A non-invasive diabetes ketoacidosis sensor for medium-frequency electrotherapy apparatus and its preparation method

Abstract

The present invention discloses a non-invasive diabetes ketoacidosis sensor for a medium-frequency electrotherapy instrument and a preparation method thereof, belonging to the technical field of wearable biosensors and their preparation. The present invention prepares an integrated sensor for multiple diabetes ketoacidosis markers, which can continuously monitor the levels of diabetes ketoacidosis markers in human sweat in situ under non-invasive conditions. And the present invention uses MXene@GQDs materials with high conductivity and large specific surface area to modify the working electrode, effectively reducing its detection limit, and the presence of GQDs is beneficial to the immobilization of enzymes on the electrode surface and the improvement of direct electron transfer, thereby improving the sensitivity of the sensor. Then, through a microfluidic system, sweat collection under resting conditions is realized, achieving the purpose of continuous non-invasive monitoring of multiple markers.

Description

Technical Field

[0001] The present invention relates to a non-invasive diabetes ketoacidosis sensor for a medium-frequency electrotherapy instrument and a preparation method thereof, belonging to the technical field of wearable biosensors and their preparation. Background Art

[0002] A medium-frequency electrotherapy instrument is a medical device used to treat various diseases and pains. It transmits electrical energy to the body parts of patients through medium-frequency current to promote healing, relieve pain and improve symptoms. The medium-frequency electrotherapy instrument stimulates the tissues of patients by transmitting medium-frequency current, usually in the frequency range of 1 kHz to 100 kHz. This current can promote cell metabolism, reduce inflammation, improve blood circulation and relieve pain. It can be used for the treatment of muscle pain, nerve pain and other various pain symptoms, and is widely used in the fields of rehabilitation medicine, physical therapy, sports medicine and pain management. It is usually used for rehabilitation treatment, pain management, muscle relaxation, nerve stimulation and other treatment purposes. It is a widely used tool in the medical and rehabilitation fields to help patients relieve pain, promote recovery and improve the quality of life. The diabetes ketoacidosis sensor can monitor the patient's illness situation in real time. When the patient gets ill, the medium-frequency electrotherapy instrument can promote cell metabolism and improve blood circulation to promote the absorption of drugs.

[0003] Diabetes ketoacidosis is a life-threatening complication of type 1 and type 2 diabetes. Despite long-term progress in diabetes treatment, the incidence and mortality rates remain high. Diabetes ketoacidosis is caused by a severe lack of insulin, usually accompanied by an increase in counter-regulatory hormones such as glucagon, cortisol and adrenaline. Uncontrolled diabetes leads to the catabolism of fatty acids and the production of so-called "ketone bodies" including acetone (2%), acetoacetic acid (20%) and β-hydroxybutyric acid (78%). The accumulation of these metabolites leads to acid-base imbalance in the body and a decrease in blood pH, a condition called "diabetes ketoacidosis". Diabetes ketoacidosis can cause patients to vomit, become severely dehydrated, be delirious, comatose and even die. Reports have shown that compared with their peers, pediatric patients with severe diabetes ketoacidosis have a decline in cognitive function, which can even be fatal if not detected and treated early.

[0004] Currently, the blood glucose meters widely used for blood glucose detection rely on fingertip blood sampling and cannot non-invasively and real-timely monitor the blood glucose level of the human body. At the same time, β-hydroxybutyric acid is usually tested in vitro using a biochemical analyzer in clinical practice, which can measure the level of β-hydroxybutyric acid in the blood, which is the most important indicator in the diagnosis of diabetes ketoacidosis. Although this method can evaluate the blood β-hydroxybutyric acid level, they require repeated fingertip pricks for regular measurements, so they cannot non-invasively and real-timely track the trend and dynamic fluctuations of the β-hydroxybutyric acid concentration and cannot timely understand the patient's illness situation.

[0005] Therefore, it is highly necessary to develop a device capable of continuously monitoring ketone bodies, glucose, and other diabetes biomarkers. Summary of the Invention

[0006] An object of the present invention is to provide a wearable sweat sensor capable of highly sensitively and continuously monitoring diabetes ketoacidosis markers (β-hydroxybutyric acid, glucose, and pH) in human sweat under non-invasive conditions.

[0007] Technical Solution of the Present Invention:

[0008] One object of the present invention is to provide a non-invasive diabetes ketoacidosis sensor for a medium-frequency electrotherapy apparatus, which is characterized in that it includes a microfluidic device and a detection electrode covering it, and the detection electrode is composed of a substrate and an electrode array arranged on it;

[0009] The microfluidic device includes a detection chamber, a sweat inlet area, and a sweat outlet channel that are respectively communicated with it; and the detection chamber houses the electrode array;

[0010] The electrode array includes an electrode body located in the detection chamber and an electrical interface respectively led out from each electrode body, and the electrode array is connected to a circuit main board through the electrical interface;

[0011] The electrode body includes a reference electrode located in the center of the detection chamber, three working electrodes distributed surrounding the reference electrode, and an m-shaped counter electrode composed of two semi-circles distributed surrounding two of the working electrodes;

[0012] The three working electrodes are respectively a β-hydroxybutyric acid sensing electrode, a glucose sensing electrode, and a pH electrode.

[0013] Further defined, the β-hydroxybutyric acid sensing electrode and the glucose sensing electrode are surrounded by the counter electrode, and the β-hydroxybutyric acid sensing electrode and the glucose sensing electrode are respectively located at the centers of the two semi-circles of the m-shaped counter electrode.

[0014] Further defined, the substrate is PET or PDMS with a thickness of 0.2 mm.

[0015] Further defined, the diameters of the working electrode and the reference electrode are 2 mm.

[0016] Another object of the present invention is to provide a preparation method for the non-invasive diabetes ketoacidosis sensor for the above medium-frequency electrotherapy apparatus, and this method includes the following steps:

[0017] (1) Prepare MXene-NH2@GQDs composite electrodes;

[0018] (2) Prepare β-hydroxybutyric acid sensing electrode, glucose sensing electrode and pH electrode respectively with the MXene-NH2@GQDs composite electrode as the substrate;

[0019] (3) Screen-print the reference electrode and counter electrode, and assemble the β-hydroxybutyric acid sensing electrode, glucose sensing electrode and pH electrode to obtain the detection electrode;

[0020] (4) Prepare the microfluidic device;

[0021] (5) Assemble the detection electrode, microfluidic device and circuit main board to obtain an integrated wearable sweat sensor for continuous non-invasive monitoring of diabetic ketoacidosis.

[0022] Further define that (1) the process of preparing the MXene-NH2@GQDs composite electrode is as follows:

[0023] Disperse GQDs in PBS solution, then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysulfosuccinimide. After 1 h of activation treatment, add monolayer MXene-NH2 nanosheets. After reacting for 3 h, filter, wash and freeze-dry the obtained precipitate in sequence. Configure the product into a dispersion liquid, then coat it on the surface of the electrode substrate, dry it, and cool it to obtain the MXene-NH2@GQDs composite electrode.

[0024] Further define that the preparation process of monolayer MXene-NH2 nanosheets is as follows:

[0025] First, mix lithium fluoride and hydrochloric acid, stir for 30 min at 35 °C, then slowly add Ti3AlC2, and stir and react at 35 °C for 24 h. After the reaction, centrifuge, discard the upper liquid, add deionized water and continue to centrifuge. Repeat the above centrifugation operation 3 times to obtain the slurry;

[0026] Then, add ammonia solution to the slurry, manually shake for 10 min and then ultrasonically treat for 60 min. Centrifuge the obtained suspension, ultrasonically treat under N2 protection, transfer the obtained dispersion liquid to a hydrothermal reactor for hydrothermal reaction treatment. After the reaction, centrifuge, wash with deionized water until the pH of the supernatant is neutral, and freeze-dry the precipitate to obtain monolayer MXene-NH2 nanosheets.

[0027] Further define that the preparation process of GQDs is as follows:

[0028] Mix citric acid, urea and water, heat and react, then dialyze in a dialysis bag with a molecular weight cut-off of 1000 Da for 24 h, and rotary evaporate to dryness to obtain GQDs.

[0029] Further define that (2) the process of preparing the β-hydroxybutyric acid sensing electrode is as follows:

[0030] First, immerse the MXene-NH2@GQDs composite electrode in a thionine solution and perform electro-polymerization pretreatment by cyclic voltammetry;

[0031] Then, dissolve nicotinamide adenine dinucleotide, β-hydroxybutyrate dehydrogenase, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution in PBS solution. Drop the resulting mixture onto the surface of the pretreated MXene-NH2@GQDs composite electrode, then add N-hydroxysulfosuccinimide solution, incubate at room temperature, rinse with deionized water, and dry;

[0032] Finally, add chitosan solution and dry at room temperature to obtain a β-hydroxybutyric acid sensing electrode.

[0033] Further defined, the process for preparing a glucose sensing electrode is as follows:

[0034] First, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to the PBS solution of glucose oxidase to obtain a glucose oxidase dispersion;

[0035] Then, drop the glucose oxidase dispersion onto the surface of the MXene-NH2@GQDs composite electrode, then add N-hydroxysulfosuccinimide solution, incubate at room temperature for 1 h, wash with deionized water, and dry at room temperature;

[0036] Finally, add chitosan solution and dry at room temperature to obtain a glucose sensing electrode.

[0037] Further defined, the process for preparing a pH electrode is as follows:

[0038] Drop the polyaniline nanoparticle dispersion onto the surface of the MXene-NH2@GQDs composite electrode, dry, then add chitosan solution and dry at room temperature to obtain a pH sensing electrode.

[0039] Even further defined, the preparation process of polyaniline nanoparticles is as follows:

[0040] First, mix chloroform and aniline evenly to obtain an aniline solution;

[0041] Then, dissolve ammonium persulfate in HCl to obtain an ammonium persulfate solution;

[0042] Finally, slowly pour the ammonium persulfate solution into the aniline solution. After standing for phase separation, discard the lower organic phase, filter the upper aqueous phase, wash the precipitate with HCl and acetone, and dry the filter cake in a vacuum oven at 40 °C for 24 h to obtain polyaniline nanoparticles.

[0043] Further defined, (4) The process of preparing the microfluidic device is as follows:

[0044] First, use AutoCAD to design the microfluidic channel and prepare the corresponding photomask template;

[0045] Then, spin-coat the SU8-2150 photoresist on the cleaned silicon wafer, heat it at 65 °C for 20 min, then raise the temperature to 95 °C and keep it warm for 60 min;

[0046] Then, cover the photomask template on the surface of the photoresist, expose it, heat it on a 95 °C hot plate for 30 min, and develop it to obtain the mold;

[0047] Then, pour the PDMS precursor and curing agent into the mold according to a mass ratio of 10:1.5 until the liquid level is flush with the mold, and cure it at 80 °C for 2 h to obtain the microfluidic channel;

[0048] Finally, spin-coat the PDMS mixture on a 100-μm-thick PDMS film, then attach the microfluidic channel to the PDMS film, and cure it at 80 °C for 2 h to obtain the microfluidic device.

[0049] The third object of the present invention is to provide an application of the non-invasive diabetes ketoacidosis sensor for the above-mentioned medium-frequency electrotherapy instrument, specifically for continuously monitoring the markers β-hydroxybutyric acid, glucose and pH in human sweat under non-invasive conditions.

[0050] Advantages of the present invention:

[0051] The present invention has prepared an integrated sensor for multiple diabetes ketoacidosis markers, which can continuously monitor the levels of diabetes ketoacidosis markers in human sweat in situ under non-invasive conditions. And the present invention uses the MXene@GQDs material with high conductivity and large specific surface area to modify the working electrode, effectively reducing its detection limit, and the presence of GQDs is beneficial to the immobilization of enzymes on the electrode surface and improving direct electron transfer, thereby improving the sensitivity of the sensor. Then, through the microfluidic system, sweat collection under resting conditions is realized, and the purpose of continuously and non-invasively monitoring multiple markers is achieved. In addition, the detection results can be analyzed by the on-site signal processing system and then sent to the smart device wirelessly, which has important clinical significance for the rapid diagnosis and treatment of diabetes ketoacidosis and the strict blood glucose control of diabetic patients. Compared with the prior art, the present invention also has the following advantages:

[0052] (1) The present invention separates two biomarkers detected based on the i-t method in two electrolytic cells to avoid interference between the generated currents and improve the stability of the signal. At the same time, the integration of multiple markers is realized, improving the diagnostic accuracy and greatly simplifying the detection equipment and waiting time required by patients.

[0053] (2) The present invention uses a microfluidic device to collect sweat, enabling patients to have a sufficient amount of sweat for detection even under resting conditions.

[0054] (3) The preparation of the composite material provided by the present invention has the advantages of convenient access to raw materials, low cost, and simple preparation process. It is a three-dimensional material preparation solution with low equipment investment and simple process flow. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 is a schematic structural diagram of the detection electrode provided by the present invention;

[0056] Figure 2 is a schematic structural diagram of the microfluidic device provided by the present invention;

[0057] Figure 3 is a schematic structural diagram of the integrated wearable sweat sensor for continuous non-invasive monitoring of multiple markers provided by the present invention;

[0058] Figure 4 is a schematic diagram of the wearing of the sensor provided by the present invention;

[0059] Figure 5 is the amperometric response of the sensor in Example 1 to the increase in the concentration of β-hydroxybutyric acid in real sweat;

[0060] Figure 6 is the amperometric response of the sensor in Example 1 to the increase in the concentration of glucose in real sweat;

[0061] Figure 7 is the response of the sensor in Example 1 to the change in pH in real sweat. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0062] To make the above objects, features, and advantages of the present invention more apparent and understandable, the following detailed description of the specific embodiments of the present invention will be given in conjunction with the embodiments of the specification.

[0063] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar generalizations without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0064] Secondly, the so-called "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that can be included in at least one implementation of the present invention. The appearances of "in one embodiment" in different places in this specification do not all refer to the same embodiment, nor are they separate or selectively exclusive embodiments from other embodiments.

[0065] The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in this field and can be obtained by those skilled in the art through commercial channels without special instructions.

[0066] Example 1

[0067] An integrated wearable sweat sensor for continuous non-invasive monitoring of multiple markers provided in this example is as Figures 1 to 4 shown. The sensor includes a microfluidic device and detection electrodes covering it. The detection electrodes are composed of a substrate and an electrode array arranged on it.

[0068] The microfluidic device includes a detection chamber, a sweat inlet area, and a sweat outlet channel that are respectively communicated with it. And the detection chamber houses the electrode array.

[0069] The electrode array includes electrode bodies located in the detection chamber and electrical interfaces respectively led out from each electrode body. The electrode array is connected to the circuit main board through the electrical interfaces.

[0070] The electrode bodies include a reference electrode located in the center of the detection chamber, three working electrodes distributed surrounding the reference electrode, and an m-shaped counter electrode composed of two semi-circles distributed surrounding two working electrodes.

[0071] The three working electrodes are respectively a β-hydroxybutyric acid sensing electrode, a glucose sensing electrode, and a pH electrode.

[0072] Specifically, the β-hydroxybutyric acid sensing electrode and the glucose sensing electrode are surrounded by the counter electrode, and the β-hydroxybutyric acid sensing electrode and the glucose sensing electrode are respectively located at the centers of the two semi-circles of the m-shaped counter electrode.

[0073] The preparation method of the above integrated wearable sweat sensor for continuous non-invasive monitoring of multiple markers provided in this example is as follows:

[0074] (1) Prepare a screen-printed electrode.

[0075] According to Figure 1 Design a five-electrode screen-printed electrode template, attach the template to a 0.2-mm-thick transparent PET substrate, print the electrode with carbon paste, and print a layer of Ag / AgCl electrode on the surface of the reference electrode in the same way. The size of the prepared electrode is 15×25 mm, and the diameters of the three working electrodes (respectively named the first working electrode, the second working electrode, and the third working electrode) and the reference electrode are all 2 mm.

[0076] (2) Prepare monolayer MXene-NH2.

[0077] Add 2 g of lithium fluoride and 40 mL of 9 M hydrochloric acid to a 100 mL polytetrafluoroethylene beaker, and stir at 35 °C for 30 min. Then, slowly add 2 g of Ti3AlC2 to the polytetrafluoroethylene beaker and stir at 35 °C for 24 h. After the reaction is completed, centrifuge the resulting product (3500 rpm, 5 min), pour off the upper liquid, add deionized water and continue to centrifuge (3500 rpm, 5 min), repeat 3 times. Next, add 40 mL of ammonia solution with pH = 9 to the resulting slurry, then manually shake for 10 min and sonicate for 60 min. Centrifuge the suspension again at 3500 rpm for 20 min to remove the unexfoliated MXene. In an environment protected by N2, sonicate the suspension containing few-layer aminated MXene for a period of time. Subsequently, transfer the dispersion to a 100 mL hydrothermal reactor and stir at 70 °C for 4 h. Finally, centrifuge at 10000 rpm and continuously wash the resulting mixture with deionized water until the pH of the supernatant becomes approximately 7. Freeze-dry the precipitate to obtain monolayer MXene-NH2 nanosheets.

[0078] (3) Prepare GQDs.

[0079] Add 3 g of citric acid, 2 g of urea and 75 mL of water to a hydrothermal reactor, and heat at 160 °C for 6 h. Then dialyze in a dialysis bag with a molecular weight cut-off of 300 Da for 24 h, and rotary evaporate to dryness for later use.

[0080] (4) Prepare polyaniline nanoparticles.

[0081] Add 50 mL of chloroform and 1 mL of aniline to a beaker, and stir to mix evenly. Add 2.85 g of ammonium persulfate to 50 mL of 1 M HCl, and stir to dissolve it completely. Then slowly pour the ammonium persulfate solution into the aniline solution. After standing, the solution is layered, with the upper layer being the aqueous phase and the lower layer being the organic phase. After standing for 3 h, the entire aqueous phase is uniformly filled with dark green polyaniline. Filter the product and wash it with 1 M HCl and acetone to remove unreacted reagents and aniline oligomers. Finally, dry the filter cake in a vacuum oven at 40 °C for 24 h.

[0082] (5) Prepare MXene@GQDs composite electrodes.

[0083] Disperse GQDs into 0.1 M PBS with pH = 7 to prepare a dispersion of 10 mg / mL. Take 10 mL of the dispersion, add 1 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.5 mg of N-hydroxysulfosuccinimide to activate for 1 h, then add 50 mg of MXene-NH2, react fully for 3 h, and finally filter, wash, and freeze-dry. Prepare a 1 mg / mL dispersion, coat it on the surface of the working electrode, and after coating evenly, place it in an oven at 50 °C to dry for 10 min and cool naturally to room temperature.

[0084] (6) Prepare a modified electrode to obtain a detection electrode.

[0085] ① β-Hydroxybutyric acid sensing electrode.

[0086] First, electrodeposit polythionine on the first working electrode. Immerse the screen-printed electrode into a 1 mM thionine solution and perform thionine electropolymerization by cyclic voltammetry (-0.2 - 0.5 V vs. Al / AgCl).

[0087] Then, prepare a solution: Dissolve nicotinamide adenine dinucleotide (oxidized form), β-hydroxybutyric acid dehydrogenase, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in 0.1 M PBS with pH 7.2 to obtain a mixed solution with concentrations of nicotinamide adenine dinucleotide (oxidized form), β-hydroxybutyric acid dehydrogenase, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide being 1 mg / mL, 200 U / mL, and 0.6 mg / mL respectively.

[0088] Next, add 1.1 mg of N-hydroxysulfosuccinimide to 1 mL of ultrapure water. Then take 2 μL of the above mixed solution and coat it on the surface of the working electrode, then add 0.5 μL of N-hydroxysulfosuccinimide solution, incubate at room temperature for 1 h, rinse with deionized water, and dry at 4 °C for 6 h.

[0089] Finally, continue to add 1 μL of 0.5 wt.% chitosan solution (the solvent is 1 wt.% glacial acetic acid) on the surface and dry at room temperature for 6 h to obtain a β-hydroxybutyric acid sensing electrode.

[0090] ② Glucose sensing electrode.

[0091] Disperse 1 mg of glucose oxidase into 1 mL of 0.1 M PBS at pH 7.2, then add 0.6 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and then add 1.1 mg of N-hydroxysulfosuccinimide to 1 mL of ultrapure water. Drop 5 μL of the glucose oxidase dispersion onto the surface of the second working electrode, then drop 1 μL of the N-hydroxysulfosuccinimide solution, incubate at room temperature for 1 h, and rinse with deionized water. Then dry at 4 °C for 6 h, and finally drop 1 μL of a 0.5 wt.% chitosan solution (solvent is 1 wt.% glacial acetic acid) onto the surface and dry at room temperature for 6 h to obtain the glucose sensing electrode.

[0092] ③ pH electrode.

[0093] Disperse 1 mg of polyaniline nanoparticles in 1 mL of deionized water to form a 1 mg / mL polyaniline dispersion. Take 2 μL of the dispersion and coat it on the surface of the third working electrode, dry at 50 °C for 1 h, and finally drop 1 μL of a 0.5% chitosan solution (1 wt.% glacial acetic acid) onto the surface and dry at room temperature for 6 h to obtain the pH sensing electrode.

[0094] (7) Preparation of the microfluidic device.

[0095] <1> Design the microfluidic channel using AutoCAD and then transfer it to a photomask template.

[0096] <2> Spin-coat SU8-2150 photoresist on the cleaned silicon wafer, rotate at 500 rpm for 5 s, and then rotate at 1000 rpm for 20 s.

[0097] <3> Heat the photoresist at 65 °C for 20 min and then at 95 °C for 60 min.

[0098] <4> Cover the photoresist surface with the photomask template.

[0099] <5> Exposure.

[0100] <6> Heat on a 95 °C hot plate for 30 min.

[0101] <7> Develop to obtain the mold.

[0102] <8> Pour the PDMS precursor and curing agent into the mold at a mass ratio of 10:1.5 until the liquid level is flush with the mold, and cure at 80 °C for 2 h.

[0103] <9> Spin-coat the PDMS mixture on a 100-μm-thick PDMS film, rotate at 200 rpm for 10 s, and then rotate at 3000 rpm for 20 s.

[0104] Attach the microfluidic channel to the PDMS film and cure it at 80 °C for 2 h to obtain a microfluidic device.

[0105] (8) Assembly

[0106] Assemble the detection electrode, the microfluidic device and the circuit main board to obtain an integrated wearable sweat sensor for continuous non-invasive monitoring of diabetic ketoacidosis.

[0107] To characterize the performance of the above-obtained wearable sweat sensor, real sweat containing different concentrations of β-hydroxybutyric acid, medium glucose concentration and pH was used as a sample for testing. First, the levels of various markers in the real sweat were measured using a gas chromatography-mass spectrometry analyzer, then diluted to the required minimum concentration, and then various required concentrations were configured. The standard curves of the current of each electrode versus the concentration were determined using the configured solutions with different concentrations as standard solutions. The test results are as Figures 5 to 7 shown. As can be seen from the figure, Figure 5 is the response of the β-hydroxybutyric acid sensing electrode to different concentrations of β-hydroxybutyric acid in real sweat, Figure 6 is the response of the glucose sensing electrode to different concentrations of glucose in real sweat, Figure 7 is the response of the pH sensing electrode to the pH of real sweat.

[0108] The above are only the preferred embodiments of the present invention. In view of the fact that those skilled in the art to which the present invention pertains can make appropriate changes and modifications to the above-described embodiments, therefore, the present invention is not limited to the specific embodiments described above, and some modifications and changes to the present invention should also fall within the protection scope of the claims of the present invention.

Claims

1. A preparation method of a non-invasive diabetes ketoacidosis sensor for a medium-frequency electrotherapy instrument, characterized in that, The non-invasive diabetes ketoacidosis sensor for medium-frequency electrotherapy instrument includes a microfluidic device and a detection electrode covering it. The detection electrode consists of a substrate and an electrode array arranged on it; The microfluidic device includes a detection chamber, a sweat inlet area and a sweat outlet channel respectively communicating with it; and the detection chamber accommodates the electrode array; The electrode array includes an electrode body located in the detection chamber and an electrical interface respectively led out from each electrode body. The electrode array is connected to the circuit main board through the electrical interface; The electrode body includes a reference electrode located in the center of the detection chamber, three working electrodes distributed surrounding the reference electrode, and an M-shaped counter electrode composed of two semi-circles distributed surrounding two of the working electrodes; The three working electrodes are respectively a β-hydroxybutyric acid sensing electrode, a glucose sensing electrode and a pH electrode; The preparation method of the non-invasive diabetes ketoacidosis sensor for medium-frequency electrotherapy instrument includes the following steps: (1) Prepare the MXene-NH2@GQDs composite electrode; Disperse GQDs in PBS solution, then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysulfosuccinimide. After 1 h of activation treatment, add monolayer MXene-NH2 nanosheets. After reacting for 3 h, filter. Wash and freeze-dry the obtained precipitate in sequence. Configure the product into a dispersion liquid, then coat it on the surface of the electrode substrate, dry it, and cool it to obtain the MXene-NH2@GQDs composite electrode; The preparation process of the monolayer MXene-NH2 nanosheets is as follows: First, mix lithium fluoride and hydrochloric acid, stir at 35 °C for 30 min, then slowly add Ti3AlC2, stir and react at 35 °C for 24 h. After the reaction, centrifuge, discard the upper liquid, add deionized water and continue centrifuging. Repeat the above centrifugation operation 3 times to obtain a slurry; Then, add ammonia solution to the slurry, manually shake for 10 min and then ultrasonically treat for 60 min. Centrifuge the obtained suspension, ultrasonically treat under N2 protection, and transfer the obtained dispersion liquid to a hydrothermal reactor for hydrothermal reaction treatment. After the reaction, centrifuge, wash with deionized water until the pH of the supernatant is neutral, and freeze-dry the precipitate to obtain monolayer MXene-NH2 nanosheets; (2) Respectively prepare a β-hydroxybutyric acid sensing electrode, a glucose sensing electrode and a pH electrode with the MXene-NH2@GQDs composite electrode as the substrate; (3) Screen-print the reference electrode and the counter electrode, and assemble the β-hydroxybutyric acid sensing electrode, the glucose sensing electrode and the pH electrode to obtain the detection electrode; (4) Prepare the microfluidic device; (5) Assemble the detection electrode, the microfluidic device and the circuit main board to obtain an integrated wearable sweat sensor for continuous non-invasive monitoring of diabetes ketoacidosis.

2. The preparation method according to claim 1, wherein The β-hydroxybutyric acid sensing electrode and the glucose sensing electrode are surrounded by the counter electrode, and the β-hydroxybutyric acid sensing electrode and the glucose sensing electrode are respectively located at the centers of the two semi-circles of the M-shaped counter electrode.

3. The preparation method according to claim 1, characterized in that The substrate is PET or PDMS with a thickness of 0.2 mm.

4. The preparation method according to claim 1, characterized in that, The diameters of the working electrode and the reference electrode are 2 mm.

5. The preparation method according to claim 1, characterized in that, (2) The process for preparing the β-hydroxybutyric acid sensing electrode is as follows: First, immerse the MXene-NH2@GQDs composite electrode in a thionine solution and perform electro-polymerization pretreatment by cyclic voltammetry; Then, in a PBS solution, mix nicotinamide adenine dinucleotide, β-hydroxybutyric acid dehydrogenase, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution. Drop the resulting mixture onto the surface of the pretreated MXene-NH2@GQDs composite electrode, then add N-hydroxysulfosuccinimide solution, incubate at room temperature, rinse with deionized water, and dry; Finally, dropwise add a chitosan solution and dry at room temperature to obtain the β-hydroxybutyric acid sensing electrode.

6. The preparation method according to claim 1, characterized in that, (2) The process for preparing the glucose sensing electrode is as follows: First, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to a PBS solution of glucose oxidase to obtain a glucose oxidase dispersion; Then, drop the glucose oxidase dispersion onto the surface of the MXene-NH2@GQDs composite electrode, then add N-hydroxysulfosuccinimide solution, incubate at room temperature for 1 h, wash with deionized water, and dry at room temperature; Finally, dropwise add a chitosan solution and dry at room temperature to obtain the glucose sensing electrode.

7. The preparation method according to claim 1, characterized in that, (2) The process for preparing the pH electrode is as follows: Drop the polyaniline nanoparticle dispersion onto the surface of the MXene-NH2@GQDs composite electrode, dry, then add a chitosan solution and dry at room temperature to obtain the pH sensing electrode.

8. Use of a non-invasive diabetes ketoacidosis sensor for medium-frequency electrotherapy apparatus prepared by the method according to claim 1, characterized in that It is used for continuously monitoring markers β-hydroxybutyric acid, glucose, and pH in human sweat under non-invasive conditions.

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