CN115747200A - Application of three-electrode detection structure in preparation of adenosine sensor - Google Patents
Application of three-electrode detection structure in preparation of adenosine sensor Download PDFInfo
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Abstract
The invention provides application of a three-electrode detection structure in preparation of an adenosine sensor, and application of the three-electrode detection structure in acupuncture. The three-electrode detection structure is applied to the preparation of an adenosine sensor, a glucose sensor, a lactic acid sensor, a glutamic acid sensor, a uric acid sensor, an ascorbic acid sensor or a dopamine sensor; the adenosine sensor is an implanted adenosine continuous detection sensor. The flexible implanted adenosine sensor with the three-electrode structure has the characteristics of good stability, high sensitivity, wide linear detection range, short response time, strong anti-interference performance and the like, can realize in-vivo continuous detection on the concentration change of adenosine in animal bodies and brains, is successfully applied to in-vivo real-time monitoring of acupuncture-induced local adenosine release in acupuncture points, and provides a brand-new modern detection means for the deep research of acupuncture onset mechanisms.
Description
Technical Field
The invention belongs to the field of electrochemistry, and particularly relates to an application of a three-electrode detection structure in preparation of an adenosine sensor.
Background
Acupuncture and moxibustion has definite therapeutic effect, but the onset mechanism and biological foundation of acupuncture and moxibustion need to be elucidated urgently. In recent years, a number of studies have shown that adenosine signaling plays a key role in the initiation of acupuncture. Adenosine is produced primarily by the hydrolysis of Adenosine Triphosphate (ATP), an endogenous neuromodulator, and is involved in a variety of physiological and pharmacological processes, such as neurotransmission, inflammation, ischemic injury and pain. As an extracellular messenger in most body fluids, adenosine passes through four G protein-coupled receptor subtypes (A) 1 、A2 A 、A 2B And A 3 ) The interactions play their role and there are significant differences in the affinities of these four receptors for adenosine. Importantly, different adenosine concentrations can mediate the activation of different adenosine receptor subtypes, thereby exerting different physiological regulatory functions. Therefore, the immediate detection of local adenosine release induced under different conditions or stimuli is a real need in the adenosine research field. At present, most of in vivo adenosine detection methods need to treat taken blood or specific tissues under an in vitro condition and then adopt a traditional high performance liquid chromatography technology or an immunological analysis method for detection, and the like, so that the defects of discontinuous, delayed, large difference, unstable and the like exist in an experimental result. Although the microdialysis technology is also used for in-vivo sampling detection in recent years, each sampling needs to be continuously sampled for 5-20 minutes, which is actually accumulated amount rather than real-time amount, and other technologies such as high performance liquid chromatography and the like are still needed for detection after sampling, so that the method cannot be truly performed in the first time,Accurately reflects the concentration change rule of adenosine in the whole acupuncture effect taking process and how the adenosine participates in real-time regulation and control.
Disclosure of Invention
In view of the above, the present invention is directed to overcoming the drawbacks of the prior art, and providing an application of a three-electrode detection structure in the preparation of an adenosine sensor.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
the invention provides an enzyme immobilization accelerant, which comprises polyethyleneimine functionalized graphene oxide and genipin in a mass ratio of 1-5.
The invention also provides an enzyme crosslinking stationary liquid, which comprises a polyethyleneimine functionalized graphene oxide solution and a multienzyme cascade reaction liquid in a volume ratio of 1-3.
Further, the multi-enzyme cascade reaction solution comprises xanthine oxidase, purine nucleoside phosphorylase, adenosine deaminase and genipin in a volume ratio of 1-2.
Further, the concentration of the polyethyleneimine-functionalized graphene oxide solution is 1-10mg/ml; the concentration of the xanthine oxidase in the multienzyme cascade reaction liquid is 0.5-5U/ml; the concentration of purine nucleoside phosphorylase in the multi-enzyme cascade reaction liquid is 0.5-5U/ml; the concentration of adenosine deaminase in the multi-enzyme cascade reaction liquid is 0.5-5U/ml; the concentration of genipin in the multienzyme cascade reaction liquid is 1-10mg/ml.
The invention also provides a method for functionally modifying the surface of the conductive reaction area of the working electrode, which comprises the following steps:
(1) Immersing the conductive reaction area into an electrodeposition solution for electrodeposition to form an electronic medium layer on the conductive reaction area;
(2) Dripping a polyethyleneimine functionalized graphene oxide solution onto the surface of the electronic medium body layer, airing, dripping the multienzyme cascade reaction solution onto the surface of the electronic medium body layer, and airing to form an enzyme reaction layer;
(3) And immersing the conductive reaction area modified with the electronic medium layer and the enzyme reaction layer into an anti-interference solution for electrodeposition, and drying to form the anti-interference layer.
Further, the electrodeposition solution in the step (1) comprises hydrochloric acid with the concentration of 2-5mg/mL, a potassium chloride solution with the concentration of 5-10mg/mL, an iron chloride solution with the concentration of 2-5mg/mL, a potassium ferricyanide solution with the concentration of 2-10mg/mL and graphite alkyne nano-particles with the concentration of 0.1-1 mg/mL; the anti-interference solution in the step (3) comprises o-phenylenediamine with the concentration of 10-50mg/mL and bovine serum albumin with the concentration of 1-10mg/mL.
The invention also provides a three-electrode detection structure, which comprises a silver foil and 2 flexible film substrates, wherein the flexible film substrates are respectively fixed on the upper surface and the lower surface of the silver foil through adhesive layers, conductive layers are arranged on the flexible film substrates, and insulating layers are arranged on the conductive layers;
the length of the insulating layer is less than that of the conducting layer;
the exposed part of the conducting layer relative to the insulating layer is a conducting reaction area, and one conducting reaction area is subjected to surface functional modification by using the method of claim 5 or 6. The modified conductive reaction area is used as a working electrode, and the unmodified conductive reaction area is used as a counter electrode.
Further, the length of the conducting layer is smaller than that of the silver foil; the conducting layer is made of at least one of gold, platinum, titanium, palladium, copper, carbon black, graphite or graphene; the flexible film substrate is at least one of polycarbonate, polytetrafluoroethylene, polyimide, polyethylene terephthalate, acrylonitrile-butadiene-styrene copolymer or polymethyl methacrylate. And after electroplating and chlorination are carried out on the exposed part of the silver foil relative to the conducting layer, a compact and stable Ag/AgCl layer is formed on the surface of the silver foil, then the silver foil is immersed in a 5% Nafion solution and stands for 1-10s, then the silver foil is taken out, and the silver foil is dried at a room temperature in a dark place to serve as a reference electrode.
The invention also provides application of the three-electrode detection structure in acupuncture.
Further, the three-electrode detection structure is applied to the preparation of an adenosine sensor, a glucose sensor, a lactate sensor, a glutamate sensor, a uric acid sensor, an ascorbic acid sensor or a dopamine sensor; the adenosine sensor is an implanted adenosine continuous detection sensor.
A flexible implantable adenosine sensor having a three-electrode configuration, said sensor comprising said three-electrode sensing configuration.
Compared with the prior art, the invention has the following advantages:
based on the technical advantages of low detection cost, no need of marking and sampling and the like of an electrochemical biosensing detection technology, the flexible implantable adenosine sensor with the three-electrode structure further adopts multiple high and new technologies such as a multienzyme cascade reaction system, composite nano material synergism, MEMS micro electro mechanical system processing and the like on the basis, so that the flexible implantable adenosine sensor has the characteristics of good stability, high sensitivity, wide linear detection range, short response time, strong anti-interference performance and the like, can realize in-vivo, in-situ, real-time and continuous measurement of the adenosine concentration in animal subcutaneous tissues and brain tissues, is successfully applied to monitoring and research of the influence of acupuncture operation with different twisting rates on the change of the local adenosine content in a cavernous region, and provides a more effective high and new technical analysis means method for the deep research of the traditional acupuncture effect mechanism.
Drawings
FIG. 1 is a perspective view of a three-electrode detection structure according to an embodiment of the present invention;
FIG. 2 is a side view of a three-electrode sensing configuration according to an embodiment of the present invention;
FIG. 3 is a plot of a linear fit of the square root of the CV scan rate to the redox peak current value in accordance with an embodiment of the present invention;
FIG. 4 is a schematic diagram illustrating the operation of the conductive reaction zone according to the embodiment of the present invention;
fig. 5 is a diagram of selecting an optimal detection voltage according to an embodiment of the present invention: 5-A is a current-time curve under different detection voltages, and 5-B is a detection sensitivity contrast diagram under different detection voltages;
FIG. 6 is a continuous detection map of adenosine in an in vitro environment according to an embodiment of the present invention: 6-A is a current-time curve, and 6-B is a linear fitting curve;
FIG. 7 is a diagram illustrating interference rejection testing according to an embodiment of the present invention;
FIG. 8 is a continuous detection of local adenosine in animals according to embodiments of the present invention;
FIG. 9 is a continuous image of regional adenosine in the brain of an animal according to an embodiment of the present invention;
FIG. 10 is a graph of in vivo real-time monitoring of acupuncture-induced local adenosine release at the crypt site, in accordance with an embodiment of the present invention.
Description of reference numerals:
1. a silver foil; 2. a flexible film substrate; 3. a conductive layer; 4. an insulating layer; 5. a conductive reaction zone; 6. and (6) a glue layer.
Detailed Description
Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, were all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
The present invention will be described in detail with reference to examples.
EXAMPLE 1 preparation of microelectrode detection Structure
1. Selecting a polyethylene glycol terephthalate flexible film with the thickness of 0.1mm as a base material, depositing a nano platinum conducting layer with the thickness of 20 mu m on one side of the flexible film by using an evaporation or magnetron sputtering method, and sticking the flexible film to specific positions of the front side and the back side of a silver foil (the purity is 99.99%) with the thickness of 0.05mm by using medical double faced adhesive tape, wherein the two sides of the silver foil can not be completely covered, and parts with conductive properties at the two ends are reserved;
2. sticking the insulated medical single-sided adhesive to the specific positions of the nano platinum conducting layers on the front and back sides of the silver foil, and reserving parts with two ends capable of conducting electricity;
3. cutting the sheet into a needle-shaped structure by using an ultraviolet laser cutting machine, wherein the size of a silver foil layer exposed on the middle layer of the tip is 1mm multiplied by 0.4mm, and nano platinum layers exposed on the front and back surfaces are respectively used as a working electrode and a counter electrode and are both 1mm multiplied by 0.4mm; the other end of each layer can be exposed out of the conductive part and is used for connecting an electrochemical workstation;
4. completely immersing the silver foil layer exposed at the middle layer of the tip into dilute hydrochloric acid, performing electroplating chlorination treatment to form an Ag/AgCl layer on the surface, immersing into a 5% Nafion solution, standing for 3s, taking out, and drying at room temperature in a dark place, as shown in figure 1-2;
example 2 surface functional modification of conductive reaction zone of working electrode
The surface of the conductive reaction area of the working electrode needs to be functionally modified, and the modification layer is an electronic medium layer, an enzyme reaction layer and an anti-interference layer from inside to outside.
Preparing an electronic medium layer: mixing and dissolving 0.1M hydrochloric acid, 0.1M potassium chloride, 1.5mM ferric chloride and 1.5mM potassium ferricyanide, slowly adding 0.1mg/mL graphite alkyne nanoparticles under the condition of rapid stirring, simultaneously adding a proper amount of cosolvent, and rapidly stirring for 1-2h, wherein ultrasonic treatment can be carried out for 0.5h if the dispersion is insufficient until an evenly dispersed electrodeposition solution is obtained; immersing the cleaned conductive reaction zone into an electrodeposition solution, and scanning for 3 circles within the range of 0.2-0.4V by adopting a cyclic voltammetry method, wherein the scanning speed is 50mV/s; taking out, washing with ultrapure water, activating in deoxygenated electrolyte solution (containing 0.1M hydrochloric acid and 0.1M potassium chloride), and scanning at a rate of 100mV/s for 50 cycles in a range of 0.2-0.4V by cyclic voltammetry; taking out, washing with ultrapure water, drying at 100 deg.C for 110min, and obtaining an electronic medium layer (Prussian blue and graphite alkyne nanometer composite material layer) on the surface of the reaction zone;
preparing an enzyme reaction layer: dripping 3 mu L of polyethyleneimine functionalized graphene oxide solution (5 mg/ml) onto the surface of the electronic medium layer, and airing at room temperature; then 3 mul of crosslinking enzyme solution (containing 2U/ml xanthine oxidase, 2.5U/ml purine nucleoside phosphorylase, 2.5U/ml adenosine deaminase and 1mg/ml genipin, the volume ratio is 2;
preparing an anti-interference layer: and (3) immersing the conductive reaction area modified with the electronic medium layer and the enzyme reaction layer into an o-phenylenediamine solution (containing 100mM o-phenylenediamine and 7mg/mL bovine serum albumin), applying a voltage of 0.75V by adopting a constant voltage method, depositing for 15min, and airing at room temperature.
Example 3 verification of the voltammetry sweep Rate for adenosine sensor Performance
Taking the modified microelectrode detection structure as an adenosine sensor, and in order to verify the influence of the voltammetry scanning rate on the detection performance of the sensor, immersing the implantation end of the adenosine sensor into deoxypotassium chloride solution with the concentration of 0.1M, connecting the other end of the sensor with an electrochemical workstation, and carrying out cyclic voltammetry detection, wherein the voltage range is-0.1-1.5V, the scanning rates are respectively 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100mV/s, and the number of scanning cycles is 10. The cyclic voltammetry scanning result shows that a pair of obvious redox peaks exist at the positions of 0.18V and 0.9V respectively, and the redox peak at the position of 0.18V is formed by mutual conversion of prussian blue deposited on the surface of the conducting layer and reduced prussian white thereof; the redox peak at 0.9V is formed by interconversion of prussian blue and its fully oxidized state prussian green, consistent with previous literature reports. The peak current value of the redox peak at 0.18V and the square root of the scanning rate are respectively subjected to linear fitting, as shown in fig. 3, and a good linear relationship exists between the peak current value and the square root, so that the electrochemical signal generated by the prepared modified layer follows a diffusion control surface reaction model.
Example 4 optimal detection Voltage selection
The detection principle of the prepared adenosine sensor is based on a multienzyme cascade reaction system in an enzyme reaction layer, namely adenosine to be detected is catalyzed and deaminated by adenosine deaminase to generate inosine, the inosine is further phosphorylated under the action of purine nucleoside phosphorylase to generate hypoxanthine and ribose, and the hypoxanthine is catalyzed by xanthine oxidase to finally produce uric acid and hydrogen peroxide. With the rapid conversion of prussian white to prussian blue in the electron mediator layer on the surface of the conductive layer, hydrogen peroxide can be rapidly reduced to OH by the transferred electrons - . At the same time, prussian blue can be reduced again to prussian white at a very low catalytic potential, typically below 0V (vs. ag/AgCl). In this process, prussian blue acts as an electron mediator to transport electrons from the surface of the conductive layer to H 2 O 2 Thereby generating a sense current. Due to the fact thatThe prepared adenosine sensor can be used for quantitatively detecting adenosine serving as a substance to be detected by a chronoamperometry method.
The adenosine detection principle of the adenosine sensor is shown by the following formula:
(Prussian white) K 4 Fe 4 II [Fe II (CN) 6 ] 3 +2H 2 O 2 → iron (Prussian blue) 4 III [Fe II (CN) 6 ] 3 +4K + +4OH -
(Prussian blue) Fe 4 III [Fe II (CN) 6 ] 3 +4K + +4e - → Prussian white K 4 Fe 4 II [Fe II (CN) 6 ] 3
The operation principle of each functional modification layer is shown in fig. 4.
To select the optimal detection voltage, the implanted end of the prepared adenosine sensor was immersed in a moderately stirred phosphate buffer solution (0.1M, pH 7.4) and the other end of the sensor was connected to an electrochemical workstation for chronoamperometric detection. The applied detection voltages are-0.1, -0.05, 0, 0.1 and 0.2V respectively, when the baseline current is stable, a certain volume of high-concentration adenosine solution is dripped into the buffer solution, the concentration gradient of the adenosine to be detected is 5 mu M, and the adenosine solution is dripped once every 100s, so that a plurality of current-time detection curves are finally obtained, as shown in figure 5-A. The adenosine concentration and the detection current value were subjected to linear fitting to obtain detection sensitivities at different detection voltages and compared, and as can be seen from fig. 5-B, when the detection voltage was-0.05V, the detection sensitivity of the sensor to adenosine was the highest, so-0.05V was selected as the optimum adenosine detection voltage.
Example 5 continuous detection of adenosine in an in vitro Environment
In order to verify the continuous detection performance of the prepared adenosine sensor on adenosine in an in vitro environment, the implanting end of the adenosine sensor is immersed into a phosphate buffer solution (0.1M, pH value 7.4) which is properly stirred, the other end of the sensor is connected with an electrochemical workstation, and timing current detection is carried out, wherein the detection voltage is-0.05V. After the baseline current is stabilized, a certain volume of high-concentration adenosine solution is dripped into the buffer solution to make the concentration gradient of the adenosine to be detected respectively be 0.5 and 5 mu M, the adenosine is dripped once every 100s, and after the adenosine is dripped for a plurality of times, the final concentration of the adenosine reaches 50 mu M, and a current-time detection curve is obtained, as shown in fig. 6-A. Linear fitting was performed on the adenosine concentration and the detected current value (fig. 6-B), resulting in a linear regression equation y = -1.1x-45.09, linear correlation coefficient (R) 2 ) The adenosine sensor has the advantages that the adenosine sensor is 0.997, the detection sensitivity is 1.1 nA/mu M, and the good linear relation between the adenosine concentration and the detection current is shown when the adenosine concentration is between 0 and 50 mu M, so that the prepared adenosine sensor has good continuous detection capability in an in-vitro environment.
Example 6 interference rejection test
In order to verify the detection specificity of the prepared adenosine sensor, the anti-interference capability of the adenosine sensor is tested. The implanted end of the adenosine sensor was immersed in moderately stirred phosphate buffered saline (0.1M, pH 7.4) and the other end of the sensor was connected to an electrochemical workstation for timed amperometric detection at-0.05V. After the baseline current is stable, dropwise adding a high-concentration adenosine solution twice into the buffer solution to increase the concentration of adenosine to be detected by 5 mu M, sequentially adding 5 mu M dopamine, 400 mu M ascorbic acid and 100 mu M uric acid into the buffer solution according to the physiological concentration of common electrochemical interferents in an in-vivo environment, and finally adding adenosine twice. As can be seen from fig. 7, compared with the detection current generated by adenosine, the interference currents generated by dopamine, ascorbic acid and uric acid, which are common in vivo interferents at three physiological concentrations, are negligible, which indicates that the adenosine sensor has good anti-interference capability, and this is a result of using the prussian blue nanocomposite to significantly reduce the detection voltage and simultaneously using the poly-o-phenylenediamine anti-interference layer in combination.
Example 7 continuous detection of local adenosine in animals
The experiment selects SPF grade healthy male Wistar rats with the body weight of 230 +/-20 g. After anesthetizing the rats by intraperitoneal injection of phenobarbital sodium, the right thighs were shaved and skin prepared, and the rats were fixed on an operating plate. The prepared adenosine sensor implanting end is implanted into local muscle tissue of thigh by a disposable sterile syringe needle or a needle without wall, and the outside is fixed by medical plaster to prevent the rat from pulling out after waking up. The other end of the sensor is connected with an electrochemical workstation for timing current detection, and the detection voltage is-0.05V. When the baseline current reaches a stable value (20 min), 50 mu L of adenosine solution (4 mM) and 50 mu L of physiological saline (9%) are slowly injected into the position 3-5mM away from the implanted position of the sensor by using an injection needle, and the change process of the detection current is recorded in real time. As shown in fig. 8, when higher concentration adenosine was injected, the local adenosine concentration increased rapidly, the detection current intensity also increased rapidly, reached a peak value after about 30s, and then the detection current intensity decreased gradually, which is probably the result of the slow decrease of the local adenosine concentration under the action of diffusion and metabolism, and the detection current peak values after two adenosine injections were substantially consistent, further demonstrating the better in vivo detection repeatability. In contrast, the injection of saline is significantly different and can only cause a transient fluctuation in the measured current, but a rapid return to baseline levels, probably because the mechanical damage caused by the injection procedure itself induces a rapid release and breakdown of local adenosine in the body, a nervous system protective action. The results prove that the prepared adenosine sensor has good in-vivo adenosine continuous detection capability.
Example 8 continuous detection of local adenosine in animal brain
The experiment selects SPF grade healthy male Wistar rat with the weight of 230 +/-20 g, after the rat is anesthetized by intraperitoneal injection of phenobarbital sodium, the top end of the head is shaved and the skin is prepared, and then the back of the rat is upwards fixed on a brain stereotaxic apparatus. The craniofacial skin of the head is cut by a surgical scissors, the skin at four corners is fixed by a vascular clamp, the cranial wound is completely exposed, and periosteum is removed by blunt separation. Finding a bregma point according to the skull suture, and taking the bregma point as a central point of a three-dimensional coordinate; operating the three-dimensional operating arm to position the three-dimensional operating arm to a bregma point, and marking the three-dimensional operating arm as a zero point; the three-dimensional operation arm is continuously operated, the three-dimensional operation arm is moved to the position of (X.Y.Z) = (3.0.0), the marking is carried out by using a marking pen, the operation arm is moved away, and a cranial drill is used for drilling holes. The prepared adenosine sensor implanting end is fixed on a holder, moved to the cranial hole and moved down to the position with Z value =5 (striatum). The other end of the sensor is connected with an electrochemical workstation for timing current detection, and the detection voltage is-0.05V. When the baseline current reaches a stable value (10 min), 4mM and 10mM adenosine solutions are sequentially injected into the brain by using a micro-injector, the injection volumes are both 20 mu L, and the change process of the detection current is recorded in real time. As shown in figure 9, the detection current intensity can be increased by two times of adenosine injection, and the current increment caused by the adenosine with higher concentration is more obvious, and the results prove that the prepared adenosine sensor can realize the in-vivo real-time detection of the concentration change of the adenosine in the brain of the animal.
Example 9 real-time monitoring of acupuncture-induced local adenosine release from the crypt site in vivo
The experiment selects SPF grade healthy male Wistar rats with the weight of 230 +/-20 g. The Zusanli acupoints are selected according to the animal acupoint positioning method from Experimental acupuncture. Zusanli: in the muscle groove about 5mm below the fibula capitulum below the outer side of the knee joint of the hind limb of the rat. First, rats were anesthetized by intraperitoneal injection of phenobarbital sodium, after complete anesthesia, the right "tsusanli" acupoints were shaved locally, and then the rats were fixed on an operating board. The implanting end of the prepared adenosine sensor is implanted into the muscle tissue of Zusanli acupoint of the lower leg of the rat by using a disposable sterile syringe needle or a wall-lacking needle, and the adenosine sensor is externally fixed by using a medical plaster to prevent the rat from being pulled out by moving after waking up. The other end of the sensor is connected with an electrochemical workstation for timing current detection, and the detection voltage is-0.05V. When the base line current reaches a steady state (20-40 min), a rat Zusanli acupoint is punctured by adopting disposable sterile acupuncture (0.35 multiplied by 25 mm), a twisting and rotating method is performed, the tonic and the laxation are performed smoothly, the frequency is 180 times/min, the duration is 2min, then, the acupuncture is performed once every 8min, the acupuncture is performed 3 times in total, the total acupuncture time is 30min, and the change process of the detection current is recorded in real time. The frequency of the twisting is controlled by a metronome, and all needling operations are completed by one person. To ensure the stability of the acupuncture manipulation, the operator repeatedly performs the practice of the acupuncture manipulation on the acupuncture manipulation parameter instrument before the start of the actual experiment and during the experiment. As shown in FIG. 10, the twist causes the rapid fluctuation and increase of the detection current, after the twist stops, the detection current is slowly reduced, the detection current changes in the three twisting processes are consistent, and the detection current intensity is higher than the initial current value in the whole needling process, which fully proves that the needling can cause the sustained release of the local adenosine in the acupoint area, and the degree and duration of the adenosine concentration change are correlated with the needling manipulation and the application intensity. The experimental results preliminarily verify that adenosine plays a certain mediating role as neurotransmitter in the acupuncture onset mechanism, and further prove that the prepared adenosine sensor can be effectively applied to the research on the acupuncture mechanism.
For comparison, the influence of acupuncture on the concentration of local adenosine on the Zusanli acupoint of a rat was examined by a conventional microdialysis-HPLC chromatography-mass spectrometry method. Rat anesthesia and fixation process as described above, microdialysis equipment is used to check whether the pipe system is intact, then connect each pipe, cut open the local skin of Zusanli acupoint with surgical scissors, pierce the syringe needle covered with the tear tube into the local muscle tissue of acupoint, the tear tube is left in the body, and the syringe needle is slowly withdrawn. Then the probe is implanted into the tear tube, the tear tube is retreated while the probe is advanced, and finally the probe is implanted into the local muscle tissue of the acupuncture point, and the probe can be fixed by using an adhesive tape or an operation suture. Perfusing with ringer's solution at constant speed (2 μ L/min), balancing for 2h, collecting tissue fluid formally, collecting a tube of sample every 30min because of extremely small amount of local tissue fluid, and collecting tissue fluid samples 30min before acupuncture and 30min after acupuncture, wherein the acupuncture method is as described above. The collecting pipe is required to be arranged on an ice box in the process of collecting the dialysate, the collecting pipe is immediately arranged in a refrigerator at the temperature of 20 ℃ below zero after the collection is finished, and the collecting pipe is transferred to the refrigerator at the temperature of 80 ℃ below zero after the dialysis is finished to be tested. The detection is carried out by adopting a high performance liquid chromatography-mass spectrometry method, the collected sample is unfrozen at room temperature, the sample is centrifuged for 10min at 13000r/min, the supernatant is removed, and the sample is detected by a computer. The final experimental results show that 30min needle insertion can cause a local adenosine concentration at the acupoint to increase about 4-fold compared to before needle stick.
Compared with the conventional detection method, although two groups of experimental results prove that acupuncture can induce the local adenosine concentration in the acupoint region to rise, the conventional method of microdialysis-high performance liquid chromatography-mass spectrometry needs to strictly and accurately correct the taken sample, and the defect that the probe recovery rate determination process is complicated exists; and the sampling time interval is long, and other technologies are still needed to assist detection after sampling, so that the change rule of adenosine in the acupuncture process cannot be truly and accurately reflected at the first time. The prepared adenosine sensor is a flexible micro composite sensing needle structure integrating a working electrode, a counter electrode and a reference electrode, has high detection sensitivity, high response speed, simple and convenient operation and convenient carrying, can realize in-vivo, in-situ, real-time and dynamic detection of local adenosine concentration change of a acupoint area in the whole acupuncture process, provides a more intuitive and effective modern biological detection method for acupuncture effect principle research, and further provides powerful scientific basis for guiding clinic and improving clinical curative effect.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. An enzyme immobilization promoter characterized by: the accelerant comprises polyethyleneimine functionalized graphene oxide and genipin in a mass ratio of 1-5.
2. An enzyme crosslinking fixative solution, which is characterized in that: the enzyme crosslinking stationary liquid comprises a polyethyleneimine functionalized graphene oxide solution and a multi-enzyme cascade reaction liquid in a volume ratio of 1-3.
3. The enzyme-crosslinked fixative solution of claim 2, wherein: the multi-enzyme cascade reaction solution comprises xanthine oxidase, purine nucleoside phosphorylase, adenosine deaminase and genipin in a volume ratio of 1-2.
4. The enzyme-crosslinked fixative solution of claim 3, wherein: the concentration of the polyethyleneimine functionalized graphene oxide solution is 1-10mg/ml; the concentration of the xanthine oxidase in the multi-enzyme cascade reaction liquid is 0.5-5U/ml; the concentration of purine nucleoside phosphorylase in the multi-enzyme cascade reaction liquid is 0.5-5U/ml; the concentration of adenosine deaminase in the multi-enzyme cascade reaction liquid is 0.5-5U/ml; the concentration of genipin in the multi-enzyme cascade reaction liquid is 1-10mg/ml.
5. A method for functionally modifying the surface of a conductive reaction zone of a working electrode is characterized in that: the method comprises the following steps:
(1) Immersing the conductive reaction area into an electrodeposition solution for electrodeposition to form an electronic medium layer on the conductive reaction area;
(2) Dripping a polyethyleneimine-functionalized graphene oxide solution onto the surface of the electronic medium body layer, airing, dripping the multienzyme cascade reaction solution of any one of claims 2-4 onto the surface of the graphene oxide solution, and airing to form an enzyme reaction layer;
(3) And immersing the conductive reaction area modified with the electronic medium layer and the enzyme reaction layer into an anti-interference solution for electrodeposition, and drying to form the anti-interference layer.
6. The method for the surface functional modification of the conductive reaction area of the working electrode as claimed in claim 5, wherein: the electrodeposition solution in the step (1) comprises hydrochloric acid with the concentration of 2-5mg/mL, a potassium chloride solution with the concentration of 5-10mg/mL, an iron chloride solution with the concentration of 2-5mg/mL, a potassium ferricyanide solution with the concentration of 2-10mg/mL and graphite alkyne nano-particles with the concentration of 0.1-1 mg/mL; the anti-interference solution in the step (3) comprises o-phenylenediamine with the concentration of 10-50mg/mL and bovine serum albumin with the concentration of 1-10mg/mL.
7. A three-electrode detection structure is characterized in that: the flexible film comprises a silver foil and 2 flexible film substrates, wherein the flexible film substrates are respectively fixed on the upper surface and the lower surface of the silver foil through adhesive layers, conductive layers are arranged on the flexible film substrates, and insulating layers are arranged on the conductive layers;
the length of the insulating layer is less than that of the conducting layer;
the exposed part of the conducting layer relative to the insulating layer is a conducting reaction area, and one conducting reaction area is subjected to surface functional modification by using the method of claim 5 or 6.
8. The three-electrode detection structure according to claim 7, wherein: the length of the conductive layer is less than that of the silver foil; the conducting layer is made of at least one of gold, platinum, titanium, palladium, copper, carbon black, graphite or graphene; the flexible film substrate is at least one of polycarbonate, polytetrafluoroethylene, polyimide, polyethylene terephthalate, acrylonitrile-butadiene-styrene copolymer or polymethyl methacrylate.
9. Use of a three-electrode detection structure according to claim 7 or 8, wherein: the three-electrode detection structure is applied to acupuncture.
10. Use of a three-electrode detection structure according to claim 9, characterized in that: the three-electrode detection structure is applied to the preparation of an adenosine sensor, a glucose sensor, a lactate sensor, a glutamate sensor, a uric acid sensor, an ascorbic acid sensor or a dopamine sensor; the adenosine sensor is an implanted adenosine continuous detection sensor.
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