A micro biosensor and its preparation method

Abstract

The present invention relates to the field of biosensor technology, and more specifically, to a micro-biosensor and a method for preparing the same. The present invention provides a micro-biosensor that loads a functional bioactive substance onto a designated location on the sensor. A drug-loaded film liquid of a specific viscosity is used to form a drug-loaded layer, i.e., a drug-loaded polymer coating, on the sensor surface. The drug-loaded layer, while not affecting the normal function of the biosensor, enables long-term, effective release of the drug at a specific location. This reduces foreign body reactions caused by implantation of the biosensor in the body, mitigates the problem of decreased sensitivity of the biosensor due to protein accumulation and fiber encapsulation, and effectively extends the sensor's service life.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and in particular to a micro biosensor and a preparation method thereof. Background Art

[0002] Biosensors, through direct contact with tissue fluids such as blood or body fluids, can quickly and sensitively detect relevant bodily indicators. However, implantation of these sensors often triggers a foreign body reaction (FBR) in the host. This is a phenomenon in which, after implantation, the sensor's functionality is compromised through leukocyte reactions, biofouling, and fiber encapsulation. This ultimately leads to decreased sensor sensitivity or device failure, shortening the sensor's lifespan. This FBR impairs sensor performance, limiting its further development and becoming a critical issue that urgently needs to be addressed in the clinical application of implantable (including minimally invasive) medical devices.

[0003] Therefore, biosensors are loaded with bioactive substances that can prevent foreign body reactions, enabling the functional coating to release the bioactive substances at specific tissue sites over a long period of time, thereby extending the lifespan of the biosensor. However, the addition of a drug-loaded coating may affect the sensor's original detection function. For example, in electrochemical continuous glucose monitoring systems, drugs that reduce foreign body reactions are mostly hydrophobic. Polymers that have good compatibility with hydrophobic drugs and achieve optimal sustained drug release are generally hydrophobic materials. However, glucose can only pass through the hydrophilic membrane layer to reach the working electrode, where a series of reactions convert the chemical signal into an electrical signal. The combined application of these two conflicting materials on the same tiny (micrometer-scale diameter) biosensor surface presents significant technical challenges and risks. Therefore, coating the sensor surface with a drug layer without altering the sensor's original electrochemical performance has become a pressing challenge. Summary of the Invention

[0004] The present invention aims to at least partially address one of the technical problems in the related art. To this end, one object of the present invention is to provide a micro-biosensor that utilizes a drug-loaded film solution of a specific viscosity to form a drug-loaded layer (drug-loaded polymer coating) on ​​the sensor surface. This drug-loaded layer, while not affecting the normal function of the biosensor, enables long-term, effective release of the drug at a specific location. This prevents foreign body reactions caused by biosensor implantation, reduces the potential for decreased sensitivity later in the biosensor due to protein accumulation and fiber encapsulation, and effectively extends the sensor's service life.

[0005] To this end, the first aspect of the present invention provides a micro biosensor for continuously monitoring subcutaneous analyte concentration. According to an embodiment of the present invention, the micro biosensor comprises:

[0006] An insulating substrate comprising a first side surface and a second side surface disposed opposite to each other;

[0007] a conductive layer, disposed on the first side surface and / or the second side surface of the insulating substrate;

[0008] an electrode, disposed on a side of the conductive layer away from the insulating substrate, the electrode being coated with a reactant;

[0009] The drug-carrying layer is disposed on the conductive layer and spaced apart from the electrode.

[0010] Wherein, the drug-loaded layer is made of a drug-loaded film liquid with a viscosity of 50 cP-100 cP.

[0011] To extend the lifespan of biosensors, bioactive substances that can prevent foreign body reactions are loaded onto their surfaces, enabling the functional coating to release the bioactive substances over a long period of time at specific tissue locations. However, drug-loading coating solutions often contain volatile organic solvents. Rapid evaporation of these organic solvents or excessive viscosity of the drug-loading layer can easily lead to rapid crusting and wrinkling of the outermost layer, resulting in an uneven surface and even excessive thickness in certain areas of the biosensor, rendering it inoperable and impairing its detection function. The present invention provides a micro-biosensor that utilizes a drug-loading coating solution of a specific viscosity to form a drug-loading layer (drug-loading polymer coating) on ​​the sensor surface. This coating loads the functional bioactive substance to specific locations on the sensor, achieving a long-term, slow release over the sensor's lifespan without affecting the normal function of the biosensor, thereby extending the sensor's lifespan. The viscosity of the drug-loading coating solution directly affects the thickness and uniformity of the dip coating. If the viscosity of the drug-loading coating solution is too low, more coating passes are required to achieve the target film thickness. If the viscosity of the drug-loading coating solution is too high, controlling the film thickness consistency of the drug-loading layer is difficult.

[0012] According to an embodiment of the present invention, the reactant includes any one of an enzyme, an antigen, an antibody or an aptamer.

[0013] According to an embodiment of the present invention, the thickness of the drug-carrying layer is 5 μm-100 μm.

[0014] If the thickness of the drug-loading layer is too small, the release amount of bioactive substances will be insufficient, the release time will be insufficient, and the service life cannot be extended; the thicker the drug-loading layer, the more drug it can carry, but due to the limitation of the inner diameter of the half-wall needle, the thickness of the drug-loading layer does not exceed 100 μm.

[0015] According to a preferred embodiment of the present invention, the thickness of the drug-carrying layer is 20 μm-60 μm.

[0016] According to an embodiment of the present invention, the drug-loaded film solution includes a bioactive substance, a carrier material and a solvent.

[0017] The mass ratio of the bioactive substance to the carrier material is 1:(1-9); or

[0018] The mass fraction of the sum of the mass of the bioactive substance and the carrier material in the drug-loaded membrane liquid is 1%-20%.

[0019] When the mass ratio of the bioactive substance to the carrier material is 1:(1-9), the release amount and release time of the bioactive substance meet the requirements, thereby extending the service life of the micro-biosensor. If the bioactive substance content is too low, the loading capacity is too small at the same thickness, resulting in insufficient bioactive substance release amount and release time, and the service life cannot be extended. If the bioactive substance content is too high and the carrier material content is too low, the quality (uniformity and smoothness) of the formed drug-loading layer will be affected, and the bioactive substance will be wasted. According to a preferred embodiment of the present invention, the combined mass of the bioactive substance and the carrier material in the drug-loading film solution is 5%-10%.

[0020] According to an embodiment of the present invention, the carrier material includes at least one of natural polysaccharide materials (such as chitosan), natural polypeptide materials, bioglass, bioceramics, aliphatic polyesters, aliphatic polycarbonates, polyanhydrides, polyorthoesters, polyurethanes, and hydroxypropyl methylcellulose;

[0021] The bioactive substance includes at least one of glucocorticoid drugs, aspirin drugs, growth factors, cytokines, and nitric oxide;

[0022] The solvent includes at least one of ethanol, tetrahydrofuran and water.

[0023] According to an embodiment of the present invention, the glucocorticoid drugs include dexamethasone drugs;

[0024] The solvent is a mixture of anhydrous ethanol and tetrahydrofuran.

[0025] According to an embodiment of the present invention, the dexamethasone-based drug includes at least one of dexamethasone acetate, dexamethasone, dexamethasone phosphate and dexamethasone sodium phosphate;

[0026] In the solvent mixture, the volume ratio of anhydrous ethanol to tetrahydrofuran is 1:9-9:1.

[0027] The second aspect of the present invention provides a method for preparing the micro biosensor for continuously monitoring subcutaneous analyte concentration according to the first aspect. According to an embodiment of the present invention, the method comprises:

[0028] The drug-loaded film liquid is coated on the surface of the conductive layer of the micro biosensor by at least one of dipping, brushing, spot coating or spraying.

[0029] The present invention provides a method for preparing a micro-biosensor. Bioactive substances are loaded onto a high molecular weight polymer to form a uniform drug-loading layer on the biosensor. The drug-loading layer, while not affecting the normal function of the biosensor, enables long-term, effective release at specific locations. This prevents foreign body reactions caused by biosensor implantation, reduces the problem of decreased sensitivity due to protein accumulation and fiber encapsulation, and effectively extends the sensor's service life. The drug in the drug-loading layer can achieve safe, effective, and sustained release of the drug in the body for up to 21 days, reducing foreign body reactions and enabling longer-term, stable detection in the micro-biosensor compared to unloaded sensors.

[0030] According to an embodiment of the present invention, the electrode comprises a working electrode,

[0031] When the drug-carrying layer is further away from the end of the implantation section of the micro-biosensor than the working electrode, the drug-carrying film liquid is applied by spraying, spot coating or brushing;

[0032] When the drug-loaded layer is closer to the end of the implanted section of the micro-biosensor than the working electrode, the drug-loaded film liquid is coated by dipping;

[0033] When the drug-carrying layer is located on both sides of the working electrode, the drug-carrying film liquid is coated by combining dipping and spraying.

[0034] The present invention applies a polymer coating loaded with functional substances to a biosensor. By regulating the loading position and method, the polymer coating is added to ensure long-term, effective release at specific locations without affecting the normal function of the biosensor. This can avoid foreign body reactions caused by biosensor implantation, reduce the problem of protein accumulation and fiber encapsulation that can lead to decreased sensitivity in the biosensor later in life, and effectively extend the sensor's service life. By regulating the sensor drug-loaded coating process, the present invention provides a method for extending the sensor's service life by spatially separating and dividing functional areas, extending the sensor's life to 21 days or even longer.

[0035] According to an embodiment of the present invention, when the drug-loaded film liquid is coated by dipping, the parameters are set as follows:

[0036] Dipping time is 0.5 s -3 s;

[0037] The speed of the micro biosensor without drug-loaded layer immersed in the drug-loaded film solution was 500 mm / s-1500 mm / s, and the speed of leaving the drug-loaded film solution was 50 mm / s-200 mm / s;

[0038] The number of dipping passes is 2-10 times;

[0039] Dip coating thickness is 5 μm-100 μm;

[0040] The depth of immersion of the micro biosensor without drug loading layer into the drug loading film solution is 1 mm -2 mm.

[0041] According to an embodiment of the present invention, when spraying is used to apply the drug-loaded film liquid, the parameters are set as follows:

[0042] Molding air valve pressure: 0.01 psi -0.5 psi;

[0043] The speed of the nozzle relative to the micro biosensor is 1 mm / s-10 mm / s.

[0044] The speed of spraying the drug-loaded film liquid from the nozzle is: 0.05 mL / min -1 mL / min;

[0045] The number of spraying times is 2-50 times;

[0046] The spraying thickness is 5 μm-100 μm.

[0047] Within the above-mentioned specific coating parameter range, the uniformity of the drug-loaded layer on the sensor surface can be further improved, thereby extending the life of the sensor.

[0048] The third aspect of the present invention provides a use of the micro biosensor described in the first aspect. According to an embodiment of the present invention, the micro biosensor is implanted subcutaneously in a user to continuously monitor physiological indicators in the user's blood or tissue fluid.

[0049] According to an embodiment of the present invention, the physiological indicators include blood sugar, blood oxygen, ketones, cholesterol, lactic acid, and temperature.

[0050] Compared with the prior art, the present invention has the following advantages:

[0051] (1) The present invention mixes the bioactive substance with the polymer by a blending method and coats it at a specific position of the biosensor using different processes. The operation is simple and efficient, the coating is smooth and uniform, and the coating thickness is controllable;

[0052] (2) The bioactive substances used in the present invention can be continuously and slowly released during the wearing period of the biosensor, achieving an ideal controlled release effect;

[0053] (3) The present invention can effectively avoid biofouling and fiber encapsulation caused by the host's foreign body reaction after the biosensor is implanted in the body, improve the problem of decreased sensitivity of the biosensor in the later stage of use, and thus extend the service life of the sensor to 21 days or even longer.

[0054] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0056] Figure 1 The partial structure of the drug-loaded sensor 1# prepared by the dip coating process in Example 1 is shown;

[0057] Figure 2 The partial structure of the drug-loaded sensor 2# prepared by the spraying process in Example 2 is shown;

[0058] Figure 3 (a) shows the drug loading capacity of drug-loaded sensor 2# prepared by spray coating process; (b) shows the drug loading capacity of drug-loaded sensor 1# prepared by dip coating process. Five sensor samples were taken from each batch for testing.

[0059] Figure 4 (a) shows the in vitro drug release rate of drug-loaded sensor 2# prepared by the spray coating process; (b) shows the in vitro drug release rate of drug-loaded sensor 1# prepared by the dip coating process. Each line in the figure represents the drug release result of a sensor, which are parallel drug-loaded sensor samples.

[0060] Figure 5 (a) shows the in vitro cumulative drug release of drug-loaded sensor 2# prepared by the spray coating process; (b) shows the in vitro cumulative drug release of drug-loaded sensor 1# prepared by the dip coating process;

[0061] Figure 6 The survival curves of the conventional sensor and the drug-loaded sensor 2# during wearing are shown. CT5-DL in the figure represents the drug-loaded sensor, and the conventional CT5 represents the non-drug-loaded sensor.

[0062] Figure 7 Shown are (a) the working electrode surface of a conventional non-drug-loaded sensor and (b) the working electrode surface of the drug-loaded sensor of the present invention after 21 days of clinical wearing by a subject;

[0063] Figure 8Shown are (a) scanning electron micrographs of the working electrode surface of a conventional non-drug-loaded sensor and (b) scanning electron micrographs of the working electrode surface of the drug-loaded sensor of the present invention after 21 days of clinical wearing by a subject;

[0064] Figure 9 The current graphs of the conventional sensor and the drug-loaded sensor are shown after the subjects wore them for 21 days. The current data of the two sensors in the figure are from the same subject. The CT4-DL is the drug-loaded sensor of the present invention, and the conventional CT4 is a non-drug-loaded sensor.

[0065] Figure 10 A cross-sectional view of the drug-loaded sensor with wrinkled surface prepared in Comparative Example 1 is shown in FIG. DETAILED DESCRIPTION

[0066] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0067] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0068] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0069] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present invention belongs.

[0070] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0071] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0072] The present invention addresses the shortcomings of existing technologies and provides a method for maintaining the sensitivity of implantable (including minimally invasive) biosensors during late-life use and extending their service life. A bioactive substance / material mixture is prepared by blending and used as a matrix material for the sensor's drug-loaded coating. By modifying the processing technique, the drug-loaded coating is successfully applied to the biosensor's non-working electrode region. By further optimizing the polymer type, polymer concentration, and bioactive substance ratio, the release rate of the bioactive substance is regulated, achieving continuous and slow release throughout the sensor's lifespan, thereby extending the biosensor's in vivo service life.

[0073] According to a specific embodiment of the present invention, the present invention provides a micro biosensor for continuously monitoring subcutaneous analyte concentration, comprising:

[0074] An insulating substrate comprising a first side surface and a second side surface disposed opposite to each other;

[0075] a conductive layer, disposed on the first side surface and / or the second side surface of the insulating substrate;

[0076] an electrode, disposed on a side of the conductive layer away from the insulating substrate, the electrode being coated with a reactant;

[0077] The drug-carrying layer is disposed on the conductive layer and spaced apart from the electrode.

[0078] Wherein, the drug-loaded layer is made of a drug-loaded film liquid with a viscosity of 50 cP-100 cP.

[0079] According to a specific embodiment of the present invention, the drug-loading layer is made of a drug-loading film liquid having a viscosity of any value within the range of 50 cP to 100 cP. For example, the drug-loading layer is made of a drug-loading film liquid having a viscosity of 50 cP, 55 cP, 60 cP, 65 cP, 70 cP, 75 cP, 80 cP, 85 cP, 90 cP, 95 cP, or 100 cP.

[0080] According to a specific embodiment of the present invention, the conductive layer of the micro-biosensor provided herein includes three electrodes: a working electrode, a counter electrode, and a reference electrode, to ensure detection accuracy and stability. The working electrode comprises an electrode base material and a modified layer. The surface of the working electrode of the micro-biosensor provided herein is coated with a substance that reacts with the analyte (i.e., the modified layer). Based on the contact reaction between the analyte and the substance that reacts with the analyte, the chemical signal is converted into an electrical signal, thereby enabling continuous monitoring of physiological indicators in blood or tissue fluid.

[0081] According to a specific embodiment of the present invention, the reactant includes any one of an enzyme, an antigen, an antibody or an aptamer.

[0082] According to a specific embodiment of the present invention, when the analyte is blood glucose, glucose oxidase or glucose dehydrogenase is coated on the electrode surface of the micro-biosensor. When the micro-biosensor of the present invention is implanted subcutaneously in a subject, the glucose oxidase can convert the glucose in the interstitial fluid into hydrogen peroxide, and then the electrode surface is oxidized, generating a current signal proportional to the blood glucose concentration, thereby realizing real-time continuous monitoring.

[0083] According to a specific embodiment of the present invention, when the analyte is a protein or peptide, a detection antibody targeting the protein or peptide is coated on the electrode surface of the microbiosensor. However, since the antibody-antigen binding itself does not generate electroactive products or electron transfer that can be detected by the electrode, a simple antibody coating cannot achieve the conversion of chemical signals to electrical signals. Therefore, a "transduction" step must be introduced, using a labeled enzyme (such as horseradish peroxidase (HRP)) or nanomaterials to indirectly generate electrical signals.

[0084] According to a specific embodiment of the present invention, when the analyte is some small molecules or ions, an aptamer that binds to the small molecules or ions can be coated on the electrode surface of the micro-biosensor. The conformational change of the aptamer before and after binding to the target object triggers a change in the electrochemical signal, thereby realizing the detection of the target object.

[0085] According to a specific embodiment of the present invention, the thickness of the drug-carrying layer is 5 μm-100 μm.

[0086] It should be noted that the thickness of the drug-carrying layer is any value within the range of 5-100 μm, for example, it can be 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, etc.

[0087] According to a preferred embodiment of the present invention, the thickness of the drug-carrying layer is 20 μm-60 μm.

[0088] According to a specific embodiment of the present invention, the drug-loaded film solution includes a bioactive substance, a carrier material and a solvent.

[0089] According to a specific embodiment of the present invention, the mass ratio of the bioactive substance to the carrier material is 1:(1-9).

[0090] For example, the mass ratio of the bioactive substance to the carrier material is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, etc.

[0091] Due to the small size of the micro-biosensor presented herein, the area available for drug loading is limited, and its thickness is constrained by the diameter of the semi-wall needle. Therefore, to maximize drug loading within this limited volume, the drug-to-polymer ratio in the drug layer must be controlled within an appropriate range. Furthermore, as the primary contributor to the mechanical strength of the drug-loading layer and the drug-loading layer, the polymer mass ratio should be greater than 50%. Taking these two factors into account, the present invention has developed a bioactive substance to carrier material mass ratio of 1:(1-9). Under this condition, the drug and polymer are completely miscible. Subsequent clinical trials and in vitro drug release experiments demonstrated excellent mechanical properties of the drug-loading layer. Furthermore, drug residue was still detectable in the drug-loaded sensor after 21 days of continuous wear, indicating that sufficient drug loading was achieved.

[0092] According to a more preferred embodiment of the present invention, the mass ratio of the bioactive substance to the carrier material is 1:1.

[0093] According to a specific embodiment of the present invention, the mass fraction of the sum of the mass of the bioactive substance and the carrier material in the drug-loaded film solution is 1%-20%.

[0094] For example, the mass fraction of the sum of the mass of the biologically active substance and the carrier material in the drug-loaded film liquid is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, etc.

[0095] According to a preferred embodiment of the present invention, the mass fraction of the sum of the mass of the bioactive substance and the carrier material in the drug-loaded film solution is 5%-10%.

[0096] According to a specific embodiment of the present invention, the carrier material can be a hydrophobic or non-hydrophobic material, or a polar or non-polar material.

[0097] According to a specific embodiment of the present invention, the carrier material includes at least one selected from natural polysaccharide materials, natural polypeptide materials, bioglass, bioceramics, aliphatic polyesters (APs), aliphatic polycarbonates (APCs), polyanhydrides (PAs), polyorthoesters (POEs), polyurethanes (PUs), and hydroxypropyl methylcellulose.

[0098] It should be noted that modified polysaccharide materials, polypeptide materials, bioglass, bioceramics, etc. can all be used as carrier materials of the present invention and used to be mixed with biologically active substances to prepare drug-loaded membrane liquids, and are also covered by the protection scope of the present invention.

[0099] According to a specific embodiment of the present invention, natural polysaccharide materials include hyaluronic acid (HA), alginate (Alg), and chitosan (CS), while natural polypeptide materials include fibrin, collagen, or silk fibroin. Aliphatic polyesters include polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), and polycaprolactone (PCL). Aliphatic polycarbonates include polyethylene carbonate (PEC), polypropylene carbonate (PPC), and polytrimethylene carbonate (PTMC). All of these materials can be used as drug-loaded polymer materials, either alone or in blends or copolymers.

[0100] According to a specific embodiment of the present invention, the bioactive substance includes at least one of glucocorticoid drugs, aspirin drugs, growth factors, cytokines, and nitric oxide.

[0101] According to a specific embodiment of the present invention, in the micro biosensor provided by the present invention, the bioactive substance contained in the drug-loaded membrane liquid is a steroidal anti-inflammatory drug or a non-steroidal anti-inflammatory drug.

[0102] According to a specific embodiment of the present invention, the bioactive substances contained in the drug-loaded membrane liquid include but are not limited to glucocorticoids, non-steroidal anti-inflammatory drugs (aspirin, etc.), growth factors (such as vascular endothelial growth factor (VEGF)), cytokines, nitric oxide, etc. These bioactive substances can be mixed with carrier materials alone in a solvent to prepare the drug-loaded membrane liquid, or multiple bioactive substances can be blended in the drug-loaded membrane liquid, all of which are covered within the scope of protection of the present invention.

[0103] According to a specific embodiment of the present invention, the solvent includes at least one of ethanol, tetrahydrofuran, and water.

[0104] It should be noted that, in the micro biosensor provided by the present invention, the solvent contained in the drug-loaded membrane liquid includes but is not limited to at least one of ethanol, tetrahydrofuran, and water.

[0105] According to a preferred embodiment of the present invention, the solvent is a mixture of anhydrous ethanol and tetrahydrofuran.

[0106] According to a preferred embodiment of the present invention, the glucocorticoid drugs include dexamethasone drugs.

[0107] It should be noted that in the micro-biosensor provided by the present invention, the bioactive substance contained in the drug-loaded membrane liquid is a glucocorticoid drug, and the glucocorticoid drug includes but is not limited to a dexamethasone drug, and the dexamethasone drug includes at least one of dexamethasone acetate, dexamethasone, dexamethasone phosphate and dexamethasone sodium phosphate. Other dexamethasone drugs or pharmaceutically acceptable salts thereof not listed are all covered within the scope of protection of the present invention.

[0108] According to a preferred embodiment of the present invention, the carrier material is a hydrophobic polymer (such as polyurethane (PUs)), and the bioactive substance is a hydrophobic drug (such as dexamethasone). By loading the hydrophobic drug onto the hydrophobic polymer, a drug-loading layer is formed on the biosensor, thereby obtaining an implantable micro biosensor with a longer service life.

[0109] According to a preferred embodiment of the present invention, when the solvent is a mixture of anhydrous ethanol and tetrahydrofuran, the volume ratio of anhydrous ethanol to tetrahydrofuran in the solvent mixture is 1:9-9:1.

[0110] For example, when the solvent is a mixture of anhydrous ethanol and tetrahydrofuran, the volume ratio of anhydrous ethanol to tetrahydrofuran in the solvent mixture is 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, etc.

[0111] According to a specific embodiment of the present invention, the present invention also provides a method for preparing the aforementioned micro-biosensor for continuously monitoring subcutaneous analyte concentration, comprising:

[0112] The drug-loaded film liquid is coated on the surface of the conductive layer of the micro biosensor by at least one of dipping, brushing, spot coating or spraying.

[0113] The preparation method provided by the present invention dissolves specific bioactive substances and carrier materials in a solvent to form a drug-loaded film liquid, and then coats the drug-loaded film liquid on the surface of the conductive layer of the micro-biosensor by at least one of dipping, brushing, spot coating or spraying. The micro-biosensor prepared in this way can achieve safe and effective sustained release of drugs in the body for up to 21 days, reduce foreign body reactions, and achieve longer-term stable detection.

[0114] It should be noted that there is no particular limitation on the mixing method used when the bioactive substance and the carrier material are dissolved in the solvent. For example, stirring, ultrasound or vortexing can be used until the mixture solution becomes transparent.

[0115] According to a specific embodiment of the present invention, the electrode comprises a working electrode,

[0116] When the drug-carrying layer is further away from the end of the implantation section of the micro-biosensor than the working electrode, the drug-carrying film liquid is applied by spraying, spot coating or brushing;

[0117] When the drug-loaded layer is closer to the end of the implanted section of the micro-biosensor than the working electrode, the drug-loaded film liquid is coated by dipping;

[0118] When the drug-carrying layer is located on both sides of the working electrode, the drug-carrying film liquid is coated by combining dipping and spraying.

[0119] According to a specific embodiment of the present invention, when the drug-loaded film liquid is coated by dipping, the parameters are set as follows:

[0120] Dipping time is 0.5 s -3 s;

[0121] The speed of the micro biosensor without drug-loaded layer immersed in the drug-loaded film solution was 500 mm / s-1500 mm / s, and the speed of leaving the drug-loaded film solution was 50 mm / s-200 mm / s;

[0122] The number of dipping passes is 2-10 times;

[0123] Dip coating thickness is 5 μm -100 μm;

[0124] The depth of immersion of the micro biosensor without drug loading layer into the drug loading film solution is 1 mm -2 mm.

[0125] According to a specific embodiment of the present invention, when spraying is used to apply the drug-loaded film liquid, the parameters are set as follows:

[0126] Molding air valve pressure: 0.01 psi -0.5 psi;

[0127] The speed of the nozzle relative to the micro biosensor is 1 mm / s-10 mm / s.

[0128] The speed of spraying the drug-loaded film liquid from the nozzle is: 0.05 mL / min -1 mL / min;

[0129] The number of spraying times is 2-50 times;

[0130] The spraying thickness is 5 μm-100 μm.

[0131] The present invention also provides a use of the aforementioned micro biosensor, wherein the micro biosensor can be implanted subcutaneously in a user to continuously monitor physiological indicators in the user's blood or tissue fluid.

[0132] It should be noted that the physiological indicators include all physiological indicators in the blood or tissue fluid of the user that need to be detected in the medical field. Based on different detection purposes, different reactants need to be coated on the electrodes.

[0133] According to a specific embodiment of the present invention, the physiological indicators include blood glucose, blood oxygen, ketones, cholesterol, lactic acid, and temperature.

[0134] According to a specific embodiment of the present invention, the micro biosensor includes an implantable biosensor such as a continuous glucose monitoring system (CGM) and a continuous ketone monitoring system (CKM) that is in direct contact with blood or tissue fluid.

[0135] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0136] Example 1 A polyurethane-based drug-loaded sensor prepared using a dip coating process 1#

[0137] When the drug-carrying layer is closer to the end of the implantable segment of the microbiosensor than the working electrode, it is applied by dip coating. A 55% mass fraction of dexamethasone acetate is added to a polyurethane solution and mixed thoroughly to obtain a first solution. Before dip coating, the first solution is diluted with a mixture of tetrahydrofuran and anhydrous ethanol (1:9 by volume) to obtain a second solution. In this second solution, the mass ratio of dexamethasone acetate to polyurethane is 1:1, and the combined mass fraction of dexamethasone acetate and polyurethane in the second solution is 5%. The viscosity of the second solution is 50 cp. The second solution is placed in a polytetrafluoroethylene mold for sensor dip coating.

[0138] The drug-loaded coating depth and thickness were controlled by adjusting the second solution, the sensor's descent height, the sensor's descent speed, and the dwell time. Specifically, at room temperature, with each descent lasting 1 s, a sensor's ascent speed of 100 mm / s, and a sensor's descent speed of 900 mm / s, six coating passes, and a coating depth of 2 mm, a drug-loaded coating with a thickness of 30 μm was achieved. Figure 1The figure shows the sensor tip prepared by dip coating. It can be observed that there is a clear boundary line of the drug-loaded layer on the dip-coated tip.

[0139] Example 2: A polyurethane-based drug-loaded sensor prepared using a spray coating process 2#

[0140] When the drug-carrying layer is further away from the end of the implantable segment of the microbiosensor than the working electrode, the drug-carrying layer is applied by spraying or spot coating, and the drug-carrying layer is applied to the upper or back surface of the sensor. A 55% mass fraction of dexamethasone acetate is added to the polyurethane solution and mixed thoroughly to obtain a first solution. Before spraying, the first solution is diluted with a mixture of tetrahydrofuran and anhydrous ethanol (9:1 by volume) to obtain a second solution. In the second solution, the mass ratio of dexamethasone acetate to polyurethane is 9:11, and the combined mass fraction of dexamethasone acetate and polyurethane in the second solution is 10%. The viscosity of the second solution is 60 cp.

[0141] The spraying area and thickness of the drug-loaded coating were controlled by controlling the spraying speed, number of spray passes, and spray trajectory. Specifically, the spraying parameters were as follows: forming air valve pressure: 0.01 psi, nozzle movement speed relative to the biosensor: 2 mm / s, nozzle discharge rate: 0.1 mL / min, and 20 spray passes to obtain a drug-loaded coating thickness of 20 μm. Figure 2 The figure shows the sensor surface prepared by spray coating, where a clear boundary line of the drug-loaded layer can be observed.

[0142] Example 3: A polyurethane-based drug-loaded sensor prepared using a spray / dip coating process 3#

[0143] When the drug-loading layer is located on both sides of the working electrode, the drug coating is applied using a combination of dip-coating and spray-coating. The sensor drug coating can be achieved by applying a single drug or multiple drugs simultaneously. For this type of sensor, single or multiple drug-loading regions can be used to achieve functional effects.

[0144] Aspirin (5% by mass) was sprayed onto the polyurethane solution above and behind the working electrode. After mixing thoroughly, a first solution was obtained. Before spraying, the first solution was diluted with a mixture of tetrahydrofuran and anhydrous ethanol (1:1 by volume) to obtain a second solution. The mass ratio of aspirin to polyurethane in the second solution was 2:3, and the combined mass fraction of aspirin and polyurethane in the second solution was 1%. The viscosity of the second solution was 80 cp.

[0145] The spraying area and thickness of the drug-loaded coating were controlled by controlling the spraying speed, number of spray passes, and spray trajectory. Specifically, the spraying parameters were as follows: forming air valve pressure: 0.1 psi, nozzle movement speed relative to the biosensor: 5 mm / s, nozzle discharge rate: 0.5 mL / min, and 15 spray passes to obtain a drug-loaded coating thickness of 15 μm.

[0146] For the area below the working electrode, a 55% mass fraction of dexamethasone acetate was added to the polyurethane solution by dip coating. After mixing thoroughly, the first solution was obtained. Before dip coating, the first solution was diluted with a mixture of tetrahydrofuran and anhydrous ethanol (1:5 by volume) to obtain a second solution. In this second solution, the mass ratio of dexamethasone acetate to polyurethane was 1:9, and the combined mass fraction of dexamethasone acetate and polyurethane in the second solution was 20%. The second solution was placed in a polytetrafluoroethylene mold and dip-coated onto the sensor. The viscosity of the second solution was 100 cp.

[0147] The coating depth and thickness were controlled by adjusting the second solution, the sensor's descent height, the sensor's descent speed, and the dwell time. At room temperature, with a 1-second dip time per pass, a sensor's ascent speed of 100 mm / s, and a descent speed of 1000 mm / s, three coating passes were performed, and the coating depth was 2 mm, resulting in a 15 μm-thick coating.

[0148] Example 4: A cellulose-based drug-loaded sensor prepared using a spray / dip coating process 4#

[0149] The preparation steps of a cellulose-based drug-loaded sensor 4# prepared using a spray / dip coating process are the same as those in Example 3, except that the drug-loaded polymer polyurethane is replaced with hydroxypropyl methylcellulose, and the selected diluent is ethanol and water (volume ratio of 1:9). The remaining steps are the same as those in Example 3.

[0150] The inventors conducted microscopic observations on the drug-loaded cross-sections of the drug-loaded sensors 1#, 2#, 3#, and 4# prepared in Examples 1-4 and found that the drug-loaded layers had no wrinkles, a uniform texture, and a smooth surface.

[0151] Experimental example:

[0152] The drug-loaded sensors 1# and 2# prepared in Examples 1 and 2 were subjected to sensor drug loading test, in vitro drug release rate test, and in vivo clinical wearing test, respectively. The test methods are as follows:

[0153] Drug loading capacity of the sensor: Place the drug-loaded sensor in a 1.5 mL centrifuge tube, add 1 mL of acetonitrile / water mixed solvent as the extraction solution, perform water bath ultrasonic extraction for 30 min, and detect the drug loading capacity. The results are as follows: Figure 3 As shown, (a) shows the drug loading capacity of drug-loaded sensor 2# prepared by spray coating process; (b) shows the drug loading capacity of drug-loaded sensor 1# prepared by dip coating process. The results show that both drug-loaded layer coating processes meet the predetermined target of drug loading capacity ≥5 μg / sensor.

[0154] In vitro drug release rate: Drug-loaded sensors prepared using both dip-coating and spray-coating processes were placed in a release medium (phosphate buffer) to verify in vitro drug release experiments. After 8, 24, 48, 72, 120, 168, 216, 264, 312, and 360 hours, the sensors were placed in new release medium. The prepared biosensor drug coating can achieve sustained and effective release during the use period. The in vitro cumulative drug release rate is as follows: Figure 4 As shown, (a) shows the in vitro drug release rate of drug-loaded sensor 2# prepared by spray coating process; (b) shows the in vitro drug release rate of drug-loaded sensor 1# prepared by dip coating process. The cumulative amount of drug released in vitro is shown in Figure 5 As shown, (a) shows the in vitro drug release of drug-loaded sensor 2# prepared by spray coating process; (b) shows the in vitro drug release of drug-loaded sensor 1# prepared by dip coating process, indicating that both spray coating or dip coating drug-loaded layer can make the drug release in a sustained manner within a 14-day period, and on the last day of drug release, the drug release rate is about 80%.

[0155] The calculation formula for the cumulative drug release is as follows:

[0156]

[0157] Where V is the volume of the test solution and C is the concentration of the test solution.

[0158] In vivo clinical wearing test: compared with conventional non-drug-loaded sensors prepared by the same process (Example 1), the purpose is to evaluate whether the current of the drug-loaded sensor is maintained at a high level in the later stage of wearing, specifically whether there is no attenuation or the attenuation is alleviated compared with the control group. We made survival curves of conventional sensors and drug-loaded sensors during the wearing process of the subjects, and used these curves for quantitative analysis. Figure 6As shown in the figure, the optimal Ks of each sensor for 3-7 days was considered 1, and the Ks (IW / BG) of each sensor was normalized. Sensors with a daily average Ks less than 80% after normalization for two consecutive days of wearing were considered dead. The results show that on the 16th day of wearing, 97.5% of the drug-loaded sensors survived, while the survival rate of the non-drug-loaded sensors was only 50%. In addition, the sensors were microscopically observed after the subjects wore them for 21 days to explore the cause of their current decay. The results are shown in the figure. Figure 7 、 8 As shown, the working electrode of the drug-loaded sensor of the present invention is smooth and has no obvious biofouling, while the working electrode of the conventional sensor without drug loading is not smooth and has obvious biofouling. Figure 9 The current diagrams of conventional sensors and drug-loaded sensors worn by subjects for 21 days are shown. The results show that the current of the drug-layer-loaded sensor provided by the present invention remains at a high level after 21 days of wear, with no attenuation observed. However, the current signal of the conventional sensor shows significant attenuation after 15 days of wear.

[0159] Comparative Example 1:

[0160] The preparation process of the drug-loaded sensor is the same as that of Example 1, except that the drug-loaded membrane solution with a viscosity of 110 cp (polyurethane viscosity is 110 cp) is dip-coated three times. Figure 10 As shown, it was found that the thickness of the obtained drug loading layer was not uniform.

[0161] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", "some implementation plans" or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0162] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A micro biosensor for continuous monitoring of subcutaneous analyte concentration, characterized in that include: An insulating substrate comprising a first side surface and a second side surface disposed opposite to each other; a conductive layer, disposed on the first side surface and / or the second side surface of the insulating substrate; an electrode, disposed on a side of the conductive layer away from the insulating substrate, the electrode being coated with a reactant; The drug-carrying layer is disposed on the conductive layer and spaced apart from the electrode. Wherein, the drug-loaded layer is made of a drug-loaded film liquid with a viscosity of 50 cP-100 cP. The drug-loaded membrane solution includes a bioactive substance, a carrier material and a solvent. The carrier material includes at least one of natural polysaccharide materials, natural polypeptide materials, bioglass, bioceramics, aliphatic polyesters, aliphatic polycarbonates, polyanhydrides, polyorthoesters, polyurethanes, and hydroxypropyl methylcellulose; The bioactive substance includes at least one of glucocorticoid drugs, aspirin drugs, growth factors, cytokines, and nitric oxide; The solvent includes at least one of ethanol, tetrahydrofuran and water.

2. The micro biosensor according to claim 1, wherein The reactant includes any one of an enzyme, an antigen, an antibody or an aptamer.

3. The micro biosensor according to claim 1, wherein The thickness of the drug-carrying layer is 5 μm-100 μm.

4. The micro biosensor according to claim 1, wherein The mass ratio of the bioactive substance to the carrier material is 1:(1-9); or The mass fraction of the sum of the mass of the bioactive substance and the carrier material in the drug-loaded membrane liquid is 1%-20%.

5. The micro biosensor according to claim 1, wherein The glucocorticoid drugs include dexamethasone drugs; The solvent is a mixture of anhydrous ethanol and tetrahydrofuran.

6. The micro biosensor according to claim 5, characterized in that The dexamethasone-based drug includes at least one of dexamethasone acetate, dexamethasone, dexamethasone phosphate and dexamethasone sodium phosphate; In the solvent mixture, the volume ratio of anhydrous ethanol to tetrahydrofuran is 1:9-9:

1.

7. The method for preparing a micro biosensor for continuously monitoring subcutaneous analyte concentration according to any one of claims 1 to 6, characterized in that: include: The drug-loaded film liquid is coated on the surface of the conductive layer of the micro biosensor by at least one of dipping, brushing, spot coating or spraying.

8. The preparation method according to claim 7, characterized in that The electrodes include a working electrode, When the drug-carrying layer is further away from the end of the implantation section of the micro-biosensor than the working electrode, the drug-carrying film liquid is applied by spraying, spot coating or brushing; When the drug-loaded layer is closer to the end of the implanted section of the micro-biosensor than the working electrode, the drug-loaded film liquid is coated by dipping; When the drug-carrying layer is located on both sides of the working electrode, the drug-carrying film liquid is coated by combining dipping and spraying.

9. The preparation method according to claim 8, characterized in that When applying the drug-loaded film liquid by dipping, set the parameters as follows: Dipping time is 0.5 s -3 s; The speed of the micro biosensor without drug-loaded layer immersed in the drug-loaded film solution was 500 mm / s-1500 mm / s, and the speed of leaving the drug-loaded film solution was 50 mm / s-200 mm / s; The number of dipping passes is 2-10 times; Dip coating thickness is 5 μm-100 μm; The depth of immersion of the micro biosensor without drug loading layer into the drug loading film liquid is 1mm-2mm.

10. The preparation method according to claim 8, characterized in that When spraying the drug-loaded film, set the parameters as follows: Molding air valve pressure: 0.01 psi -0.5 psi; The speed of the nozzle relative to the micro biosensor is 1 mm / s-10 mm / s. The speed of spraying the drug-loaded film liquid from the nozzle is: 0.05 mL / min -1 mL / min; The number of spraying times is 2-50 times; The spraying thickness is 5 μm-100 μm.

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