in a 0.5mol/L sulfuric acid solution at a scanning speed of 0.05V/s and within a range of-0.2-1.0V;
(5) soaking the carbon fiber substrate treated in the step (4) in 5mmol/L NaHS solution for 2 minutes, and washing the soaked carbon fiber substrate and the nano gold particles by adopting secondary distilled water;
(6) coating perfluorosulfonic acid-polytetrafluoroethylene copolymer on the surfaces of the carbon fiber substrate treated in the step (5) and the gold nanoparticles to form H2S permeates the membrane to obtain said electrode.
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