Enzyme protective agent for continuous glucose sensor and continuous glucose sensor

Abstract

The invention provides an enzyme protective agent for a continuous glucose sensor and the continuous glucose sensor, the enzyme protective agent comprises the following components: poloxamer and a zwitterionic polymer, and the mass percent concentration of the poloxamer is 0.1%-2%, and the mass percent concentration of the zwitterionic polymer is 0.5%-5%. By adopting the technical scheme provided by the invention, the poloxamer and the zwitterionic polymer are introduced, so that an enzyme layer coating process can be optimized, a coffee ring effect can be inhibited, and the consistency and monitoring accuracy of the sensor can be improved; and on the premise of keeping the signal transmission performance of the sensor unchanged, the activity and long-term stability of the biological enzyme preparation in the production, storage, transportation and use processes are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to an enzyme protectant for a continuous glucose sensor and the continuous glucose sensor. Background Art

[0002] Diabetes is a metabolic disease characterized by abnormally elevated blood sugar levels. During treatment, it can cause hypoglycemic coma. If not treated promptly, it can lead to long-term complications such as stroke, amputation, and kidney failure. Continuous glucose monitoring systems (CGMS) are key tools for blood sugar management. They monitor glucose levels in real time or near real time to optimize insulin administration and reduce the risk of blood sugar fluctuations. Currently, mainstream CGMS (such as the FreeStyle Libre TM ) uses a subcutaneous electrochemical sensor based on "wired enzyme electrode" technology. The core principles include: (1) glucose oxidase (GOx) catalyzes the oxidation of glucose to produce hydrogen peroxide and gluconic acid; (2) osmium-based redox polymers act as electron mediators, promoting electron transfer to the electrode through a hydrogel conductive layer to generate a current signal; (3) the polymer membrane regulates the glucose permeation rate to match the blood glucose response. However, this technology has the following drawbacks:

[0003] (1) Insufficient enzyme stability: GOx is easily affected by factors such as temperature, pH, and ionic strength during storage, transportation, and use, resulting in decreased activity or aggregation and inactivation;

[0004] (2) Uneven distribution of enzyme layer: In the traditional spot coating process, the enzyme-mediator mixture produces a "coffee ring effect" due to solvent evaporation, resulting in uneven enzyme layer thickness and poor signal consistency;

[0005] (3) Oxygen dependence: GOx relies on oxygen as a natural electron acceptor, which affects detection accuracy in the subcutaneous hypoxic environment;

[0006] (4) Limitations of traditional stabilizers: Stabilizers such as albumin, sugars, and polyols have insufficient protective effects under high temperature, high salt, or extreme pH conditions and cannot maintain enzyme activity for a long time.

[0007] The above problems restrict the detection accuracy and service life of CGMS, and it is urgent to achieve technological breakthroughs through material innovation and process optimization. Summary of the Invention

[0008] In response to the above technical problems, the present invention discloses an enzyme protectant for a continuous glucose sensor and a continuous glucose sensor. By improving the formula of the enzyme protectant and optimizing the enzyme layer coating process, the coffee ring effect is suppressed, and the sensor consistency and monitoring accuracy are improved.

[0009] To this end, the technical solution adopted in the present invention is:

[0010] An enzyme protectant for a continuous glucose sensor, comprising poloxamer and a zwitterionic polymer, with the mass percentages of the poloxamer and zwitterionic polymer being 0.1% to 2% and 0.5% to 5%, respectively. This technical solution achieves improved enzyme stability, coating uniformity, signal response speed, and long-term storage performance.

[0011] As a further improvement of the present invention, the mass percentage concentrations of the poloxamer and the zwitterionic polymer are 1% and 2% respectively.

[0012] As a further improvement of the present invention, the zwitterionic polymer is at least one of polymethacrylate sulfobetaine, polyethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer, polyethylene glycol-polylactic acid, polyethylene glycol-polycaprolactone, polyacrylic acid amphiphilic polymer, natural amphiphilic polymer, zwitterionic-hydrophobic block copolymer, polysorbate 80, and polyoxyethylene lauryl ether.

[0013] As a further improvement of the present invention, the zwitterionic polymer is polymethacrylate sulfobetaine.

[0014] As a further improvement of the present invention, the polyethylene glycol-polylactic acid includes methoxy PEG-polylactic acid, and the polyethylene glycol-polycaprolactone includes polyethylene glycol-polycaprolactone;

[0015] The polyacrylic acid amphiphilic polymer includes at least one of polyacrylic acid-b-polystyrene and polymethyl methacrylate-b-polyethylene glycol; the natural amphiphilic polymer includes at least one of chitosan derivatives and hyaluronic acid-hydrophobic modifiers, and the hyaluronic acid-hydrophobic modifier includes HA-octadecylamine; the zwitterion-hydrophobic block copolymer includes at least one of polysulfobetaine-b-polypropylene glycol and polycarboxybetaine-b-polystyrene; the polyethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer includes F68, P123, T904.

[0016] The present invention discloses a continuous glucose sensor. The components of the enzyme layer include the enzyme protectant for the continuous glucose sensor, a redox mediator, glucose oxidase, and a crosslinking agent for solidifying the components on the electrode surface.

[0017] As a further improvement of the present invention, the redox mediator is an osmium polymer, and the cross-linking agent includes one or more of polyethylene glycol diglycidyl ether, polyethylene glycol triglycidyl ether, or polyethylene glycol tetraglycidyl ether.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] By adopting the technical solution of the present invention and introducing poloxamer and zwitterionic polymers, the enzyme layer coating process can be optimized, the coffee ring effect can be suppressed, and the consistency of the sensor and the accuracy of monitoring can be improved. Moreover, while maintaining the sensor signal transmission performance unchanged, the activity and long-term stability of the bioenzyme preparation during production, storage, transportation and use (such as in extreme pH, high temperature or high ionic strength environments) can be significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 These are the enzyme coating uniformity test results of Example 1 and Comparative Examples 1 to 3 of the present invention.

[0021] Figure 2 This is the cyclic voltammetry curve of the enzyme electrode of Example 1 of the present invention.

[0022] Figure 3 This is a graph showing the test results of the enzyme electrode coated with the enzyme membrane according to Example 1 of the present invention at different glucose concentrations.

[0023] Figure 4 3 is a residual diagram of the test results of the enzyme electrode coated with the enzyme membrane in Example 1 of the present invention at different glucose concentrations. DETAILED DESCRIPTION

[0024] The preferred embodiments of the present invention are described in further detail below.

[0025] An enzyme protectant for a continuous glucose sensor, comprising poloxamer F127 and a zwitterionic polymer PSBMA, with the mass percentages of the poloxamer and zwitterionic polymer in the enzyme layer being 0.1% to 2% and 0.5% to 5%, respectively. The poloxamer is used to inhibit the coffee ring effect, improve the uniformity of the enzyme layer, and help stabilize the enzyme conformation; while the zwitterionic polymer PSBMA forms a super-strong hydration layer to prevent enzyme aggregation, resist nonspecific adsorption, and improve the signal-to-noise ratio.

[0026] Further preferably, the mass percentage concentrations of the poloxamer F127 and the zwitterionic polymer PSBMA in the enzyme layer are 0.5% to 1% and 1% to 3%, respectively. Using this technical solution, the mass percentage concentration of the poloxamer is 0.5% to 1%, which can form a micellar structure. Dynamic light scattering (DLS) verification of particle size distribution results shows that the addition of this enzyme protectant can evenly disperse the enzyme molecules. Enzyme contact angle tests show that the contact angle is reduced by 20% to 30%, which can regulate the droplet drying dynamics. The mass percentage concentration of the zwitterionic polymer PSBMA is 1% to 3%. Compared with PEG, the zeta potential test shows that the surface charge is neutralized, the enzyme aggregation is reduced, and the particle size distribution is narrowed; fluorescent labeling experiments confirm that non-specific protein adsorption is reduced by more than 90%.

[0027] The enzyme protectant is used in the enzyme layer of a continuous glucose sensor, which also includes 5 mg / mL of glucose oxidase (GOx), which serves as the core enzyme component that catalyzes the glucose reaction; 10 mg / mL of a redox mediator (such as an osmium polymer) that plays an electron transfer role; and 7.5 mg / mL of a crosslinker that solidifies each component on the electrode surface.

[0028] When the mass percentage concentration of poloxamer (F127) is less than 0.5%, it is insufficient to suppress the coffee ring effect, and the enzyme layer remains uneven. When the mass percentage concentration is greater than 2%, the solution viscosity may be too high, affecting the accuracy of inkjet printing or dispensing.

[0029] For the zwitterionic polymer PSBMA, when the mass percentage concentration is less than 0.5%, the hydration layer is incomplete, the enzyme protection effect is limited, and the activity loss at high temperature is greater than 50%. When the mass percentage concentration is greater than 5%, electron transfer may be hindered (CV cyclic voltammetry shows a decrease in the mediator redox peak current).

[0030] The following describes the details in conjunction with specific embodiments.

[0031] Example 1

[0032] Poloxamer F127 and zwitterionic polymer PSBMA are used as enzyme protectants for continuous glucose sensor, and the mass percentage concentrations of poloxamer F127 and zwitterionic polymer PSBMA are 1% and 2%, respectively.

[0033] The enzyme protectant is used in the enzyme layer of a continuous glucose sensor, which also includes 5 mg / mL of glucose oxidase (GOx), which serves as the core enzyme component that catalyzes the glucose reaction; 10 mg / mL of a redox mediator (such as an osmium polymer) that plays an electron transfer role; and 7.5 mg / mL of a cross-linker, polyethylene glycol diglycidyl ether, which is used to solidify each component on the electrode surface.

[0034] Comparative Example 1

[0035] Only F127 was used as the enzyme protectant with a mass percentage concentration of 1%. Others were the same as in Example 1.

[0036] Comparative Example 2

[0037] Only PSBMA was used as the enzyme protective agent with a mass percentage concentration of 2%. Other steps were the same as those in Example 1.

[0038] Comparative Example 3

[0039] Albumin is added as an enzyme protectant using existing technology.

[0040] The continuous glucose sensors of Example 1 and Comparative Examples 1-3 were subjected to accelerated aging experiments, and the results of enzyme activity retention are shown in Table 1. It can be seen that the technical solution of Example 1 has higher activity retention and good enzyme stability.

[0041] Table 1

[0042]

[0043] The continuous glucose sensors of Example 1, Comparative Example 1 and Comparative Example 2 were tested for relevant parameters such as osmotic pressure, and the results are shown in Table 2. It can be seen that the technical solution of Example 1 has higher local water activity, glucose diffusion coefficient, and lower enzyme conformation fluctuation amplitude. In the composite system of Example 1, PSBMA locks water to stabilize the enzyme, and F127 constructs a mass transfer channel. The two work together to significantly optimize the enzyme microenvironment, and the effect far exceeds the superposition of single components. PSBMA is responsible for creating and maintaining a high water activity, dynamically stable hydrated microenvironment around the enzyme molecules. F127 is responsible for constructing a low-resistance, hydrophilic, fast mass transfer channel around the enzyme complex to ensure efficient circulation of reactants and products. The combination of the two, the "water environment" locked by PSBMA provides an ideal operating basis for the "channel" of F127, and the "channel" of F127 ensures that the microenvironment optimized by PSBMA can efficiently exchange substances, and together optimize the enzyme microenvironment to a level far exceeding that of a single component.

[0044] Table 2

[0045]

[0046] The sensors of Example 1, Comparative Example 1, and Comparative Example 2 were implanted for 14 days and continuously monitored. The reliability data comparison is shown in Table 3. A sensor containing 5% of a commercial PEG group was used as Comparative Example 3.

[0047] Table 3

[0048] system MARD(%) Drift rate (% / day) Calibration times / week Comparative Example 3 10.2 1.8 3.2 Comparative Example 1 7.5 0.9 1.5 Comparative Example 2 8.3 1.1 1.8 Example 1 4.2 0.1 0.3

[0049] The coating uniformity of the enzymes of Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 3 was tested by laser confocal microscopy. The results are as follows: Figure 1 shown.

[0050] Figure 1 The lower right corner shows the enzyme spot image of Comparative Example 3, which exhibits a distinct coffee ring effect with a thin center and thick edges. The lower left, middle, and upper right corners correspond to images of Comparative Example 1 using only F127 (1%), Comparative Example 2 using only PSBMA (2%), and Example 1 using F127 (1%) + PSBMA (2%), respectively. It can be seen that the enzyme spot of Example 1 exhibits uniform thickness at the center and edges. While the enzyme in Comparative Example 1, which uses only F127, can reduce edge accumulation during coating, slight unevenness still exists (coefficient of thickness variation ~15%). However, the combined use of F127 and PSBMA in Example 1 reduces the coefficient of thickness variation to <5%.

[0051] It can be seen that the combination of poloxamer and zwitterionic polymers can further optimize enzyme stability and signal consistency, and promote the development of CGMS towards higher reliability.

[0052] The cyclic voltammetry curve of the enzyme electrode of Example 1 is as follows: Figure 2 As shown in FIG, a cyclic voltammetry curve (CV curve) is obtained by changing the electrode potential and measuring the current response on the electrode. After multiple scans, the cyclic voltammetry curves of the enzyme electrode of Example 1 still highly overlap, indicating that the electrode reaction is reversible.

[0053] The enzyme electrode coated with the enzyme of Example 1 was tested at different glucose concentrations. The results are as follows: Figure 3 As shown in the results, the signal was uniform and consistent during the test, and the linear R 2 >0.99, the consistency of the enzyme electrode is good. Figure 4 As shown, it can be seen that the data points of the enzyme electrode coated with the outer membrane in Example 1 are closely clustered together, indicating that the difference between the data is small, the discreteness is low, and the consistency of the sensor is good.

[0054] Example 2

[0055] Based on Example 1, the difference of this example is that the mass percentage concentration of F127 is 4%, and the mass percentage concentration of PSBMA is 3%.

[0056] Example 3

[0057] Based on Example 1, the difference of this example is that the mass percentage concentration of F127 is 6%, and the mass percentage concentration of PSBMA is 5%.

[0058] The performance comparison of Examples 1 to 3 and Comparative Example 3 is shown in Table 4.

[0059] From the structures in Table 4, it can be seen that the technical solution of Example 1 has better enzyme stability, good deposition uniformity, and good signal consistency.

[0060] Table 4

[0061]

[0062] The above is a further detailed description of the present invention in conjunction with specific preferred embodiments, and the present invention includes but is not limited to the above specific implementation contents. For those skilled in the art of the present invention, without departing from the concept of the present invention, several simple deductions or substitutions can be made, which should be considered to fall within the scope of protection of the present invention.

Claims

1. An enzyme protectant for a continuous glucose sensor, characterized in that: The components include poloxamer and zwitterionic polymer, and the mass percentage concentrations of the poloxamer and zwitterionic polymer are 0.1% to 2% and 0.5% to 5% respectively.

2. The enzyme protectant for a continuous glucose sensor according to claim 1, characterized in that: The mass percentage concentrations of the poloxamer and the zwitterionic polymer are 1% and 2% respectively.

3. The enzyme protectant for continuous glucose sensor according to claim 2, characterized in that: The zwitterionic polymer is at least one of polymethacrylate sulfobetaine, polyethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer, polyethylene glycol-polylactic acid, polyethylene glycol-polycaprolactone, polyacrylic acid amphiphilic polymer, natural amphiphilic polymer, zwitterionic-hydrophobic block copolymer, polysorbate 80, and polyoxyethylene lauryl ether.

4. The enzyme protectant for a continuous glucose sensor according to claim 3, characterized in that: The zwitterionic polymer is polymethacrylate sulfobetaine.

5. The enzyme protectant for a continuous glucose sensor according to claim 3, characterized in that: The polyethylene glycol-polylactic acid includes methoxy PEG-polylactic acid, the polyethylene glycol-polycaprolactone includes polyethylene glycol-polycaprolactone; the polyacrylic acid amphiphilic polymer includes at least one of polyacrylic acid-b-polystyrene and polymethyl methacrylate-b-polyethylene glycol, the natural amphiphilic polymer includes at least one of chitosan derivatives and hyaluronic acid-hydrophobic modifiers, and the hyaluronic acid-hydrophobic modifier includes HA-octadecylamine; the zwitterion-hydrophobic block copolymer includes at least one of polysulfobetaine-b-polypropylene glycol and polycarboxybetaine-b-polystyrene; the polyethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer includes 6. A continuous glucose sensor, characterized in that: The components of the enzyme layer include the enzyme protectant for the continuous glucose sensor according to any one of claims 1 to 5, glucose oxidase, a redox mediator, and a cross-linking agent for solidifying the components on the electrode surface.

7. The continuous glucose sensor according to claim 6, wherein: The redox mediator is an osmium polymer, and the cross-linking agent includes one or more of polyethylene glycol diglycidyl ether, polyethylene glycol triglycidyl ether, or polyethylene glycol tetraglycidyl ether.

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