WO2023134762A1 - 一种连续监测或检测体内分析物的微针贴片、其制备方法及相关装置 - Google Patents

一种连续监测或检测体内分析物的微针贴片、其制备方法及相关装置 Download PDF

Info

Publication number
WO2023134762A1
WO2023134762A1 PCT/CN2023/072321 CN2023072321W WO2023134762A1 WO 2023134762 A1 WO2023134762 A1 WO 2023134762A1 CN 2023072321 W CN2023072321 W CN 2023072321W WO 2023134762 A1 WO2023134762 A1 WO 2023134762A1
Authority
WO
WIPO (PCT)
Prior art keywords
microneedle
transdermal
patch
detection
glucose
Prior art date
Application number
PCT/CN2023/072321
Other languages
English (en)
French (fr)
Inventor
金拓
Original Assignee
鲁烁
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 鲁烁 filed Critical 鲁烁
Publication of WO2023134762A1 publication Critical patent/WO2023134762A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/1468—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using chemical or electrochemical methods, e.g. by polarographic means
    • A61B5/1473—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using chemical or electrochemical methods, e.g. by polarographic means invasive, e.g. introduced into the body by a catheter
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/14503—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue invasive, e.g. introduced into the body by a catheter or needle or using implanted sensors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/14532—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue for measuring glucose, e.g. by tissue impedance measurement
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/1486—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using enzyme electrodes, e.g. with immobilised oxidase
    • A61B5/14865—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using enzyme electrodes, e.g. with immobilised oxidase invasive, e.g. introduced into the body by a catheter or needle or using implanted sensors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/68—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient
    • A61B5/6846—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient specially adapted to be brought in contact with an internal body part, i.e. invasive
    • A61B5/6847—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient specially adapted to be brought in contact with an internal body part, i.e. invasive mounted on an invasive device
    • A61B5/685—Microneedles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B2503/00—Evaluating a particular growth phase or type of persons or animals
    • A61B2503/40—Animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B2503/00—Evaluating a particular growth phase or type of persons or animals
    • A61B2503/42—Evaluating a particular growth phase or type of persons or animals for laboratory research

Definitions

  • the present disclosure relates to a microneedle patch capable of continuously monitoring or detecting in vivo analytes such as blood sugar content without causing pain and skin damage, a preparation method of the microneedle patch, and related tests including the microneedle patch device.
  • analytes substances that can reflect the above biological information are called analytes, such as the concentration of glucose in blood.
  • traditional analyte detection methods need to invade human skin, contact blood or at least endocrine body fluids, and realize the determination of analyte and its content. This method is generally called Invasive testing methods or minimally invasive testing methods.
  • Invasive detection methods can be listed as a blood sugar detection test paper disclosed in the patent CN209979545U.
  • the blood sugar detection test paper detects blood sugar, it is necessary to use the blood collection needle on the test paper to take blood on the fingertip, and then cooperate with the detection instrument to detect blood. blood sugar concentration.
  • This type of detection device has been industrialized, and the price of use can be accepted by the vast majority of diabetic patients.
  • Minimally invasive detection methods can be cited such as the Chinese patent CN102472719B applied by the large multinational pharmaceutical company Abbott Diabetes Care, which claims the priority of the US patent US12495798, and discloses a device and method for monitoring analytes in vivo. It includes a housing, a radio frequency receiver, an analyte sensor, a memory, and a signal processor, wherein the components of the analyte sensor include a working electrode and a reference electrode, glucose oxidase coated on the working electrode, and glucose oxidase immobilized
  • the semipermeable membrane on the working electrode because the material of the semipermeable membrane is relatively soft, it is difficult for the analyte sensor to be implanted in the skin.
  • an insertion device equipped with a 5-7mm rigid tip.
  • Implanted in the skin, and the rigid tip is removed from the skin by the ejection mechanism of the insertion device.
  • the analyte sensor is implanted in the skin for a long time, which can continuously and accurately monitor the blood glucose concentration in the user's body, which solves the problem that the invasive detection method can only measure the blood glucose level intermittently, and has high detection accuracy.
  • the principle of continuous blood glucose monitoring devices such as the Abbott Free Style Libre series has also been industrialized, but the high price of use can only be accepted by few diabetics, and cannot be popularized among the majority of diabetics.
  • the device for continuously monitoring the analyte level based on this detection mechanism also has the following disadvantages: (1) The electrochemical sensor needs to be implanted into the skin with the help of an insertion device, and the insertion device has a complicated structure and is only for one-time use, and the cost is high; (2) The semi-permeable membrane cannot cover the glucose oxidase well, and the glucose oxidase will gradually leak out with the prolongation of the use time, reducing the detection signal; (3) The 5-7mm rigid tip piercing the skin will also cause the use of (4) The long-term placement of the electrochemical sensor in the subcutaneous tissue will cause inflammation and rejection of the tissue, and the high cost of one-time use requires the device to stay in the subcutaneous tissue for as long as possible. .
  • non-invasive detection and monitoring methods In order to avoid pain and damage, the scientific community has carried out research on non-invasive detection and monitoring methods.
  • the non-invasive detection and monitoring methods under research are roughly divided into two categories: (1) non-invasive detection and monitoring methods that can penetrate human tissue and interstitial fluid Light sources, such as near-infrared, etc. are used to irradiate, and the concentration of the target component is estimated from the diffuse reflection or Raman spectrum of the light source; (2) Exocrine body fluids, such as tears, saliva, sweat, and urine, are collected for detection.
  • the above methods have many limitations.
  • near-infrared is a rare frequency among various light sources that can penetrate tissues and body fluids, it does not have any molecular motion (rotation, vibration, energy level transition, etc.) It is difficult for reflections to carry chemical information.
  • the near-infrared Raman effect falls into the molecular vibration energy level, there are not many molecular vibration modes with Raman activity.
  • the vibration structures of biomolecules are similar.
  • the Raman spectrum of an object is extremely difficult to distinguish from the mixed spectrum - even by Fourier transform. Aside from relying on specific chemical mechanisms, some research teams are trying to realize the monitoring of blood sugar levels and their changes by near-infrared diffuse reflectance method through big data comparison.
  • microneedle-based analyte detection devices are divided into two types in terms of detection methods: the first type uses the piercing properties of microneedles to form micropores on the skin, and then draws out body fluids through the micropores for detection; the second type It is to assemble the microneedle into a biosensor and insert it into the body to detect the concentration level of the analyte.
  • Patent CN109199400B discloses a microneedle array-based blood glucose electrochemical sensor, which is designed based on the above-mentioned first type of detection method.
  • the sensor includes a microneedle array electrode, reference electrode 1, reference electrode 2 and stored biometric Conductive electrochemical detection zone for molecules.
  • the microneedle array electrode pierces the skin, and then rebounds and exits the skin.
  • the micropores formed on the skin by the microneedle array lead the body fluid to the conductive elastic hydrogel that stores biorecognition molecules in the conductive electrochemical detection area. On the glue, carry out the detection of blood sugar.
  • Diabetic patients are prone to high blood sugar after a meal or patients with insulin secretion disorders need real-time monitoring of blood sugar in order to cope with the unstable blood sugar caused by insulin secretion disorders.
  • this method will have a monitoring delay due to the need to draw out body fluids , not suitable for real-time monitoring, and this method is even more unsuitable for continuous monitoring or high-frequency detection of biological substance levels in the body, which are necessary for precision drug therapy; such as blood sugar level-physical state-insulin The relationship between dose and rate of administration.
  • the inventors conducted research on the monitoring or detection technology of analytes in the body. After entering the dermis, touch as little or no nerve endings as possible. Because the nerve endings in the dermis are mainly distributed in the deep part of the dermis away from the epidermis, the inventors believe that the length of the detection tool that penetrates the skin is short enough, and the shallow part of the dermis close to the epidermis can be in contact with body fluids and No pain is felt without touching the nerve endings. The inventor believes that the damage to the skin caused by the detection tool piercing the skin must be within the scope of reversible damage.
  • the microneedles with small length and diameter can achieve non-invasive and painless effects when used as a detection tool to penetrate the skin.
  • most of the existing microneedles are made of metal, although they can pierce the skin, but have poor affinity with the skin, and cannot effectively fix the detection components inside it. There is a gap between the metal microneedle and the skin. Severe rejection.
  • the inventor considers using a polymer material with better bio-affinity to make microneedles, but the polymer material needs to meet the requirements of having enough hardness to penetrate the skin in a dry state, and be able to accommodate or embed the detection components, and the body fluid The analyte should be able to contact the detection component immobilized in the polymer material.
  • the polymer material needs to have a network structure, and the detection components are fixed in the mesh to avoid the leakage of the detection components, and also allow the analyte in the body fluid to penetrate into the network structure and contact the detection components.
  • In a dry state it also needs to have a certain hardness to penetrate the skin.
  • polyvinyl alcohol has good bioaffinity, but it is necessary to make the polyvinyl alcohol form a gel with a network structure in order to realize the immobilization of the detection components and enable the analyte to diffuse into it. internal, and polyvinyl alcohol dissolves in body fluids.
  • the inventor unexpectedly discovered that polyvinyl alcohol that has undergone freezing-thawing treatment can form a network structure that swells and does not dissolve after absorbing water, and has the hardness to penetrate the skin in a dry state. Based on the above ideas, the inventor launched a method of detecting blood sugar Research on microneedle patches.
  • the first aspect of the present invention relates to a microneedle patch for continuous monitoring or detection of analytes in the body, including at least one first transdermal microneedle standing on the skin-facing surface of the patch substrate layer, and the microneedles are far away from the patch substrate layer
  • One end of the first transdermal microneedle has a sharp shape; an electrochemical reaction cell is integrated in the first transdermal microneedle, and the analyte electrochemically reacts with the detection component contained in the matrix of the first transdermal microneedle in the electrochemical reaction cell or The action forms an electrical signal, and the concentration level of the analyte is reflected by detecting the magnitude of the electrical signal.
  • the matrix of the first transdermal microneedle has a network structure that swells and does not dissolve after absorbing water,
  • the mesh structure allows the analyte to diffuse into or move out of the matrix of the first transdermal microneedle, while preventing the detection component therein from moving away.
  • the second transdermal microneedle also includes at least one second transdermal microneedle standing on the skin surface of the patch substrate layer, the second transdermal microneedle does not contain detection components, and the electrochemical reaction in the first transdermal microneedle
  • the reaction pool includes a working electrode
  • the second transdermal microneedle includes a reference electrode
  • the reference electrode in the second transdermal microneedle is connected with the working electrode in the first transdermal microneedle to form a measurement circuit.
  • the electrical signal generated in the measurement circuit includes an electrical quantity that is quantitatively related to the chemical balance and a current value that is quantitatively related to the electrochemical reaction rate.
  • the length of the first transdermal microneedle is in the range of 600-1500 ⁇ m, and the diameter at the maximum cross-sectional area is in the range of 200-700 ⁇ m.
  • one end of the working electrode in the electrochemical reaction cell is inserted into the first transdermal microneedle matrix, and the other end extends out of the back surface of the patch substrate layer to form the first electrical contact.
  • one end of the reference electrode is inserted into the second transdermal microneedle matrix, and the other end extends out of the back surface of the patch substrate layer to form a second electrical contact.
  • the working electrode in the electrochemical reaction cell at least includes an inert conductor embedded in the first transdermal microneedle matrix, and the inert conductor is selected from platinum, gold, copper, silver, and carbon materials. one or a combination of several.
  • the working electrode further includes a terminal that is electrically connected to the inert conductor and transmits the electrical signal formed on the inert conductor to the outside of the first transdermal microneedle.
  • the raw material for preparing the first transdermal microneedle matrix in the first transdermal microneedles is a hydrophilic polymer.
  • the raw material for preparing the first transdermal microneedle matrix includes at least polyvinyl alcohol as the main material.
  • the raw material polyvinyl alcohol for the preparation of the first transdermal microneedle matrix is completely alcoholyzed polyvinyl alcohol and/or a mixture of completely alcoholyzed polyvinyl alcohol and partially alcoholyzed polyvinyl alcohol.
  • the raw materials for the preparation of the first transdermal microneedle matrix can also include auxiliary materials selected from the group consisting of dextran, chitosan, alginate, hyaluronic acid, sodium hyaluronate, carboxymethyl fiber One or more mixtures of Sodium Sulfate and Polyethylene Glycol.
  • a second aspect of the present invention relates to an apparatus for processing electrical signals generated by a microneedle patch for continuous monitoring or detection of an analyte in the body, comprising at least a signal processor which is compatible with the aforementioned continuous monitoring or detection
  • the measurement loop formed by the microneedle patch within the analyte is electrically connected and monitored in the measurement loop
  • the electrical signal is converted into a digital signal related to the analyte level.
  • the third aspect of the present invention relates to a device for continuously monitoring or detecting analytes in the body, comprising at least the above-mentioned microneedle patch, the microneedle patch is optionally bonded with an adhesive layer that adheres to the skin; and
  • a signal processor electrically connected to the measurement circuit between the first transdermal microneedle and the second transdermal microneedle, monitoring the electrical signal in the measurement circuit and converting the electrical signal into an analyte level-related Digital signal.
  • the device for continuously detecting analytes in the body further includes a connecting plate, and plugging elements are provided on both sides of the connecting plate, and the microneedle patch can be connected to the connecting plate through the plugging elements on one side of the connecting plate.
  • the signal processor is detachably connected to the connection board through the plug-in element on the other side of the connection board, and the measurement circuit formed by the signal processor and the microneedle patch is electrically connected through the connection board.
  • the device for continuously monitoring or detecting analytes in the body described in the present invention is a wearable device.
  • a fourth aspect of the present invention relates to a method for preparing a microneedle patch for continuous monitoring or detection of an analyte in the body, comprising the following steps:
  • Step 1 Casting the molding preparation solution of the microneedle matrix into a casting mold with a sharp end placed on a running suction device, and then continuing to pour the molding preparation solution of the microneedle patch substrate layer into the casting mold, The casting mold is placed in the suction device in operation;
  • Step 2 Freeze-thaw the above-mentioned molding preparation solution in the casting mold
  • Step 3 inserting the electrode into the casting mold, the end of which protrudes from the surface of the molding preparation liquid;
  • Step 4 Repeat Step 2 several times.
  • Step 5 The preparation of the microneedle patch is dried at room temperature to shrink, and then dried until completely cured.
  • the casting mold used in the method for preparing a microneedle patch for continuous monitoring or detection of in vivo analytes has microneedle holes for microneedle molding, and the molding of the microneedle matrix is performed by a suction device.
  • the preparation solution is aspirated into the microneedle orifice.
  • the electrochemical reaction pool is concentrated in the first transdermal microneedle, and the matrix of the first transdermal microneedle can penetrate the epidermis and reach the dermis, and penetrate the skin through the first transdermal microneedle
  • the electrochemical reaction cell is brought into the skin, and the electrochemical reaction cell is implanted into the human body without complicated one-time insertion devices or application devices, which simplifies the structure of the analyte detection device, saves the cost of use, and realizes instant measurement , to meet the low-price demand of ordinary patients for continuous monitoring of blood sugar, to resolve the inconvenience caused by the cost of patients having to attach it for a long time, and to reduce the resistance of the testers during the test.
  • the first transdermal microneedle matrix in this technical solution can not only bring the electrochemical reaction cell into the skin, but also allow the analyte to diffuse into or move away at the same time, and prevent the detection components from moving away, which can better
  • the detection components are fixed in it, and the detection components will not leak during the long-term monitoring process, which can ensure long-term and accurate monitoring of analytes.
  • the first transdermal microneedle matrix in the present invention forms a network structure through freezing-thawing, which will not reduce or affect the activity of the detection components and enzymes, nor will it hinder the peristalsis of the enzyme when it is catalyzed, so it can better Maintain the activity of the enzyme, improve the detection accuracy of the microneedle patch, ensure the practicability of the microneedle patch and the accuracy of continuous measurement, make the microneedle patch for continuous monitoring or detection of analytes in the body have industrialization prospects, and enable the majority of Generally accepted and suitable for diabetic patients.
  • the macromolecule material used in the microneedle patch of the present invention is an affinity material, and the human body has almost no discomfort even if it is worn for a long time, which improves the user's cooperation degree for long-term wearing according to the doctor's request.
  • Figure 1 Schematic diagram of the measurement circuit formed by the first transdermal microneedle and the second transdermal microneedle.
  • Figure 2 Schematic diagram of the electrochemical reaction occurring in the first transdermal microneedle when the detection component is glucose oxidase.
  • Figure 3 The change curve of the current with time after the microneedle patch measurement circuit is turned on.
  • Figure 4 The constant response current-glucose concentration relationship diagram of the microneedle patch loaded with different glucose oxidases.
  • Figure 5 The constant response current-glucose concentration fitting linear plot of the microneedle patch loaded with 10wt% glucose oxidase.
  • Figure 6 The relationship between the constant response current value and the glucose solution when the microneedle patch is used for different days.
  • Figure 7 Comparison of the measured glucose value and the real glucose value between the microneedle patch and the competitor Abbott Free Style Libre 3 (Abbott millimeter needle patch) at different amounts of glucose added.
  • Figure 8 The relationship between the value of the enrichment charge on the working electrode and the time when the circuit is disconnected.
  • Fig. 9 Relationship curves of the saturation charge value of the microneedle patch loaded with different glucose oxidases versus glucose concentration.
  • Figure 10 Relationship curves of saturation charge-off time at different glucose concentrations.
  • Figure 11 The graph of the relationship between the enrichment saturation electricity value and the concentration of glucose solution at different temperatures.
  • Fig. 12 The graph of the relationship between the saturation electricity value and the glucose concentration of the microneedle patch loaded with 10wt% glucose oxidase.
  • Figure 13 Linear fitting diagram of the relationship between the saturated electric quantity value of the microneedle patch and the glucose concentration with 10wt% glucose oxidase loading.
  • Figure 14 The relationship between the saturated electricity value enriched on the working electrode and the glucose concentration in different days
  • Figure 15 Microneedle patch with a film applied.
  • Figure 16 Curves of current-time relationship when the microneedle patch tests the blood sugar level of rabbits.
  • Figure 17 The blood glucose value-time relationship curve of the rabbit when the Roche blood glucose meter tests different glucose injection volumes.
  • Figure 18 The relationship curve of microneedle patch and Roche blood glucose meter testing rabbit blood glucose value-time.
  • Figure 19 The relationship between microneedle patch and Roche blood glucose meter for 3 consecutive days testing rabbit blood glucose value-time.
  • the present invention describes a microneedle patch for continuous monitoring or detection of analytes in the body, including at least one first transdermal microneedle standing on the skin-adhering surface of the patch substrate layer, and one end of the microneedle away from the patch substrate layer It has a sharp shape; an electrochemical reaction cell is integrated in the first transdermal microneedle, and the analyte electrochemically reacts or interacts with the detection components contained in the matrix of the first transdermal microneedle in the electrochemical reaction cell to form
  • the electrical signal reflects the concentration level of the analyte by detecting the magnitude of the electrical signal.
  • An electrochemical reaction cell is integrated in the first transdermal microneedle of the present invention.
  • the electrochemical reaction cell of the present invention has elements of an electrochemical reaction.
  • the electrochemical reaction cell includes a participating The working electrode that reacts with the analyte, and the enzyme or other active components that catalyze the reaction, the enzyme or other active components are referred to as detection components in this technical solution.
  • the first transdermal microneedle When it is necessary to detect the analytes in the body, the first transdermal microneedle can be directly inserted into the body fluid of the skin, and the analytes in the body An electrical signal is formed in the chemical reaction cell, and the concentration level of the analyte is determined based on the quantitative relationship between the electrical signal and the concentration of the analyte.
  • the first transdermal microneedle can directly insert the electrochemical reaction cell into the skin without resorting to a complicated insertion device. Among them, the complexity of the monitoring device is simplified, and the production cost of the monitoring device is reduced.
  • the first transdermal microneedle includes a first transdermal microneedle matrix. The microneedle shallow-insertion attachment process has no pain, which reduces the patient's resistance.
  • the sharp guides and relatively expensive disposable ejection attachment devices used in the market's minimally invasive blood glucose monitoring attachment devices are no longer needed.
  • the duration of attachment is not limited by cost, but can be optimized according to the living habits of users.
  • the low cost of use makes it possible for microneedle patches for continuous monitoring or detection of analytes in the body to be widely used in diabetic patients.
  • the microneedle with a sharp shape at the end away from the patch substrate layer can include but not limited to one or more of conical, bipyramidal, and pyramidal shapes.
  • the double-cone combination shape can be a double-cone combination with a tip, or a cone-pyramid combination with a tip.
  • the matrix of the first transdermal microneedle has a network structure that swells and does not dissolve after absorbing water, and the network structure allows the analyte to diffuse into or move away from the first transdermal microneedle. matrix, and prevent the detection components in it from moving away.
  • the first transdermal microneedle matrix When the first transdermal microneedle matrix is inserted into the skin, it penetrates the epidermis of the skin to reach the dermis, and absorbs the body fluid in the dermis to form a network structure that swells and does not dissolve. Through the network structure of the first transdermal microneedle matrix Immobilization of detection components and free diffusion of analytes into and out of the assay are achieved. The analyte diffuses into the first transdermal microneedle matrix, and a detectable electrical signal is generated in the electrochemical reaction cell, and the concentration of the analyte is determined according to the quantitative relationship between the electrical signal and the analyte.
  • the first transdermal microneedle matrix allows the analyte to diffuse into or move away, while preventing the detection component from moving away, so that the analyte and the detection component can be effectively contacted when the first transdermal microneedle is in the internal environment of the analyte, And it can ensure that the detection components exist in the first transdermal microneedle stably for a long time, prolonging the service time of the electrochemical reaction pool, and realizing guarantee for continuous monitoring.
  • the second transdermal microneedle also includes at least one second transdermal microneedle standing on the skin surface of the patch substrate layer, the second transdermal microneedle does not contain detection components, and the electrochemical reaction in the first transdermal microneedle
  • the reaction pool includes a working electrode
  • the second transdermal microneedle includes a reference electrode
  • the reference electrode in the second transdermal microneedle is connected with the working electrode in the first transdermal microneedle to form a measurement circuit.
  • the second transdermal microneedle includes a second transdermal microneedle matrix and a reference electrode located in the second transdermal microneedle matrix.
  • the structure and material of the second transdermal microneedle matrix are the same as those of the first transdermal microneedle matrix or similar.
  • the detection component interacts with the analyte in the body fluid to generate an electrical signal that can be detected, such as the enriched electrons or positive charges or electricity on the working electrode; or the analyte interacts with the detection component and the working electrode in the electrochemical reaction cell.
  • the electrons or electricity enriched on the working electrode of the electrochemical reaction cell are converted into detectable electrical signals such as current, electricity, or electromotive force by forming a loop with the reference electrode.
  • the number of the first transdermal microneedles can be 2, 3, 4 or even more, and the number of the second transdermal microneedles can be 2, 3, 4
  • the setting of multiple first transdermal microneedles can not only increase the strength of the electrical signal, but also increase the stability of the microneedle patch, and the setting of multiple second transdermal microneedles can improve the strength of the electrical signal. stability.
  • two first transdermal microneedles and one second transdermal microneedle are taken as examples for illustration, and three microneedles can form a relatively stable structure.
  • the detection mechanism of the microneedle patch is described.
  • the detection component as glucose oxidase as an example
  • the process of electrochemical reaction and electrical signal generation is described.
  • the first transdermal microneedle with the working electrode and the glucose oxidase immobilized and the second transdermal microneedle with the reference electrode pass through the epidermis of the skin to reach the body fluid in the dermis.
  • the first transdermal microneedle and the second transdermal microneedle absorb the water in the body fluid to swell, and the glucose in the body fluid diffuses into the first transdermal microneedle to undergo an electrochemical reaction as shown in Figure 2.
  • glucose is adsorbed on the catalytic site of glucose oxidase and reacts to form gluconolactone, which is then hydrolyzed into gluconic acid; while the catalytic site of glucose oxidase is converted into a reduced state, and the reduced glucose oxidase is released in body fluids.
  • oxygen in the electrode Under the action of oxygen in the electrode, it is re-oxidized to a catalytically active oxidation state, and a molecule of hydrogen peroxide is released at the same time; hydrogen peroxide loses two electrons on the surface of the working electrode to generate a molecule of oxygen and two hydrogen ions.
  • Oxygen participates in the conversion of glucose oxidase, hydrogen ions become the constituents of gluconic acid, and the products and reactants realize local circulation.
  • the electrons are absorbed by the working electrode in the first transdermal microneedle. Since the electrochemical reaction has occurred on the working electrode but no electrochemical reaction has occurred on the reference electrode, the working environment of the working electrode and the reference electrode are different, and the electrode potential is different.
  • a potential difference is generated, and the working electrode and the reference electrode are electrically connected to form a circuit to generate a current, and the concentration level of the analyte is estimated through the quantitative relationship between the current and the analyte.
  • the glucose on the left side of the reaction formula is oxidized and loses electrons, and the working electrode on the right side gains electrons, while the oxidation state and reduction state of glucose oxidase, as well as hydrogen peroxide and oxygen are all in the The reaction system is internally circulated, so the supply of oxygen will not be a factor affecting the reaction rate.
  • the detection components are selected from enzymes, hormones, antibodies, DNA, A mixture of one or more of RNA and modified enzymes.
  • Enumerable enzymes include, but are not limited to, one or more of glucose oxidase, catalase, amylase, creatine kinase, lactate oxidase, and cholesterol oxygenase.
  • Exemplary hormones include, but are not limited to, growth hormone and/or thyroid stimulating hormone.
  • the enzyme that can be enumerated is glucose oxidase modified by nano gold.
  • the modification method of the glucose oxidase modified by nano-gold in this scheme can be: the nano-gold particle is fixed on the working electrode through thiol, then the coenzyme (FAD) of glucose oxidase is taken out and fixed on the nano-gold particle Finally, the glucose oxidase from which the coenzyme has been removed is recombined with the coenzyme-gold nanoparticle composition, and the active center of the modified glucose oxidase is directly connected to the gold nanoparticle and then connected to the working electrode to improve the electron transfer efficiency.
  • FAD coenzyme
  • the nano-gold modified glucose oxidase catalyzes glucose, it oxidizes the glucose into gluconolactone and turns itself into a reduced state, and the reduced-state gold nano-modified glucose oxidase is oxidized on the working electrode and becomes an oxidized state again. And form two molecules of hydrogen ions and two electrons, and the generated electrons are detected by the working electrode to form corresponding electrical signals.
  • the detection component is glucose oxidase.
  • the electrical signal generated in the measurement circuit includes an electrical quantity that is quantitatively related to the chemical balance and a current value that is quantitatively related to the electrochemical reaction rate.
  • the reaction rate has nothing to do with the load of glucose oxidase, but has a first-order relationship with the glucose concentration. Therefore, the entire reaction rate, that is, the rate of electron generation, is also linearly related to the magnitude of the current and the glucose concentration, and becomes a constant current when the current is stable.
  • the glucose concentration in the body fluid can be obtained by generating a constant current between the working electrode and the reference electrode.
  • the glucose concentration in the body fluid is similar to the glucose concentration in the blood, so the blood glucose concentration can be known.
  • the above detection mechanism and the following electrical signal measurement methods are also applicable to the rest of the detection components.
  • the detection component is glucose oxidase
  • the following two electrical signal measurement methods are described in detail:
  • the constant current in the continuous circuit can be measured to reflect the glucose concentration in the body fluid, and then reflect the blood sugar level.
  • the current in the measurement circuit can be continuously recorded, read and processed to realize real-time monitoring of the blood glucose level.
  • the collection of blood sugar data can also be done intermittently, for example, the interval can be controlled at 5-10 minutes, and the continuous multiple intermittent blood sugar values can reflect the change trend of blood sugar, so as to realize the continuous blood sugar value monitoring, and intermittent data Acquisition can reduce data memory storage requirements.
  • the glucose concentration can be obtained under the condition of known constant current through the empirical formula between constant current and glucose concentration.
  • the electricity can be used to reflect the concentration of glucose.
  • the power is measured, it is expressed as the integral of the instantaneously changing current to time, that is, when the disconnected circuit is connected, although the current in the discharge process decays with time, the absolute value of the instantaneous current is far greater than that when the circuit is continuously connected. constant current. Therefore, when the current signal is weak, the power in the disconnected circuit can be recorded, read and data processed intermittently, so as to realize the continuous monitoring of the blood sugar level.
  • the glucose concentration can be obtained under the condition of known electric quantity through the empirical formula between electric quantity and glucose concentration.
  • the electricity value or current value in the circuit is measured to accurately calculate the blood glucose concentration, which can better ensure the accuracy of the blood glucose concentration measurement value and ensure the microneedle patch. practicality.
  • the detection component is immobilized on the working electrode.
  • the detection component is fixed or encapsulated in the first transdermal microneedle matrix.
  • the detection components in the electrochemical reaction cell can be fixed by coating on the working electrode in the electrochemical reaction cell, or more preferably, the detection components are mixed with the matrix material of the first transdermal microneedle and integrally formed in the microneedle . Different detection components have different fixed amounts in the microneedle patch.
  • an optional method is coating the detection component on the working electrode, and then embedding the working electrode coated with the detection component in the first transdermal microneedle matrix.
  • the selective physical encapsulation of the detection components by the first transdermal microneedle matrix is realized through post-processing such as freezing-thawing cross-linking after microneedle casting or 3D printing.
  • the fixing methods of the detection components in the existing electrochemical reaction cell include the following: (1) adsorption The adsorption method achieves the purpose of immobilization through the interaction between the carrier surface and the secondary bond on the detection surface, but the binding force between the detection component and the carrier is weak, and it is easy to fall off and lose; (2) the embedding method, the embedding method is the carrier After mixing with the solution of the detection component, the polymerization reaction is carried out with the help of the initiator, and the detection component is limited in the network of the carrier by physical action, but the size of the carrier network is difficult to control, and it is easy to cause leakage of the detection component, and the initiator needs to pass through ultraviolet rays.
  • cross-linking method is to use a bifunctional or multifunctional reagent to perform a cross-linking reaction between the detection molecules, between the detection molecule and the carrier, and the detection molecule
  • the method of cross-linking each other to form a network structure, the commonly used cross-linking agent is glutaraldehyde, but this method easily leads to partial inactivation of the enzyme.
  • the detection components of enzymes will gradually be hydrolyzed and inactivated in body fluids.
  • the content of the detection components is not enough to maintain the reaction rate and the analyte concentration is one order, the measured value of the analyte concentration level will deviate. Therefore, when making a microneedle patch, the effective life of the patch can be regulated by the load of the detection component in the microneedle, and the load of the detection component is adjusted according to the concentration level of the analyte.
  • the detection component is fixed in the first transdermal microneedle matrix, and the fixed amount of the detection component is determined according to the actual range concentration of the analyte.
  • fixed amounts such as 1wt%, 2wt%, 3wt%, 4wt%, 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, 10wt%, 11wt%, 12wt%, 13wt%, 14wt%, 15wt% , 16wt%, 17wt%, 18wt%, 19wt%, 20wt%
  • the content ratio is the ratio of the detection component weight to the matrix weight and detection component weight sum of the first transdermal microneedle, and the content ratio can be even higher .
  • the length of the first transdermal microneedle is in the range of 600-1500 ⁇ m, such as in the range of 700-1300 ⁇ m, 750-1250 ⁇ m, 800-1200 ⁇ m, 850-1100 ⁇ m, and the value is, for example, 900 ⁇ m, 950 ⁇ m, 1000 ⁇ m, 1050 ⁇ m, 1150 ⁇ m, 1250 ⁇ m, etc.
  • the length of the first transdermal microneedle includes but is not limited to the above numerical ranges and points.
  • the diameter at the largest cross-sectional area of the first transdermal microneedle is in the range of 200-700 ⁇ m, for example, in the range of 250-600 ⁇ m, 300-550 ⁇ m, 350-500 ⁇ m, and the value is, for example, 400 ⁇ m, 650 ⁇ m, etc., the first transdermal microneedle
  • the diameter at the maximum cross-sectional area of the dermal microneedles includes, but is not limited to, the above numerical ranges and points.
  • the length of the first transdermal microneedle of the present invention is in the range of 600-1500 ⁇ m. After piercing the epidermis with a thickness of about 75-150 ⁇ m, it reaches the dermis with a thickness of about 1-2 mm. Since the length of the first transdermal microneedle does not touch Or less contact with the nerves, the puncture of the epidermis by the first transdermal microneedle basically does not cause pain. Through the design of the length and diameter of the first transdermal microneedle, the epidermis of the first transdermal microneedle also belongs to reversible deformation. chemical reaction pool Inserted into the skin, greatly reducing the pain and resistance of the patient to be tested.
  • one end of the working electrode in the electrochemical reaction cell is inserted into the first transdermal microneedle matrix, and the other end extends out of the patch to form the first electrical contact.
  • the operation of the microneedle patch is more convenient, faster and simpler when it is connected with an external electrical signal collection device.
  • one end of the reference electrode is inserted into the second transdermal microneedle matrix, and the other end extends out of the patch to form a second electrical contact.
  • the operation of the microneedle patch is also more convenient, faster and simpler when it is connected to an external electrical signal collection device.
  • the working electrode in the electrochemical reaction cell at least includes an inert conductor embedded in the first transdermal microneedle matrix.
  • the inert conductor in the present invention refers to a conductor that does not dissolve in an electrochemical reaction.
  • the inert conductor is selected from one or a combination of platinum, gold, copper, silver and carbon materials.
  • the working electrode further includes a terminal post that is electrically connected to the inert conductor and transmits the electrical signal formed on the inert conductor to the outside of the first transdermal microneedle.
  • the inert conductor is a platinum wire
  • the terminal is a copper rod
  • one end of the platinum wire is inserted into the copper rod to form a working electrode
  • the copper rod is far away from the tip of the first transdermal microneedle
  • One end of the chip extends out of the chip substrate layer to form a first electrical contact.
  • the diameter of the platinum wire is 0.1-0.18 mm, and the diameter of the copper rod is 0.8-1.4 mm.
  • the composition of the reference electrode may be the same as or different from that of the working electrode.
  • the material of the reference electrode includes, but is not limited to, one or a combination of Ag/AgCl, platinum, gold, copper, silver, and carbon materials.
  • the working electrode Due to the small size of the microneedle, the working electrode is smaller, and the operation is more complicated when the working electrode is connected to the external detection equipment.
  • the introduction of the terminal post improves the convenience of the connection between the working electrode and the external detection equipment and the efficiency of electrical signal transmission. Stability, when the binding post is set on the reference electrode, it also improves the convenience of connecting the reference electrode and external detection equipment.
  • the preparation raw material of the first transdermal microneedle matrix is hydrophilic polymer.
  • the first transdermal microneedle matrix made of hydrophilic polymer materials has better biocompatibility in the skin, and can better maintain the activity of the detection components immobilized therein, prolonging the time of detection components in the first transdermal microneedle matrix.
  • the survival time in the needle matrix realizes the continuous and accurate monitoring of blood glucose in body fluids by microneedles.
  • the raw material for the preparation of the first transdermal microneedle matrix includes at least polyvinyl alcohol as the main material.
  • the inventor considers using polymer materials with better bio-affinity to make microneedles, but polyvinyl alcohol needs to form a gel with a network structure to achieve the fixation of the detection components and enable the analyte to diffuse into its interior, and the polymer Vinyl alcohol dissolves in body fluids.
  • the way to make polyvinyl alcohol form a network structure gel is divided into chemical cross-linking method and physical cross-linking method.
  • the chemical bond realizes cross-linking, while the physical cross-linking method is divided into ionic cross-linking and microcrystalline construction. Microcrystals are formed inside to achieve cross-linking.
  • the inventor believes that the use of chemical cross-linking needs to add a cross-linking agent or initiator to the polyvinyl alcohol, and most of the cross-linking agents or initiators are highly toxic and harmful to the human body, and ultraviolet rays will affect the detection of ingredients. Therefore, the inventors cannot use chemical cross-linking based on the above considerations. Ionic cross-linking needs to make polyvinyl alcohol charged, and it also needs to occur in a multivalent counter-ion solution, and this cross-linking method is not suitable for the human body.
  • polyvinyl alcohol can have a network structure that swells but does not dissolve after absorbing body fluids through the freeze-thaw process, and is in a hard glass state in a dry state, with the hardness to penetrate the skin.
  • the inventors also found that the network structure achieved by the freeze-thaw process, compared to the cross-linking of the network structure by UV curing, is safe for the active detection components, and the material is implanted under this process.
  • the human body is also safe and will not affect the activity of the detection components, which improves the effective utilization of the detection components, provides a reliable guarantee for the accuracy of the detection process, and ensures the practicability of the microneedle patch.
  • the microneedle patch in the present invention has simple structure, high detection accuracy, and can continuously monitor analytes with a long service time in the body, so that the microneedle patch has industrialization prospects in the field of continuous and accurate monitoring of analytes .
  • the inventors have found that the chain segments of polyvinyl alcohol can form ordered micro-regions called microcrystals or microcrystalline domains through freezing-thawing treatment, and microcrystals or microcrystalline domains will not separate when they are in a thawed state, thereby After absorbing water, it can form a network structure that swells and does not dissolve, which improves the hardness of the microneedle when it is dry.
  • Embedding the active detection components in polyvinyl alcohol with a network structure will not affect the activity of the detection components, and the physical process of freezing and thawing will not affect the activity of the detection components, so that the detection components will undergo catalytic reactions. It can maintain the natural conformation and improve the effective utilization rate of the detected components. pass The times of freezing-thawing treatment are increased to increase the density of the microcrystalline domain, so that the cross-linking density of the first transdermal microneedle matrix can be adjusted.
  • polyvinyl alcohol is a chemically inert pharmaceutical excipient with good biocompatibility, and has even been used as a material for soft contact lenses. When microneedles are inserted into the skin, it can reduce inflammation and rejection in body tissues reaction.
  • the optional range of freezing temperature in the present invention is from -5°C to -25°C, or from -10°C to -20°C.
  • the thawing temperature described in the present invention can be regarded as at room temperature.
  • the polyvinyl alcohol is completely alcoholyzed polyvinyl alcohol and/or a mixture of completely alcoholyzed polyvinyl alcohol and partially alcoholyzed polyvinyl alcohol.
  • the complete alcoholysis of PVA has a degree of alcoholysis of 99% ⁇ 2%, the degree of alcoholysis of the partially alcoholyzed PVA is at 88% ⁇ 2%, and the degree of alcoholysis of a small amount of alcoholyzed PVA is at 78% ⁇ 2%. %.
  • microneedles made of completely alcoholyzed polyvinyl alcohol will have problems such as excessive microneedle deformation and uncontrolled shape during dehydration, drying and shrinkage.
  • excessive microneedle shrinkage leads to too short microneedles , too thin or bent, which makes it difficult for the electrode to be inserted into the microneedle or the formed microneedle is difficult to penetrate the skin.
  • the inventor unexpectedly found that the use of partial alcoholysis and complete alcoholysis of PVA for mixing can effectively improve the deformation of the microneedles, and the microneedles can penetrate the skin better, and the use of a small amount of alcoholysis and complete alcoholysis of PVA can also be mixed.
  • the microneedle It can improve the deformation problem of the microneedle, but it will reduce the ease of the microneedle penetrating the skin, and it is more difficult for the microneedle to penetrate the skin.
  • the inventor believes that the possible reason is that by adding partially alcoholysed PVA, the amount of hydrogen bonds in the microneedles can be reduced, which can reduce the problem of excessive internal stress caused by hydrogen bond interactions during the drying process of the microneedles, and reduce the amount of microneedles.
  • the deformation rate of the needle matrix keeps the microneedles in good shape, while too little hydrogen bond affects the crosslinking degree of the microneedle matrix, which reduces the crosslinking density and hardness of the microneedle matrix.
  • the weight average molecular weight of polyvinyl alcohol commonly used in this field is in the range of 10KDa-130KDa.
  • the polyvinyl alcohol when preparing the matrix of the first transdermal microneedle, is configured as a polyvinyl alcohol solution, and the concentration of the polyvinyl alcohol solution is in the range of 18wt%-40wt%.
  • the concentration of polyvinyl alcohol solution is, for example, 20wt%, 22wt%, 25wt%, 27wt%, 29wt%, 30wt%, 32wt%, 34wt%, 36wt%, 38wt%, the concentration of polyvinyl alcohol includes but not limited to the above numerical points .
  • the raw materials for the preparation of the first transdermal microneedle matrix may also include auxiliary materials selected from the group consisting of dextran, chitosan, alginate, hyaluronic acid, sodium hyaluronate , sodium carboxymethylcellulose, polyethylene glycol or a mixture of several.
  • the high The molecules are configured as an auxiliary polymer solution, and the concentration of the auxiliary polymer solution is in the range of 5wt%-40wt%, such as 7wt%, 9wt%, 11wt%, 13wt%, 15wt%, 17wt%, 20wt%, 22wt% , 25wt%, 27wt%, 29wt%, 30wt%, 32wt%, 34wt%, 36wt%, 38wt%, etc.
  • the concentration of excipient polymers includes but not limited to the above numerical points.
  • the mass ratio between the polyvinyl alcohol solution and the polymer solution of the auxiliary material is between 3/1- 8/1 range.
  • the mass ratio between the polyvinyl alcohol solution and the polymer solution of the auxiliary material is, for example, a value of 4/1, 5/1, 6/1, 7/1 or a range between the above values.
  • the mass ratio between the polyvinyl alcohol solution and the polymer solution of the auxiliary material is 3/1-8/1, the hardness of the transdermal microneedle is better, the affinity to biomolecules is better, and it is easy to cast.
  • the material of the first transdermal microneedle matrix can form non-covalent cross-linking and realize the characteristics of water absorption swelling and insolubility, and can be added by adding other polymers such as dextran, chitosan, alginate, hyaluronic acid, Sodium hyaluronate, sodium carboxymethylcellulose, and polyethylene glycol regulate the crosslinking density, that is, the mesh size of the crosslinking structure and the swelling property of the microneedles, and optimize the working environment of the detection components.
  • These materials are not only compatible with PVA, but also friendly to biomolecules, providing a good immobilization environment for active detection molecules.
  • a device for processing electrical signals generated by a microneedle patch for continuous monitoring or detection of internal analytes at least comprising a signal processor, the signal processor being compatible with the aforementioned microneedle patch for continuous monitoring or detection of internal analytes
  • the resulting measurement loop is electrically connected, and the electrical signal in the measurement loop is monitored and converted into a digital signal related to the level of the analyte.
  • a device for continuously monitoring or detecting analytes in the body comprising at least the microneedle patch as described above, the microneedle patch is bonded with an adhesive layer that adheres to the skin;
  • a signal processor electrically connected to the measurement circuit between the first transdermal microneedle and the second transdermal microneedle, monitoring the electrical signal in the measurement circuit and converting the electrical signal into an analyte level-related Digital signal.
  • the signal processor in this technical solution is input with a quantitative relationship between the electrical signal and the level of the analyte, and the electrical signal in the detection circuit of the signal processor can be converted into a digital signal related to the level of the analyte, which can realize the above-mentioned functions.
  • Any processor can be used as an embodiment of the technical solution, and the signal processor that can be listed includes an A/D converter.
  • the device for continuously detecting analytes in the body further includes a connection plate, and plug-in components are arranged on both sides of the connection plate, and the microneedle patch is plugged in on one side of the connection plate.
  • the components are detachably connected to the connection board, and the signal processor is connected with the plug-in components on the other side of the connection board.
  • the connection board is detachably connected, and the measurement circuit formed by the signal processor and the microneedle patch is electrically connected through the connection board.
  • the plug-in element in the present invention is an original element that can be inserted and removed detachably, and there are elements with plug-in functions such as elastic clips and plug joints that can be enumerated.
  • the microneedle patch and the connection plate are set to be detachably connected. When the microneedle patch needs to be replaced, the microneedle patch is directly removed from the connection plate and replaced with a new microneedle patch to form a new continuous
  • a device for detecting analytes in the body, and the signal processor on the connection board can be reused many times.
  • the signal processor is detachably connected to the connection board. When a fault occurs and needs to be replaced, it can be directly removed and replaced with a new device without affecting the use of other devices.
  • the microneedle patch and related signal reading device of the present invention can be worn on the body to realize continuous monitoring or detection of the content or concentration of biological substances in the body.
  • a microneedle patch method for continuous monitoring or detection of in vivo analytes of the present invention at least includes the following steps:
  • Step 1 casting the matrix forming preparation liquid of the first transdermal microneedle and the matrix forming preparation liquid of the second transdermal microneedle into casting molds with sharp ends respectively, and casting the matrix forming preparation liquid of the first transdermal microneedle
  • the matrix forming preparation solution of the second transdermal microneedle is cast on the microneedle hole for the second transdermal microneedle forming on the casting mold
  • the casting mold is placed in the running suction device, and the molding preparation solution of the microneedle matrix is sucked by the suction device Suction into microneedle holes;
  • Step 2 freezing and thawing the matrix forming preparation solution of the first transdermal microneedle and the matrix forming preparation solution of the second transdermal microneedle in the casting mold;
  • Step 3 Insert the working electrode and the reference electrode into the casted first transdermal microneedle and the second transdermal microneedle, the ends of which protrude from the surface of the molding preparation solution;
  • Step 4 Repeat Step 2 several times.
  • Step 5 The preparation of the microneedle patch is dried at room temperature to shrink, and then dried until completely cured.
  • the electrochemical reaction when the detection component is glucose oxidase is taken as an example, and the detection principle when the first transdermal microneedle and the second transdermal microneedle constitute a measurement circuit are explained in detail.
  • the detection principle is also applicable to the detection components that undergo electrochemical reactions.
  • a method for preparing a microneedle patch for continuously detecting an analyte in the body comprising the following steps:
  • the first transdermal microneedle and the second transdermal microneedle are conical with a tip, and the casting mold of the breathable polymer material is placed on the running suction device, and the breathable
  • the polymer casting mold has microneedle holes for casting microneedles, casting the casting solution of the first transdermal microneedle and the casting solution of the second transdermal microneedle on different microneedle holes of the mold, and passing The suction device sucks the casting solution into the microneedle hole, and when the microneedle hole is filled with the casting liquid, stop the suction device, then place an auxiliary mold on the casting mold to prevent the loss of the chip substrate layer solution, and Cast the casting solution of the patch substrate layer in the auxiliary mold, the casting mold has 21 microneedle holes, and every three microneedle holes form a microneedle patch for continuous monitoring or detection of analytes in the body , one of the microneedle holes is the second transdermal
  • Insertion of electrodes Insert the working electrode with the platinum wire into the copper rod into the center of the first transdermal microneedle, and insert the reference electrode with the platinum wire into the copper rod into the center of the second transdermal microneedle, wherein the platinum wire is inserted into the center of the second transdermal microneedle.
  • the diameter is 0.15mm, the length is 1.1mm, the length in the microneedle is 0.7mm, the length in the copper rod is 0.4mm, the diameter of the copper rod is 1.2mm, and the length of the copper rod needs to protrude from the patch substrate layer ;
  • Re-freezing-thawing treatment put the microneedle connection mold inserted into the electrode into the -20°C refrigerator for 21 hours, then take it out and thaw it at room temperature for 3.5 hours, and repeat the freezing and thawing 3 times;
  • Glucose solutions of different concentrations are prepared by dissolving the required amount of glucose in a pH of 7.0 Prepared in PBS solution, where PBS solution was purchased from ThermoFisher, and glucose was purchased from Sigma.
  • microneedle patches used in the following examples are all based on the examples of the above-mentioned preparation process, and are specifically adjusted according to the required content of glucose oxidase, wherein the proportion of glucose oxidase is: the weight of glucose oxidase accounts for the first A ratio of the weight of the transdermal microneedle matrix to the weight of glucose oxidase.
  • hyperglycemia means that the glucose concentration is greater than 7mmol/L
  • normal blood sugar means that the glucose concentration is between 3.9-7mmol/L
  • hypoglycemia means that the glucose concentration is less than 3.9mmol/L.
  • the range of glucose concentration is 1-36mmol/L, which can meet the variation range of glucose concentration in actual human test.
  • a detection device as shown in Figure 1 which includes two first transdermal microneedles 1, a second transdermal microneedle 6, all inserted
  • the working electrode of the first transdermal microneedle 1, the working electrode is formed by inserting a platinum wire 2 into a copper rod 3, wherein the copper rod 3 extends out of the chip substrate layer 4 to form the first electrical contact, and the side wall of the copper rod 3
  • a groove 12 is provided on the top, and the groove 12 is snapped into the patch 4 to improve the bonding strength between the copper rod 12 and the patch 4 .
  • the first transdermal microneedle 1 contains glucose oxidase
  • the second transdermal microneedle 6 is inserted with a reference electrode.
  • the reference electrode is also formed by inserting a platinum wire into a copper rod, wherein the copper rod protrudes from the patch 4 to form a second electrode.
  • two parallel first electric contact points and second electric contact points are electrically connected to form a measurement circuit
  • the nanoampere meter 5 is electrically connected to the circuit to measure the current in the circuit, and the nanoampere meter can be selected Keysight digital multimeter.
  • the microneedle patch in the detection device was inserted into the 0.5 mm thick polyvinyl alcohol electrospun fiber simulated dermis layer, wherein the polyvinyl alcohol electrospun fiber simulated dermis layer was placed on a carrier On the transdermal pool with solutions of different glucose concentrations.
  • the polyvinyl alcohol electrospun fiber contacts and absorbs the glucose solution, allowing the first transdermal microneedle and the second transdermal microneedle inserted therein to swell.
  • Glucose in the solution quickly diffuses into the first transdermal microneedle and the second transdermal microneedle that are swollen into a hydrogel state, and the glucose that enters the first transdermal microneedle is catalyzed and oxidized by the glucose oxidase contained therein to generate After gluconolactone, it is hydrolyzed into gluconic acid; while the catalytic site of glucose oxidase is converted into a reduced state, and the reduced state of glucose oxidase is re-oxidized to a catalytically active oxidation state under the action of oxygen in body fluids.
  • the change value of the current with time after the measurement circuit is connected is investigated, and the microneedle patch with the content of glucose oxidase in the first transdermal microneedle is 10wt%, and the configuration concentration is The 12mM glucose solution was reacted at 37°C for 4 minutes, then the measurement circuit was connected, and the current curve with time was measured through the microneedle patch, and the current-time curve shown in Figure 3 was obtained. It can be seen from Figure 3 that when the measurement circuit is not connected, the electrons in the electrochemical reaction are enriched on the surface of the working electrode and are not removed, forming a chemical potential that drives the reverse reaction.
  • the electrons enriched on the surface of the working electrode are quickly moved to the reference electrode, forming a high instantaneous current of 158nA, and the current continues to decay to 35nA as time goes on, and remains constant at this value after 3s.
  • This constant current value can reflect Glucose concentration
  • the constant current value at any time point after 3s can be selected as the current value to reflect the glucose concentration.
  • a microneedle patch with a glucose oxidase content of 3 wt% in the first transdermal microneedle and a first transdermal microneedle were prepared respectively.
  • the glucose concentration value can be calculated with this empirical formula.
  • the microneedle patch with a glucose oxidase content of 10wt% still has a good linear relationship between the current and the glucose concentration in a larger glucose concentration.
  • the 6mm sensor needle is placed under the skin for 14 days; we can also attach it for 14 days and read data intermittently. But shortening the attachment time is more convenient for patients - such as coping with summer heat, sweating, showering, etc. Since the microneedle patch in the technical solution has a lower use cost, the microneedle patch can be replaced when the patient performs activities such as taking a shower. In order to investigate the time of accurate monitoring of glucose concentration by the microneedle patch loaded with 10wt% glucose oxidase.
  • the microneedle patch was continuously placed on the transdermal pool at 37°C for 14 days; the response of the current to the glucose concentration was measured on the 1st, 2nd, 5th, 8th, and 14th day, and the 8th time after the circuit was connected was taken.
  • the current value was a constant current value, and the relationship curve between current and glucose concentration was drawn for different days, and Figure 6 was obtained. It can be seen from Figure 6 that when the microneedle patch was continuously placed in the transdermal pool at 37°C, the constant current maintained an accurate linear relationship with the glucose concentration within 1 to 8 days, and when it reached 14 days, the measured value was somewhat low, but generally maintains a linear relationship.
  • Glucose concentration ranges from 3mM to 24mM, which is the most common range for diabetic patients.
  • Glucose solutions with concentrations of 3mM, 6mM, 9mM, 12mM, 18mM and 24mM were prepared respectively, and then the prepared glucose solutions were placed in a constant temperature water bath at 37°C to obtain Glucose solution at 37°C.
  • the in vitro transdermal drug release pools were placed in a 37°C water bath, and then glucose solutions of different concentrations at 37°C were added to different transdermal drug release pools.
  • the same in vitro experiment of glucose concentration detection was carried out using Abbott Free Style Libre3, a commercially available Abbott continuous monitoring blood glucose meter.
  • the transdermal drug release cell is placed in a constant temperature shallow water bath at 37°C, and then 3-24mM glucose solution at 37°C is added thereto.
  • 3-24mM glucose solution at 37°C is added thereto.
  • the sensor is installed on a disposable ejection device, and the ejection device is pressed to make the sensor probe instantly pierce the 0.5mm thick PVA fiber membrane of simulated dermal tissue.
  • Place the sensor probe with PVA fiber membrane on the transdermal drug release pool to react with the glucose solution, measure the glucose concentration by scanning the sensor with the scanning detector that comes with the Abbott continuous monitoring blood glucose meter, and record the glucose concentration after the value is stable.
  • the sensor with the PVA fiber membrane is taken out and placed in the glucose solution of the next concentration, and the measured glucose concentration value is recorded.
  • the microneedle patch with the glucose oxidase content of 3wt% in the first transdermal microneedle, the microneedle patch with the glucose oxidase content of 5wt% in the first transdermal microneedle, and the glucose oxidase in the first transdermal microneedle were used respectively.
  • Microneedle patch with oxidase content of 10wt% configure glucose solution with glucose concentration from 1mM to 36mM, and use microneedle patches with different glucose oxidase loadings to place in glucose solutions with different concentrations under the condition of 37°C
  • the reaction is carried out in the middle, connect the measurement circuit and integrate the area under the curve within 2s of the current release curve to obtain the value of the enriched electric quantity on the working electrode, change the circuit disconnection time, measure the enriched electric quantity on the electrode under different disconnection times, until the electrode When the enriched electricity on the battery reaches saturation, the relationship curve between the saturated electricity value and the glucose concentration is drawn, and Fig. 9 is obtained.
  • Example 8 The in vitro test in Example 8 was carried out at 25° C., because the human body temperature is around 37° C., and this example further investigated the influence of temperature on the enriched and saturated electric quantity on the working electrode. Under the conditions of 25°C and 37°C respectively, the microneedle patch with 10wt% glucose oxidase load was reacted with 3mM, 6mM, 12mM and 24mM glucose solutions, and the enrichment and saturation electricity value and the electricity enrichment saturation on the working electrode were measured. time, and draw the relationship curve between enrichment saturation electricity and glucose concentration at different temperatures, and Figure 11 is obtained. It can be seen from Fig. 11 that there is no significant difference (P>0.05) in the enrichment saturation electric quantity value on the working electrode measured at different temperatures.
  • an electrochemical reaction cell microneedle was carried out in a glucose solution at 37°C for 14 consecutive days using the coulometric method. Glucose concentration detection in vitro experiments. The microneedle patches loaded with 10wt% glucose oxidase were reacted with 3mM, 9mM, 18mM and 30mM glucose solutions respectively, and the microneedle patches were continuously placed on the transdermal pool with different glucose concentrations for 14 days. On the 2nd day, the 5th day, the 8th day and the 14th day, the enrichment saturation electric quantity value on the working electrode was tested.
  • the intermittent galometric method can be used to solve the problem by prolonging the time of the electricity enrichment.
  • the glucose solution will evaporate and decrease as the experiment progresses, and the corresponding concentration of glucose solution should be replenished in real time during the experiment.
  • microneedle patch with 6.1wt% glucose oxidase loading was applied to living rabbits for in vivo testing.
  • the specific implementation process and experimental results are as follows:
  • Glucose was purchased from: Sigma; multimeter and software: keysight; blood glucose meter: Roche, Luokang Quanqi; test paper: Roche; medical sterile cotton balls: Oujie; blood collection needle: Roche; hair removal cream: Veet.
  • FIG. 15 is an exemplary embodiment, wherein the application film 9 is pasted to the substrate layer of the microneedle patch 7, leaking out the first electrical contact 8 and The second electrical contact is for electrical connection with external devices.
  • the blood glucose monitoring microneedle patch After the blood glucose monitoring microneedle patch reacts with the glucose in the body fluid for 4 minutes, turn on the "Start" button of the keysight software, and the multimeter starts to collect data.
  • the working electrode is reconnected, measure for 40s, press the "stop” button, and export the data;.
  • a Roche blood glucose meter to measure blood sugar by taking blood from the ear vein on the opposite ear to the ear with the microneedle patch.
  • the method of using a Roche blood glucose meter to measure blood sugar Disinfect the rabbit's ear with 75% alcohol cotton, The special blood collection needle punctures the skin and squeezes the bleeding.
  • the blood sugar level rose rapidly and returned to normal blood sugar level within 2 hours.
  • the blood sugar value measured by the microneedle patch is slightly lower than that obtained by blood collection. The concentration of glucose reacts later than blood glucose.
  • the rabbit blood glucose monitoring results for 3 consecutive days show that the microneedle patch has a good correlation with the blood glucose value measured by the commercially available Roche blood glucose meter. It is proved that the blood glucose monitoring microneedle patch can continuously monitor blood glucose, and the results are stable and reliable.
  • the variance of the blood glucose values measured by the electrochemical blood glucose monitoring microneedle patch and the blood glucose meter was first compared using the two-sample variance F-test. If the p value is less than 0.05, it means that there is a significant difference in the variance of the blood glucose values measured between the two groups, and a two-sample heteroscedasticity t-test is required for testing; if the p value is greater than 0.05, it means that the blood glucose values measured between the two groups There was no significant difference in variance, and a two-sample equal variance t-test was used for testing.
  • the obtained two-sided test p>0.05, it means that there is no significant difference in the blood sugar values of the two groups compared with each other; if 0.01 ⁇ p ⁇ 0.05, it means that there is a significant difference in the blood sugar values measured between the two groups sexual difference; if p ⁇ 0.01, it means that there is a very significant difference in the measured blood glucose values between the two groups.
  • Table 2 shows the blood glucose value and t-test results measured by the microneedle patch and the blood glucose meter after the fasting injection of glucose in the healthy rabbits on the first day
  • Table 3 shows the microneedle patch and the blood glucose of the healthy rabbits after the fasting injection of glucose on the second day
  • Table 4 shows the blood glucose value and t test results measured by the microneedle patch and the blood glucose meter after the healthy rabbits were injected with glucose on an empty stomach on the third day.
  • Glucose was injected into the ear vein of rabbits fasting for 12 hours to determine the changes in blood sugar, and the measurement was continued for 3 days. From the statistical results in Table 2, Table 3, and Table 4, it can be seen that there is no significant difference in the blood glucose measurement values between the microneedle patch and the blood glucose meter. Therefore, our microneedle patch for blood glucose monitoring can be used for continuous monitoring of blood glucose in vivo.
  • a rabbit was randomly selected, and the blood glucose monitoring microneedle patch was firmly attached to one ear of the rabbit, and the blood glucose value was measured after fasting for 12 hours every day, and the blood glucose was measured by Roche blood sampling instrument as the control group.
  • the results of continuous daily monitoring of fasting blood glucose in rabbits showed that the blood glucose monitoring microneedle patch can continuously monitor blood glucose for 14 days and the measured blood glucose value is stable. After 15 days, the blood glucose value measured by the blood glucose monitoring microneedle patch was significantly lower than that measured by the blood glucose meter.
  • the possible reason is that the glucose oxidase in the blood glucose monitoring microneedle patch has undergone long-term electrode action and Under the influence of physiological environment, the activity of partial oxidase will be invalidated. It can be seen that the microneedle patch used for monitoring or detecting analytes in the body of the present invention can exist stably and for a long time in a living body and can accurately measure related signals.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Medical Informatics (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Biophysics (AREA)
  • Pathology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Animal Behavior & Ethology (AREA)
  • Molecular Biology (AREA)
  • Surgery (AREA)
  • Optics & Photonics (AREA)
  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Emergency Medicine (AREA)
  • Measurement Of The Respiration, Hearing Ability, Form, And Blood Characteristics Of Living Organisms (AREA)

Abstract

本发明涉及一种不造成痛感与皮肤损伤便能连续监测或检测体内分析物如血糖含量的微针贴片，所述检测体内分析物的微针贴片至少包括立于贴片上的一个第一透皮微针，所述第一透皮微针中集成有电化学反应池，分析物与电化学反应池中的检测成份反应或作用形成电信号，通过检测电信号的大小反映分析物的浓度水平；涉及微针贴片的制备方法；还涉及包括所述微针贴片的检测装置，其将电信号转化为指示分析物的浓度水平的数字信号；所述微针贴片为可穿戴的，使用过程无痛、简易、经济。

Description

一种连续监测或检测体内分析物的微针贴片、其制备方法及相关装置 技术领域
本公开涉及一种不造成痛感与皮肤损伤便能连续监测或检测体内分析物如血糖含量的微针贴片、所述微针贴片的制备方法、及包括所述微针贴片的相关检测装置。
背景技术
人体的许多生物信息，如血糖、血脂等需要通过对血液或内分泌体液的检测得知，本发明中将能够反应上述生物信息的物质称为分析物，如血液中的葡萄糖的浓度。为保证检测到的分析物浓度的准确度和精确度，传统的分析物检测方法须要侵入人体皮肤，接触血液或至少接触内分泌体液，实现对分析物及其含量的测定，这种方法一般称为有创式检测方法或微创式检测方法。
有创式的检测方法可列举的如专利CN209979545U中公开的一种血糖检测试纸，该血糖检测试纸检测血糖时需要使用试纸上的采血针头在指尖上进行取血，再配合检测仪器检测血液中的血糖浓度。此类型的检测装置已经实现了产业化，使用价格也能被绝大多数糖尿病患者所接受，通过检测血液获得血糖值的精确数据，但与该技术方案类似的有创式的测试方法存在如下缺点：(1)这种方法会造成皮肤疼痛以及皮肤的损伤，容易使测试者产生抵触情绪；(2)测试者通常每天只能间歇测量两三次，以至于不能及时发现血糖值过高或过低的情况，无法根据血糖变化趋势来调整胰岛素或降糖药的使用量，反而可能增加低血糖症发生的可能性。
微创式的检测方法可列举的如大型跨国药企雅培糖尿病护理公司申请的中国专利CN102472719B，该专利要求了美国专利US12495798的优先权，公开了一种体内监测分析物的装置和方法，该装置包括有壳体、射频接收器、分析物传感器、存储器和信号处理器，其中分析物传感器的组成部件包括有工作电极和参考电极，涂敷于工作电极上的葡萄糖氧化酶，将葡萄糖氧化酶固定于工作电极上的半透膜，由于半透膜的材质较柔软分析物传感器难以植入皮肤中，需要借助装配有5-7mm的刚性尖端的插入装置将分析物传感器全部或部分的 植入皮肤中，并通过插入装置的弹射装置将刚性尖端从皮肤中移除。分析物传感器长期植入皮肤中，可进行连续准确的监测使用者体内的血糖浓度，解决了有创式检测方法只能间歇测量血糖值的问题，并且具有较高的检测精确度，基于该设计原理的连续监测血糖的装置如Abbott Free Style Libre系列也实现了产业化，但是过高的使用价格仅能被很少糖尿病患者所接受，不能在广大的糖尿病患者中普及。并且基于该检测机理的连续监控分析物水平的装置还存在如下缺点：(1)电化学传感器需要借助插入装置才能植入皮肤中，而插入装置结构复杂，仅供一次性使用，成本较高；(2)半透膜不能很好的包覆葡萄糖氧化酶，随着使用时间的延长葡萄糖氧化酶会逐渐泄露出来，降低检测信号；(3)5-7mm的刚性尖端刺入皮肤同样会给使用者带来异物感或不适感；(4)电化学传感器长时间置于皮下组织中会引起组织的炎症反应和排异反应，而较高的一次性使用成本则要求装置尽可能长时间滞留皮下。
美国德康医疗申请的专利EP3524150B1、EP3641636B1、US11166657B2、US10932709B2、US10932699B2公开的技术方案中以及美敦力医疗器械、微泰医疗、凯立特医疗等多家大型医疗器械公司商业化的连续监控分析物水平的装置均是基于该微创式的检测原理设计的，但是由于过高的使用价格以及使用舒适度方面存在问题，商业化的产品也未能在广大糖尿病患者中普及。
为避免痛感和损伤，科学界开展了无创式的检测和监测方法的研究，在研的无创检测和监测的方法大致分为两类：(1)用可穿透人体组织及组织液的非损伤性光源，如近红外等进行照射，从光源的漫反射或拉曼光谱推测目标成份的浓度；(2)采集外分泌体液，如眼泪、唾液、汗液、尿液等进行检测。上述方法存在着诸多局限，比如近红外虽然是各种光源中少有的可穿透组织和体液的频率，其本身却没有任何分子运动(转动、振动、能级跃迁等)与之对应，漫反射很难携带化学信息。近红外的拉曼效应虽然落入分子振动能级，具有拉曼活性的分子振动模式本来就不多，加之生物分子的振动结构相近，在无法对分析物进行体内原位提纯的条件下，分析物的拉曼光谱很难极难从混合谱中分辨出来——即使通过傅里叶变换。一些研究团队抛开对于特异性化学机制的依赖，尝试通过大数据比对实现近红外漫反射法对血糖水平及其变化的监测。但人体影响近红外漫反射的非特异性因素太多，科技界至今尚未得到精确而有规律的结果，不能通过近红外照射的方法连续的获取准确的血糖浓度数据，该方 法虽然能避免痛感和损伤，但不能准确监测血糖浓度，不具备实际使用前景和产业化前景。
上述方法的后者也不能准确测量分析物的浓度水平，原因在于其所采用的外分泌体液与血液及内分泌体液之间被组织膜隔离，目标成分的浓度与内分泌系统有着数量级的差别。换句话说，如此大的浓度差使得测量上的绝对误差带来数量级放大的相对误差。如此差别使得测量精度难以为一些疾病的药物治疗提供准确的参考；比如难以为血糖调控给出精准的胰岛素剂量。
如何才能连续的获得血糖或其他体内分析物的精准检测，而又不因针刺痛感让待测试者产生抵触感，给体内分析物指标监测领域带来了新的挑战。微针在刺入皮肤时不会产生刺痛感，科学界开始关注基于微针的分析物检测装置的研究。现有基于微针的分析物检测装置从检测方式上分为两类：第一类是利用微针的刺穿性在皮肤上形成微孔，通过微孔将内体液引出继而检测；第二类是将微针组装成生物传感器插入体内对分析物的浓度水平进行检测。
专利CN109199400B中公开了一种基于微针阵列的血糖电化学传感器，是基于上述第一类检测方式设计的，该传感器包括微针阵列电极、参比电极一、参比电极二以及储存有生物识别分子的导电电化学检测区。在使用时，微针阵列电极刺入皮肤，继而回弹、退出皮肤，通过微针阵列在皮肤上形成的微孔将体液引到导电电化学检测区的储存有生物识别分子的导电弹性水凝胶上，进行血糖的检测。该方法需要将体液引出再测量，会存在较长时间的物理延时，降低检测实时性；体液渗出过程中会出现掺杂汗液等干扰成分的情况，降低检测准确度；而且容易使体液外渗处皮肤起湿疹，产生炎症等问题。糖尿病患者在餐后容易出现血糖偏高的状况或胰岛素分泌失调患者为应对胰岛素分泌失调引起的血糖不稳定的状况都需要进行血糖的即时监测，而该方法由于需要将体液引出会存在监测延时，不适于即时监测，该方法更不适用于对于体内生物物质水平的连续监测或高频率检测连续，而监测或高频率检测对于精准药物治疗是必要的；如血糖水平——身体状态——胰岛素给药剂量及速率的相关性。
如何能够无创、无痛、即时连续的进行分析物监测或检测且进行监测或检测时与人体具有较好的亲和性，成为了科学研究新的挑战。
发明内容
发明人立足于现有监测或检测体内分析物的现状，对体内分析物的监测或检测技术进行研究，发明人发现要实现监测或检测过程中的无创、无痛的效果需要使检测工具在刺入真皮层后尽可能少的接触神经末梢或不接触神经末梢。由于真皮层中的神经末梢主要分布于真皮层中远离表皮层的深处，发明人认为需要检测工具刺入皮肤的长度足够短，在真皮层中靠近表皮层的浅处便能与体液接触且不触及神经末梢便不造成痛感。发明人认为检测工具刺入皮肤时对皮肤造成的损伤要属于可逆损伤范围内，当检测工具从皮肤中脱离时，检测工具造成的皮肤损伤能够短时间内自行复原，才能达到无创的效果。发明人认为这种情况下长度和直径均较小的微针作为检测工具刺入皮肤时能够达到无创、无痛的效果。但是现有的微针大多数为金属材质的虽然能够刺入皮肤但是与皮肤的亲和性较差，并且不能有效的将检测成份固定于其内部，金属材质的微针与皮肤之间存在较为严重的排异反应。发明人考虑采用生物亲和性较好的高分子材料做成微针，但是高分子材料需要满足在干燥状态下具有足够的硬度刺入皮肤，且能够容纳或包埋检测成份，并且体液中的分析物要能够和固定于高分子材料中的检测成分相接触。鉴于以上问题发明人认为高分子材料需要具备网状结构，将检测成份固定于网眼之中，才能避免检测成份的泄露，并且也能使体液中的分析物穿入网状结构中与检测成份相接触，在干燥的状态下还需具有一定的硬度能够刺入皮肤。
高分子合成领域技术人员已知聚乙烯醇(PVA)具有较好的生物亲和性，但是需要使聚乙烯醇形成网状结构的凝胶才能实现检测成份的固定且能够使分析物扩散进入其内部，且聚乙烯醇在体液中会发生溶解。发明人意外发现经过冷冻-解冻处理的聚乙烯醇能够在吸收水后形成溶胀而不溶解的网状结构，且在干燥状态下具有刺入皮肤的硬度，基于以上思路发明人展开了检测血糖的微针贴片的研究。
本发明第一方面涉及一种连续监测或检测体内分析物的微针贴片，包括立于贴片衬底层贴肤面的至少一个第一透皮微针，所述微针远离贴片衬底层的一端具有尖锐形状；所述第一透皮微针中集成有电化学反应池，分析物在电化学反应池中与容纳于第一透皮微针的基质中的检测成份发生电化学反应或作用形成电信号，通过检测电信号的大小反映分析物的浓度水平。
优选的，所述第一透皮微针的基质具有吸水后溶胀而不溶解的网状结构， 所述网状结构允许分析物扩散进入或移动离开第一透皮微针的基质，而阻止其中的检测成份移动离开。
优选的，还包括至少一个立于贴片衬底层贴肤面的第二透皮微针，所述第二透皮微针中不含有检测成份，所述第一透皮微针中的电化学反应池中包括有工作电极，所述第二透皮微针中包括有参考电极，第二透皮微针中的参考电极与第一透皮微针中的工作电极连接成测量回路。
优选的，所述测量回路中产生的电信号包括与化学平衡成定量关系的电量值和与电化学反应速率成定量关系的电流值。
优选的，所述第一透皮微针的长度在600-1500μm的范围，最大横截面积处的直径在200-700μm的范围。
优选的，所述电化学反应池中的工作电极一端插入第一透皮微针基质中，另一端延伸出贴片衬底层贴肤面背面形成第一电接触处。
优选的，所述参考电极一端插入第二透皮微针基质中，另一端延伸出贴片衬底层贴肤面背面形成第二电接触处。
优选的，所述电化学反应池中的工作电极至少包括包埋于第一透皮微针基质中的惰性导体，所述惰性导体选自选自铂、金、铜、银、碳材料中的一种或几种组合。
优选的，所述工作电极还包括与惰性导体电连接并将惰性导体上形成的电信号传递到第一透皮微针外部的接线柱。
优选的，所述第一透皮微针中的第一透皮微针基质的制备原料为亲水性高分子。
优选的，所述第一透皮微针基质的制备原料至少包括有主料聚乙烯醇。
优选的，所述第一透皮微针基质的制备原料聚乙烯醇为完全醇解的聚乙烯醇和/或完全醇解的聚乙烯醇与部分醇解的聚乙烯醇的混合物。
优选的，所述第一透皮微针基质的制备原料还可包括辅料，所述辅料选自葡聚糖、壳聚糖、海藻酸盐、透明质酸、透明质酸钠、羧甲基纤维素钠、聚乙二醇中的一种或几种的混合物。
本发明的第二个方面涉及一种用于处理连续监测或检测体内分析物的微针贴片产生的电信号的装置，至少包括信号处理器，所述信号处理器与前述的连续监测或检测内分析物的微针贴片形成的测量回路电连接，监测测量回路中 的电信号并将电信号转换成与分析物水平相关的数字信号。
本发明的第三个方面涉及一种连续监测或检测体内分析物的装置，至少包括上述的微针贴片，所述微针贴片上可选粘结有与皮肤贴合的粘贴层；和
信号处理器，所述信号处理器与第一透皮微针和第二透皮微针之间的测量回路电连接，监测测量回路中的电信号并将电信号转换成与分析物水平相关的数字信号。
优选的，所述连续检测体内分析物的装置还包括连接板，所述连接板的两侧均设有插接元件，所述微针贴片通过连接板一侧的插接元件与连接板可拆卸相接连，所述信号处理器通过连接板另一侧的插接元件与连接板可拆卸相连接，所述信号处理器和微针贴片构成的测量回路通过连接板实现电连接。
本发明所述的连续监测或检测体内分析物的装置为可穿戴装置。
本发明的第四个方面涉及用于制备连续监测或检测体内分析物的微针贴片的方法，包含如下步骤：
步骤1：将微针基质的成型制备液浇铸于放于运行的抽吸装置上的具有尖锐末端的浇铸模具内，之后继续将微针贴片衬底层的成型制备液浇铸于该浇铸模具内，所述浇铸模具放于运行的抽吸装置内；
步骤2：将上述成型制备液在浇铸模具内冷冻-解冻；
步骤3：将电极插入所述浇铸模具内，其末端伸出成型制备液表面；
步骤4：重复步骤2若干次；以及
步骤5：将微针贴片制备物于室温干燥收缩，后干燥至完全固化。
优选的，所述用于制备连续监测或检测体内分析物的微针贴片的方法所用的浇铸模具具有用于微针成型的微针孔眼，通过抽吸装置将所述微针基质的成型制备液抽吸进入微针孔眼。
有益效果：
(1)本技术方案将电化学反应池集中于第一透皮微针中，且第一透皮微针的基质能够刺入表皮而到达真皮层处，通过第一透皮微针刺入皮肤的同时将电化学反应池带入皮肤中，无需复杂的一次性插入装置或施加装置将电化学反应池植入人体中，简化了检测分析物装置的结构，节约了使用成本，实现了即时测量，满足普通患者对连续监测血糖的低价格需求，化解了成本造成的患者不得不长时间贴附的不便，减轻了待测试者测试时的抵触情绪。
(2)本技术方案中的第一透皮微针基质不仅能够将电化学反应池带入皮肤中，而且同时允许被分析物扩散进入或移动离开，而阻止检测成份移动离开，能够较好的将检测成份固定于其中，长期监测过程中检测成份不会泄露，能够保证对分析物的长期精准的监测。
(3)本发明中的第一透皮微针基质通过冷冻-解冻形成网状结构，不会降低或影响检测成份及酶的活性，也不会阻碍酶在催化时发生的蠕动，能较好的保持酶的活性，提高微针贴片的检测精度，保证微针贴片的实用性和连续测量的精准性，使连续监测或检测体内分析物的微针贴片具有产业化前景，使广大糖尿病患者普遍接受和适用。
(4)本发明微针贴片所用高分子材料为亲和性材料，人体即使佩戴时间较长，也几乎没有不适感，提高了用户应医生要求长期佩戴的配合度。
本发明带来的有益技术效果不显于上述效果，更多细节说明具体请参见具体实施方式和实施例。
附图说明
附图仅说明了一些实施方式，因此不应视为对范围的限制。
1-第一透皮微针、12-凹槽、2-铂丝、3-铜棒、4-贴片衬底层、5-纳安表、6-第二透皮微针、7-微针贴片衬底层、8-第一电接触处。
图1：第一透皮微针与第二透皮微针构成的测量回路示意图。
图2：当检测成份为葡萄糖氧化酶时，在第一透皮微针中发生的电化学反应示意图。
图3：微针贴片测量回路接通后电流随时间的变化曲线。
图4：担载不同葡萄糖氧化酶的微针贴片的恒定反应电流-葡萄糖浓度关系图。
图5：担载10wt％葡萄糖氧化酶的微针贴片的恒定反应电流-葡萄糖浓度拟合线性图。
图6：微针贴片使用不同天数时，恒定反应电流值-葡萄糖溶液关系图。
图7：在不同葡萄糖添加量下，微针贴片与竞品Abbott Free Style Libre 3(雅培毫米针贴片)的葡萄糖测量值以及葡萄糖真实值比较。
图8：工作电极上的富集电量值-电路断开时间的关系曲线。
图9：担载不同葡萄糖氧化酶的微针贴片的饱和电量值-葡萄糖浓度的关系曲线。
图10：不同葡萄糖浓度下的饱和电量-断开时间的关系曲线。
图11：不同温度下，富集饱和电量值-葡萄糖溶液浓度关系图。
图12：10wt％葡萄糖氧化酶载量的微针贴片饱和电量值-葡萄糖浓度的关系曲线图。
图13：10wt％葡萄糖氧化酶载量的微针贴片饱和电量值-葡萄糖浓度关系线性拟合图。
图14：不同天数下工作电极上富集的饱和电量值-葡萄糖浓度之间的关系图
图15：带有贴敷膜的微针贴片。
图16：微针贴片测试兔子血糖值时电流-时间关系曲线图。
图17：罗氏采血血糖仪测试不同葡萄糖注射量时兔子血糖值-时间的关系曲线图。
图18：微针贴片与罗氏采血血糖仪测试兔子血糖值-时间的对比关系曲线图。
图19：微针贴片与罗氏采血血糖仪连续3天测试兔子血糖值-时间的对比关系曲线图。
具体实施方式
本发明描述了一种连续监测或检测体内分析物的微针贴片，包括立于贴片衬底层贴肤面的至少一个第一透皮微针，所述微针远离贴片衬底层的一端具有尖锐形状；所述第一透皮微针中集成有电化学反应池，分析物在电化学反应池中与容纳于第一透皮微针的基质中的检测成份发生电化学反应或作用形成电信号，通过检测电信号的大小反映分析物的浓度水平。
本发明的第一透皮微针中集成有电化学反应池，本发明的电化学反应池中有电化学反应的要素，作为一种电化学反应池可列举的方案，电化学反应池包含参与反应与分析物发生作用的工作电极、以及催化该反应的酶或其他活性成分，所述酶或其他活性成分在本技术方案中称为检测成份。需要对体内的分析物进行检测时，可直接将第一透皮微针插入皮肤的体液中，体内分析物在电 化学反应池中形成电信号，依据电信号与分析物浓度之间的定量关系判定分析物的浓度水平，第一透皮微针无需借助于结构复杂的插入装置可直接将电化学反应池插入皮肤中，简化了监测装置的复杂度，降低了监测装置的生产成本。第一透皮微针中包括有第一透皮微针基质。微针浅插入式的贴附过程没有痛感，降低了患者的抵触情绪，市场上的血糖微创监测贴附装置所用的尖锐引导物和相对昂贵的一次性弹射贴附装置不再需要，一次贴附的持续时间不受成本制约，而可随使用者的生活习惯而优化，较低的使用成本使连续监测或检测体内分析物的微针贴片在广大糖尿病患者中普遍使用成为可能。
作为一种优选的技术方案，所述远离贴片衬底层的一端具有尖锐形状的微针可列举的形状包括但不限于圆锥形、双锥体组合形、棱锥形中的一种或几种的组合，所述的双锥体组合形可以为具有一个尖端的双圆锥组合体也可以为具有一个尖端的圆锥和棱锥组合体。
作为一种优选的技术方案，所述第一透皮微针的基质具有吸水后溶胀而不溶解的网状结构，所述网状结构允许分析物扩散进入或移动离开第一透皮微针的基质，而阻止其中的检测成份移动离开。
第一透皮微针基质插入皮肤时是刺透皮肤的表皮层到达真皮层，吸收真皮层中的体液形成溶胀而不溶解的网状结构，通过第一透皮微针基质具有的网状结构实现检测成分的固定和分析物的自由扩散进入和移动离开。分析物扩散进入第一透皮微针基质，在电化学反应池中产生可被检测到的电信号，依据电信号与分析物之间的定量关系判定分析物浓度。通过第一透皮微针基质允许分析物扩散进入或移动离开，而阻止检测成份移动离开的特性，使第一透皮微针处在分析物体内环境中时分析物与检测成份能够有效接触，且能保证检测成份长期稳定的存在于第一透皮微针中，延长了电化学反应池的使用时间，为连续监测实现保障。
优选的，还包括至少一个立于贴片衬底层贴肤面的第二透皮微针，所述第二透皮微针中不含有检测成份，所述第一透皮微针中的电化学反应池中包括有工作电极，所述第二透皮微针中包括有参考电极，第二透皮微针中的参考电极与第一透皮微针中的工作电极连接成测量回路。
第二透皮微针包括第二透皮微针基质和位于第二透皮微针基质中的参考电极。第二透皮微针基质的结构、材料与第一透皮微针基质的结构、材料相同 或相似。检测成份与体液中的分析物相作用，产生可以被检测到的电信号，如工作电极上富集的电子或正电荷或者电量；或分析物与电化学反应池中的检测成份及工作电极相作用在电化学反应池的工作电极上富集的电子或电量通过与参比电极形成回路，而转化为电流、电量或电动势等可被检测到的电信号。
在电化学反应测量回路中第一透皮微针的个数可以是2根、3根、4根甚至于更多根，第二透皮微针的个数可以是2根、3根、4根甚至于多根，多根第一透皮微针的设置不仅可以增加电信号强度，而且还可以增加微针贴片的稳定性，多根第二透皮微针的设置可以提高电信号的稳定性。本技术方案中以两根第一透皮微针和一根第二透皮微针为例进行阐述，且三根微针能够构成较稳定的结构。
以图1中两根第一透皮微针和一根第二透皮微针之间电连接成测量回路为例，阐述微针贴片的检测机理。如图2所示，以检测成份为葡萄糖氧化酶时为例，阐述电化学反应和电信号产生的过程。将带有工作电极并固定有葡萄糖氧化酶的第一透皮微针、带有参考电极的第二透皮微针穿过皮肤的表皮层而到达真皮层中的体液中。此时，第一透皮微针和第二透皮微针吸收体液中的水分发生溶胀，体液中的葡萄糖扩散进入第一透皮微针中发生如图2所述的电化学反应，在电化学反应中葡萄糖在葡萄糖氧化酶的催化位点上吸附并反应生成葡萄糖酸内酯后，水解为葡萄糖酸；而葡萄糖氧化酶的催化位点则转化为还原态，还原态的葡萄糖氧化酶在体液中的氧气的作用下重新被氧化为具有催化活性的氧化态，同时放出一分子的过氧化氢；过氧化氢在工作电极表面失去两个电子，生成一分子的氧气，两个氢离子。氧气参与葡萄糖氧化酶的转化，氢离子成为葡萄糖酸的组成部分，产物与反应物实现局部循环。电子被第一透皮微针中的工作电极吸收，由于工作电极上发生了电化学反应，而参考电极上未发生电化学反应，工作电极与参考电极的工作环境不同，而电极电位不同，其间产生电位差，工作电极和参考电极电连接构成回路后其间产生电流，通过电流与分析物之间的定量关系推算分析物的浓度水平。如图2所示，在电化学反应中，反应式左侧的葡萄糖被氧化失去电子，右侧的工作电极获得电子，而葡萄糖氧化酶的氧化态与还原态、以及过氧化氢和氧气均在反应体系内部循环，因此氧气的供给不会成为影响反应速率的因素。
作为一种优选的技术方案，所述检测成份选自酶、激素、抗体、DNA、 RNA、改性酶中的一种或多种的混合物。
可列举的酶包括但不限于葡萄糖氧化酶、过氧化氢酶、淀粉酶、肌酸激酶、乳酸氧化酶、胆固醇氧气酶中的一种或多种。
可列举的激素包括但不限于生长激素和/或促甲状腺激素。
可列举的该性酶为纳米金改性的葡萄糖氧化酶。
本方案中的纳米金改性的葡萄糖氧化酶的改性方法可以为：将纳米金颗粒通过硫醇固定于工作电极上，然后将葡萄糖氧化酶的辅酶(FAD)取出，固定于纳米金颗粒上，最后将除去辅酶的葡萄糖氧化酶重新与辅酶-纳米金颗粒的组合物相结合，改性后的葡萄糖氧化酶的活性中心直接与纳米金连接进而和工作电极相连接，提高电子传递效率。纳米金改性的葡萄糖氧化酶在催化葡萄糖时，将葡萄糖氧化为葡萄糖内酯，自身变为还原态，还原态的纳米金改性的葡萄糖氧化酶在工作电极上被氧化重新变为氧化态，并形成两分子的氢离子和两个电子，生成的电子被工作电极检测到形成相对应的电信号。
作为一种优选的技术方案，所述检测成份为葡萄糖氧化酶。
作为一种优选的技术方案，所述测量回路中产生的电信号包括与化学平衡成定量关系的电量值和与电化学反应速率成定量关系的电流值。当内分泌体液扩散到第一透皮微针中的葡萄糖浓度远远低于葡萄糖氧化酶的催化位点时，反应速率与葡萄糖氧化酶的载量无关，而对葡萄糖浓度呈一级关系。于是整个反应速率，即电子生成的速率也是电流的大小与葡萄糖浓度之间呈线性相关，当电流稳定时会变为恒定电流。于是，通过工作电极和参考电极之间产生的恒定电流便可得知体液中的葡萄糖浓度。而体液中的葡萄糖浓度与血液中的葡萄糖浓度相近，从而可得知血糖浓度。以上检测机理以及以下电信号的测量方式同样适用于其余检测成分，现以检测成份为葡萄糖氧化酶时，具体描述以下两种电信号的测量方式：
(1)当电化学反应中的恒定电流信号较强，可进行稳定的测量时，可以测量持续接通电路中的恒定电流来反应体液中的葡萄糖浓度，进而反应血糖值。在测量回路持续接通的情况下，对测量回路中的电流可以进行持续的记录、读取和进行数据处理，以实现血糖值的实时监控。从给药角度看，对血糖值数据的采集也可以是间歇进行的，比如间隔时间可控制在5-10分钟，通过连续的多个间歇的血糖值反应血糖的变化趋势，实现血糖值的连续监控，且间歇数据的 采集可以降低数据存储器的存储量要求。可以通过恒定电流与葡萄糖浓度之间的经验公式，在已知恒定电流的条件下获取葡萄糖浓度。
(2)当电化学反应中的电流信号较弱，不足以维持稳定的测量时，可以测量断开电路中工作电极上富集的电量来代替电流值的测量。在测量回路断开的情况下，电化学反应中的电子停留在工作电极的表面，随着电化学反应的持续进行，电子不断富集，如图2所示的反应方程式右侧的化学势不断升高，逐步与反应方程式左侧的化学势相等，到达反应平衡状态，在图2描述各个单元反应中，葡萄糖氧化酶的氧化态和还原态；以及过氧化氢和氧气在反应过程中自我循环；葡萄糖、葡萄糖酸、以及富集在工作电极上的电子的密度(即电量)决定着反应方程式两侧的化学势。于是，葡萄糖和电子之间存在着相互关系，电量可用来反映葡萄糖浓度。电量在测量时表现为瞬间变化的电流对时间的积分，即断开的电路接通时，虽然放电过程中的电流随时间衰减，瞬间电流的绝对值远远大于电路处于持续接通状态下的恒定电流。因此当电流信号较弱时，可以间歇的对断开电路中的电量进行记录、读取和数据处理，以实现血糖值的连续监测。可以通过电量与葡萄糖浓度之间的经验公式，在已知电量的条件下获取葡萄糖浓度。
依据葡萄糖浓度与电量值或电流值之间的动力学关系，测量回路中的电量值或电流值，精确算出血糖浓度，能较好的确保血糖浓度测量值的精确度，保证微针贴片的实用性。
作为一种优选的技术方案，所述检测成份固定于工作电极上。
作为一种优选的技术方案，所述检测成份固定于或包封于第一透皮微针基质中。
电化学反应池中的检测成份可通过涂敷于电化学反应池中的工作电极上进行固定，或更优选的检测成份与第一透皮微针的基质材料相混合而一体成型于微针中。不同的检测成份，在微针贴片中的固定的量有所不同。当检测成份固定于工作电极上时，可选的方法有将检测成份涂敷于工作电极上，然后将涂敷有检测成份的工作电极包埋于第一透皮微针基质中。第一透皮微针基质对于检测成份的选择性物理包封通过微针浇注成形或3D打印成形后的冷冻-解冻交联等后续处理实现。
现有的电化学反应池中的检测成份的固定方式包括以下几种：(1)吸附 法，吸附法是通过载体表面和检测表面的次级键相互作用而达到固定化目的，但是检测成份和载体之间结合力弱，容易脱落流失；(2)包埋法，包埋法是载体与检测成份的溶液混合后，借助引发剂进行聚合反应，通过物理作用将检测成份限定在载体的网络中，但是载体网络的大小较难调控，容易使检测成份发生泄漏，且引发剂需要通过紫外光进行固化交联，也容易导致酶部分失活；(3)交联法，交联法是利用双功能或多功能试剂在检测分子间、检测分子与载体间进行交联反应，把检测分子彼此交叉连接起来，形成网络结构的方法，常用的交联剂是戊二醛，但该方法容易导致酶的部分失活。
如酶类的检测成份在体液中会逐步水解失活，当检测成份的含量下降到不足以维持反应速率对分析物浓度呈一级时，分析物浓度水平的测量值会发生偏差。所以，制作微针贴片时可以通过微针中检测成份的载量调控贴片的有效寿命，检测成份的载量根据分析物的浓度水平进行调整。
作为一种优选的技术方案，所述检测成份固定于第一透皮微针基质中，检测成份的固定量根据分析物的实际范围浓度进行确定。可列举的固定量如1wt％、2wt％、3wt％、4wt％、5wt％、6wt％、7wt％、8wt％、9wt％、10wt％、11wt％、12wt％、13wt％、14wt％、15wt％、16wt％、17wt％、18wt％、19wt％、20wt％，所述含量比为检测成份重量占第一透皮微针的基质重量和检测成份重量和的比例，所述含量比甚至可以更高。
作为一种优选的技术方案，所述第一透皮微针的长度在600-1500μm的范围，例如在700-1300μm、750-1250μm、800-1200μm、850-1100μm的范围，数值例如为900μm、950μm、1000μm、1050μm、1150μm、1250μm等，第一透皮微针的长度包括但不限于以上数值范围和点。
所述第一透皮微针的最大横截面积处的直径在200-700μm的范围，例如在250-600μm、300-550μm、350-500μm的范围，数值例如为400μm、650μm等，第一透皮微针的最大横截面积处的直径包括但不限于以上数值范围和点。
本发明的第一透皮微针的长度在600-1500μm的范围，刺穿厚度约75-150μm的表皮层后到达厚度约1-2mm的真皮层，由于第一透皮微针的长度不接触或较少接触神经，第一透皮微针对表皮的刺穿基本不引起痛感。通过对第一透皮微针长度和直径的设计，第一透皮微针对表皮的也属于可逆形变，移去后皮肤数小时后便可恢复原状，无需借助带有5-7mm刚性尖端使电化学反应池 插入皮肤，大幅度减轻待测试患者的痛感和抵触情绪。
作为一种优选的技术方案，所述电化学反应池中的工作电极一端插入第一透皮微针基质中，另一端延伸出贴片形成第一电接触处。
通过形成第一电接触处，使微针贴片在与外部电信号采集设备相连接时，操作更加方便、快捷、简单。
作为一种优选的技术方案，所述参考电极一端插入第二透皮微针基质中，另一端延伸出贴片形成第二电接触处。
通过形成第二电接触处，同样使微针贴片在与外部电信号采集设备相连接时，操作更加方便、快捷、简单。
作为一种优选的技术方案，所述电化学反应池中的工作电极至少包括包埋于第一透皮微针基质中的惰性导体。
本发明中的惰性导体指在电化学反应中不发生溶解的导体。
作为一种优选的技术方案，所述惰性导体选自选自铂、金、铜、银、碳材料中的一种或几种组合。
作为一种优选的技术方案，所述工作电极还包括与惰性导体电连接并将惰性导体上形成的电信号传递到第一透皮微针外部的接线柱。
作为一种优选的技术方案，所述惰性导体为铂丝，所述接线柱为铜棒，所述铂丝的一端插入铜棒中构成工作电极，所述铜棒远离第一透皮微针尖端的一端延伸出贴片衬底层形成第一电接触处。
作为一种优选的技术方案，所述铂丝的直径在0.1-0.18mm，铜棒的直径在0.8-1.4mm。
作为一种优选的技术方案，所述参考电极的构成可与工作电极的构成相同或不相同。
作为一种优选的技术方案，所述参考电极的材质包括但不限于Ag/AgCl、铂、金、铜、银、碳材料中的一种或几种组合。
由于微针的体积较小，其中的工作电极更小，在工作电极与外部检测设备连接时操作较复杂，通过接线柱的引入，提高工作电极与外部检测设备连接的便利性和电信号传导的稳定性，参考电极上设置接线柱时也提高参考电极与外部检测设备连接的便利性。
作为一种优选的技术方案，所述第一透皮微针基质的制备原料为亲水性 高分子。
亲水性高分子材料制备的第一透皮微针基质在皮肤中具有较好的生物相容性，能更好的保持固定于其中的检测成份的活性，延长检测成份在第一透皮微针基质中的存活时间，实现微针在体液中对血糖的连续精准监测。
作为一种优选的技术方案，所述第一透皮微针基质的制备原料至少包括有主料聚乙烯醇。发明人考虑采用生物亲和性较好的高分子材料做成微针，但是需要使聚乙烯醇形成网状结构的凝胶才能实现检测成份的固定且能够使分析物扩散进入其内部，且聚乙烯醇在体液中会发生溶解。使聚乙烯醇形成网状结构的凝胶的方式分为化学交联法和物理交联法，化学交联法需要在聚乙烯醇中添加交联剂或引发剂在紫外线的作用下，形成新的化学键实现交联，而物理交联法分为离子交联和微晶构建，离子交联需要使聚乙烯醇带电然后和多价反离子溶液作用形成交联，微晶构建是使聚乙烯醇内部形成微晶体从而实现交联。发明人认为，采用化学交联需要在聚乙烯醇中加入交联剂或引发剂，而绝大多数交联剂或引发剂的毒性较高，对人体的伤害较大，且紫外线会对检测成份的活性造成较大的影响，基于以上考虑发明人不能采用化学交联。而离子交联需要使聚乙烯醇带电，而且还需要在多价的反离子溶液中才能发生，而人体内不适用该交联方式。发明人意外发现聚乙烯醇通过冷冻-解冻的工艺能够在吸收体液后具有溶胀而不溶解的网状结构，且在干燥状态下呈坚硬的玻璃态，具有刺入皮肤的硬度。而且发明人还发现通过冷冻-解冻工艺实现的网状结构，相较于通过紫外固化实现网状结构的交联，该工艺对具有活性的检测成份是安全的，该材料在该工艺下植入人体也是安全的，不会对检测成份的活性造成影响，提高了检测成份的有效利用率，对检测过程的精确性提供了可靠的保障，保证微针贴片的实用性。本发明中的微针贴片结构简单、检测精确度高、在体内具有较长的使用时间能对分析物进行连续监测，使微针贴片在连续、精确监测分析物领域中具备产业化前景。发明人发现聚乙烯醇的链段通过冷冻-解冻处理，能形成有序的微区称为微晶体或曰微晶域，微晶体或微晶域在解冻状态时也不会分开，从而在吸水后能够形成溶胀而不溶解的网状结构，提高微针干燥时的硬度。将居于活性的检测成份包埋于网状结构的聚乙烯醇中，不会对检测成份的活性造成影响，冷冻-解冻的物理过程也不会影响检测成份的活性，使检测成份在发生催化反应时能够保持自然构象，提高检测成份的有效利用率。通过 增加冷冻-解冻处理的次数，增加微晶域的密度，从而使得第一透皮微针基质的交联密度可以调控。并且聚乙烯醇为化学惰性的药用辅料，具有较好的生物相容性，甚至曾被用作软式隐形眼镜的材料，当微针插入皮肤时能降低机体组织中的炎症反应和排异反应。
本发明所述的冷冻温度可选的范围在-5℃到-25℃、或-10℃到-20℃。本发明所述的解冻温度可视为在室温下。
作为一种优选的技术方案，所述聚乙烯醇为完全醇解的聚乙烯醇和/或完全醇解的聚乙烯醇与部分醇解的聚乙烯醇的混合物。
所述完全醇解的PVA为醇解度在99％±2％，所述部分醇解的PVA的醇解度在88％±2％，少量醇解的PVA的醇解度在78％±2％。
发明人发现以完全醇解的聚乙烯醇为材质的微针在脱水干燥收缩的过程中，会出现微针变形过大、形态不受控制的问题，比如微针收缩过多导致微针过短、过细或发生弯曲，使电极在插入微针时的操作难度加大或形成的微针难以刺入皮肤。发明人意外发现使用部分醇解与完全醇解的PVA进行混合能够有效改善微针的变形问题，而且微针能较好的刺入皮肤，而使用少量醇解和完全醇解的PVA混合虽也能改善微针的变形问题，却会降低微针刺入皮肤的容易度，微针比较难刺入皮肤。发明人认为可能的原因是通过添加添加部分醇解的PVA，减少微针中的氢键量，能够降低微针干燥过程中由于氢键相互作用而引起的内应力过大的问题，减小微针基质的变形率，使微针保持较好的形态，而氢键过少对微针基质的交联度有影响，降低微针基质的交联密度和硬度。
本领域中常用的聚乙烯醇的重均分子量在10KDa-130KDa的范围。
作为一种优选的技术方案，在制备第一透皮微针的基质时，聚乙烯醇配置成聚乙烯醇溶液，聚乙烯醇溶液的浓度在18wt％-40wt％的范围。
聚乙烯醇溶液的浓度例如为20wt％、22wt％、25wt％、27wt％、29wt％、30wt％、32wt％、34wt％、36wt％、38wt％，聚乙烯醇的浓度包括但不限于以上数值点。
作为一种优选的技术方案，所述第一透皮微针基质的制备原料还可包括辅料，所述辅料选自葡聚糖、壳聚糖、海藻酸盐、透明质酸、透明质酸钠、羧甲基纤维素钠、聚乙二醇中的一种或几种的混合物。
作为一种优选的技术方案，在制备第一透皮微针的基质时，所述辅料高 分子配置成辅料高分子溶液，所述辅料高分子溶液的浓度在5wt％-40wt％的范围，例如为7wt％、9wt％、11wt％、13wt％、15wt％、17wt％、20wt％、22wt％、25wt％、27wt％、29wt％、30wt％、32wt％、34wt％、36wt％、38wt％等。辅料高分子的浓度包括但不限于以上数值点。
作为一种优选的技术方案，当第一透皮微针的基质的制备原料为聚乙烯醇和辅料高分子的混合物时，聚乙烯醇溶液与辅料高分子溶液之间的质量比在3/1-8/1的范围。聚乙烯醇溶液与辅料高分子溶液之间的质量比例如为数值4/1、5/1、6/1、7/1或在以上数值之间的范围内。优选的，聚乙烯醇溶液与辅料高分子溶液之间的质量比在3/1-8/1时透皮微针的硬度、对生物分子的亲和性较好且易于浇铸。第一透皮微针基质的材料除了可形成非共价交联，实现吸水溶胀不溶解的特性外，可通过添加其他聚合物如葡聚糖、壳聚糖、海藻酸盐、透明质酸、透明质酸钠、羧甲基纤维素钠、聚乙二醇，调控交联密度即交联结构的网眼大小和微针的溶胀性，优化检测成份的作用环境。这些材料不但与PVA兼容，而且对生物分子友好，为具有活性的检测分子提供良好的固定环境。
一种用于处理连续监测或检测体内分析物的微针贴片产生的电信号的装置，至少包括信号处理器，所述信号处理器与上述的连续监测或检测内分析物的微针贴片形成的测量回路电连接，监测测量回路中的电信号并将电信号转换成与分析物水平相关的数字信号。
一种连续监测或检测体内分析物的装置，至少包括如上所述的微针贴片，所述微针贴片上粘结有与皮肤贴合的粘贴层；和
信号处理器，所述信号处理器与第一透皮微针和第二透皮微针之间的测量回路电连接，监测测量回路中的电信号并将电信号转换成与分析物水平相关的数字信号。
本技术方案中的信号处理器中输入有电信号与分析物水平相关的定量关系式，信号处理器检测回路中的电信号可转换为与分析物水平相关的数字信号，能够实现上述功能的信号处理器都可作为本技术方案的一种实施方式，可列举的信号处理器有A/D转换器。
作为一种优选的技术方案，所述连续检测体内分析物的装置还包括连接板，所述连接板的两侧均设有插接元件，所述微针贴片通过连接板一侧的插接元件与连接板可拆卸相接连，所述信号处理器通过连接板另一侧的插接元件与 连接板可拆卸相连接，所述信号处理器和微针贴片构成的测量回路通过连接板实现电连接。
本发明中的插接元件为可以实现可拆卸插入和拆除的原件，可列举的有弹性夹和插接头等具有插接功能的元件。将微针贴片与连接板设置成可拆卸的连接，当微针贴片需要更换时，直接将微针贴片从连接板上拆下，更换为新的微针贴片，组成新的连续检测体内分析物的装置，而处于连接板上的信号处理器可重复多次使用。而信号处理器可拆卸的连接于连接板上，当发生故障需要更换时，可直接拆下换成新的器件，而不影响其余器件的使用。
本发明中的微针贴片及相关信号读取装置可穿戴于身，以实现对体内生物物质的含量或浓度的连续监测或检测。
可选地，本发明的一种连续监测或检测体内分析物的微针贴片的方法至少包含如下步骤：
步骤1：将第一透皮微针的基质成型制备液和第二透皮微针的基质成型制备液分别浇铸于具有尖锐末端的浇铸模具内，第一透皮微针的基质成型制备液浇铸于浇铸模具上用于第一透皮微针成型的微针孔眼内，第二透皮微针的基质成型制备液浇铸于浇铸模具上用于第二透皮微针成型的微针孔眼内，之后继续将微针贴片衬底层的成型制备液浇铸于该浇铸模具内，所述浇铸模具放于运行的抽吸装置内，通过抽吸装置将所述微针基质的成型制备液抽吸进入微针孔眼；
步骤2：将上述第一透皮微针的基质成型制备液和第二透皮微针的基质成型制备液在浇铸模具内冷冻-解冻；
步骤3：将工作电极和参考电极插入所述浇铸的第一透皮微针和第二透皮微针内，其末端伸出成型制备液表面；
步骤4：重复步骤2若干次；以及
步骤5：将微针贴片制备物于室温干燥收缩，后干燥至完全固化。
制备过程示例
本发明阐述具体实施方式时，以检测成份为葡萄糖氧化酶时的电化学反应为例，具体阐述当第一透皮微针与第二透皮微针构成测量回路时的检测原理，对于其余可发生电化学反应的检测成份，其检测原理同样适用。
连续检测体内分析物的微针贴片的制备方法，包括以下步骤：
(1)配置第一透皮微针的浇铸溶液：将4g浓度为20wt％PVA水溶液、1g 浓度为10wt％葡聚糖水溶液和一定量的葡萄糖氧化酶混合并搅拌，直至混合溶液均一稳定无相分离，所述PVA的牌号：国药集团124，所述葡聚糖购于Sigma-Aldrich；
(2)配置第二透皮微针的浇铸溶液：所述第二透皮微针的浇铸溶液与第一透皮微针的浇铸溶液不同的点在于，不含有葡萄糖氧化酶，其余原料和制备方法相同；
(3)微针贴片的贴片衬底层的浇铸溶液：贴片衬底层采用浓度为22wt％的PVA水溶液，所述PVA的牌号：国药集团124；
(4)微针贴片的浇铸：第一透皮微针和第二透皮微针为具有尖端的圆锥状，将透气的高分子材料的浇铸模具放于运行的抽吸装置上，透气的高分子浇铸模具上具有用于浇铸微针成型的微针孔眼，将第一透皮微针的浇铸溶液与第二透皮微针的浇铸溶液浇铸于模具不同的微针孔眼上，通过抽吸装置将浇铸溶液抽吸进入微针孔眼，待微针孔眼被浇铸液填满时，停止运行抽吸装置，然后在浇铸模具上放置防止贴片衬底层溶液流失的辅助模具，并在辅助模具中浇铸贴片衬底层的浇铸溶液，所述浇铸模具上具有21个微针孔洞，每三个微针孔洞浇铸完成后构成一个连续监测或检测体内分析物的微针贴片，其中一个微针孔洞为第二透皮微针，两个微针孔洞为第一透皮微针；
(5)微针贴片的冷冻-解冻：将浇铸完溶液的浇铸模具和防止溶液流失的辅助模具放入-20℃的冰箱中冷冻30h，于室温下解冻30min；
(6)电极的插入：将铂丝插入铜棒中的工作电极插入第一透皮微针中心处，铂丝插入铜棒中的参考电极插入第二透皮微针中心处，其中铂丝的直径为0.15mm，长度为1.1mm，其中在微针中的长度为0.7mm，在铜棒中的长度为0.4mm，铜棒的直径为1.2mm，铜棒的长度需伸出贴片衬底层；
(7)再次冷冻-解冻处理：将插入电极的微针连通模具再次放于-20℃冰箱中冷冻21h，之后拿出在室温下解冻3.5h，反复冻融3次；
(8)微针贴片的干燥：反复冷冻-解冻结束后，从冰箱中取出放于室温下，并自然干燥收缩24h后，将微针贴片在模具中揭下来，并借用夹具夹住微针贴片放于干燥器中干燥，直至干燥完全。其中所浇铸的第一透皮微针和第二透皮微针的干燥后的长度在900μm的范围，最大截面积处的直径为300μm的范围。
不同浓度的葡萄糖溶液的配制为将所需要的葡萄糖的量溶于PH为7.0的 PBS溶液中配置得到，其中PBS溶液购于ThermoFisher，葡萄糖购于Sigma。
以下实施例中所使用的微针贴片均是基于上述制备过程示例进行的的，根据所需要的葡萄糖氧化酶的含量进行具体调整，其中葡萄糖氧化酶所占比例为：葡萄糖氧化酶重量占第一透皮微针基质重量和葡萄糖氧化酶重量和的比例。
一般对血糖值的界定分为三种情况，高血糖即葡萄糖浓度大于7mmol/L，正常血糖即葡萄糖浓度在3.9-7mmol/L，低血糖即葡萄糖浓度小于3.9mmol/L。本方案在进行实验时，葡萄糖浓度的范围在1-36mmol/L，能够满足实际人体测试中葡萄糖浓度的变化范围。
实施例1
为验证本技术方案中的微针贴片的检测和监测效果，构造如图1所示的检测装置，其中包括两个第一透皮微针1、一个第二透皮微针6、均插入第一透皮微针1的工作电极，工作电极由铂丝2插入铜棒3中构成，其中铜棒3伸出贴片衬底层4形成第一电接触处，所述铜棒3的侧壁上设有凹槽12，通过凹槽12卡接于贴片4中，提高铜棒12与贴片4之间的结合强度。第一透皮微针1中含有葡萄糖氧化酶，第二透皮微针6中插入有参考电极，参考电极同样由铂丝插入铜棒中构成，其中铜棒伸出贴片4形成第二电接触处，两个并联的第一电接触处和第二电接触处之间电连接，构成测量回路，纳安表5电连接于所述回路中，测量回路中的电流，纳安表可选择Keysight数字万用表。以下具体实施方式在操作过程中，将该检测装置中的微针贴片插入0.5mm厚的聚乙烯醇电纺纤维模拟的真皮层中，其中聚乙烯醇电纺纤维模拟的真皮层安放于载有不同葡萄糖浓度溶液的透皮池上。聚乙烯醇电纺纤维接触并吸收葡萄糖溶液，让插入其中的第一透皮微针和第二透皮微针溶胀。溶液中的葡萄糖快速扩散进入溶胀为水凝胶状态的第一透皮微针和第二透皮微针，进入第一透皮微针中的葡萄糖被载于其中的葡萄糖氧化酶催化氧化，生成葡萄糖酸内酯后，水解为葡萄糖酸；而葡萄糖氧化酶的催化位点则转化为还原态，还原态的葡萄糖氧化酶在体液中的氧气的作用下重新被氧化为具有催化活性的氧化态，同时放出一分子的过氧化氢；过氧化氢在工作电极的铂丝表面失去两个电子，生成一分子的氧气，两个氢离子，其反应原理如图2所示，工作电极将电子传输至参考电极的过程中被纳安表5检测到，形成可以读取的电流值。
实施例2
在保证葡萄糖氧化酶担载量充足的情况下，考察测量回路接通后电流随时间的变化值，制备第一透皮微针中葡萄糖氧化酶含量为10wt％的微针贴片，配置浓度为12mM的葡萄糖溶液，在37℃的条件下，反应4min之后接通测量回路，通过微针贴片测量电流随时间的变化曲线，得到如图3所示的电流-时间曲线。通过图3可以看出，当测量回路未接通时，电化学反应中的电子富集在工作电极的表面不被移走，形成了驱动反向反应的化学势，测量回路在接通的瞬间，富集在工作电极表面的电子被迅速移到参考电极，形成较高的瞬间电流158nA，随时间的延长电流持续衰减至35nA，并在3s后恒定于该值，该恒定的电流值可以反应葡萄糖浓度，在检测过程中可以选定3s后的任一时间点的恒定电流值作为反应葡萄糖浓度的电流值，后续实验中均选择测量回路接通8s后的电流值为恒定电流值，通过该实验可以验证本发明中的测试原理，当电信号较强时，可以通过测量回路中的恒定电流反应葡萄糖浓度。
实施例3
为考察第一透皮微针中的葡萄糖氧化酶的载量对血糖值监测精确度的影响，分别制备第一透皮微针中葡萄糖氧化酶含量为3wt％的微针贴片、第一透皮微针中葡萄糖氧化酶含量为5wt％的微针贴片、第一透皮微针中葡萄糖氧化酶含量为10wt％的微针贴片，配置葡萄糖浓度在1mM到36mM的多个不同浓度的葡萄糖溶液，在37℃的条件下使用不同葡萄糖氧化酶担载量的微针贴片分别放置于不同浓度的葡萄糖溶液中进行反应，因微针在溶液中4min能够达到较好溶胀的效果，待反应4min后接通测量回路，取电流接通8s时的稳态电流值作为恒定反应电流值，绘制恒定反应电流-葡萄糖浓度曲线，得到图4中的曲线。从图4中可以看出，当葡萄糖氧化酶的载量降低后，较高的葡萄糖浓度下，电流不再线性响应葡萄糖浓度的增加。这一结果支持了我们设计思路，即当葡萄糖氧化酶的催化位点的密度不大幅超出葡萄糖浓度时，电化学反应速率不再对葡萄糖浓度呈一级。第一透皮微针中葡萄糖氧化酶含量为10wt％的微针贴片，恒定电流值在37℃下，葡萄浓度为1mM到36mM之间的线性关系良好，对该测试结果进行线性拟合，得到如图5所示的拟合后的直线，R2＝0.9968，线性关系良好，拟合方程为：y＝2.467x+5.6154，对拟合方程进行变形得出葡萄糖浓度与恒定反应电流之间的关系，即CGlu＝(I-5.6154)/2.467，其中CGlu为葡萄糖浓度，单位为mol/L，I为恒定反应电流值，单位为nA。在已知恒定电流的条件下，利 用该经验公式可以算得葡萄糖浓度值。葡萄糖氧化酶含量为10wt％的微针贴片在较大的葡萄糖浓度内电流与葡萄糖浓度仍具有较好的线性关系，在后续实验中，若无特殊声明则采用葡萄糖氧化酶含量为10wt％的微针贴片进行实验。
实施例4
雅培等公司的产品即是一次贴附，6mm传感器针长置于皮下14天；我方也可贴附14天，间歇读取数据。但缩短贴附时间更方便患者——如应对夏季的炎热、出汗、冲凉等。由于本技术方案中的微针贴片具有较低的使用成本，当患者进行冲凉等活动时，可更换微针贴片。为考察载有10wt％葡萄糖氧化酶的微针贴片精确监测葡萄糖浓度的时间。微针贴片在37℃下连续置于透皮池上14天；分别在第1、2、5、8、14天测定了电流对葡萄糖浓度的响应，并取电路接通后的第8s时的电流值为恒定电流值，绘制不同天数时，电流-葡萄糖浓度之间的关系曲线，得到图6。由图6可见，微针贴片连续置于37℃的透皮池时，恒定电流与葡萄糖浓度在1～8天内保持了精确的线性关系，到达14天时，测量值有所偏低，但大体上维持了线性关系。这一测试结果说明，在37℃的条件下，葡萄糖氧化酶随着孵育时间的增加逐步降解失活，使得其有效载量低于达到线性电流响应所需的量。由于第14天的电流测定值虽然仍然有所偏差，仍然保持着对于葡萄糖浓度的线性，可以预期适当增加葡萄糖氧化酶的载量，便可实现两周的葡萄糖浓度精准监测。葡萄糖溶液随着实验的进行会蒸发减少，在实验过程中要实时补充对应浓度的葡萄糖溶液。
实施例5
葡萄糖浓度糖尿病患者最常见的3mM到24mM的变化范围内，分别配制浓度为3mM、6mM、9mM、12mM、18mM和24mM的葡萄糖溶液，然后将配制好的葡萄糖溶液放置于37℃恒温水浴中，得到37℃的葡萄糖溶液。将体外实验透皮释药池放置于37℃水浴中，然后向不同透皮释药池中分别加入不同浓度的37℃葡萄糖溶液。采用10wt％葡萄糖氧化酶载量的微针贴片进行葡萄糖浓度检测，测得恒定电流值，将测得的数值代入得到的恒定电流与葡萄糖浓度的关系CGlu＝(I-5.6154)/2.467计算得到葡萄糖浓度。
利用市售雅培连续监测血糖仪Abbott Free Style Libre3进行同样的葡萄糖浓度检测体外实验。将透皮释药池置于37℃的恒温浅水浴中，然后向其分别加入3-24mM的37℃葡萄糖溶液。按照雅培连续监测血糖仪上的说明将葡萄糖传 感器安装在一次性的弹射装置上，按压弹射装置，使传感器探针瞬间刺穿0.5mm厚的模拟真皮组织PVA纤维膜。将带有PVA纤维膜的传感器探针放置于透皮释药池上与葡萄糖溶液反应，用雅培连续监测血糖仪自带的扫描检测仪扫描传感器测得葡萄糖浓度，待数值稳定后记录葡萄糖浓度。将连带PVA纤维膜的传感器取出放置于下一个浓度的葡萄糖溶液中，记录测得的葡萄糖浓度数值。
将微针贴片测试的葡萄糖浓度值-真实葡萄糖浓度值之间绘制图线、市售雅培连续监测血糖仪测试的葡萄糖浓度值-真实葡萄糖浓度值之间绘制图线、真实葡萄糖浓度值绘制图线作为对比图线，得到图7。从图7中可以看出使用微针贴片得到的葡萄糖浓度值与实际添加的葡萄糖浓度值更加接近，本发明中的微针贴片具有较高的检测精准度。
实施例6
为进一步考察第一透皮微针工作电极上富集的电量与测量回路断开时间的关系。在25℃恒定室温条件下，采用10wt％葡萄糖氧化酶载量的微针贴片与12mM葡萄糖溶液反应，改变电路断开时间，测量不同断开时间下的电流-时间曲线，对曲线下面积积分得到工作电极上的富集电量值，绘制工作电极上的富集电量值与电路断开时间的关系曲线，得到图8。从图8可知，随着测量回路断开时间的增长，工作电极上的富集电量值也随之增加，最终达到一个平衡值，本实施例中的反应条件下工作电极上的富集电量值约在4min达到平衡。
实施例7
分别采用第一透皮微针中葡萄糖氧化酶含量为3wt％的微针贴片、第一透皮微针中葡萄糖氧化酶含量为5wt％的微针贴片、第一透皮微针中葡萄糖氧化酶含量为10wt％的微针贴片，配置葡萄糖浓度在1mM到36mM的葡萄糖溶液，在37℃的条件下使用不同葡萄糖氧化酶担载量的微针贴片分别放置于不同浓度的葡萄糖溶液中进行反应，连通测量回路对电流释放曲线2s内的曲线下面积积分得到工作电极上的富集电量值，改变电路断开电路时间，测定不同断开时间下电极上的富集电量，直至电极上的富集电量达到饱和，作该饱和电量值与葡萄糖浓度的关系曲线，得到图9。从图9可以看出，随着酶担载量的降低，反应达到平衡时工作电极上的蓄积饱和电量值无显著性变化。实验中我们发现，酶载量的变化影响着一次电量测量后，反应达到平衡的工作电极上富集电量达到饱和的时间。当第一透皮微针中葡萄糖氧化酶担载量为10wt％时，工作电极 上电量达到饱和的时间为4min。当第一透皮微针中葡萄糖氧化酶担载量降到5wt％和3wt％时，电极上电量富集饱和时间为7min和11min。由此看出葡萄糖氧化酶担载量的多少影响化学反应到达平衡的时间。
实施例8
为进一步考察葡萄糖的浓度对工作电极上电量富集达到饱和的时间的影响，在25℃下，分别测试葡萄糖浓度为6mM、12mM、24mM和36mM时，微针贴片工作电极上的富集电量值与电路断开时间的关系，并绘制不同葡萄糖浓度下的工作电极饱和富集电量与断开时间的关系曲线，得到图10。从图10可知随葡萄糖浓度的升高，工作电极上的饱和电量值也随之升高。
实施例9
实施例8中的体外测试是在25℃的条件下进行的，因为人体温度在37℃左右，本实施例进一步考察了温度对工作电极上的富集饱和电量值的影响。分别在25℃和37℃条件下，采用10wt％葡萄糖氧化酶载量的微针贴片与3mM、6mM、12mM和24mM葡萄糖溶液反应，测定工作电极上的富集饱和电量值以及电量富集饱和的时间，并绘制不同温度下富集饱和电量-葡萄糖浓度之间的关系曲线，得到图11。从图11可知不同温度下测得的工作电极上的富集饱和电量值也无显著性差异(P>0.05)。
实施例10
由实施例2可知，当断开的电路接通后的2s内电流呈衰减趋势，2s之后电流达到稳定状态。为考察葡萄糖浓度与反应达到平衡时微针贴片工作电极上的富集饱和电量值的关系。将10wt％葡萄糖氧化酶载量的微针贴片与不同浓度的37℃葡萄糖溶液反应4min以上，电荷在工作电极上富集达到饱和，连通电路，测定电流衰减曲线，对2秒内曲线下的面积积分得到工作电极上的富集饱和电量值，绘制该饱和电量值与葡萄糖浓度的关系曲线，得到图12，并对图12进行线性拟合，得到如图13所示的曲线，图13中的R2＝0.9804，具有良好的线性关系，通过得到图13中的拟合曲线获得经验公式，通过经验公式即可在已知工作电极饱和电量的条件下计算所对应的葡萄糖浓度值。
实施例11
电化学反应池微针在使用的过程中受环境等因素的影响酶会失活。本实施例采用电量法在37℃的葡萄糖溶液中进行了连续14天的电化学反应池微针 葡萄糖浓度检测体外实验。10wt％葡萄糖氧化酶载量的微针贴片分别与3mM、9mM、18mM和30mM的葡萄糖溶液反应，将微针贴片连续放置于不同葡萄糖浓度的透皮池上14天，分别于第1天、第2天、第5天、第8天和第14天时测试工作电极上的富集饱和电量值。并绘制不同天数下工作电极上富集的饱和电量值与葡萄糖浓度之间的关系图，得到图14。从图14中可以看出，在14天内，不同的测量天数测得的电极上的富集饱和电量值变化不显著(P>0.05)。通过该试验可以看出微针贴片可用于通过监测电量值的方式连续监测葡萄糖浓度变化情况。我们同时考察了一次电量测定后，铂丝工作电极上富集电量达到饱和的时间，在前8天内电极电量富集饱和的时间为4分钟，而在第14天，电量富集饱和的时间延长为5分钟。对于电化学反应池微针使用过程中酶的降解，采用间歇性电量法可以通过延长电量富集的时间来解决。葡萄糖溶液随着实验的进行会蒸发减少，在实验过程中要实时补充相对应浓度的葡萄糖溶液。
实施例12
将6.1wt％葡萄糖氧化酶载量的微针贴片应用于活体兔子中进行活体测试，具体实施过程和实验结果如下所示：
一、试剂以及相关仪器
葡萄糖购于：Sigma；万用表及软件：keysight；；血糖仪：罗氏，罗康全活力型；试纸：罗氏；医用消毒棉球：欧洁；采血针：罗氏；脱毛膏：薇婷。
二、动物实验
(1)动物：
成年新西兰大白兔，购自上海甲干生物科技有限公司。生产许可证号：SCXK(沪)2010-0028。体重：2-2.5kg。数量6只。级别：普通级。性别：雌性。动物饲养和实验均在上海交通大学实验动物中心专用动物房进行，每只单笼饲养，兔子适应性饲养一周后开始实验，普通兔子饲料正常喂养。
(2)动物实验方法：
适应性饲养后，进行以下实验：
1、兔子单耳用脱毛膏去毛，将微针贴片贴附在脱毛的兔子耳朵上，将具有一侧粘结层的贴敷膜粘贴到微针贴片上，至少保证电接触处部分露出贴敷膜，通过贴敷膜的使用使微针贴片牢固的贴敷于被监测体上，图15为一种可列举的实施方式，其中贴敷膜9粘贴至微针贴片衬底层7上，漏出第一电接触处8以及 第二电接触处以便与外部器件电连接，。使用时大拇指按着微针贴片中间部位用力将3根微针刺入兔子单耳皮肤内，然后再将贴敷膜揭去将微针贴片牢牢贴在兔子耳朵上；
2、兔子单笼饲养，实验前空腹12h(禁食不禁水)；
3、第一天：空腹血糖测量；
血糖监测微针贴片与体液中葡萄糖反应4分钟后，将keysight软件“开始”按钮打开，万用表开始采集数据，同时将仪器上的接线笔正负极分别与微针贴片上的参考电极和工作电极重新连通，测量40s，按“停止”按钮，导出数据；。
4、同时，在贴微针贴片的耳朵的对侧耳朵采用耳缘静脉取血用罗氏血糖仪进行血糖测量，使用罗氏血糖仪进行血糖测量的方法：对兔子耳朵用75％酒精棉消毒，专用的采血针刺破皮肤挤压出血，用罗氏专业血糖试纸前端吸取微量血液之后插入血糖仪，即可读出血糖值；。
5、在兔子耳缘静脉注射一定量的50wt％浓度的葡萄糖溶液；。
6、注射葡萄糖结束后15min、30min、1h、2h、3h、4h分别如上步骤测定血糖值；。
测量结束，给兔子添加饲料正常进食；。
7、需要多天测试时，后续的每天重复步骤2-步骤6。
微针贴片测试新西兰大白兔的电信号的选择
在六只新西兰大白兔中任选一只，于空腹12h之后，将微针贴片贴附于兔子单耳，4min之后开始测量血糖值，通过测试软件得到电流与时间之间的关系曲线，如图16所示。从图16中可以看出采用微针贴片对兔子血糖进行测量，测得的电流值在2秒内快速降低然后达到恒定电流，且恒定电流较小，为0.0X nA。原因为兔子实验中测量的是组织液中的葡萄糖含量，动物体内组织液量较体外葡萄糖溶液量相比明显减少，微针溶胀和葡萄糖扩散速度变慢，从而导致了葡萄糖氧化酶与葡萄糖反应变缓。从图16中可以看出微针贴片在测量兔子组织液中的葡萄糖浓度时，检测到的恒定电流值非常小，所以后续试验中我们通过对瞬间电流时间积分，获得电量值，结合电量值与葡萄糖浓度的化学动力学关系以及实施例10中的经验公式，计算出葡萄糖浓度。
新西兰大白兔注射葡萄糖剂量的筛选
任选一只兔子，兔子空腹12h后测空腹血糖，紧接着分别耳缘静脉按兔 子重量注射3g/kg、2g/kg、1.25g/kg 50wt％葡萄糖溶液，分别于15、30、60、120、180、240min采用罗氏血糖仪测定血糖值，不同葡萄糖注射量兔子血糖变化如下表1所示，将表1绘制成曲线，得到图17。从表1和图17中可以看出，当注射量为3g/kg时，15min兔子血糖值超出了采血血糖仪的检测上限，因而没有读数。为了更贴合人体正常血糖范围区间，我们选定1.25g/kg的50wt％葡萄糖注射量作为接下来动物实验的实验条件。
微针贴片与罗氏采血血糖仪兔子血糖监测结果以1.25g/kg的50wt％葡萄糖分别注射量到六只兔子耳缘静脉处，按照动物实验方法进行微针贴片和罗氏采血血糖仪的血糖值测试，其中通过微针贴片测试得到的电路接通时变化的瞬间电流，通过实施例10中的电量与葡萄糖浓度之间的经验公式，计算用微针贴片和罗氏采血血糖仪分别进行测量的六只兔子的血糖平均值，并绘制血糖-时间曲线，得到图18。从图18中可以看出微针贴片测的血糖值与市售罗氏采血血糖仪所测血糖值的变化趋势相同，存在小范围的差值。兔子耳缘静脉注射葡萄糖之后血糖值快速升高，在2小时恢复正常血糖水平。在血糖快速升高时，微针贴片测得的血糖值略低于采血的血糖值，可能的原因为，采血血糖仪采取的是毛细血管的血，而微针贴片监测的是体液中的葡萄糖浓度反应较血糖迟。
微针贴片与罗氏采血血糖仪连续3天兔子血糖监测
为进一步比较电化学血糖监测微针贴片与采血血糖仪测得的血糖值，本研究对6只健康兔子空腹12h后注射50wt％葡萄糖(1.25g/kg)，对测得的血糖值进行显著性差异检测。并通过微针贴片和罗氏采血血糖仪连续3天进行监测血糖，并分别计算测试得到的平均血糖值，测试数据如图19所示。连续进行3天实验，对3天测得的血糖值进行显著性差异检测。从图中可以看出连续3天的兔子血糖监测结果显示微针贴片与市售罗氏血糖仪测得的血糖值相关性良好。证明了血糖监测微针贴片可以连续的对血糖进行监测，且结果稳定可靠。
基于数理统计原则，首先使用双样本方差F-检验比较电化学血糖监测微针贴片和采血血糖仪测得的血糖值的方差。若p值小于0.05，说明两组间测得的血糖值的方差存在显著性差异，需用双样本异方差t检验进行检验；若p值大于0.05，则说明两组间测得的血糖值的方差没有显著性差异，使用双样本等方差t检验进行检验。对于进一步t检验，若所得的双侧检验的p＞0.05，则说明相互比较的两组血糖数值没有显著性差异；若0.01＜p＜0.05，则说明两组之间测得的血糖值存在显著性差异；若p＜0.01，则说明两组之间测得的血糖值存在极显著性差异。其中表2为第一天健康兔子空腹注射葡萄糖后微针贴片与采血血糖仪测得的血糖值与t检验结果、表3为第二天健康兔子空腹注射葡萄糖后微针贴片与采血血糖仪测得的血糖值与t检验结果、表4为第三天健康兔子空腹注射葡萄糖后微针贴片与采血血糖仪测得的血糖值与t检验结果。




对空腹12h的兔子耳缘静脉注射葡萄糖测定其血糖变化，连续测量3天。从表2、表3、表4中的统计结果可以看出微针贴片与采血血糖仪血糖测量值的没有显著性差异。因此，我们的血糖监测微针贴片可用于活体血糖连续监测。
任意选择一只兔子，将血糖监测微针贴片牢牢帖附于兔子单耳，分别于每天空腹12h之后测定血糖值，同时罗氏采血仪采血测定血糖作为对照组。每天连续对兔子空腹血糖进行监测的结果显示，血糖监测微针贴片可以连续进行14天的血糖监测且测得的血糖值稳定。15天之后血糖监测微针贴片测得的血糖值较采血血糖仪测得的血糖值显著降低，可能的原因为，血糖监测微针贴片中的葡萄糖氧化酶经过长时间的电极作用和体内生理环境的影响，部分氧化酶的活性失效。由此可知本发明中的用于监测或检测体内分析物的微针贴片能够在活体中稳定、长期的存在并精确进行相关信号的测量。
虽然已经根据所示的实现方式提供了本公开，但是本领域普通技术人员将容易地认识到，可以存在对实施例的变型，并且这些变型将在本公开的范围内。因此，在不脱离所附权利要求的范围的情况下，本领域普通技术人员可以进行许多修改。

Claims (18)

  1. 一种连续监测或检测体内分析物的微针贴片，其特征在于，包括立于贴片衬底层贴肤面的至少一个第一透皮微针，所述微针远离贴片衬底层的一端具有尖锐形状；
    所述第一透皮微针中集成有电化学反应池，分析物在电化学反应池中与容纳于第一透皮微针的基质中的检测成份发生电化学反应或作用形成电信号，通过检测电信号的大小反映所述分析物的体内浓度水平。
  2. 根据权利要求1所述的连续监测或检测体内分析物的微针贴片，其特征在于，所述第一透皮微针的基质具有吸水后溶胀而不溶解的网状结构，所述网状结构允许分析物扩散进入或移动离开第一透皮微针的基质，而阻止其中的检测成份移动离开。
  3. 根据权利要求2所述的连续监测或检测体内分析物的微针贴片，其特征在于，还包括至少一个立于贴片衬底层贴肤面的第二透皮微针，所述第二透皮微针中不含有检测成份，所述第二透皮微针中包括有参考电极，
    所述第一透皮微针中的电化学反应池中包括有工作电极，
    第二透皮微针中的参考电极与第一透皮微针中的工作电极连接成测量回路。
  4. 根据权利要求3中所述的连续监测或检测体内分析物的微针贴片，其特征在于，所述测量回路中产生的电信号包括与化学平衡成定量关系的电量值和与电化学反应速率成定量关系的电流值。
  5. 根据权利要求1所述的连续监测或检测体内分析物的微针贴片，其特征在于，所述第一透皮微针的长度在600-1500μm的范围，最大横截面积处的直径在200-700μm的范围。
  6. 根据权利要求3所述的连续监测或检测体内分析物的微针贴片，其特征在于，所述电化学反应池中的工作电极一端插入第一透皮微针基质中，另一端延伸出贴片衬底层贴肤面背面形成第一电接触处。
  7. 根据权利要求6所述的连续监测或检测体内分析物的微针贴片，其特征在于，所述参考电极一端插入第二透皮微针基质中，另一端延伸出贴片衬底层贴肤面背面形成第二电接触处。
  8. 根据权利要求3所述的连续监测或检测体内分析物的微针贴片，其特征在于，所述电化学反应池中的工作电极至少包括包埋于第一透皮微针基质中的惰性导体，所述惰性导体选自选自铂、金、铜、银、碳材料中的一种或几种组合。
  9. 根据权利要求8所述的连续监测或检测体内分析物的微针贴片，其特征在于，所述工作电极还包括与惰性导体电连接并将惰性导体上形成的电信号传递到第一透皮微针外部的接线柱。
  10. 根据权利要求1所述的连续监测或检测体内分析物的微针贴片，其特征在于，所述第一透皮微针中的第一透皮微针基质的制备原料为亲水性高分子。
  11. 根据权利要求10所述的连续监测或检测体内分析物的微针贴片，其特征在于，所述第一透皮微针基质的制备原料至少包括有主料聚乙烯醇。
  12. 根据权利要求11所述的连续监测或检测体内分析物的微针贴片，其特征在于，所述第一透皮微针基质的制备原料还可包括辅料，所述辅料选自葡聚糖、壳聚糖、海藻酸盐、透明质酸、透明质酸钠、羧甲基纤维素钠、聚乙二醇中的一种或几种的混合物。
  13. 一种用于处理连续监测或检测体内分析物的微针贴片产生的电信号的装置，其特征在于，至少包括信号处理器，所述信号处理器与如权利要求3-12中所述的连续监测或检测体内分析物的微针贴片形成的测量回路电连接，监测测量回路中的电信号并将电信号转换成与分析物水平相关的数字信号。
  14. 一种连续监测或检测体内分析物的装置，其特征在于，至少包括如权利要求3-12中所述的微针贴片，所述微针贴片上可选粘结有与皮肤贴合的粘贴层；和
    信号处理器，所述信号处理器与第一透皮微针和第二透皮微针之间的测量回路电连接，监测测量回路中的电信号并将电信号转换成与分析物水平相关的数字信号。
  15. 根据权利要求14中所述的连续监测或检测体内分析物的装置，其特征在于，所述装置还包括连接板，所述连接板的两侧均设有插接元件，所述微针贴片通过连接板一侧的插接元件与连接板可拆卸相接连，所述信号处理器通过连接板另一侧的插接元件与连接板可拆卸相连接，所述信号处理器和微针贴片构成的测量回路通过连接板实现电连接。
  16. 根据权利要求14-15所述的连续监测或检测体内分析物的装置，其为可穿戴装置。
  17. 一种用于制备连续监测或检测体内分析物的微针贴片的方法，其特征在于，包含如下步骤：
    步骤1：将微针基质的成型制备液浇铸于具有尖锐末端的浇铸模具内，之后继续将微针贴片衬底层的成型制备液浇铸于该浇铸模具内，所述浇铸模具放于运行的抽吸装置内；
    步骤2：将上述成型制备液在浇铸模具内冷冻-解冻；
    步骤3：将电极插入所述浇铸模具内，其末端伸出成型制备液表面；
    步骤4：重复步骤2若干次；以及
    步骤5：将微针贴片制备物于室温干燥收缩，后干燥至完全固化。
  18. 根据权利要求17所述的用于制备连续监测或检测体内分析物的微针贴片的方法，其特征在于，所述浇铸模具具有用于微针成型的微针孔眼，通过抽吸装置将所述微针贴片基质的成型制备液抽吸进入微针孔眼。
PCT/CN2023/072321 2022-01-17 2023-01-16 一种连续监测或检测体内分析物的微针贴片、其制备方法及相关装置 WO2023134762A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210049073.4 2022-01-17
CN202210049073.4A CN114366091B (zh) 2022-01-17 2022-01-17 一种连续监测或检测体内分析物的微针贴片、其制备方法及相关装置

Publications (1)

Publication Number Publication Date
WO2023134762A1 true WO2023134762A1 (zh) 2023-07-20

Family

ID=81144602

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/072321 WO2023134762A1 (zh) 2022-01-17 2023-01-16 一种连续监测或检测体内分析物的微针贴片、其制备方法及相关装置

Country Status (2)

Country Link
CN (1) CN114366091B (zh)
WO (1) WO2023134762A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114366091B (zh) * 2022-01-17 2023-08-22 礼诚(北京)国际生物医药科技有限公司 一种连续监测或检测体内分析物的微针贴片、其制备方法及相关装置

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102202720A (zh) * 2008-10-07 2011-09-28 金拓 相转化聚合物微针
US20160157764A1 (en) * 2014-12-03 2016-06-09 IMAST scarl Microneedle array device and method of making
CN110090044A (zh) * 2019-04-23 2019-08-06 西安交通大学 一种用于采集皮肤间质液的水凝胶微针贴片及其制备方法和使用方法
CN110123340A (zh) * 2019-04-12 2019-08-16 南通纺织丝绸产业技术研究院 丝素蛋白微针电极及其制备和应用
CN110522460A (zh) * 2019-09-29 2019-12-03 清华大学深圳国际研究生院 一种基于透皮微针阵列的多指标检测传感器及制备方法
CN111150407A (zh) * 2020-02-20 2020-05-15 苏州大学 可穿戴血糖仪
WO2021118431A1 (en) * 2019-12-11 2021-06-17 Gaston Adrian Crespo Paravano Methods of modifying microneedles and needles for transdermal electrochemical detection of ions and (bio)molecules
CN113842142A (zh) * 2021-09-27 2021-12-28 上海电机学院 一种穿戴式连续血糖检测装置及方法
CN114366091A (zh) * 2022-01-17 2022-04-19 礼诚(北京)国际生物医药科技有限公司 一种连续监测或检测体内分析物的微针贴片、其制备方法及相关装置

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3416620B1 (en) * 2016-02-15 2024-04-03 Shanghai Jiao Tong University Method to print microneedle patches rapidly
CN109199400B (zh) * 2018-09-10 2021-08-31 中山大学 基于微针阵列的血糖电化学传感器
CN111481470A (zh) * 2020-06-09 2020-08-04 无锡元旭生物技术有限公司 用于可溶性微针的水凝胶贴片及其制备方法
US20230302266A1 (en) * 2020-06-30 2023-09-28 Lg Household & Health Care Ltd. Microneedle comprising microfiber network structure

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102202720A (zh) * 2008-10-07 2011-09-28 金拓 相转化聚合物微针
US20160157764A1 (en) * 2014-12-03 2016-06-09 IMAST scarl Microneedle array device and method of making
CN110123340A (zh) * 2019-04-12 2019-08-16 南通纺织丝绸产业技术研究院 丝素蛋白微针电极及其制备和应用
CN110090044A (zh) * 2019-04-23 2019-08-06 西安交通大学 一种用于采集皮肤间质液的水凝胶微针贴片及其制备方法和使用方法
CN110522460A (zh) * 2019-09-29 2019-12-03 清华大学深圳国际研究生院 一种基于透皮微针阵列的多指标检测传感器及制备方法
WO2021118431A1 (en) * 2019-12-11 2021-06-17 Gaston Adrian Crespo Paravano Methods of modifying microneedles and needles for transdermal electrochemical detection of ions and (bio)molecules
CN111150407A (zh) * 2020-02-20 2020-05-15 苏州大学 可穿戴血糖仪
CN113842142A (zh) * 2021-09-27 2021-12-28 上海电机学院 一种穿戴式连续血糖检测装置及方法
CN114366091A (zh) * 2022-01-17 2022-04-19 礼诚(北京)国际生物医药科技有限公司 一种连续监测或检测体内分析物的微针贴片、其制备方法及相关装置

Also Published As

Publication number Publication date
CN114366091A (zh) 2022-04-19
CN114366091B (zh) 2023-08-22

Similar Documents

Publication Publication Date Title
KR100328654B1 (ko) 바이오센서, 이온영동 샘플링 시스템 및 그것의 사용방법
US8224414B2 (en) System and method for analyte sampling and analysis with hydrogel
AU2005302584B2 (en) System and method for analyte sampling and analysis with hydrogel
US20090312622A1 (en) Device And Method For Determining A Value Of A Physiological Parameter Of A Body Fluid
JP2005514091A (ja) 非侵襲性または最小限侵襲性のモニタリング法
WO2023134762A1 (zh) 一种连续监测或检测体内分析物的微针贴片、其制备方法及相关装置
Zhang et al. Microneedle-assisted technology for minimally invasive medical sensing
CN111956951B (zh) 离子导入微针药贴及制备方法
He et al. Reverse iontophoresis generated by porous microneedles produces an electroosmotic flow for glucose determination
Liu et al. Transdermal amperometric biosensors for continuous glucose monitoring in diabetes
CN113546294A (zh) 一种微针自助检测治疗装置
Liu et al. A microtube-based wearable closed-loop minisystem for diabetes management
CN101274118B (zh) 夹合自粘贴载药膜的理疗电极片及其制备方法
CN100588437C (zh) 夹合自粘贴载药膜的理疗电极片及其制备方法
CN110123340A (zh) 丝素蛋白微针电极及其制备和应用
CN114674885A (zh) 一种柔性可穿戴式汗液葡萄糖电化学传感器的制备方法
CN100588438C (zh) 夹合自粘贴载药膜的理疗电极片及其制备方法
Ghoreishizadeh et al. Continuous Glucose Monitoring Sensors for Management of Diabetes: State-of-the-Art and Future Perspectives
CN113907753B (zh) 一种无创血糖检测电极贴片和反离子电渗体外实验装置
Srineela et al. A Review on Continuous Glucose Monitoring (CGM): Perspectives on Glucose Biosensors
WO2023215131A1 (en) Flexible electronics for analyte detection
CN115969372A (zh) 一种可实现血糖连续监测和控制的血糖监控设备和方法
CN114569124A (zh) 一种微针阵列酶复合电极及其制备方法
CN113974615A (zh) 组织液检测装置及其系统
CN113289237A (zh) 基于离子导入驱动的触控微针阵列诊疗一体化可穿戴设备

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23740103

Country of ref document: EP

Kind code of ref document: A1