Microneedle patch for wearable electrochemical luminescence sensing equipment and application of microneedle patch

Abstract

The invention belongs to the technical field of electrochemical luminescence detection equipment, and particularly relates to a microneedle patch for wearable electrochemical luminescence sensing equipment and application of the microneedle patch. The microneedle patch comprises a microneedle base and a microneedle array, the microneedle base is divided into a left area, a middle area and a right area through two parallel grooves; the middle area is conductive and light-transmitting, and the left and right areas are conductive and light-proof; the microneedle arrays are arranged in the three areas and are all subjected to conductive treatment; the three areas are separated by grooves and are insulated from one another and are not conductive; and the surface in each area has conductive performance, so that electric signals can be conducted. The light-emitting sensing equipment takes the microneedle patch as a real-time acquisition tool of a tissue fluid sample, detects the concentration of cTnI in the tissue fluid on line, realizes real-time monitoring of a marker cTnI of myocardial injury and acute myocardial infarction in vivo, has the characteristics of high detection sensitivity, portability, simplicity in operation, low cost and the like, and realizes online in-situ monitoring of wearable electrochemical luminescence of a patient.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemiluminescence detection equipment, and particularly relates to a microneedle patch for wearable electrochemiluminescence sensing equipment and its application. Background Art

[0002] Electrochemiluminescence (ECL) is the most commonly used biomarker detection method due to its advantages of precise quantification, high sensitivity and specificity, wide detection range, and ease of high-throughput automation. Currently, ECL detection of relevant biomarkers is performed in vitro, offline, and incapable of real-time, online dynamic monitoring of these biomarkers. This prevents ECL from fully leveraging its rapid and highly sensitive advantages in disease risk prevention, effectively preventing the serious consequences of disease onset.

[0003] To achieve online real-time monitoring of relevant markers in the body, the help of wearable biosensor devices is needed. Wearable biosensor devices refer to wearable electronic devices that integrate biosensor technology to monitor human physiological or biochemical indicators in real time. As an ideal detection object for wearable biosensor devices, tissue fluid contains a rich variety of biomarkers. A microneedle patch is a patch containing a microneedle array structure. When the microneedle patch acts on the skin surface, it can safely and effectively contact the tissue fluid and then detect the markers therein. The microneedle patch acts on the skin surface without pain and is simple and convenient to operate. At present, wearable biosensor devices based on electrochemiluminescence are in a blank stage. In the face of the daily monitoring needs of patients with acute diseases, it is urgent to develop a wearable electrochemiluminescence sensing device based on microneedle patches to achieve real-time in situ monitoring of relevant markers in tissue fluid. This device facilitates online, in-situ monitoring of wearable ECL. Its portability, ease of use, and low cost make it a versatile tool for diverse applications: it can be installed in ambulances to facilitate early diagnosis and disease progression monitoring during transport; it can also be used for daily monitoring of myocardial injury markers in patients recovering from viral infections at home, allowing prompt medical attention if elevated concentrations are detected. To prevent the risk of myocardial injury in patients recovering from viral infections, achieving early diagnosis of acute illnesses and improving their survival rates is crucial. Summary of the Invention

[0004] The purpose of the present invention is to provide a microneedle patch for a wearable electrochemiluminescent sensing device that is easy to carry, simple to operate, and low in cost, and its application.

[0005] The microneedle patch for a wearable electrochemiluminescent sensing device provided by the present invention comprises a microneedle base and a microneedle array;

[0006] The microneedle base is divided into three areas: left, middle, and right by two parallel grooves. The left and right areas have the same area, and the middle area has the largest area. The middle area is conductive and light-transmissive, while the left and right areas are conductive but not light-transmissive.

[0007] The microneedle array is arranged in three areas, all of which are treated to be conductive: the middle area is gold-plated, and the microneedle array serves as a working electrode; the left and right areas are gold-plated and silver-coated, respectively, with the microneedle array in the gold-plated area serving as a counter electrode, and the microneedle array in the silver-coated area serving as a reference electrode; the three areas are separated by grooves, insulated from each other and non-conductive; the surface of each area has conductive properties and can conduct electrical signals;

[0008] The microneedles of the microneedle array are conical;

[0009] The microneedle array in the middle area is subjected to a chemical modification treatment after gold plating: specifically, an electrochemiluminescent substance, gold nanoparticles and chitosan are mixed and coated on the surface of the microneedles, a thiol-containing stem-loop structure is formed by annealing single-stranded DNA, and the stem-loop structure is modified on the surface of the microneedles through a gold-sulfur bond.

[0010] Preferably, the microneedle height is 1 mm, the microneedle base diameter is 250 μm, and the tip diameter is 80 μm; the microneedle spacing in the microneedle array is 0.5 mm;

[0011] Preferably, the size of the microneedle base is 15×15×1.5 mm, and the size of the groove is not limited, and is defined according to actual conditions while ensuring separation and insulation of the three microneedle array areas.

[0012] In the present invention, the gold plating adopts an ion vacuum plating method to form a gold nanoparticle coating on the surface, and the silver coating is a coating method to form a conductive silver coating.

[0013] In the present invention, the specific steps of gold plating the middle area of the microneedle base (microneedle array) are as follows:

[0014] a) Clean the microneedles to ensure that their surfaces are clean and free of contamination;

[0015] b) Fix the microneedle on the sample holder 2 to 3 cm away from the gold target, place it in a vacuum chamber and evacuate to a high vacuum state;

[0016] c) cleaning and activating the surface of the microneedles by argon ion bombardment after the vacuum degree reaches the standard;

[0017] d) Subsequently, the gold target is bombarded with high-energy ions, causing the gold atoms to sputter and evenly deposit on the surface of the microneedles. The bombardment is repeated 5 to 7 times to obtain a conductive and light-transmitting gold film;

[0018] Preferably, the ions bombard the gold target 2 cm away from the gold target and repeat the bombardment 6 times;

[0019] e) After coating, cool the sample and remove it.

[0020] After gold plating using the above-mentioned specific method and parameters, the light transmittance of the gold-plated middle area can be guaranteed to be greater than 80%.

[0021] In the present invention, the microneedle base and the microneedle array are integrally formed; the microneedles are provided with equally spaced perforations on the side of the cone, parallel to the axis of the cone, for increasing the contact area between the microneedles and the tissue fluid in the skin.

[0022] Preferably, the perforation diameter is 0.1 mm.

[0023] In the present invention, the electrochemiluminescent material is one of terpyridine ruthenium (iridium), luminol and its derivatives, quantum dots, noble metal nanoclusters and hydrogen bond organic frameworks.

[0024] The microneedle patch of the present invention can be used to prepare a wearable electrochemiluminescent sensing device.

[0025] A wearable electrochemiluminescent sensing device based on the above-mentioned microneedle patch, comprising a wearable microneedle detection module and a control and display module;

[0026] The wearable microneedle detection module includes a micro photomultiplier tube, a micro electrochemical workstation, a housing, a flexible circuit board, a microneedle patch, and a wristband;

[0027] The micro photomultiplier tube detects the electrochemiluminescence signal and converts it into an electronic pulse signal;

[0028] The micro electrochemical workstation provides the electrical signal required for the electrochemiluminescence reaction;

[0029] The micro photomultiplier tube and the micro electrochemical workstation are arranged in the housing;

[0030] A light-transmitting hole is provided at the bottom of the housing, and the detection port of the micro photomultiplier tube faces the light-transmitting hole;

[0031] A darkroom sealing ring is provided around the light-transmitting hole on the outer bottom surface of the shell;

[0032] The flexible circuit board is disposed at the light-transmitting hole on the outer bottom surface of the housing and is connected to the micro-electrochemical workstation; the microneedle patch can be detachably mounted on the flexible circuit board, with the microneedle array in the microneedle patch facing outward, and the detection port of the micro-photomultiplier tube facing the light-emitting area on the back of the microneedle patch through the light-transmitting hole; the flexible circuit board transmits electrical signals to different areas on the microneedle patch to perform an electrochemical luminescence reaction;

[0033] When the microneedle patch contacts the surface of the skin, it can penetrate the skin so that the microneedle tips are in full contact with the tissue fluid;

[0034] The wristband is fixed to the housing, and the housing is tied and fixed to the wrist, so that the darkroom sealing ring can be in close contact with the skin to form an independent darkroom, eliminating the interference of background light;

[0035] The control and display module includes a photon counter, a host computer control unit and a display unit; the host computer control unit is connected to the photon counter and the display unit;

[0036] The photon counter is also connected to the micro photomultiplier tube data. The photon counter counts the number of electronic pulses generated by the photomultiplier tube each time it emits light, obtains the electrochemical luminescence intensity, and sends it to the host computer control unit;

[0037] The host computer control unit processes the electronic pulse data to form a test result; and can display the test result in real time on the display unit to show the change trend of the marker concentration over time.

[0038] In the present invention, the flexible circuit board matches the shape of the microneedle patch, and a through hole for light signals to pass through is provided at the center, which matches the position of the light-transmitting hole of the housing;

[0039] The flexible circuit board is provided with three upright metal contacts, and the side of the microneedle base is also provided with a conductive layer. The three metal contacts separately contact the side surfaces of the left, middle and right areas of the microneedle base to achieve electrical conduction; in this way, the power-on status of the three areas can be independently controlled by the host computer control unit.

[0040] In the present invention, the data connection between the photon counter and the micro photomultiplier tube is achieved by Bluetooth connection or cable connection.

[0041] When the present invention is used, a microneedle is inserted into the skin. When a marker is present in tissue fluid, the marker is recognized and captured by the probe modified on the microneedle surface, thereby freeing the ferrocene-modified single-stranded DNA that was originally complementary to the recognition end, thereby restoring the light signal of the electrochemiluminescent material. The aptamer chain composed of some bases on the thiol-modified stem-loop structure modified on the microneedle surface can specifically recognize the marker, thereby eliminating interference from other components in the tissue fluid and achieving specific detection of the marker in the tissue fluid; tissue fluid contains a rich variety of biomarkers, including proteins, nucleic acids, small molecules, etc.

[0042] The wearable microneedle detection module is designed to adjust the wristband's tightness when it determines that the target subject's physiological state meets preset conditions, allowing the microneedle patch to embed into the target subject's subcutaneous tissue and ensure full contact between the microneedle tip and the tissue fluid. Chemical modification on the microneedle surface enables specific identification of markers, triggering an electrochemiluminescent reaction. The electrochemiluminescent light signal is then converted to the marker concentration based on the light signal intensity, enabling real-time monitoring of the marker in the tissue fluid. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Schematic diagram of the three-dimensional model of the microneedle patch, (a) is the overall intention diagram, and (b) is the schematic diagram of the microneedle monomer structure.

[0044] Figure 2 Figure 1 shows the electrically driven electrochemiluminescent, wash-free microfluidic chip device of the present invention. (a) is a schematic diagram of the overall assembly, and (b) is a schematic diagram of the explosion of the wearable microneedle detection module.

[0045] Figure 3 It is a schematic diagram of the surface modification method of the microneedle patch and the principle of marker detection.

[0046] Figure 4 Schematic diagram of the working process of wearable electrochemiluminescence sensing device based on microneedle patch.

[0047] Figure 5 This is a schematic diagram of the practical application of wearable electrochemiluminescence sensing devices based on microneedle patches.

[0048] Figure 6 The finished product of 3D printing of microneedle patch.

[0049] Figure 7 SEM characterization of the microneedle patch.

[0050] Figure 8 Characterize the light transmittance of microneedle patches.

[0051] Figure 9 It is a microneedle patch that has been vacuum coated.

[0052] Figure 10 Designed for flexible PCB.

[0053] Figure 11 The timeline axis for the SD rat experiment.

[0054] Figure 12(a) Real-time monitoring of cTnI concentrations in rats in the AMI model group, healthy group, and AMI sham-operated group within 6 hours after ECLD placement. (b) Real-time monitoring of cTnT concentrations in rats in the AMI model group, healthy group, and AMI sham-operated group within 6 hours after ECLD placement. (c) Four-parameter fitting curve of ELISA standard test. (d) Serum cTnI concentrations measured by ELISA kit every 30 minutes.

[0055] Figure 13 The changes in cTnI concentration in Bama pigs in healthy and AMI states were continuously monitored within 16 hours after ECLD was worn.

[0056] The numbers in the figure are: 1 is the wearable microneedle detection module, 2 is the control and display module, 3 is the micro photomultiplier tube, 4 is the micro electrochemical workstation, 5 is the shell, 6 is the darkroom sealing ring, 7 is the flexible circuit board, 8 is the microneedle patch, 9 is the wristband, 10 is the display unit, 11 is the photon counter, 12 is the host computer control unit, 13 is the microneedle base, 14 is the microneedle, 15 is the groove, and 16 is the perforation. DETAILED DESCRIPTION

[0057] The present invention is further described in detail below with reference to the accompanying drawings.

[0058] The microneedle patch of the present invention comprises a microneedle base 13 and a microneedle array;

[0059] The microneedle base 13 is divided into three areas: left, middle, and right by two parallel grooves 15. The left and right areas have the same area, and the middle area has the largest area.

[0060] The microneedle array is arranged in three regions, with the middle region being gold-plated as a working electrode microneedle array, the left and right regions being gold-plated and silver-coated, respectively. The gold-plated region serves as a counter electrode microneedle array, and the silver-coated region serves as a reference electrode microneedle array. The microneedle arrays in the three regions are treated to be conductive, and the three regions are separated by grooves 15, which are insulated and non-conductive. The surface of each region has conductive properties and can conduct electrical signals. The spacing between the microneedles 14 in the array is 0.5 mm, and the dimensions of the microneedle base 13 are 15×15×1.5 mm.

[0061] The gold plating adopts ion vacuum plating method to form a gold nanoparticle coating on the surface, and the silver coating is a coating method to form a conductive silver coating;

[0062] After the middle area is gold-plated, it is chemically modified:

[0063] An electrochemiluminescent material, gold nanoparticles and chitosan are mixed and coated on the surface of the microneedle. A thiol-containing stem-loop structure is formed by annealing single-stranded DNA, and the stem-loop structure is modified on the surface of the microneedle through a gold-sulfur bond.

[0064] The microneedle 14 is basically conical in shape, with a microneedle height of 1 mm, a microneedle bottom diameter of 250 μm, and a tip diameter of 80 μm; on the side of the cone, there are four perforations 16 arranged at equal intervals and parallel to the axis of the cone, with a diameter of 0.1 mm, which are used to increase the contact area between the microneedle 14 and the tissue fluid in the skin.

[0065] The microneedle base 13 and the microneedle array are integrally formed and manufactured by 3D printing technology.

[0066] Stereolithography-based 3D printing technology is widely used in a variety of high-precision applications. It utilizes a digital light projector to emit ultraviolet light, controlled by a digital micromirror device (DMD) to control the light pattern, and then cures the photosensitive resin layer by layer, ultimately creating a highly precise three-dimensional object. Stereolithography-based 3D printing offers the advantages of high precision and speed, making it suitable for the fabrication of microneedle patches. Stereolithography-based 3D printing technology achieves micron-level resolution, ensuring highly consistent size and shape for each microneedle. It can also meet the demands of producing microneedle models with complex geometries. The biocompatible photosensitive resin material used in 3D printing can prevent toxic microneedles from damaging the skin.

[0067] Since the thickness of the dermis and epidermis of human skin is in the range of 0.5-2 mm, in order to ensure that the microneedle patch can fully contact with the tissue fluid, the designed microneedle patch model is as follows Figure 4 As shown, the microneedle patch model is printed on a 3D printer. After printing, the excess photosensitive resin on the microneedle surface is cleaned with ethanol and then cured under ultraviolet light. Subsequently, the microneedle surface is sprayed with gold using ion vacuum coating technology to make the microneedle surface conductive.

[0068] The surface of the microneedle patch is divided into three areas by grooves, namely the working electrode microneedles, the counter electrode microneedles and the reference electrode microneedles; it is necessary to ensure that the microneedles in each area are isolated from each other and the conduction within the area is good; when the microneedles in a certain area are subjected to conductive treatment, a clamp is used to block the microneedles in other areas.

[0069] The specific steps of conducting conductive treatment using ion vacuum coating technology are as follows:

[0070] a) Clean the microneedles to ensure that their surfaces are clean and free of contamination;

[0071] b) Fix the microneedle on the sample holder 2 cm away from the gold target, place it in a vacuum chamber and evacuate to a high vacuum state;

[0072] c) cleaning and activating the surface of the microneedles by argon ion bombardment after the vacuum degree reaches the standard;

[0073] d) Subsequently, the gold target is bombarded with high-energy ions, causing the gold atoms to sputter and evenly deposit on the surface of the microneedles. The bombardment is repeated six times to obtain a conductive and light-transmitting gold film;

[0074] e) After coating, cool the sample and remove it.

[0075] After gold plating using the above-mentioned specific method and parameters, the light transmittance of the gold-plated middle area can be guaranteed to be greater than 80%.

[0076] The surface morphology of the microneedle was observed using SEM, AFM, etc. The mechanical strength of the microneedle was examined using rheometers, universal electronic testing machines, etc. The conductivity of the microneedle surface and whether the different areas of the microneedle were isolated from each other were measured using a multimeter.

[0077] The surface morphology of the microneedles was observed using SEM, proving that the 3D printing accuracy can meet the requirements of microneedle patches, such as Figure 7 The transmittance of microneedles plays a key role in the conduction of electrochemiluminescence signals. To this end, the transmittance of the microneedle patch was tested and it was found that the transmittance of both the front and back sides of the microneedles could reach over 80%.

[0078] A wearable electrochemiluminescent sensing device based on the above-mentioned microneedle patch includes a wearable microneedle detection module 1 and a control and display module 2;

[0079] The wearable microneedle detection module 1 includes a micro photomultiplier tube 3, a micro electrochemical workstation 4, a housing 5, a flexible circuit board 6, a microneedle patch 8 and a wristband 9;

[0080] The micro photomultiplier tube 3 detects the electrochemical luminescence signal and converts it into an electronic pulse signal; an existing product is selected;

[0081] The micro electrochemical working station 4 provides the electrical signal required for the electrochemiluminescence reaction; the existing product is selected;

[0082] The micro photomultiplier tube 3 and the micro electrochemical workstation 4 are arranged in the housing 5;

[0083] The bottom of the housing 5 is provided with a light-transmitting hole, and the detection port of the micro photomultiplier tube 3 faces the light-transmitting hole;

[0084] A microneedle mounting seat is provided around the light-transmitting hole on the outer bottom surface of the housing 5. The microneedle mounting seat is a circle of integrally formed square enclosure around the light-transmitting hole. A circle of rubber darkroom sealing ring 6 is provided inside the microneedle mounting seat along the square enclosure.

[0085] The flexible circuit board 7 is fitted to the light-transmitting hole on the outer bottom surface of the housing 5. A metal guide wire is provided in each of the three corresponding areas on the flexible circuit board 7. One end of the metal guide wire is connected to the conductive surface of the corresponding area, and the other end is connected to three pre-set electrical signal contacts on the micro-electrochemical workstation 4. The microneedle patch 8 can be detachably mounted on the flexible circuit board 7. The detection port of the micro-photomultiplier tube 3 faces the light-emitting area on the back of the microneedle patch 8 through the light-transmitting hole. The flexible circuit board 7 transmits the electrical signal to different areas on the microneedle patch 8 to perform an electrochemical luminescence reaction.

[0086] The flexible circuit board 7 and the microneedle patch 8 match in shape, and a through hole for light signals to pass through is provided at the center, which matches the position of the light-transmitting hole of the housing 5;

[0087] The flexible circuit board 7 is provided with three upright metal contact pieces, such as Figure 10 As shown, a conductive layer is also provided on the side of the microneedle base 13, and three metal contacts separately contact the side surfaces of the left, middle and right areas of the microneedle base 13 to achieve electrical conduction; thereby, the power-on status of the three areas can be independently controlled by the host computer control unit.

[0088] When the microneedle patch 8 contacts the surface skin, it can penetrate the skin so that the tips of the microneedles 14 are in full contact with the tissue fluid;

[0089] The wristband 9 is fixed to the housing, and the housing 5 is tied and fixed to the wrist, so that the darkroom sealing ring 6 can be in close contact with the skin to form an independent darkroom, eliminating the interference of background light;

[0090] The control and display module 2 includes a photon counter 11, a host computer control unit 12 and a display unit 10; the host computer control unit 12 is connected to the photon counter 11 and the display unit 10;

[0091] The photon counter 11 is also connected to the micro photomultiplier tube 3. The photon counter 11 counts the number of electronic pulses generated by the photomultiplier tube 3 each time it emits light, obtains the electrochemical luminescence intensity, and sends it to the host computer control unit 12.

[0092] The host computer control unit 12 processes the electronic pulse data to form a test result; and can display the test result in real time on the display unit 10 to show the change trend of the marker concentration over time.

[0093] The data connection between the photon counter 11 and the micro photomultiplier tube 3 can be realized by wireless connection by adding a Bluetooth module, or can be directly connected by a cable.

[0094] The present invention verifies the feasibility of the design scheme of the wearable microneedle detection module. The flexible circuit board can transmit the electrical signal to the microneedle patch. The flexible circuit board is designed as follows Figure 10 As shown, a window for optical signal transmission is reserved in the middle of the flexible circuit board. Because the edges of the microneedle patch also exhibit good conductivity during vacuum coating, gold-exposed areas are provided on three edges of the flexible circuit board, where brass springs can be welded. When the springs at the edges of the flexible circuit board come into contact with the sides of the microneedle patch, electrical conduction is achieved between the flexible circuit board and the microneedle patch.

[0095] To investigate the microneedle installation scheme for the microneedle mounting port of the wearable microneedle detection module and the feasibility of constructing a local darkroom in the test area during real-time detection, a local cavity was machined for testing. A rubber sealing ring was customized for the microneedle mounting port, and double-sided tape was used to achieve a tight fit between the sealing ring and the microneedle mounting port. A microneedle fixing spring was welded to the flexible circuit board, and the flexible circuit board was affixed to the microneedle mounting port using double-sided tape. The microneedle patch was evaluated for ease of installation and tested for its conductivity after it was in place. This demonstrated that the design met the requirements, that the microneedle patch could be quickly and easily installed, and that conductivity between the flexible circuit board and each area of the microneedle could be achieved after it was in place.

[0096] Application example reference content:

[0097] Evaluation of in situ monitoring capabilities in a rat myocardial infarction model

[0098] To further verify the monitoring potential of ECLD, an SD rat AMI model group, a sham operation group, and a healthy group were constructed to verify its feasibility of in situ real-time monitoring. Different response platforms were constructed using Y-shaped probes targeting cTnI and cTnT to verify the universality of ECLD. The AMI group, sham operation group, and healthy control group rats were raised under the same conditions for four weeks, and then the corresponding model construction and operation were applied. ECLD was inserted into the skin through a microneedle patch and then monitored for a period of time after equilibration. At the same time, serum collected from the mouse eyeballs during the same period was used to control the ELISA signal. According to the traditional ELISA process in the instructions, the equilibration incubation time is 5 hours and the detection time is 5 minutes.

[0099] Since AMI is an acute disease, its early rapid detection and early treatment intervention by doctors are crucial to improving the survival rate of patients. Therefore, we mainly studied the changes in the concentration of myocardial infarction-related markers (cTnI and cTnT) in vivo by in situ online monitoring of the microneedle patch within 6 hours after the establishment of the AMI model. After the rat's abdomen was depilated, the ECLD was continuously fixed on the rat's waist and abdomen. The microneedle was inserted into the abdominal skin to contact the ISF. After equilibrium, the ECLD test was started every 30 minutes, and blood was collected from the eyeball at the same time. The ECL test signal obtained at the moment of the first insertion into the skin was used as the baseline for subtraction. The microneedle left a pinhole mark after insertion, indicating that the microneedle had successfully pierced the epidermis.

[0100] The ST segment elevation observed in the real-time electrocardiogram after the AMI model was established was used as the starting point for timing. Due to the differences between different rats, the change in cTnI concentration was recorded as the slope change relative to the baseline. The change rate formula is (I1-I0) / I0×100%, which is obtained from Figure 12 It can be seen from a that the change rate of the healthy group and the sham operation group was small within 6 hours, while the AMI model group showed a significant upward trend after 3 hours, and the change rate began to show a stable trend after 5.5 hours. Figure 12 As shown in b, the concentration of cTnT changes significantly at the beginning, and the change rate of the healthy group and the sham operation group is relatively stable within 6 hours. The concentration of the cTnI standard is used as the horizontal axis, and the OD value of the microplate reader test result is used as the vertical axis. The ELISA standard curve is fitted using a logistic four-parameter curve ( Figure 12 c). Figure 12 As shown in Figure d, rat serum cTnI concentrations peaked at 6 hours and began to decline after 6 hours. Serum cTnI concentrations had returned to their original levels by around 12 hours. This is consistent with the peak time of serum cTnI concentrations reported in the literature. The in situ ECLD monitoring results showed the same trend as the ELISA spot test results within 6 hours. In situ ECLD monitoring is simple and non-invasive, allowing for more rapid detection of dynamic changes in ISF cTnI concentrations and real-time monitoring of AMI risk. It is suitable for rapid monitoring in settings such as home monitoring, ambulances, and ICUs, thereby assisting physicians in making rapid assessments and appropriate interventions to reduce the risk of mortality. These results demonstrate that our constructed ECLD has a certain level of real-time monitoring capability in small live animals. Whether this wearable device can ultimately be applied to humans requires further ECLD real-time monitoring experiments in larger live animals.

[0101] 4.3.12 Evaluation of ECLD in situ monitoring capabilities in the Bama pig myocardial infarction model

[0102] In this study, we used the same Bama pig as the experimental subject. First, it was set as the healthy group in its healthy state, and the group was monitored continuously for 16 hours. Subsequently, it was set as the AMI (acute myocardial infarction) model group on the next day, and was also monitored continuously for 16 hours. In both groups of experiments, the same part of the same ear was selected for operation each time. After the ECLD was inserted into the pig's ear through a microneedle patch, it was continuously monitored at intervals of 30 minutes after a period of equilibrium. At the same time, during the same time period, blood was collected from the anterior vena cava of the Bama pig, and a control test was performed according to the traditional process of the ELISA kit instructions. The process includes 5 hours of equilibrium incubation and 5 minutes of detection time.

[0103] After the microneedle was inserted into the Bama pig's ear, it left a pinhole mark, indicating that the microneedle had successfully pierced the pig's epidermis. The healthy Bama pigs showed a lower ECL stable signal, while the AMI model pigs showed a higher ECL stable signal, indicating a certain signal difference between the two groups. This also shows that ECLD can still resist interference and produce a certain signal output in the complex fluid environment of large living organisms.

[0104] The data monitoring, recording and processing methods of the Bama pig myocardial infarction model group and the healthy group were the same as those of the rats, and the monitoring was continuous for 16 hours. Figure 13 As can be seen, the healthy group showed a relatively small rate of change within 16 hours, with a relatively stable curve. The AMI model group showed a significant upward trend within 6 hours of the next day, after which the rate of change remained relatively stable until 16 hours. This demonstrates the universality of the constructed ECLD, enabling timely and rapid response and real-time monitoring capabilities for both small and large live animals.

[0105] The above description of the exemplary embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. Those skilled in the art can readily make various modifications to these exemplary embodiments and apply the general principles described herein to other embodiments without requiring creative effort. Therefore, the present invention is not limited to the above exemplary embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A microneedle patch for a wearable electrochemiluminescent sensing device, characterized in that: including a microneedle base and a microneedle array; The microneedle base is divided into three areas: left, middle, and right by two parallel grooves. The left and right areas have the same area, and the middle area has the largest area. The middle area is conductive and light-transmissive, while the left and right areas are conductive but not light-transmissive. The microneedle array is arranged in three areas, all of which are treated to be conductive: the middle area is gold-plated, and the microneedle array serves as a working electrode; the left and right areas are gold-plated and silver-coated, respectively, with the microneedle array in the gold-plated area serving as a counter electrode, and the microneedle array in the silver-coated area serving as a reference electrode; the three areas are separated by grooves, insulated from each other and non-conductive; the surface of each area has conductive properties and can conduct electrical signals; The microneedles of the microneedle array are conical; The microneedle array in the middle area is subjected to a chemical modification treatment after gold plating: specifically, an electrochemiluminescent substance, gold nanoparticles and chitosan are mixed and coated on the surface of the microneedles, a thiol-containing stem-loop structure is formed by annealing single-stranded DNA, and the stem-loop structure is modified on the surface of the microneedles through gold-sulfur bonds.

2. The microneedle patch according to claim 1, wherein The microneedle has a height of 1 mm, a base diameter of 250 μm, and a tip diameter of 80 μm; and a microneedle spacing in the microneedle array is 0.5 mm.

3. The microneedle patch according to claim 1, wherein The gold plating adopts an ion vacuum plating method to form a gold nanoparticle coating on the surface, and the silver coating is a coating method to form a conductive silver coating.

4. The microneedle patch according to claim 3, wherein The specific steps of gold plating the middle area of the microneedle base are as follows: a) Clean the microneedle to ensure that its surface is clean and free of contamination; b) Fix the microneedle on the sample holder 2 to 3 cm away from the gold target, place it in a vacuum chamber and evacuate to a high vacuum state; c) cleaning and activating the surface of the microneedles by argon ion bombardment after the vacuum degree reaches the standard; d) Subsequently, the gold target is bombarded with high-energy ions, causing the gold atoms to sputter and evenly deposit on the surface of the microneedles. The bombardment is repeated 5 to 7 times to obtain a conductive and light-transmitting gold film; Preferably, the ions bombard the gold target 2 cm away from the gold target and repeat the bombardment 6 times; e) After coating, cool the sample and remove it.

5. The microneedle patch according to claim 1, wherein The microneedle base and the microneedle array are integrally formed; the microneedles are provided with equally spaced perforations on the side of the cone, which are parallel to the axis of the cone, so as to increase the contact area between the microneedles and the tissue fluid in the skin.

6. The microneedle patch according to claim 5, wherein The perforation diameter is 0.1 mm.

7. A wearable electrochemiluminescent sensing device using the microneedle patch according to any one of claims 1 to 6, characterized in that: Includes a wearable microneedle detection module and a control and display module; The wearable microneedle detection module includes a micro photomultiplier tube, a micro electrochemical workstation, a housing, a flexible circuit board, a microneedle patch, and a wristband; The micro photomultiplier tube detects the electrochemiluminescence signal and converts it into an electronic pulse signal; The micro electrochemical workstation provides the electrical signal required for the electrochemiluminescence reaction; The micro photomultiplier tube and the micro electrochemical workstation are arranged in the housing; A light-transmitting hole is provided at the bottom of the housing, and the detection port of the micro photomultiplier tube faces the light-transmitting hole; A darkroom sealing ring is provided around the light-transmitting hole on the outer bottom surface of the shell; The flexible circuit board is arranged at the light-transmitting hole on the outer bottom surface of the housing and is connected to the micro electrochemical workstation; the microneedle patch can be detachably mounted on the flexible circuit board, with the microneedle array in the microneedle patch facing outward, and the detection port of the micro photomultiplier tube facing the light-emitting area on the back of the microneedle patch through the light-transmitting hole; The flexible circuit board transmits electrical signals to different areas on the microneedle patch, causing electrochemiluminescence reactions; When the microneedle patch contacts the surface of the skin, it can penetrate the skin so that the microneedle tips are in full contact with the tissue fluid; The wristband is fixed to the housing, and the housing is tied and fixed to the wrist, so that the darkroom sealing ring can be in close contact with the skin to form an independent darkroom, eliminating the interference of background light; The control and display module includes a photon counter, a host computer control unit and a display unit; the host computer control unit is connected to the photon counter and the display unit; The photon counter is also connected to the micro photomultiplier tube data. The photon counter counts the number of electronic pulses generated by the photomultiplier tube each time it emits light, obtains the electrochemical luminescence intensity, and sends it to the host computer control unit; The host computer control unit processes the electronic pulse data to form a test result; The test results can be displayed in real time on the display unit, showing the trend of changes in marker concentration over time.

8. The wearable electrochemiluminescent sensing device according to claim 7, wherein: The flexible circuit board matches the shape of the microneedle patch, and a through hole for light signals to pass through is provided at the center, which matches the position of the light-transmitting hole in the housing; The flexible circuit board is provided with three upright metal contacts, and the side of the microneedle base is also provided with a conductive layer. The three metal contacts separately contact the side surfaces of the left, middle and right areas of the microneedle base to achieve electrical conduction; in this way, the power-on status of the three areas can be independently controlled by the host computer control unit.

9. The wearable electrochemiluminescent sensing device according to claim 8, wherein: The data connection between the photon counter and the micro photomultiplier tube is achieved by Bluetooth connection or cable connection.

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