Microneedle diagnosis and treatment device

Abstract

The microneedle diagnosis and treatment device comprises a sensing microneedle, a photo-thermal microneedle, a light source module, an electrochemical sensing module, a main control module and an upper computer, the sensing microneedle and the photo-thermal microneedle are respectively and electrically connected with the electrochemical sensing module, the light source module and the electrochemical sensing module are respectively and electrically connected with the main control module, and the main control module is in communication connection with the upper computer. The sensing microneedle disclosed by the invention is matched with the electrochemical sensing module and the main control module, so that the change of the glucose level in joint tissue fluid can be monitored in real time, and monitored data is transmitted to an upper computer through the main control module; a user can utilize the host computer to cooperate with the master control module to control the operation of the light source module, so that the photo-thermal microneedle absorbs light energy and generates high temperature, thereby inhibiting synovial hyperplasia of tissue fluid and achieving the purpose of integration of monitoring and treatment, the structure is simple, and closed-loop management of monitoring-evaluation-treatment can be realized.

Description

Technical Field

[0001] The present invention relates to the technical field of medical devices, and particularly to a microneedle diagnosis and treatment device. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic, progressive, systemic autoimmune disease. Its typical features are symmetrical joint inflammation, accompanied by cartilage damage, bone erosion, joint swelling and pain. The disease can cause joint deformity and impairment of body function in the early stage, and as the disease progresses, it will affect other organs. Genetic, environmental and autoimmune factors are the three core inducing factors for the onset of RA. Its complex pathogenesis involves a variety of inflammatory mediators, such as tumor necrosis factor-α, C-reactive protein, interleukin 18, etc. These factors play important roles in the disease development process. The global prevalence of RA is about 1%, which brings a heavy burden to patients, the medical system and society. Therefore, early accurate diagnosis and effective treatment are extremely crucial.

[0003] Interstitial fluid (ISF) is the transmission medium of metabolites between capillaries and tissue cells. Its composition is similar to that of plasma, rich in biological relevant information such as glucose, cholesterol, uric acid, sodium ions, pH value, etc., and can reflect local inflammation and metabolic conditions. Specifically, glucose metabolism is a key link in the pathogenesis of RA, and its local changes can reflect the degree of inflammatory activity of RA patients. Photothermal therapy is a commonly used treatment method for RA. By exciting nanomaterials with near-infrared light to generate local heat, it can effectively inhibit synovial hyperplasia. However, how to achieve minimally invasive and in-situ extraction of ISF remains a key problem in RA diagnosis.

[0004] Existing microneedle technology, as an emerging transdermal technology, can achieve painless and minimally invasive interstitial fluid collection due to its characteristic of being able to penetrate the stratum corneum without touching nerve endings, and has a drug loading function. However, currently, microneedle technology is mostly only used for interstitial fluid collection to monitor RA, and additional devices are still required during treatment. There is still a lack of a device that can achieve "integration of diagnosis and treatment" for RA. Summary of the Invention

[0005] The purpose of the present invention is to provide a microneedle diagnosis and treatment device that can perform real-time monitoring and on-demand treatment of RA, and achieve the integration of diagnosis and treatment.

[0006] To achieve the above purpose, the solution of the present invention is: a microneedle diagnosis and treatment device, including a sensing microneedle, a photothermal microneedle, a light source module, an electrochemical sensing module, a main control module and a host computer;

[0007] The sensing microneedle and the photothermal microneedle are respectively electrically connected to the electrochemical sensing module, the light source module and the electrochemical sensing module are respectively electrically connected to the main control module, and the main control module establishes a communication connection with the host computer.

[0008] Preferred solution, the preparation of the sensing microneedles includes:

[0009] Dissolve 2 g of gelatin powder in 10 ml of distilled water, stir for 30 min at 70 °C and 1000 r / min to obtain a gelatin aqueous solution; add 0.2 g of carbon nanotube powder to the gelatin aqueous solution, stir for 30 min at 70 °C and 1000 r / min to prepare a carbon nanotube / gelatin composite hydrogel.

[0010] Heat the carbon nanotube / gelatin composite hydrogel solution to 70 °C and then pour it into a PDMS mold with multiple microneedle arrays, evacuate for 10 minutes under a pressure of 1 MPa to fill the tips of the microneedles, and then place the PDMS mold at 50 °C to dry for 6 h. After the carbon nanotube / gelatin composite hydrogel solution is completely solidified, peel the microneedles from the PDMS mold to obtain carbon nanotube / gelatin sensing microneedles.

[0011] Preferred solution, the preparation of the sensing microneedles further includes:

[0012] Process the three electrodes of the carbon nanotube / gelatin sensing microneedles: do not process the counter electrode, spray Ag / AgCl ink on the surface of the reference electrode, and modify the working electrode with aptamer.

[0013] Among them, the modification process of the working electrode includes: use a micro syringe to drop 20 μL of glucose oxidase solution with a concentration of 10 mg / mL on the surface of the carbon nanotube / gelatin sensing microneedles. After natural drying for 10 min, then drop 20 μL of cross-linking agent GA solution with a concentration of 5%. After reacting for 2 h, then drop 5 μL of Nafion solution. The concentration ratio of Nafion to ethanol in this Nafion solution is 1:10. After natural drying for 5 min, store it at 4 °C for standby.

[0014] Name the processed carbon nanotube / gelatin sensing microneedles as TCNG MN, where T represents three electrodes, C represents carbon, N represents nanotubes, G represents gelatin, and MN represents microneedles.

[0015] Preferred solution, the preparation of the photothermal microneedles includes:

[0016] Dissolve 0.5 g of sodium alginate powder in 10 ml of distilled water, stir for 30 min at 70 °C and 1000 r / min to obtain a sodium alginate aqueous solution; add 0.2 g of Fe3O4 powder to the sodium alginate aqueous solution, stir for 30 min at 50 °C and 1000 r / min to prepare an Fe3O4 / sodium alginate composite hydrogel.

[0017] Pour the Fe3O4 / sodium alginate composite hydrogel solution into a PDMS mold with multiple microneedle arrays, and evacuate it for 10 minutes under a pressure of 1 MPa to fill the tips of the microneedles. Then, place the PDMS mold at 50 °C and dry it for 1 h. After the Fe3O4 / sodium alginate composite hydrogel solution is completely solidified, peel the microneedles from the PDMS mold to obtain Fe3O4 / sodium alginate photothermal microneedles.

[0018] Name the Fe3O4 / sodium alginate photothermal microneedles as FA MN, where F represents Fe3O4 nanoparticles, A represents sodium alginate, and MN represents microneedles.

[0019] In a preferred embodiment, the PDMS mold consists of an 11x11 microneedle array, and the length of the microneedles is 800 microns.

[0020] In a preferred embodiment, the light source module uses an infrared array lamp.

[0021] In a preferred embodiment, the electrochemical sensing module uses an LMP91000 electrochemical front-end chip.

[0022] In a preferred embodiment, the main control module uses a CC2541 system-on-chip.

[0023] In a preferred embodiment, the host computer is a computer or a mobile phone.

[0024] After adopting the above scheme, the beneficial effects of the present invention are as follows: The sensing microneedles of the present invention, in conjunction with the electrochemical sensing module and the main control module, can monitor the change in glucose level in joint tissue fluid in real time, and transmit the monitored data to the host computer through the main control module. When the glucose level exceeds the set threshold, the user can use the host computer in conjunction with the main control module to control the operation of the light source module, so that the photothermal microneedles absorb light energy and generate high temperature, thereby inhibiting synovial hyperplasia of tissue fluid and achieving the purpose of integrated monitoring and treatment. The structure is simple and can realize a closed loop of monitoring-evaluation-treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the system block diagram of the microneedle diagnosis and treatment device in the embodiment of the present invention;

[0026] Figure 2 is the circuit schematic diagram of the infrared array lamp in the embodiment of the present invention;

[0027] Figure 3 is the circuit schematic diagram of the electrochemical front-end in the embodiment of the present invention;

[0028] Figure 4 is the circuit schematic diagram of the CC2541 system-on-chip in the embodiment of the present invention;

[0029] Figure 5It is the system design flow block diagram of adopting the CC2541 system-on-chip in the embodiment of the present invention;

[0030] Figure 6 It is the scanning electron microscope single-needle diagram of TCNG MN in the embodiment of the present invention;

[0031] Figure 7 It is the scanning electron microscope array diagram of TCNG MN in the embodiment of the present invention;

[0032] Figure 8 It is the scanning electron microscope single-needle diagram of FA MN in the embodiment of the present invention;

[0033] Figure 9 It is the scanning electron microscope array diagram of FA MN in the embodiment of the present invention;

[0034] Figure 10 It is the rat back skin diagram after puncture by TCNG MN in the embodiment of the present invention;

[0035] Figure 11 It is the rat back skin diagram after puncture by FA MN in the embodiment of the present invention;

[0036] Figure 12 It is the process diagram of the disappearance of the needle holes in the puncture area on the rat back after puncture with TCNG MN in the embodiment of the present invention;

[0037] Figure 13 It is the process diagram of the disappearance of the needle holes in the puncture area on the rat back after puncture with FA MN in the embodiment of the present invention;

[0038] Figure 14 It is the tissue section diagram of the rat back skin after puncture by TCNG MN in the embodiment of the present invention;

[0039] Figure 15 It is the tissue section diagram of the rat back skin after puncture by FA MN in the embodiment of the present invention;

[0040] Figure 16 It is the repeatability test curve diagram of TCNG MN in the embodiment of the present invention;

[0041] Figure 17 It is the repeatability standard curve diagram of TCNG MN in the embodiment of the present invention;

[0042] Figure 18 It is the detection limit test curve diagram of TCNG MN in the embodiment of the present invention;

[0043] Figure 19 It is the selectivity test curve diagram of TCNG MN in the embodiment of the present invention;

[0044] Figure 20It is the long-term stability test curve graph of TCNG MN in the embodiment of the present invention;

[0045] Figure 21 It is the test graph of the influence of three electrodes on cell toxicity of TCNG MN in the embodiment of the present invention;

[0046] Figure 22 It is the photothermal curve graph of FA MN loaded with different concentrations of Fe3O4 nanoparticles in the embodiment of the present invention;

[0047] Figure 23 It is the cyclic photothermal curve graph of FA MN in the embodiment of the present invention;

[0048] Figure 24 It is the microscope image of FA MN after being inserted into the body in the embodiment of the present invention;

[0049] Figure 25 It is the dissolution height curve graph of FA MN in the embodiment of the present invention;

[0050] Figure 26 It is the in vitro drug release curve graph of FA MN in the embodiment of the present invention;

[0051] Figure 27 It is the microscope image of normal macrophages in the embodiment of the present invention;

[0052] Figure 28 It is the microscope image of macrophages treated with LPS in the embodiment of the present invention;

[0053] Figure 29 It is the comparison graph of the cell survival rate of RAW 246.7 with different concentrations of FA MN in the embodiment of the present invention.

[0054] Label description:

[0055] 1. Sensing microneedle; 2. Photothermal microneedle; 3. Light source module; 4. Electrochemical sensing module; 5. Main control module; 6. Host computer. Detailed implementation manners

[0056] This embodiment provides a microneedle diagnosis and treatment device, as Figure 1 shown, including a sensing microneedle 1, a photothermal microneedle 2, a light source module 3, an electrochemical sensing module 4, a main control module 5 and a host computer 6;

[0057] The sensing microneedles 1 and the photothermal microneedles 2 are electrically connected to the electrochemical sensing module 4 respectively. The sensing microneedles 1 are used to monitor the glucose level in joint tissue fluid, and the photothermal microneedles 2 are used for the rapid dissolution and delivery of subcutaneous photothermal agents. The light source module 3 and the electrochemical sensing module 4 are electrically connected to the main control module 5 respectively. The main control module 5 establishes a communication connection with the host computer 6. The main control module 5 is built-in with a signal conversion unit and a wireless transmission unit, and can feedback the monitored data to the host computer 6 in real time. When the monitored data exceeds the set threshold, the host computer 6 can be used in cooperation with the main control module 5 to control the operation of the light source module 3, so that the photothermal microneedles 2 absorb light energy and generate high temperature, thereby inhibiting the synovial hyperplasia of tissue fluid and treating RA, and finally achieving the purpose of integrating monitoring and treatment.

[0058] Further, the preparation of the sensing microneedles includes:

[0059] Dissolve 2 g of gelatin powder in 10 ml of distilled water, and stir for 30 min at 70 °C and 1000 r / min to obtain a gelatin aqueous solution; add 0.2 g of carbon nanotube powder to the gelatin aqueous solution, and stir at 70 °C and 1000 r / min for 30 min to prepare a carbon nanotube / gelatin composite hydrogel.

[0060] Heat the carbon nanotube / gelatin composite hydrogel solution to 70 °C, then pour it into a PDMS mold with multiple microneedle arrays, and evacuate for 10 minutes under a pressure condition of 1 MPa to fill the microneedle tips. Then place the PDMS mold at 50 °C and dry for 6 h until the carbon nanotube / gelatin composite hydrogel solution is completely solidified, and then peel the microneedles from the PDMS mold to obtain carbon nanotube / gelatin sensing microneedles.

[0061] Gelatin has good biocompatibility and degradability. When the carbon nanotube / gelatin sensing microneedles are implanted into the joint, the gelatin component can reduce the rejection reaction of the immune system and ensure the smooth progress of monitoring. Carbon nanotubes have excellent electrical conductivity, which is beneficial to accurately monitor bioelectric signals and transmit them to the host computer through the electrochemical sensing module and the main control module, improving the accuracy of detection. In addition, the entire preparation process of the carbon nanotube / gelatin sensing microneedles is relatively simple, and can be mass-produced at one time through the PDMS mold, with low cost.

[0062] Further, the preparation of the sensing microneedles also includes:

[0063] Process the three electrodes of the carbon nanotube / gelatin sensing microneedles: Do not process the counter electrode, spray Ag / AgCl ink on the surface of the reference electrode, which can provide a stable potential reference for the working electrode and make the measurement results more accurate and reliable. The working electrode is modified with aptamer.

[0064] Among them, the modification process of the working electrode includes: using a micro syringe to drop 20 μL of glucose oxidase solution with a concentration of 10 mg / mL on the surface of the carbon nanotube / gelatin sensing micro needle. After natural drying for 10 min, 20 μL of cross-linking agent GA solution with a concentration of 5% is dropped. After reacting for 2 h, 5 μL of Nafion solution is dropped. The concentration ratio of Nafion to ethanol in this Nafion solution is 1:10. After natural drying for 5 min, it is stored for standby under the condition of 4 °C;

[0065] The catalytic reaction of glucose oxidase can significantly enhance the detection signal and improve the detection sensitivity of the sensing micro needle to glucose. The cross-linking agent GA solution can improve the stability and reusability of the sensing micro needle. Nafion can form a protective film on the working electrode, which can reduce the interference of other interfering substances in the tissue fluid on the monitoring results.

[0066] The processed carbon nanotube / gelatin sensing micro needle is named TCNG MN. Among them, T represents three electrodes, C represents carbon, N represents nanotube, G represents gelatin, and MN represents micro needle.

[0067] Furthermore, the preparation of the photothermal micro needle includes:

[0068] Dissolve 0.5 g of sodium alginate powder in 10 ml of distilled water, and stir for 30 min at 70 °C and 1000 r / min to obtain a sodium alginate aqueous solution; add 0.2 g of Fe3O4 powder to this sodium alginate aqueous solution, and stir at 50 °C and 1000 r / min for 30 min to prepare Fe3O4 / sodium alginate composite hydrogel;

[0069] Pour the Fe3O4 / sodium alginate composite hydrogel solution into a PDMS mold with multiple micro needle arrays, and evacuate for 10 minutes under a pressure condition of 1 MPa to fill the tips of the micro needles. Then place the PDMS mold at 50 °C and dry for 1 h until the Fe3O4 / sodium alginate composite hydrogel solution is completely solidified, and then peel the micro needles from the PDMS mold to obtain Fe3O4 / sodium alginate photothermal micro needles;

[0070] The Fe3O4 / sodium alginate photothermal micro needle is named FA MN. Among them, F represents Fe3O4 nanoparticles, A represents sodium alginate, and MN represents micro needle.

[0071] Furthermore, the PDMS mold consists of an 11x11 micro needle array, and the length of the micro needle is 800 microns, but not limited to this, which can effectively improve the preparation efficiency of the micro needle and ensure the production consistency.

[0072] Furthermore, the light source module uses an infrared array lamp. Specifically, the infrared array lamp has strong tissue penetration ability, low light scattering effect, and high energy absorption efficiency. The photo-thermal microneedle of this embodiment inhibits synovial hyperplasia of tissue fluid by absorbing near-infrared light of the infrared array lamp. Its principle is to irradiate the target site with near-infrared light, so that the photo-thermal agent delivered to the target site absorbs light energy and generates local high temperature, thereby realizing precise ablation or treatment of local tissue, that is, photo-thermal therapy.

[0073] As the light source for photo-thermal therapy, the light intensity of the infrared array lamp directly determines the treatment effect. A stable infrared array lamp can not only provide sufficient light energy, but also precisely control the wavelength and power to ensure the safety and effectiveness of the treatment. Compared with a single light source, the infrared array lamp, as an integrated light source system, has higher light energy output and more uniform light field distribution, which can meet the needs of large-area treatment. At the same time, the infrared array lamp can realize independent adjustment of each lamp unit, so as to affect the photo-thermal temperature by changing the unit radiation intensity, thereby improving the treatment effect and reducing side effects. The specific circuit schematic diagram of the infrared array lamp is as Figure 2 shown.

[0074] Furthermore, the electrochemical sensing module of this embodiment uses an LMP91000 electrochemical front-end chip, but is not limited thereto. Based on the constant potential chronoamperometry method, it provides a complete signal path solution between the sensor and the main control module, including a transimpedance amplifier (TIA), an I2C interface, and a voltage bias output, and can output a voltage signal proportional to the input current.

[0075] Specifically, the sensing microneedle of this embodiment adopts a three-electrode system. Therefore, in actual use, the OP_MODE register of the LMP91000 electrochemical front-end chip needs to be adjusted to the 3-lead amperometric cell mode to meet the sensing requirements. In addition, the LMP91000 electrochemical front-end chip sets a bias voltage (the bias voltage range is 0% to 24% of the voltage source) between the working electrode WE and the reference electrode RE, and constructs a constant potential circuit based on a differential amplifier to realize the potential comparison between the working electrode WE and the reference electrode RE. At the same time, the impedance change between the working electrode WE and the reference electrode RE causes the amplified error signal to be applied to the counter electrode CE, thereby realizing the constancy of the voltage between the working electrode WE and the reference electrode RE. Under the action of the constant voltage, the electron mediator modified on the working electrode WE can convert molecules into electrons, and then form a flowing redox current between the reference electrode RE and the counter electrode CE. In addition, the generated redox current needs to use the TIA circuit to realize the proportional conversion of the input current to the output voltage, and its gain depends on R TIA. The LMP91000 electrochemistry front-end chip provides 7 built-in gain resistances to meet different requirements, ranging from 2.75 kΩ to 350 kΩ. Subsequently, the redox current generated on the working electrode WE is amplified proportionally to an analog voltage signal by the TIA circuit and output to the main control module through the VOUT pin for analog / digital signal conversion.

[0076] To achieve the stability of the voltage input to the LMP91000 electrochemistry front-end chip, in this embodiment, the low-dropout linear regulator AMS1117-3.3 is selected as the external reference voltage source, but not limited to this. Its output voltage is 3.3 V and the accuracy is 1.5%. In addition, since hydrogen peroxide generated by glucose oxidase catalyzing glucose needs to be catalyzed at 700 mV to generate current, therefore, in this embodiment, the bias voltage of the LMP91000 electrochemistry front-end chip is set to 22% of the voltage source (about 720 mV) to serve as the effective potential for catalyzing hydrogen peroxide. Subsequently, the gain resistance of the TIA circuit is set to 120 kΩ, and the load resistance is set to 33 Ω, which can convert a current of 10 nA into 39.6 mV. The specific electrochemistry front-end circuit schematic diagram is as Figure 3 shown.

[0077] Furthermore, the main control module in this embodiment uses the CC2541 system-on-chip, but not limited to this. It incorporates an enhanced 8051 microcontroller, a power amplifier, a 2.4 GHz wireless transceiver, RAM, and other peripherals. In terms of power consumption, the CC2541 has three operating modes: high-speed mode PM1 (270 μA), low-power mode PM2 (1 μA), and ultra-low-power mode PM3 (0.5 μA), and users can change according to different requirements. In addition, this CC2541 system-on-chip supports a wireless transmission rate of up to 1 Mbps, which makes it suitable for signal transmission scenarios with fewer parameters. The specific circuit schematic diagram is as Figure 4 shown.

[0078] Furthermore, the upper computer is a computer or a mobile phone, but not limited to this. Taking the mobile phone side as an example, in this embodiment, the development of the embedded program is realized on the IAR Embedded Workbench development program software, which is mainly used for code writing, compilation, debugging to testing, etc. of the CC2541 system-on-chip. The specific system design flow block diagram is as Figure 5 shown.

[0079] Specifically, it is first necessary to perform initialization configuration on each module and port of the system. This process includes the initialization of the clock, GPIO, ADC, timer, and Bluetooth module. The clock initialization ensures the basic operating frequency of the system, and the GPIO port configuration ensures the communication between the MCU of the CC2541 system-on-chip and external hardware (such as shift registers, electrochemistry front-end chips, and infrared array lights). The ADC initialization prepares for subsequent electrochemical monitoring (including parameter configurations such as resolution, channels, and time). The timer initialization ensures that the system schedules tasks at predetermined time intervals, maintaining the accuracy and stability of the system operation. In addition, the Bluetooth module needs to be configured with services and characteristics for pairing and data transmission with the mobile phone. The CC2541 system-on-chip is set to the low-power mode to reduce unnecessary power consumption. When the timer is triggered, the microcontroller is woken up from the low-power mode and starts real-time monitoring of biomarkers, that is, monitoring the glucose level in interstitial fluid. At the same time, the built-in ADC of the CC2541 system-on-chip starts the continuous sampling mode. In this mode, the ADC continuously reads data from the electrochemistry front-end chip, enabling the CC2541 system-on-chip to more accurately capture subtle signal fluctuations and changes. Furthermore, it is necessary to calculate the mean value of the collected electrochemical data to filter the collected data to eliminate the influence of noise on the data, thereby obtaining a more stable and representative electrochemical detection result. Subsequently, the processed data is sent to the mobile phone terminal in hexadecimal format with the assistance of the Bluetooth module. When the mobile phone APP receives the data, it decodes and converts the hexadecimal data according to the predetermined conversion algorithm, converting the original signal into actual electrochemical parameters. Subsequently, the APP uses a visualization program to plot the change trend of the electrochemical signal as a line graph, enabling users to intuitively see the fluctuations of the electrochemical detection signal.

[0080] Experimental tests of sensing microneedles and photothermal microneedles:

[0081] 1. Morphological characterization of microneedles

[0082] The appearances of TCNG MN and FAMN were observed using a scanning electron microscope (SEM). First, TCNG MN and FAMN were freeze-dried at -60 °C for 48 h, then the freeze-dried TCNG MN and FAMN were fixed on the sample column with conductive tape and gold-plated, and then SEM characterization was performed.

[0083] As Figures 6 to 9As shown, the needle body height of TCNG MN is 650 ± 25 μm, the needle tip diameter is 8 ± 2 μm, the needle base width is 252 ± 12 μm, and the needle spacing is 292 ± 26 μm. The height of FAMN is 628 ± 22 μm, the needle tip diameter is 7 ± 2 μm, the needle base width is 243 ± 11 μm, and the needle spacing is 290 ± 23 μm. These microneedles are arranged neatly, with consistent height and diameter dimensions and uniform spacing, indicating that the preparation process is precise and well-controlled.

[0084] 2. Penetration performance test of microneedles

[0085] SD rats were anesthetized with 3% sodium pentobarbital. The longer hairs on the rats' backs were shaved off using a pet-specific hair clipper, and the remaining short hairs were shaved off with an electric shaver to ensure that the skin in the experimental area was smooth and hairless. TCNGMN and FAMN were pressed onto the shaved back skin of the rats and kept in contact for 5 minutes, and then the rats were sacrificed by cervical dislocation. Then, the skin area that had been treated was cut off with surgical instruments and placed in 4% paraformaldehyde solution for fixation. After 24 hours, the excised skin was paraffin-embedded and sectioned, stained with HE, and observed under a microscope for the insertion sites of TCNG MN and FAMN into the skin.

[0086] As Figure 10 and Figure 11 shown, it can be observed that almost every microneedle pierced the rat skin, leaving neatly distributed holes on its surface, confirming that TCNG MN and FAMN have sufficient penetration performance. In addition, as Figure 12 and Figure 13 shown, 2 minutes after the microneedles were removed, the slight redness and swelling caused by the application of the microneedles gradually disappeared. After 4 minutes, most of the microneedle holes were hardly recognizable. Until 6 minutes later, the needle holes completely disappeared, and the skin surface returned to flatness without obvious traces, indicating that the use of TCNG MN and FA MN did not cause permanent damage to the rat skin.

[0087] In addition, to further evaluate the penetration performance of TCNG MN and FA MN, the rat skin was sectioned and the penetration situation was observed. As Figure 14 and Figure 15 shown, regular needle hole patterns can be seen in the cross-section of the skin in the figure, and the penetration depths are approximately 250 μm and 150 μm, exceeding the width of the rat stratum corneum (about 20 - 30 μm), indicating that TCNG MN and FAMN can successfully penetrate the skin.

[0088] 3. Electrochemical performance test of TCNG MN

[0089] In this embodiment, the CHI660E electrochemical workstation was used in combination with the i-t method to explore the electrochemical performance of TCNG MN. The electrochemical test was carried out in PBS solution, and a three-electrode electrochemical detection system was adopted. By continuously dropping high-concentration glucose (step: 1 mM) into the PBS solution, a glucose concentration gradient was established. The detection range of glucose was set to 0-10 mM, which covered the normal physiological concentration range of human glucose. In addition, to test the glucose detection limit of TCNG MN, PBS solutions with different glucose concentrations (0 μM, 10 μM, 20 μM, 40 μM, and 80 μM) were prepared, and the prepared TCNG MN was immersed into these glucose solutions with different concentrations respectively. Subsequently, the i-t method was used to measure the electrochemical data, and the concentration-current curve of glucose was plotted.

[0090] Specifically, 10 μL of 1 M glucose solution (step = 1 mM) was successively added dropwise into a beaker containing 10 mL of 0.01 M PBS solution using a pipette gun to make it have a corresponding concentration gradient, and the electrochemical workstation was used to record the current curves generated at different concentrations. As Figure 16 shown, the electrode would respond rapidly after adding the glucose sample, and as the glucose concentration increased, it could be observed that the current generated on the microneedle sensor increased successively. After adding the sample 10 times, the i-t curve showed excellent linear response, and its correlation coefficient reached 0.99. The calibration curve of glucose concentration and current is as Figure 17 shown, which indicates that the TCNG MN prepared by this method has good repeatability.

[0091] The glucose concentration gradients tested in this embodiment all covered the change range of human glucose concentration. The linear range of the prepared glucose sensor was as high as 10 mM (the normal physiological range of human glucose usually does not exceed 10 mM), and the linear equation was i = 0.0353c - 0.0455, where i is the current in μA and c is the glucose concentration in mM.

[0092] In addition, the detection limit, as another index to measure the monitoring ability of the sensor, represents the lowest concentration that the sensor can detect, which is usually equivalent to 3 times the sensor noise concentration in the blank solution. The standard deviation of the noise response of the blank solution was about 0.1 μA (S / N = 3). From this, the detection limit of the TCNG MN prepared in this embodiment was calculated to be about 10 μM, as Figure 18 shown, and the lower detection limit is helpful for accurately detecting trace glucose.

[0093] In practical applications, glucose monitoring in body fluids needs to face a complex biological fluid environment. These biological fluids may contain various interferents, such as uric acid (UA), lactate, and ascorbic acid (AA). These substances may compete with glucose for the active sites of enzymes, thus interfering with the signal and causing deviations in the detection results. Therefore, the anti-interference performance of TCNG MN is the key to ensuring the accuracy of detection results.

[0094] Specifically, a solution containing interfering substances was added dropwise to the PBS solution, namely 0.1 mM sodium chloride (NaCl), 2 mM ascorbic acid (AA), 2 mM uric acid (UA), and 5 mM lactate. After reacting for a period of time, the change in current was recorded. As Figure 19 shown, for TCNG MN, first, 0.1 mM glucose was added dropwise to the solution twice to observe the generated electrical signal to verify its effectiveness. Then, uric acid (UA), sodium chloride (NaCl), ascorbic acid (AA), and lactate were added in sequence. The results showed that there were only slight current fluctuations in the current curve and no obvious change in the electrical signal was generated. To further verify whether the addition of interferents would affect the monitoring of TCNG MN, different concentrations of glucose were added dropwise to the solution again, and the current curve showed obvious current changes. This indicates that the interferents do not affect the detection of glucose by the sensor, demonstrating the excellent selectivity of TCNG MN, which is attributed to the high selectivity of glucose oxidase and the selective permeability of the Nafion membrane.

[0095] In addition, the long-term stability of TCNG MN was also tested in this example. The process of TCNG MN detecting biomarkers in skin ISF usually requires continuous testing for more than 30 minutes, so continuous and stable detection is very important for TCNG MN. During monitoring, TCNG MN needs to maintain a stable relationship between the input of different concentrations of biomarkers and the output current for a long time. TCNG MN was immersed in simulated ISF (containing a certain concentration of glucose), and long-term electrical signal detection was carried out. As Figure 20 shown, the working electrode WE of TCNG MN was in a stable state in a glucose solution with a constant concentration, and the current response increased after adding glucose dropwise, and the stable detection time could reach 60 minutes, indicating that TCNG MN has the ability of long-term stable monitoring.

[0096] 4. Cytotoxicity Detection of TCNG MN

[0097] The test cells were mouse fibroblasts (L929). The cells were seeded in a 96-well plate, 1×10 5(pieces), and placed in a constant temperature and humidity incubator for 24 h. Subsequently, the old culture medium was removed, and 100 μL of three - electrode extracts with different concentrations (10 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, and 1000 μg / mL) were added to each well. After co - culturing for 24 h, the cells were cultured for another 4 h with 20 μL of 0.5 mg / mL MTT solution. Then, 200 μL of DMSO was added, and after mixing for 10 min, the absorbance at 490 nm was measured using a microplate reader. The cell viability was calculated according to the following formula. The control group was untreated L929 cells and the same volume of culture medium.

[0098]

[0099] In the formula, As represents the absorbance of the experimental group, Ab represents the absorbance of the blank control group, and Ac represents the absorbance of the negative control group.

[0100] As Figure 21 shown, after the three - electrode extracts of TCNG MN were co - cultured with L929 cells for 24 h respectively, within the concentration range of 10 - 500 μg / mL, the cell activities changed from 99.5%, 98.9% and 99.3% to 90.2%, 92.7% and 90.3% respectively, and there was no significant difference with the increase of concentration (P > 0.05). This indicates that the cell viability was not affected after the cells contacted the three - electrode extracts of TCNG MN. It directly proves that the three - electrode of TCNG MN has low toxicity to cells in vitro, that is, it has good biosafety.

[0101] 5. In vitro photothermal performance test of FAMN

[0102] During the test, an infrared thermometer gun was used to record the temperature change of FAMN containing different amounts of Fe3O4 nanoparticles (0.5%, 1%, 2% and 4%) under the irradiation of near - infrared light (808 nm, 1 W / cm 2 ) emitted by an infrared array lamp to determine the optimal content of Fe3O4 nanoparticles.

[0103] As Figure 22As shown, it can be observed that the photothermal microneedles with load concentrations of 0.5%, 1%, 2%, and 4% are heated from 25°C to 43°C, 65°C, 73°C, and 77°C respectively within 1 minute after being irradiated by near-infrared laser, and then slowly heated to 68°C, 81°C, 87°C, and 90°C at 5 minutes. It can be seen that the temperature change of the microneedles is positively correlated with the concentration of the loaded Fe3O4 nanoparticles. In actual application scenarios, the intensity of the near-infrared laser will be greatly reduced after passing through the skin tissue. Therefore, it is necessary to select a loading concentration of 2% or 4% of Fe3O4 nanoparticles with a relatively fast heating rate. However, considering that the heating rates of the 2% and 4% Fe3O4 nanoparticle loading concentrations are almost the same, and taking into account the biosafety and cost issues brought by high concentrations, the 2% Fe3O4 nanoparticle loading concentration is selected in this example.

[0104] To further study the photothermal stability of FAMN, a cyclic irradiation test was also carried out in this example. Under the condition of near-infrared light (808 nm, 1 W / cm 2 ), FAMN was irradiated for 5 minutes, and the temperature change was recorded. Then, after turning off the near-infrared irradiation for 5 minutes, the temperature change of FAMN was continuously recorded, and the cyclic test was repeated 3 times.

[0105] As Figure 23 shown, after 5 minutes of near-infrared irradiation, the temperature of FAMN rapidly rises to 87°C, and then drops sharply to 25°C after removing the near-infrared irradiation for 5 minutes. The temperature change remains consistent in three consecutive cycles, proving that FAMN has stable and reproducible photothermal performance. It should be noted that the temperature of FAMN should be controlled to prevent skin burns during clinical applications.

[0106] 6. Dissolution performance test of FAMN

[0107] SD rats were anesthetized with 3% sodium pentobarbital, and FAMN was applied to the shaved back skin of the SD rats. Subsequently, FAMN was removed from the skin at 30 s, 60 s, 90 s, and 120 s of application time, and the height and dissolution trend of FAMN were observed with an optical microscope.

[0108] Since sodium alginate is a natural polysaccharide with a linear structure and has excellent biodegradability. Therefore, FAMN based on sodium alginate will quickly melt when piercing the skin, thereby releasing the embedded Fe3O4 nanoparticles. As Figure 24 shown, FAMN quickly dissolves after being inserted into the skin of SD rats. The tip of FAMN is significantly deformed and blunt at 30 s of insertion time, the height of FAMN drops by half at 60 s, and almost all of the needle part of FAMN dissolves at 120 s.

[0109] In addition, to study the photothermal dissolution promotion property of FAMN, FAMN was also attached to the back of SD rats, and near-infrared light (808 nm, 1 W / cm 2 ) emitted by an infrared array lamp was used to irradiate FAMN, and the distance between the sample and the infrared array lamp was 10 cm. Subsequently, FAMN was removed from the skin at 10 s, 20 s, 30 s, and 40 s after the application of FAMN, and the height and dissolution trend of the microneedles were observed with an optical microscope. The remaining height (Y) of the microneedles corresponding to different times (X) was plotted as a dissolution curve.

[0110] According to the temperature sensitivity of sodium alginate, the tip of FAMN will deform with the increase in temperature. As Figure 25 shown, under the irradiation of near-infrared light, the temperature of FAMN will rapidly increase due to the heating of Fe3O4 nanoparticles, thereby making FAMN soft and further accelerating the release of nanoparticles in FAMN. After FAMN was inserted into the skin of SD rats and near-infrared irradiation was applied, the tip of FAMN became significantly blunt after 10 s of irradiation, the height of FAMN decreased by about half after 30 s of irradiation, and almost all of it dissolved after 40 s of irradiation. Therefore, under the irradiation of near-infrared light, Fe3O4 nanoparticles can accelerate the dissolution of FAMN, which makes FAMN have better dissolution performance.

[0111] 7. In vitro release performance test of FAMN

[0112] In this example, amoxicillin was selected as the model drug. First, FAMN loaded with amoxicillin was placed in a dialysis bag with a molecular weight cut-off of 8000 Da, and the dialysis bag was immersed in 20 mL of phosphate buffered saline (PBS, pH = 7.4). Subsequently, near-infrared light (808 nm, 1 W / cm 2 ) emitted by an infrared array lamp was used to irradiate the sample, and the distance between the sample and the infrared array lamp was 10 cm. The concentration of amoxicillin was measured with a UV-visible spectrophotometer at a wavelength of 234 nm as a control.

[0113] As Figure 26As shown, after 2 minutes of soaking, the amoxicillin release rate of FA MN rapidly increased to 33%, which can be attributed to the rapid dissolution property of sodium alginate. After 22 minutes of soaking, the cumulative release amount of amoxicillin reached a plateau. After 60 minutes of soaking, the amoxicillin release rate of FA MN reached 89%. Under near-infrared light irradiation, FA MN showed a faster drug release rate. After 2 minutes of irradiation, the amoxicillin release rate of FA MN rapidly increased to 46%. After 7 minutes of irradiation, the amoxicillin release rate of FAMN reached 70%, and then the release curve slowed down. After 20 minutes of irradiation, the amoxicillin release rate of FAMN reached 90%. The experimental differences indicate that under near-infrared light irradiation, FA MN can achieve relatively more efficient drug controlled release, making it applicable to rapid dissolution drug release applications.

[0114] 8. Cytotoxicity test of FAMN

[0115] RAW 246.7 cells were induced with lipopolysaccharide (LPS, 1 μg / mL). Subsequently, polarized RAW 246.7 cells were cultured with FA MN leaching solutions at different concentrations (10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 250 μg / mL, and 500 μg / mL) and exposed to near-infrared light (808 nm, 1 W / cm 2 ) emitted by an infrared array lamp for 5 minutes. The cell viability was calculated according to the following formula, and an uninduced RAW 246.7 cell control group was established.

[0116]

[0117] In the formula, As represents the absorbance of the experimental group, Ab represents the absorbance of the blank control group, and Ac represents the absorbance of the negative control group.

[0118] By taking microscopic pictures of macrophages, the phenotypic changes (polarization) of macrophages after LPS induction were studied. As Figure 27 and Figure 28 shown, normal macrophages are usually in a small round or oval shape, with smooth cell edges, uniform cytoplasmic distribution, and regular overall morphology. After LPS treatment, the morphology of macrophages changes towards the M1 type, with a significant increase in cell volume, irregular morphology, more pseudopodia, and an increase in cytoplasmic particles, and some cells showing stretching or flattening characteristics.

[0119] The near-infrared induced cytotoxicity of Fe3O4 nanoparticles on M1 macrophages comes from two pathways: the heat generated based on the photothermal effect and ferroptosis therapy achieved by the release of Fe 2+ in an acidic environment. As Figure 29As shown, after the FA MN leaching solution was cultured with RAW 246.7 cells before polarization for 24 h, the cell viability of the Control group exceeded 92% in the range of 10 - 500 μg / mL, and no significant difference was shown with the increase of concentration (P > 0.05). This data fully confirmed that FA MN had no obvious toxic and side effects on RAW 246.7 cells before polarization at a concentration of 500 μg / mL.

[0120] In addition, when the concentration of Fe3O4 nanoparticles did not exceed 500 μg / mL, the cell viability of the FA group exceeded 84%, and no significant difference was shown with the increase of concentration (P > 0.05). Further analysis showed that the cell viability of the FA group was lower than that of the Control group. It was speculated that the difference might be attributed to the inflammatory microenvironment in which the polarized RAW 246.7 cells were located, which facilitated the ferroptosis of the cells. Under near-infrared light irradiation, the cell viability of the FA + Laser group was significantly lower than that of the FA group, and the FA + Laser group showed concentration-dependent cytotoxicity. When the concentration reached 500 μg / mL, the FA + Laser group showed a stronger inhibitory effect under the combined treatment of photothermal therapy and ferroptosis, and the cell viability was 32%.

[0121] The above are only the preferred embodiments of the present invention, and do not limit the design of this case. All equivalent changes made according to the key design of this case fall within the protection scope of this case.

Claims

1. A microneedle diagnosis and treatment device, characterized in that: It includes sensing microneedles, photothermal microneedles, a light source module, an electrochemical sensing module, a main control module, and a host computer; The sensing microneedles and the photothermal microneedles are respectively electrically connected to the electrochemical sensing module, the light source module and the electrochemical sensing module are respectively electrically connected to the main control module, and the main control module establishes a communication connection with the host computer.

2. The microneedle diagnosis and treatment device according to claim 1, wherein: The preparation of the sensing microneedles includes: Dissolve 2 g of gelatin powder in 10 ml of distilled water, stir at 70 °C and 1000 r / min for 30 min to obtain a gelatin aqueous solution; add 0.2 g of carbon nanotube powder to this gelatin aqueous solution, and stir at 70 °C and 1000 r / min for 30 min to prepare a carbon nanotube / gelatin composite hydrogel; Heat the carbon nanotube / gelatin composite hydrogel solution to 70 °C and then pour it into a PDMS mold with multiple microneedle arrays, and evacuate for 10 minutes under a pressure condition of 1 MPa to fill the microneedle tips. Then place the PDMS mold at 50 °C and dry for 6 h until the carbon nanotube / gelatin composite hydrogel solution is completely solidified, and then peel the microneedles from the PDMS mold to obtain carbon nanotube / gelatin sensing microneedles.

3. The microneedle diagnosis and treatment device according to claim 2, wherein: The preparation of the sensing microneedles also includes: Process the three electrodes of the carbon nanotube / gelatin sensing microneedles: Do not process the counter electrode, spray Ag / AgCl ink on the surface of the reference electrode, and modify the working electrode with an aptamer; Among them, the modification process of the working electrode includes: Use a micro syringe to drop 20 μL of a glucose oxidase solution with a concentration of 10 mg / mL on the surface of the carbon nanotube / gelatin sensing microneedles. After natural drying for 10 min, then drop 20 μL of a cross-linking agent GA solution with a concentration of 5%. After reacting for 2 h, then drop 5 μL of a Nafion solution, and the concentration ratio of Nafion to ethanol in this Nafion solution is 1:

10. After natural drying for 5 min, store it at 4 °C for standby; Name the processed carbon nanotube / gelatin sensing microneedles as TCNG MN, where T represents three electrodes, C represents carbon, N represents nanotubes, G represents gelatin, and MN represents microneedles.

4. A microneedle diagnosis and treatment device according to claim 1, characterized in that: The preparation of the photothermal microneedles includes: Dissolve 0.5 g of sodium alginate powder in 10 ml of distilled water, stir at 70 °C and 1000 r / min for 30 min to obtain a sodium alginate aqueous solution; add 0.2 g of Fe3O4 powder to this sodium alginate aqueous solution, and stir at 50 °C and 1000 r / min for 30 min to prepare an Fe3O4 / sodium alginate composite hydrogel; Pour the Fe3O4 / sodium alginate composite hydrogel solution into a PDMS mold with multiple microneedle arrays, and evacuate for 10 minutes under a pressure condition of 1 MPa to fill the microneedle tips. Then place the PDMS mold at 50 °C and dry for 1 h until the Fe3O4 / sodium alginate composite hydrogel solution is completely solidified, and then peel the microneedles from the PDMS mold to obtain Fe3O4 / sodium alginate photothermal microneedles; The Fe3O4 / sodium alginate photothermal microneedles are named FA MN, where F represents Fe3O4 nanoparticles, A represents sodium alginate, and MN represents microneedles.

5. The microneedle diagnosis and treatment device according to claim 2 or 4, characterized in that: The PDMS mold consists of an 11x11 microneedle array, and the length of the microneedles is 800 microns.

6. The microneedle diagnosis and treatment device according to claim 1, wherein: The light source module uses an infrared array lamp.

7. The microneedle diagnosis and treatment device according to claim 1, wherein: The electrochemical sensing module uses an LMP91000 electrochemical front-end chip.

8. The microneedle diagnosis and treatment device according to claim 1, characterized in that: The main control module uses a CC2541 system-on-chip.

9. The microneedle diagnosis and treatment device according to claim 1, wherein: The host computer is a computer or a mobile phone.

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