A zwitterionic polymer for implantable biosensors, its preparation method and application

Abstract

This invention provides an amphoteric polymer for implantable biosensors and its preparation method. An amphoteric polymer precursor material is prepared by copolymerizing a siloxane monomer, two amphoteric compound precursors, a monomer for anchoring the probe surface, and a soft monomer. This yields an amphoteric polymer precursor material that improves the oxygen permeability and selective permeability of the coating, while also controlling the polymer's hardness, resulting in better bending resistance, reduced detachment, and improved biocompatibility. Coating the surface of the implantable biosensor with this amphoteric polymer precursor material to form a dense film significantly enhances the detection sensitivity of the implantable biosensor and effectively extends its effective detection time.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biocompatible selective permeable membrane, in particular, to a surface chemical coating treatment of an implantable biosensor, and more particularly to a zwitterionic polymer precursor material for an implantable biosensor and a preparation method and application thereof. BACKGROUND

[0002] The implantable biosensor refers to a sensor device which can be partially or wholly implanted into a human body, and can continuously measure important physiological and pathological parameters such as blood oxygen, blood glucose, virus antibody, lactate, cholesterol, bilirubin and amino acid, and more directly reflect the changes of the signs of the measured object due to environmental changes.

[0003] For the implantable sensor, the biocompatibility of the device is the primary problem. The rejection reaction of the organism to the foreign body starts from the moment of implantation. First, the protein is non-specifically adsorbed and wrapped on the surface of the electrode. Then, the cells are attracted by the protein and form a fibrous wrapping in the later stage. For the implantable sensor for detecting chemical signals (such as blood glucose, lactate, potassium ions, etc.), the reaction of the organism should not block the diffusion of the detected substance. Therefore, the stability of the local environment of the subcutaneous tissue directly affects the stability of the implantable sensor.

[0004] In the prior art, most of the glucose sensors include a high molecular material inner membrane layer, an enzyme membrane layer and a high molecular outer membrane layer, such as patents CN101530328 / CN201398971 / CN102243208A / CN101530327, etc. The high molecular diffusion layer mostly uses polyvinyl alcohol, polyethylene glycol, polyurethane or polytetrafluoroethylene, etc. However, these materials have insufficient anti-protein adsorption capacity in the body, and the biocompatibility problem still exists. At the same time, there are problems such as poor anti-bending capacity in the body, easy peeling of the membrane layer, etc., which limit the sensor to realize an effective detection time longer than 14 days. Therefore, how to improve the anti-protein adsorption capacity of the membrane layer, reduce the foreign body rejection reaction, improve the biocompatibility of the sensor, and at the same time improve the anti-bending capacity, make it more flexible, not easy to peel off, and reduce the foreign body rejection reaction, improve the biocompatibility of the sensor, is the key to improve the detection accuracy and service life of the sensor.

[0005] Biocompatibility, anti-protein adsorption capacity, cytotoxicity, appropriate permeation and diffusion performance for target analytes, barrier performance for potential interferents, anti-bending capacity, and resistance to hydrolysis, heat and other degradation mechanisms are major challenges faced by high molecular outer layer materials. In addition, due to the requirements of production, transportation and storage, the material also needs to have a stable chemical molecular structure to maintain stable properties for a long time before being used.

[0006] The current selection of biocompatible permeable membranes is very limited, so it is necessary to further improve or improve some disadvantages of traditional technology in implantable sensor, develop a biocompatible polymer membrane material with better protein adsorption resistance, excellent selective permeability, and good bending resistance, and not easy to fall off, which has very important significance for improving the detection sensitivity of biosensors and prolonging the effective monitoring time of biosensors. SUMMARY

[0007] The present application aims at the deficiencies of the prior art, and provides a zwitterionic polymer precursor material for coating implantable sensors and a preparation method thereof, which is prepared by copolymerizing siloxane monomers, two zwitterionic compound precursors, monomers for anchoring probe surfaces and soft monomers into a zwitterionic polymer precursor material, so that the oxygen permeability and selective permeability of the coating are improved, the softness and hardness of the polymer are controlled, the coating has better bending resistance and is not easy to fall off, has better biocompatibility, the zwitterionic polymer precursor coating is coated on the surface of the implantable biosensor to form a dense film layer, which can significantly improve the detection sensitivity of the implantable biosensor and effectively prolong the effective detection time of the implantable biosensor.

[0008] The zwitterionic polymer precursor material described in the present application is a precursor material specially developed to improve the stability of zwitterionic polymers, facilitate long-term stability before use, and facilitate production, transportation and storage; the precursor material only needs to add ethanol for hydrolysis when used for coating implantable biosensors, and can also be hydrolyzed after coating, which is very convenient to use.

[0009] In one aspect, the present application provides a zwitterionic polymer precursor material, which is polymerized from siloxane monomers, zwitterionic compound precursors, monomers for anchoring probe surfaces and soft monomers, and the zwitterionic compound precursors include methacryloyloxyethyl methylaminopropyl ethyl acrylate and 2-(dimethylamino) ethyl methacrylate.

[0010] The existing zwitterionic polymers for coating implantable biosensors generally have poor protein adsorption resistance and biocompatibility, the softness and hardness are difficult to adjust, the bending resistance is poor, and the detection sensitivity decreases after long-term use, and even the film layer falls off.

[0011] The zwitterionic polymer precursor material provided by the application is polymerized from a siloxane monomer, multiple zwitterionic compound precursors, a monomer for anchoring a probe surface and a soft monomer; the siloxane part can improve the oxygen permeability of the coating, and can selectively permeate oxygen and glucose molecules; the use of two zwitterionic compound precursors can hydrolyze to generate a zwitterionic structure and play a good synergistic effect, providing better biocompatibility and stronger anti-protein non-specific adsorption characteristics; the flexibility of the polymer can be regulated by changing the ratio of the soft monomer; the monomer for anchoring the probe surface is mainly used to crosslink and fix the zwitterionic coating and the sensor surface layer through a chemical bond, and the polymer material is bonded to the surface of the sensor through a chemical bond to form a dense and stable polymer film layer. The zwitterionic polymer coating material can significantly improve the accuracy and biocompatibility of the sensor while realizing stable transmission of electrochemical signals, and prolong the effective detection time of the sensor.

[0012] Zwitterionic polymers are a class of polymers that are overall electrically neutral and have equal amounts of anionic and cationic groups on the side chains. They have been widely studied and applied in the fields of biomedicine due to their strong hydration ability and good biocompatibility. Zwitterionic polymers have excellent anti-protein non-specific adsorption performance and biocompatibility, and are widely used in antifouling coatings, protein modification, drug delivery, membrane separation materials and other fields.

[0013] The application creatively adds 2-(dimethylamino) ethyl methacrylate, which can play a synergistic effect with the zwitterionic compound methacryloyloxyethyl methyl amino acrylate, to the zwitterionic polymer precursor material. After the copolymerization of the two zwitterionic compound monomers, the prepared polymer precursor material has more excellent biocompatibility and stronger anti-protein non-specific adsorption characteristics. Meanwhile, the soft monomer that can adjust the softness and hardness of the film layer is also added in the application, which can effectively improve the flexibility of the zwitterionic polymer. The zwitterionic polymer precursor material provided by the application can significantly improve the bending resistance and effectively prevent the film layer from falling off after being used for a long time after being used for coating an implantable sensor, thereby effectively prolonging the effective detection time of the implantable biosensor, significantly improving the detection accuracy, and ensuring the detection sensitivity during long-term use.

[0014] Further, the soft monomer is any one or more selected from lauryl acrylate, ethyl acrylate, n-butyl acrylate, 2-ethylhexyl acrylate, lauryl methacrylate and n-butyl methacrylate.

[0015] Further, the siloxane monomer is any one or more selected from the group consisting of methacryloyloxypropyl tris(trimethylsiloxy)silane, methacryloyloxypropyl triethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane, vinyltris(2-methoxyethoxy)silane, methyl-vinylsiloxane, methyl-vinylpolysiloxane, polymethylsiloxane, amino-terminated siloxane, and epoxy-terminated siloxane.

[0016] Silicones have good air permeability, softness, stability and biocompatibility, and are a common biomedical material, which can be used as medical catheters, contact lenses, and skin dressings.

[0017] In some modes, the present application uses methacryloyloxypropyl tris(trimethylsiloxy)silane (TRIS) as a silicon-containing monomer, which has good molecular chain flexibility and a main chain containing a large (CH3)3SiO- group, and has particularly excellent oxygen permeability.

[0018] In some modes, the methacryloyloxypropyl tris(trimethylsiloxy)silane (TRIS) can also be a polysiloxane monomer, or a siloxane compound that can be grafted onto a polymer by or after grafting. The siloxane can also be grafted into a zwitterionic polymer containing active functional groups, which can be replaced by polydimethylsiloxane and its derivatives, commonly used polydimethylsiloxane, cyclo-methylsiloxane, amino-siloxane, poly-methylphenylsiloxane and polyether polysiloxane copolymer, wherein n is 10-1000, and X is amino, epoxy or alkane substituent.

[0019]

[0020] TRIS amino-terminated siloxane polymethylsiloxane

[0021] In some modes, the methacryloyloxypropyl tris(trimethylsiloxy)silane (TRIS) can also be replaced by methacryloyloxypropyl triethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane, vinyltris(2-methoxyethoxy)silane or methyl-vinyl(siloxane and polysiloxane), all of which can improve the oxygen permeability of the coating to some extent.

[0022] Further, the soft monomer is lauryl acrylate; and the siloxane monomer is methacryloyloxypropyl tris(trimethylsiloxy)silane.

[0023] By using different soft monomers and adjusting their proportions, zwitterionic polymer precursor materials with suitable flexibility and bending resistance can be obtained. When lauryl acrylate is used as the soft monomer, due to its excellent chemical stability, the resulting zwitterionic polymer precursor material not only exhibits good flexibility and bending resistance but is also more stable, less prone to deterioration during long-term storage, and, when coated onto implantable biosensors, can further extend the effective detection time of the implantable biosensors and significantly improve detection accuracy, ensuring long-term detection sensitivity.

[0024] Furthermore, the monomer on the surface of the anchoring probe is selected from any one or more of the following: silane coupling agents, hydroxyl-containing compounds, amino-containing compounds or compounds that can expose amino groups through hydrolysis, and carboxyl-containing compounds or compounds that can expose carboxyl groups through hydrolysis.

[0025] In some embodiments, the monomer on the anchoring probe surface is a monomer containing an active functional group capable of binding to the probe surface; the monomer containing an active functional group capable of binding to the probe surface is selected from any one or more of silane coupling agents, hydroxyl-containing compounds, compounds containing amino groups or compounds that can expose amino groups upon hydrolysis, and compounds containing carboxyl groups or compounds that can expose carboxyl groups upon hydrolysis; the zwitterionic polymer precursor material has a structural formula as shown in formula (2):

[0026]

[0027] Equation (2)

[0028] Among them, FG is an active functional group; (the total number of n, m, l, p is 5000~100000), [n / (n+m+l+p)]×100%≈25%; [m / (n+m+l+p)]×100%≈25%; [l / (n+m+l+p)]×100%≈25%; [p / (n+m+l+p)]×100%≈25%.

[0029] Further, the silane coupling agent is any one or more of methacryloyloxypropyltriethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane, and vinyltri(2-methoxyethoxy)silane; the hydroxyl-containing compound is any one or more of polyethylene glycol methacrylate, hydroxyethyl methacrylate, polyol, polyether, polyesteramide, and castor oil; and the amino-containing compound includes one or more of tert-butylaminoethyl methacrylate, 2-aminoethyl methacrylate, and lysine methacrylate monomer LysMA.

[0030] In some methods, the monomer anchored to the probe surface can be a vinyl silane coupling agent copolymerized with other monomers, or it can be grafted onto the probe surface first by using a silane coupling agent containing an organic group (such as amino, epoxy, double bond, etc.) at one end, and then by using an alkoxy group to react with a hydroxyl group. The zwitterionic polymer is connected to the probe surface using a silane coupling agent, including one or more of methacryloyloxypropyltriethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane, vinyltri(2-methoxyethoxy)silane, etc.

[0031]

[0032] Methacryloxypropyltriethoxysilane vinyltrimethoxysilane vinyltriethoxysilane vinyltri(2-methoxyethoxy)silane

[0033] In some methods, the monomers anchored to the probe surface, in addition to the methods using silane coupling agents mentioned above, also include hydroxyl-containing compounds such as polyethylene glycol methacrylate (PEGMA) and hydroxyethyl methacrylate, as well as active hydroxyl-containing compounds such as polyols, polyethers, polyesteramides, and castor oil. Alternatively, they can be amino-containing compounds such as tert-butylaminoethyl methacrylate, 2-aminoethyl methacrylate, and lysine methacrylate monomer LysMA, as well as compounds containing carboxyl groups and those that can be hydrolyzed to expose these functional groups.

[0034] In some embodiments, the monomer anchored to the probe surface is polyethylene glycol methacrylate (PEGMA).

[0035] PEGMA primarily involves chemically cross-linking and fixing an amphoteric coating to the sensor surface. This polymer is bonded to the sensor surface, forming a dense and stable polymer film. This coating material not only achieves stable transmission of electrochemical signals but also improves sensor accuracy and biocompatibility, and extends the sensor's effective detection time.

[0036] Different types of monomers anchored to the probe surface can be anchored to different enzymes in the inner layer of implantable sensors, and the cross-linking agents used for different anchoring groups also vary slightly. For example, protein groups that can be coupled under mild conditions include: amino, carboxyl, thiol groups of cysteine, imidazole groups of histidine, phenolic groups of tyrosine, and hydroxyl groups of serine and threonine. For instance, amino and imidazole groups can be linked using cross-linking agents containing epoxy groups, such as PEGDGE (polyethylene glycol diglycidyl ether), while thiol and hydroxyl groups can be linked using isocyanates. Therefore, the choice of monomers anchored to the probe surface depends on the coating material of the enzyme in the inner layer of the implantable sensor.

[0037] Polyethylene glycol methacrylate (PEGMA) is suitable as a coating material for implantable sensors containing cysteine ​​(thiol) and threonine (hydroxyl) fragments in the inner layer of enzymes.

[0038] In some methods, when coating the prepared zwitterionic polymer precursor material onto the implantable sensor, a crosslinking agent is also required. Different crosslinking agents can be selected for different monomers anchored to the probe surface.

[0039] In some methods, the crosslinking agent can be a diisocyanate including toluene diisocyanate (TDI), isophorone diisocyanate (IPDI), diphenylmethane diisocyanate (MDI), dicyclohexylmethane diisocyanate (HMDI), hexamethylene diisocyanate (HDI), lysine diisocyanate (LDI), etc. The zwitterionic polymer precursor material is cured and crosslinked on the polyurethane surface by reacting the highly active unsaturated bonds in the isocyanate structure with active hydroxyl compounds. Alternatively, triethylene glycol dimethacrylate (TEGDMA), ethylene glycol dimethacrylate (EDMA), trimethylolpropane trimethacrylate (TMPTMA), etc., can be thermally, lightly, and irradiatedly crosslinked with organic peroxides onto the polyurethane coating on the probe's outer surface.

[0040] Furthermore, the monomer on the surface of the anchoring probe is polyethylene glycol methacrylate, and the zwitterionic polymer precursor material has the structural formula shown in formula (1):

[0041]

[0042] Equation (1);

[0043] Among them, b=5~30, (the total number of l, o, m, p, q is 5000~100000), [l / (l+o+m+p+q)]×100%≈10%; [o / (l+o+m+p+q)]×100% ≈25%; [m / (l+o+m+p+q)]×100%≈25%; [p / (l+o+m+p+q)]×100%≈25%; [q / (l+o+m+p+q)]×100%≈15%.

[0044] On the other hand, the present invention provides a method for preparing a zwitterionic polymer precursor material, the method comprising the following steps:

[0045] (1) Preparation of ethyl methacryloyloxyethyl methaminoacrylate;

[0046] (2) A zwitterionic polymer precursor material is prepared by copolymerizing methacryloyloxyethyl methylamino acrylate, 2-(dimethylamino)ethyl methacrylate, siloxane monomer, monomer on the surface of the anchoring probe, soft monomer, initiator and solvent.

[0047] Further, the siloxane monomer is methacryloyloxypropyltris(trimethylsiloxane)silane, the monomer on the anchoring probe surface is polyethylene glycol methacrylate, and the soft monomer is lauryl acrylate; wherein, the content of methacryloyloxyethyl methylamino acrylate is 1-30%, the content of 2-(dimethylamino)ethyl methacrylate is 1-30%, the content of lauryl acrylate is 1-25%, the content of polyethylene glycol methacrylate is 0.5-40%, and the content of methacryloyloxypropyltris(trimethylsiloxane)silane is 0.5-45%; the initiator is azobisisobutyronitrile, and the solvent is anhydrous tetrahydrofuran.

[0048] Furthermore, it also includes step (3): nitrogen gas is introduced to remove the air in the reaction system, and the system is placed in an oil bath at 62°C for 12 hours; after the reaction is completed, most of the organic solvent is removed by vacuum distillation, and the unpolymerized small molecule polymers in the reaction system are removed by centrifugation with solvent 2 and washing. The precipitate is collected by filtration and placed under high vacuum at 50°C for 2 days.

[0049] In some methods, the solvent is the polymerization solvent of the hydrolyzed precursor polymer, which can be dissolved in tetrahydrofuran, or in other organic solvents such as methanol, ethanol, and DMSO; the solvent in the monomer synthesis process can be anhydrous tetrahydrofuran, or in other organic solvents such as anhydrous dichloromethane, anhydrous dimethyl sulfoxide, and anhydrous acetonitrile.

[0050] In some methods, solvent 2 shown is the solvent used for precipitation, which, in addition to hexane, can also be a low-polarity organic solvent such as diethyl ether or petroleum ether.

[0051] In some methods, the initiator for the polymerization process can be azobisisobutyronitrile (AIBN), or it can be an azo compound (such as azobisisobutyronitrile and dimethyl azobisisobutyrate initiator), a peroxide (such as hydrogen peroxide, ammonium persulfate, potassium persulfate, benzoyl peroxide, tert-butyl peroxide, methyl ethyl ketone peroxide, etc.), or a composite initiation system (such as the azobisisobutyronitrile and azobisisobutyronitrile ABVN composite initiation system, the benzoyl peroxide BPO and tert-butyl peroxide composite initiation system, etc.).

[0052] In some embodiments, the initiator is azobisisobutyronitrile (AIBN), and the solvent is anhydrous tetrahydrofuran.

[0053] Further, the preparation method of the zwitterionic compound precursor in step (1) is as follows: 50.06 g (0.5 mol) of ethyl acrylate is added to a 500 mL round-bottom flask, and 37.56 g (0.5 mol) of 2-methylaminoethanol is slowly added dropwise using a constant-pressure separatory funnel under ice bath conditions. The mixture is stirred for 12 h to obtain a colorless, transparent, viscous liquid, 2-hydroxyethylmethylaminoacrylate. 90 mL of triethylamine, 125 mg of 2,6-di-tert-butyl-p-cresol, and 50 mL of anhydrous tetrahydrofuran are then added to the 2-hydroxyethylmethylaminoacrylate. Similarly, under ice bath conditions, a mixed solution containing 58 mL of methacryloyl chloride and 50 mL of tetrahydrofuran is slowly added dropwise using a constant-pressure separatory funnel. The reaction is stopped after stirring for 12 h. The product solution was filtered through a sand core funnel to remove the generated triethylamine hydrochloride. The solvent in the filtrate was removed by rotary evaporation. Finally, it was extracted and purified, and the solvent was removed by vacuum distillation to obtain a colorless oily liquid, namely methyl methacryloyloxyethyl methylamino acrylate.

[0054]

[0055] In some methods, R1 of the aforementioned 2-methylaminoethanol can be replaced not only by methyl but also by ethyl, tert-butyl, etc. For example, ethyl substitution yields 2-ethylaminoethanol, and tert-butyl substitution yields 2-tert-butylaminoethanol.

[0056]

[0057] In some embodiments, the R2 portion of the aforementioned ethyl acrylate can be replaced not only by ethyl groups, but also by methyl groups, tert-butyl groups, etc., i.e., methyl acrylate, tert-butyl acrylate, etc.

[0058] In some ways, the aforementioned ethyl acrylate can be linked to 2-methylaminoethanol via Michael addition of a tertiary amine to an acrylate containing a double bond, or via nucleophilic substitution of a carbon-halogen bond. R3 can be ethyl, methyl tert-butyl, etc., i.e., methyl bromoacetate, ethyl bromoacetate, tert-butyl bromoacetate, methyl methacrylate, ethyl methacrylate, tert-butyl methacrylate, etc., can generate carboxylic acid functional groups in carboxylate betaine analogs through hydrolysis.

[0059] In some methods, the chain lengths of the tertiary amine and ethyl acrylate are not adjustable; however, the distance between the methacryloyloxy group and the tertiary amine in methacryloyloxyethyl methylamino acrylate can be adjusted by changing the compound. The hydrolysis rate of the zwitterionic polymer precursor can be achieved by altering the length of the ester group in the carboxylic acid ester.

[0060] In another aspect, the present invention provides the use of the zwitterionic polymer precursor material as described above in the preparation of coating formulations for implantable sensors.

[0061] The beneficial effects of this invention are:

[0062] 1. By copolymerizing siloxane monomers, two zwitterionic compound precursors, monomers on the surface of anchoring probes, and soft monomers to form zwitterionic polymer precursor materials, a material with superior biocompatibility and strong resistance to non-specific protein adsorption is obtained. This material also improves the control of polymer hardness, giving the coating better bending resistance and making it less prone to peeling off.

[0063] 2. By leveraging the synergistic effect of two zwitterionic precursors, methacryloyloxyethyl methylamino acrylate and 2-(dimethylamino)ethyl methacrylate, the coating's resistance to non-specific protein adsorption is enhanced, its oxygen permeability and selective permeability are improved, and it exhibits better biocompatibility.

[0064] 3. By fully utilizing the control of soft monomers on the hardness of the polymer, the coating has better resistance to bending and is less prone to peeling, and can further improve the stability of the polymer.

[0065] 4. It can significantly improve the detection sensitivity of implantable biosensors and effectively extend the effective detection time of implantable biosensors. Attached Figure Description

[0066] Figure 1 The synthesis reaction formula for ethyl methacryloyloxyethyl methylamino acrylate in Example 1;

[0067] Figure 2 The structural formula of polymer 1 in Example 2 is shown below;

[0068] Figure 3 The curves show the fluctuations of the current signals of implantable biosensors with different membrane layer modifications as a function of glucose concentration in Example 9.

[0069] Figure 4 This is a schematic diagram of the cytotoxicity test results for different membrane layers in Example 10. Detailed Implementation

[0070] The present invention will be further described in detail below with reference to the embodiments. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0071] Example 1 Synthesis of methyl methacryloyloxyethyl methylamino acrylate (CBE)

[0072] The synthesis reaction formula for ethyl methacryloyloxyethyl methylamino acrylate provided in this embodiment is as follows: Figure 1 As shown, the main synthetic steps are as follows: 50.06 g (0.5 mol) of ethyl acrylate was added to a 500 mL round-bottom flask. Under ice bath conditions, 37.56 g (0.5 mol) of 2-methylaminoethanol was slowly added dropwise using a constant-pressure separatory funnel. The mixture was stirred for 12 h to obtain a colorless, transparent, viscous liquid, 2-hydroxyethylmethylaminoacrylate. 90 mL of triethylamine, 125 mg of 2,6-di-tert-butyl-p-cresol, and 50 mL of anhydrous tetrahydrofuran were then added to the 2-hydroxyethylmethylaminoacrylate. Again under ice bath conditions, a mixed solution containing 58 mL of methacryloyl chloride and 50 mL of tetrahydrofuran was slowly added dropwise using a constant-pressure separatory funnel. The mixture was stirred for 12 h and then the reaction was stopped. The product solution was filtered through a sintered glass funnel to remove the generated triethylamine hydrochloride. The filtrate was then evaporated to remove the solvent. Finally, the solution was extracted and purified, and the solvent was removed by vacuum distillation to obtain a colorless, oily liquid, namely methacryloyloxyethylmethylaminoacrylate.

[0073] Example 2 Synthesis and application of polymer 1

[0074] The structural formula of polymer 1 provided in this embodiment is as follows: Figure 2 As shown. Among them, the parameters of polymer 1 are: [l / (n+m+l+p)]×100%≈25%; [m / (n+m+l+p)]×100%≈25%; [o / (n+m+l+p)]×100%≈25%; [p / (n+m+l+p)]×100%≈25%.

[0075] The polymerization process of polymer 1 is as follows: Methacryloxyethyl methylamino acrylate (CBE) (12.8 g), polyethylene glycol methacrylate (PEGMA) (15 mL), methacryloyloxypropyl tris(trimethylsiloxane)silane (TRIS) (54.7 g), lauryl acrylate (soft monomer, 21 g), 2-(dimethylamino)ethyl methacrylate (23.7 g), azobisisobutyronitrile (AIBN) (0.88 g), and anhydrous tetrahydrofuran (1 L) were added to a reaction flask. After thorough stirring and dissolution, nitrogen gas was introduced to purge air from the reaction system. Finally, the reaction flask was placed in an oil bath at 62°C for 12 hours. After the reaction was completed, most of the organic solvent was removed by vacuum distillation. The precipitate was then precipitated with n-hexane, centrifuged, and washed to remove small molecule polymers from the reaction system. The precipitate was collected by filtration and placed under high vacuum at 50°C for 2 days.

[0076] The process of coating the implantable sensor with polymer 1 is as follows: Polymer 1 is dissolved in ethanol (to hydrolyze the zwitterionic polymer precursor) and shaken to prepare a polymer mixture solution of 0.5 g / L. 30 mg of hexamethylene diisocyanate (HDI) (approximately 0.55 times the molar amount of hydroxyl groups in the copolymer) is added to the solution. After mixing evenly, the probe part of the implantable biosensor is immediately immersed in the solution. After the probe is coated, it is placed in a dry environment to cure. After that, it is washed with anhydrous ethanol to remove residual components and polymer that is not bonded to the probe surface. The ethanol solvent is then evaporated in a dry environment to remove the residue, thus obtaining the probe modified with zwitterionic polymer.

[0077] Example 3: Preparation of a polymer containing only CBE, an amphoteric compound.

[0078] This embodiment uses the method provided in Example 2 to prepare the polymer. The specific preparation process is as follows: 12.8 g of methacryloyloxyethyl methylamino acrylate (CBE), 15 mL of polyethylene glycol methacrylate (PEGMA), 54.7 g of methacryloyloxypropyltris(trimethylsiloxane)silane (TRIS), 0.88 g of azobisisobutyronitrile (AIBN), and 1 L of anhydrous tetrahydrofuran were added to a reaction flask. After thorough stirring and dissolution, nitrogen gas was introduced to purge air from the reaction system. Finally, the reaction flask was placed in an oil bath at 62°C for 12 hours. After the reaction was completed, most of the organic solvent was removed by vacuum distillation. The precipitate was then precipitated with n-hexane, centrifuged, and washed to remove small molecule polymers from the reaction system. The precipitate was collected by filtration and placed under high vacuum at 50°C for 2 days.

[0079] Example 4: Preparation of a polymer containing only 2-(dimethylamino)ethyl methacrylate, an amphoteric compound.

[0080] This embodiment uses the method provided in Example 2 to prepare the polymer. The specific preparation process is as follows: 2-(dimethylamino)ethyl methacrylate (23.7 g), polyethylene glycol methacrylate (PEGMA) (15 mL), methacryloyloxypropyltris(trimethylsiloxane)silane (TRIS) (54.7 g), azobisisobutyronitrile (AIBN) (0.88 g), and anhydrous tetrahydrofuran (1 L) are added to a reaction flask. After thorough stirring and dissolution, nitrogen gas is introduced to purge air from the reaction system. Finally, the reaction flask is placed in an oil bath at 62°C and reacted for 12 hours. After the reaction is completed, most of the organic solvent is removed by vacuum distillation. The precipitate is then precipitated with n-hexane, centrifuged, and washed to remove small molecule polymers from the reaction system. The precipitate is collected by filtration and placed under high vacuum at 50°C for 2 days.

[0081] Example 5 Preparation of polymers without soft monomers

[0082] This embodiment uses the method provided in Example 2 to prepare the polymer. The specific preparation process is as follows: 25.6 g of methacryloyloxyethyl methylamino acrylate (CBE), 15 mL of polyethylene glycol methacrylate (PEGMA), 54.7 g of methacryloyloxypropyltris(trimethylsiloxane)silane (TRIS), 0.88 g of azobisisobutyronitrile (AIBN), and 1 L of anhydrous tetrahydrofuran were added to a reaction flask. After thorough stirring and dissolution, nitrogen gas was introduced to purge air from the reaction system. Finally, the reaction flask was placed in an oil bath at 62°C for 12 hours. After the reaction, most of the organic solvent was removed by vacuum distillation. The precipitate was then precipitated with n-hexane, centrifuged, and washed to remove small molecule polymers from the reaction system. The precipitate was collected by filtration and placed under high vacuum at 50°C for 2 days.

[0083] Example 6: Zwitterionic polymer precursor materials prepared using different soft monomers

[0084] This embodiment uses the preparation method of polymer 1 provided in Example 2 to prepare the polymer, wherein the soft monomers are lauryl acrylate, ethyl acrylate, n-butyl acrylate, 2-ethylhexyl acrylate, lauryl methacrylate, and n-butyl methacrylate, respectively. The polymer prepared in Example 5 without added soft monomers is used as a blank control, thus obtaining seven different polymers. These seven polymers are used to coat implantable biosensors, and the stability, flexibility, bending resistance, non-detachment, effective detection time, and long-term detection sensitivity of the seven implantable biosensors are investigated. The stability is tested by observing its properties after being placed in a 50°C high-temperature environment for 30 days. The flexibility and bending resistance are tested by subjecting the polymer membrane to 1... The creases on the membrane surface after 80º bending and whether the creases recovered were used as the criteria. The method for detecting the shedding performance was to implant the coated implantable biosensor into mice and maintain it for two months, observing whether the membrane on the sensor surface showed peeling, protrusion, or swelling. Each polymer coating membrane was repeated 5 times. The method for detecting the effective detection time was to soak the coated electrode in PBS buffer solution, remove the electrode every day for electrochemical detection of glucose gradient concentration, and continue to soak it in PBS buffer solution after the test. The method for detecting long-term detection sensitivity was to compare the electrode tested in the first test with the electrode tested after continuous soaking for 20 days, and then test its glucose detection sensitivity (here only the sensitivity exceeding 90% is considered as the effective detection time). The results are shown in Table 1.

[0085] Table 1. Effects of different soft monomers on the prepared zwitterionic polymer precursor materials

[0086]

[0087] As shown in Table 1, the polymer prepared without soft monomers exhibits poor stability, weak flexibility and bending resistance, and is prone to peeling after coating. Furthermore, the effective detection time is relatively short, and the detection sensitivity is not high. However, the addition of soft monomers significantly improves polymer stability, making it suitable for long-term storage and convenient transportation. It also enhances the flexibility and bending resistance of the sensor after coating, preventing peeling and significantly improving detection time and sensitivity. In particular, the use of lauryl acrylate as a soft monomer results in more stable performance, better overall performance, and higher detection sensitivity.

[0088] Example 7: Effect of different contents of soft monomers on the preparation of zwitterionic polymer precursor materials

[0089] In this embodiment, the polymer was prepared using the preparation method of polymer 1 provided in Example 2. The soft monomer used was lauryl acrylate, and 1%, 5%, 10%, 15%, 20%, 25%, and 30% lauryl acrylate were added respectively to obtain seven different polymers. These seven polymers were then used to coat implantable biosensors. The stability, flexibility, bending resistance, non-detachment, effective detection time, and long-term detection sensitivity of the seven implantable biosensors were investigated. The stability was tested by observing its properties after being placed in a 50°C high-temperature environment for 30 days. The flexibility and bending resistance were tested by observing the creases on the membrane surface after the polymer membrane was bent at 180°C and whether the creases were intact. To assess recovery, the detachment performance was tested by implanting the coated biosensor into mice for two months and observing whether the membrane on the sensor surface showed signs of peeling, bulging, or swelling. Each polymer coating was repeated five times. The effective detection time was tested by immersing the coated electrode in PBS buffer solution and performing electrochemical detection of glucose gradient concentrations daily. After each test, the electrode was immersed in PBS buffer solution again. The long-term detection sensitivity was tested by comparing the electrode tested in the first test with the electrode tested after 20 consecutive days of immersion, and then testing its glucose detection sensitivity (only a sensitivity exceeding 90% was considered an effective detection time). The results are shown in Table 2.

[0090] Table 2. Effect of different contents of soft monomers on the prepared zwitterionic polymer precursor materials

[0091]

[0092] As shown in Table 2, when the content of the soft monomer lauryl acrylate is less than 5%, the prepared polymer has slightly poor stability, low flexibility and bending resistance, and may peel off after coating. Furthermore, the effective detection time is short and the detection sensitivity is not high. However, when the content of the soft monomer is between 10-25%, the stability of the zwitterionic polymer is significantly improved, making it suitable for long-term storage and convenient transportation. It also improves the flexibility and bending resistance of the sensor after coating, preventing peeling and significantly increasing detection time and sensitivity. In particular, when the content of the soft monomer lauryl acrylate is 20%, the performance is more stable, the overall effect is better, and the detection sensitivity is higher. However, when the content of the soft monomer is too high, the detection sensitivity decreases. Therefore, the optimal soft monomer content is 20%.

[0093] Example 8 Comparison of the anti-adsorption properties of polymer precursor materials prepared from different zwitterionic compounds

[0094] In this embodiment, the zwitterionic polymer precursor materials prepared in Examples 2, 3, and 4 were used to coat implantable sensors, and their resistance to nonspecific protein adsorption was tested. The following three groups were examined: Group 1, prepared using two zwitterionic compounds, methacryloyloxyethyl methylamino acrylate (CBE) and 2-(dimethylamino)ethyl methacrylate; Group 2, prepared using only one zwitterionic compound, methacryloyloxyethyl methylamino acrylate (CBE); and Group 3, prepared using only one zwitterionic compound, 2-(dimethylamino)ethyl methacrylate. The resistance to nonspecific protein adsorption of the three zwitterionic polymer precursor materials was assessed. The resistance to nonspecific protein adsorption of the sensor probe coated with the polymer film was determined using a modified enzyme-linked immunosorbent assay (ELISA).

[0095] First, circular membranes cut from each zwitterionic polymer precursor material and equally sized circular polyurethane membranes were placed in 48-well plates. 1 mL of pure water was added to each well, and the plates were washed on a horizontal shaker for 5 min. The pure water was then removed. Next, 1 mL of 1×PBS buffer was added to each well, and the plates were washed twice on a horizontal shaker. The buffer solution was removed, and 1 mL of a solution containing Anti-IgG HRP conjugate was added to each well. The plates were incubated at 37°C for 75 min on a horizontal shaker. After incubation, the conjugate solution was removed. 1 mL of PBS buffer was added to each well, and the PU membrane was removed and placed in a new well. The membrane was washed with 1 mL of PBS buffer on a horizontal shaker for 5 min, repeating this process twice. Finally, the same solution was added to another empty well and washed on a horizontal shaker for 5 min as a blank control. The PBS buffer solution was removed, and 500 μL of TMB chromogenic solution was added to each well. The plates were then developed on a horizontal shaker at 37°C for 15 min. To avoid cross-contamination, rinse the tweezers with pure water immediately when handling different membranes. Then, add 500 μL of hydrochloric acid stop solution to each well and shake well. Add 200 μL of the liquid to a 96-well plate, and shake at medium speed for 5 seconds using a microplate reader to remove air bubbles while mixing. Then, measure the absorbance at 450 nm and 620 nm wavelengths, respectively. The ratio of the absorbance of the experimental result to that on the unmodified PU membrane is taken as the relative protein adsorption amount. The formula for the relative protein adsorption amount is as follows:

[0096] Relative protein adsorption capacity = ×100%

[0097] Where Asample is the absorbance of the sample to be tested; Ablank is the absorbance of the blank well; Acontrol is the absorbance of the unmodified PU membrane well; three parallel samples were set for each group. The results of the detection of relative protein adsorption are shown in Table 3.

[0098] Table 3. Comparison of anti-adsorption properties of polymer precursor materials prepared from different zwitterionic compounds

[0099]

[0100] As shown in Table 3, when polymers are prepared using either methacryloyloxyethyl methylamino acrylate (CBE) or 2-(dimethylamino)ethyl methacrylate, the resulting polymer precursor still exhibits a certain amount of protein adsorption. However, when polymers are prepared using both methacryloyloxyethyl methylamino acrylate (CBE) and 2-(dimethylamino)ethyl methacrylate, the amount of protein adsorption is significantly reduced. This indicates that methacryloyloxyethyl methylamino acrylate (CBE) and 2-(dimethylamino)ethyl methacrylate have a significant synergistic effect.

[0101] Example 9: Glucose permeation performance test of the zwitterionic polymer precursor material provided by the present invention.

[0102] In this embodiment, the zwitterionic polymer precursor material prepared in Example 2 was used to coat the implantable sensor, and it was compared with the implantable sensor coated with the zwitterionic polymer precursor material prepared in Example 3. An implantable sensor without a polymer outer membrane layer was used as a blank control. The membrane thickness was kept consistent (approximately 25 μm). PBS solutions of glucose at concentrations of 0, 5, 10, 15, 20, 25, 30, and 40 mM were prepared. Electrochemical testing was performed using a CHI660 multichannel electrochemical workstation. The amperometric it curve was selected, the voltage was set to 0.55 V, and the sensitivity (A / V) was set to 10⁻⁶. Current-time curves (it) were plotted. After the baseline stabilized, the PBS solution was changed sequentially according to the glucose concentrations of 0, 5, 10, 15, 20, 25, 30, and 40 mM. Finally, the data was exported to plot current-time curves, and the fluctuation of the current signal and its trend with glucose concentration were observed. The results are as follows: Figure 3 The current-time curve is shown.

[0103] Figure 3As shown, the probe modified with a zwitterionic polymer containing two zwitterionic compounds, methacryloyloxyethyl methylamino acrylate (CBE) and 2-(dimethylamino)ethyl methacrylate, prepared in Example 2, exhibits a stable and orderly electrochemical signal. In contrast, the unmodified probe shows a better fit below 15 mM concentration, while the electrochemical signal decreases above 15 mM. Furthermore, the probe modified with the zwitterionic polymer is superior to the probe prepared in Example 3, which uses a zwitterionic polymer precursor material containing only CBE as the outer film layer, exhibiting better signal transmission. This indicates that the coating can successfully transmit glucose and oxygen molecules and convert them into electrochemical signals.

[0104] Example 10 Cytotoxicity test of the zwitterionic polymer precursor material provided by the present invention

[0105] The cytotoxicity assay uses CCK-8, a rapid and highly sensitive assay kit widely used for cell proliferation and cytotoxicity. The intensity of the color is inversely proportional to cytotoxicity. The OD value is measured at a wavelength of 450 nm using an ELISA reader, which indirectly reflects the number of live cells.

[0106] First, HUVEC cells were cultured at 5 × 10⁻⁶. 4 100 μL of the modified probe (previously modified with zwitterionic polymer precursors prepared in Example 2 and Example 3, respectively) was seeded into sterile 96-well plates and cultured for 24 h. The culture medium was then replaced with DMEM medium containing 0.2 g / mL of the probe extract before and after modification, with blank and experimental controls included. Culture was continued for another 24 h. After culture, the culture medium was removed, and the cells were washed twice with DMEM medium. 100 μL of 100 μL / mL CCK-8 DMEM medium was added, and culture was continued for 0.5 h. After culture, absorbance was measured using a microplate reader at a detection wavelength of 450 nm and a reference wavelength of 620 nm. Figure 4 The cell viability of both the polyurethane membrane and the zwitterionic polymer membrane was higher than 95% in the cytotoxicity test results, indicating that the materials have good safety.

[0107] Example 11 Platelet adhesion experiment of the zwitterionic polymer precursor material provided by the present invention

[0108] Platelet adhesion experiment: Membranes with a diameter of approximately 0.5 cm were prepared from the zwitterionic polymer precursor materials prepared in Example 2 and Example 3, respectively, and fixed to the bottom of a 96-well plate. 100 μL of platelet-rich plasma (PRP) was added to each well, and the plates were placed in an incubator at 37°C for static adsorption for 2 h. After adsorption, the membranes were slowly rinsed twice with sterile PBS buffer solution, and then 1 mL of 2.5% (w / v) glutaraldehyde PBS solution was added and the plates were fixed at 4°C for 45 min. After fixation, the plates were slowly rinsed with pure water, and 200 μL of pure water was added to centrifuge tubes. The membranes were then placed in the centrifuge tubes, flash-frozen in liquid nitrogen, and then dried in a freeze dryer for about 24 h. The surface microstructure and morphology of the membranes were observed using a field emission scanning electron microscope SU-8010.

[0109] It can be clearly seen that the surface of the membrane modified with the zwitterionic polymer precursor material prepared in Example 3 still adsorbed a very small amount of platelets, while the surface of the membrane modified with the zwitterionic polymer precursor material prepared in Example 2 did not have any platelets adhering to it. It is evident that the zwitterionic polymer precursor material provided by the present invention can completely avoid the adhesion of platelets.

[0110] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. Use of a soft monomer for the preparation of a zwitterionic polymer precursor material for coating an implantable sensor, and for increasing the effective detection time and the detection sensitivity of the implantable sensor, characterized in that, The soft monomer is lauryl acrylate, the zwitterionic polymer precursor material is polymerized from a siloxane monomer, a zwitterionic compound precursor, a monomer that anchors the probe surface, and a soft monomer; the siloxane monomer is any one or more selected from the group consisting of methacryloyloxypropyl tris(trimethylsiloxy)silane, methacryloyloxypropyl triethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane, vinyltris(2-methoxyethoxy)silane, methyl-vinylsiloxane, methyl-vinylpolysiloxane; the zwitterionic compound precursor includes and 2-(dimethylamino)ethyl methacrylate; the monomer that anchors the probe surface is a monomer containing an active functional group that can bind to the probe surface.

2. A zwitterionic polymeric precursor material characterized in that, polymerized from a siloxane monomer, a zwitterionic compound precursor, a monomer that anchors the probe surface, and a soft monomer; the siloxane monomer is any one or more selected from the group consisting of methacryloyloxypropyl tris(trimethylsiloxy)silane, methacryloyloxypropyl triethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane, vinyltris(2-methoxyethoxy)silane, methyl-vinylsiloxane, methyl-vinylpolysiloxane; the zwitterionic compound precursor includes and 2-(dimethylamino)ethyl methacrylate; the monomer that anchors the probe surface is a monomer containing a reactive functional group that binds to the probe surface; and the soft monomer is lauryl acrylate.

3. The zwitterionic polymer precursor material of claim 2, wherein, The siloxane monomer is methacryloxypropyl tris(trimethylsiloxy)silane.

4. The zwitterionic polymer precursor material of claim 3, wherein, The monomer of the anchoring probe surface is selected from any one or more of a silane coupling agent, a hydroxyl-containing compound, an amino-containing or hydrolytically amino-exposing compound, a carboxyl-containing or hydrolytically carboxyl-exposing compound.

5. The zwitterionic polymer precursor material of claim 4, wherein, The silane coupling agent is any one or more of methacryloxypropyl triethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane, vinyltris(2-methoxyethoxysilane); the hydroxyl-containing compound is any one or more of polyethylene glycol methacrylate, hydroxyethyl methacrylate, and the amino-containing or hydrolytically amino-exposing compound is one or more of tert-butylaminoethyl methacrylate, 2-aminoethyl methacrylate.

6. The zwitterionic polymer precursor material of claim 5, wherein, The monomer of the anchoring probe surface is polyethylene glycol methacrylate, and the zwitterionic polymer precursor material has a structural formula as shown in formula (1): Formula (1); wherein b = 5-30, the total number of l, o, m, p, q is 5000-100000, [l / (l+o+m+p+q)]x100%=10%; [o / (l+o+m+p+q)]x100%=25%; [m / (l+o+m+p+q)]x100%=25%; [p / (l+o+m+p+q)]x100%=25%; [q / (l+o+m+p+q)]x100%=15%.

7. The method for preparing the zwitterionic polymer precursor material according to any one of claims 2 to 6, characterized in that, comprising the following steps: (1) Preparation ; (2) mixing , 2-(dimethylamino)ethyl methacrylate, silicone monomer, monomer anchoring probe surface, soft monomer, initiator and solvent to copolymerize to prepare zwitterionic polymer precursor material.

8. The method of claim 7, wherein, The siloxane monomer is methacryloyloxypropyl tris(trimethylsiloxy)silane, the monomer anchoring the probe surface is polyethylene glycol methacrylate, and the soft monomer is lauryl acrylate; wherein, The content of the 2-(dimethylamino)ethyl methacrylate is 1-30%, the content of the lauryl acrylate is 1-25%, the content of the polyethylene glycol methacrylate is 0.5-40%, and the content of the methacryloyloxypropyl tris(trimethylsiloxy)silane is 0.5-45%; the initiator is azobisisobutyronitrile, and the solvent is anhydrous tetrahydrofuran.

9. Use of a zwitterionic polymer precursor material according to any one of claims 2-6 for the preparation of a coating for an implantable sensor formulation.

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