A flexible enzymatic glucose sensor detectable in vitro

Abstract

The application discloses a flexible enzyme glucose sensor capable of in-vitro detection, which comprises a flexible substrate and an electrode system arranged on the flexible substrate, the electrode system comprising a working electrode, a counter electrode and a reference electrode, a functional layer formed by compounding amino-graphene quantum dots and hollow gold nanospheres is modified on the working electrode, and glucose oxidase immobilized by chitosan is further modified on the functional layer. The sensor has a good linear relationship in the range of 50 muM to 1.4 mM, has the advantages of high sensitivity and good stability, and can be used for detecting urine glucose.

Description

Technical Field

[0001] This invention relates to a flexible enzyme glucose sensor that can be detected in vitro. Background Technology

[0002] Current blood glucose testing technologies primarily rely on invasive blood sampling methods, which pose a significant challenge to frequent blood glucose monitoring. For example, in the management of type 1 diabetes in adolescents, children need to have their blood glucose levels monitored every time they eat, making invasive blood sampling methods unsuitable. Continuous glucose monitoring (CGM) technology refers to the technology of continuously monitoring the glucose concentration in subcutaneous interstitial fluid using a glucose sensor. Compared to traditional blood glucose meters, it avoids the pain and inconvenience of finger-prick blood sampling and is highly portable. However, its high cost limits its widespread application.

[0003] Currently, diabetes detection primarily relies on monitoring blood glucose levels, but other bodily fluids, such as urine, offer additional advantages for diabetes management. Kidney complications are among the most common long-term complications of diabetes, in which glucose is excreted through urine. Therefore, exploring reliable methods for detecting glucose in urine is crucial. While commercial urine strips offer a simple method for detecting urinary glucose, their poor sensitivity and low reliability limit their use. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the present invention provides a flexible enzyme glucose sensor that can be detected in vitro.

[0005] To solve the above problems, the present invention adopts the following technical solution:

[0006] A flexible enzyme glucose sensor capable of in vitro detection is characterized in that: the flexible enzyme glucose sensor includes a flexible substrate and an electrode system disposed on the flexible substrate; the electrode system includes a working electrode, a counter electrode, and a reference electrode; a functional layer is modified on the working electrode, the functional layer being composed of amino-based graphene quantum dots and hollow gold nanospheres; and chitosan-immobilized glucose oxidase is further modified on the functional layer.

[0007] Furthermore, the functional layer is obtained by first drop-coating an amino-graphene quantum dot (af-GQD) solution onto the surface of the working electrode, followed by drop-coating a hollow gold nanosphere solution. The amino groups of af-GQD interact with the hollow gold nanospheres, covalently fixing the hollow gold nanospheres to the electrode surface. Even further, the concentration of the amino-graphene quantum dot solution is 1–6 mg / mL.

[0008] Furthermore, the chitosan-immobilized glucose oxidase is formed by drop-coating a mixed solution of glucose oxidase and chitosan onto the functional layer. Even further, the mixed solution of glucose oxidase and chitosan is obtained by adding glucose oxidase to a chitosan solution and ultrasonically dispersing it uniformly; the chitosan solution has a pH value between 4.2 and 6.3 and a mass concentration of 0.1% to 1.0%, and the concentration of glucose oxidase in the resulting mixed solution is 15 to 30 mg / mL.

[0009] Furthermore, the flexible substrate is made of polyethylene terephthalate (PET).

[0010] Furthermore, the counter electrode and the working electrode are formed by printing conductive carbon paste on a flexible substrate, and the reference electrode is formed by printing conductive silver paste on a flexible substrate. The immobilization temperature of the silver paste and carbon paste is between 120 and 150°C, and the immobilization time is between 10 and 15 minutes.

[0011] Compared with existing technologies, the beneficial effects of this invention are reflected in:

[0012] 1. This invention introduces amino-based graphene quantum dots and hollow gold nanospheres into the working electrode. By utilizing the interaction between the amino groups of af-GQD and the hollow gold nanospheres, the hollow gold nanospheres are covalently fixed on the electrode surface, thereby improving the electron transfer rate and conductivity of the sensing process.

[0013] 2. Hollow gold nanospheres possess a large surface area, high conductivity, and good biocompatibility, which can effectively increase the loading of glucose oxidase and maintain its biological activity. Due to the hollow structure of the hollow gold nanospheres, glucose oxidase molecules can exist both inside and outside the shell, effectively amplifying the intensity of the oxidation current for detecting glucose.

[0014] 3. The sensor of the present invention has a good linear relationship in the range of 50μM to 1.4mM, and has the advantages of high sensitivity and good stability, and can be used for the detection of urinary glucose. Attached Figure Description

[0015] Figure 1 This is a transmission electron microscope (TEM) image of the amino-based graphene quantum dots prepared in Example 1.

[0016] Figure 2 The infrared spectrum of the amino-based graphene quantum dots prepared in Example 1 is shown.

[0017] Figure 3 This is a transmission electron microscope (TEM) image of the hollow gold nanospheres prepared in Example 1.

[0018] Figure 4 Electrochemical characterization of the electrodes modified step by step in each step of Example 1.

[0019] Figure 5 The CV comparison graphs show the af-GQD modified electrode sensors obtained using three concentrations (2, 4, and 6 mg / mL) of amino-graphene quantum dot solutions.

[0020] Figure 6 Comparison of CV values ​​for glucose detection using modified electrodes at different pH values ​​(e.g.) Figure 6 (a) as shown) and the linear relationship between oxidation current and pH (as shown in the graph). Figure 6 (b) is shown.

[0021] Figure 7 This is a cyclic voltammogram of the glucose sensor prepared in Example 1 of the present invention.

[0022] Figure 8 The linear relationship between the oxidation peak of the glucose sensor prepared in Example 1 of this invention and the glucose concentration in the range of 50 μM to 1.4 mM is shown. Detailed Implementation

[0023] The technical solution of the present invention will be described in detail below through specific embodiments. The following embodiments are implemented under the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0024] Example 1

[0025] This embodiment provides a flexible enzyme glucose sensor capable of in vitro detection, including a flexible PET substrate and an electrode system disposed on the flexible substrate; the electrode system includes a working electrode, a counter electrode, and a reference electrode; the counter electrode and the working electrode are formed by printing conductive carbon paste on the flexible substrate, and the reference electrode is formed by printing conductive silver paste on the flexible substrate.

[0026] The working electrode is modified with a functional layer composed of amino-based graphene quantum dots and hollow gold nanospheres, and chitosan-immobilized glucose oxidase is further modified onto this functional layer. The specific modification method for the working electrode is as follows:

[0027] Step 1: Activation of screen-printed electrode: Soak the electrode in 0.05 mol / mL sulfuric acid solution for 2 hours, then use sulfuric acid solution as electrolyte to scan 20 times by cyclic voltammetry. After the peak value of the CV graph is basically stable, take it out, rinse it with pure water, air dry it at room temperature, and store it at 4℃.

[0028] Step 2: Modification of Aminographene Quantum Dots (af-GQD)

[0029] Weigh 2.5g of citric acid into an Erlenmeyer flask and heat it in a homogeneous reactor at 200℃ for 40 minutes. After the reaction is complete, remove the Erlenmeyer flask; a pale yellow oily liquid will appear inside. After cooling, add 35mL of ammonium hydroxide in a fume hood. The liquid gradually turns orange-red. Due to the decrease in temperature, some of the oily liquid solidifies and needs to be sonicated until completely dissolved. Then seal and place in the dark for 1-2 days until the liquid turns dark yellow. Then place it in a 300D dialysis bag for dialyzing, changing the water every hour until the pH of the liquid in the dialysis bag reaches 7. Freeze-dry the resulting pale yellow liquid to obtain af-GQD powder.

[0030] af-GQD powder was added to deionized water to prepare a 4 mg / mL solution, which was then drop-coated onto the electrode surface and dried to obtain an af-GQD modified electrode.

[0031] Step 3: Modification of hollow gold nanospheres (HGNs)

[0032] In a 250 mL three-necked round-bottom flask, 100 mL of pure water was mixed with 100 μL of 0.4 M cobalt chloride solution and 400 μL of 0.1 M sodium citrate solution. Simultaneously, 100 μL of a 1 wt% polyvinylpyrrolidone (PVP) solution with an average molecular weight of 58,000 was added. Argon gas was used for bubbling instead of magnetic stirring, as the cobalt nanoparticles are magnetic and using a magnetic stirrer would lead to chain formation. Bubbling was performed for 40 min to remove oxygen from the solution. Then, 100 μL of 1.0 M sodium borohydride solution was added. After a few seconds, the solution changed from light pink to dark black, indicating the formation of cobalt nanoparticles. The added PVP slowed the reaction between the cobalt nanoparticles and oxygen when the system was introduced into an aerobic environment, ensuring a complete reaction between chloroauric acid and the cobalt nanoparticles.

[0033] Subsequently, 30 mL of the cobalt nanoparticle solution was transferred to a 100 mL beaker, and 10 mL of pure water and 62 μL of 1 wt% chloroauric acid solution were immediately added to reduce Au by cobalt. 3+ The solution was stirred for another five minutes under aerobic conditions to allow the oxidation and displacement reactions of cobalt to proceed simultaneously. The color slowly changing from black to dark green indicated that HGNs formation was complete and the cobalt had reacted completely. The cobalt oxide generated during the reaction, due to the addition of sodium citrate, decomposed to form cobalt ions. Finally, the solution was centrifuged at 10,000 rpm at room temperature for 15 minutes. The supernatant was removed and dispersed in 5 mL of pure water to obtain a pure HGNs solution.

[0034] In step 2, a pure HGNs solution is drop-coated onto the surface of the af-GQD modified electrode, and after drying, an af-GQD-HGNs composite material modified electrode is obtained.

[0035] Step 4: Disperse glucose oxidase in a 0.2% (pH=5) chitosan solution and ultrasonically disperse until homogeneous. Then, drop-coat the resulting mixed solution onto the electrode surface prepared in Step 3 and allow it to stand for 12 hours to allow glucose oxidase to deposit on the electrode surface, thus obtaining the modified working electrode. The concentration of glucose oxidase in the mixed solution is 30 mg / mL. The prepared electrode is stored at 4°C.

[0036] Example 2

[0037] The flexible enzyme glucose sensor provided in this embodiment has the same structure as that in Embodiment 1, the difference being the specific modification method of the working electrode as follows:

[0038] Step 1: Activation of screen-printed electrode: Soak the electrode in 0.05 mol / mL sulfuric acid solution for 2 hours, then use sulfuric acid solution as electrolyte to scan 20 times by cyclic voltammetry. After the peak value of the CV graph is basically stable, take it out, rinse it with pure water, air dry it at room temperature, and store it at 4℃.

[0039] Step 2: Modification of Aminographene Quantum Dots (af-GQD)

[0040] Weigh 5g of citric acid into an Erlenmeyer flask and heat it in a homogeneous reactor at 200℃ for 40 minutes. After the reaction is complete, remove the flask; a pale yellow oily liquid will appear inside. After cooling, add 70mL of ammonium hydroxide in a fume hood. The liquid gradually turns orange-red. Due to the decrease in temperature, some of the oily liquid solidifies and needs to be sonicated until completely dissolved. Then seal and place in the dark for 1-2 days until the liquid turns dark yellow. Then place it in a 300D dialysis bag for dialyzing, changing the water every hour until the pH of the liquid in the dialysis bag reaches 7. Freeze-dry the resulting pale yellow liquid to obtain af-GQD powder.

[0041] af-GQD powder was added to deionized water to prepare a 2 mg / mL solution, which was then drop-coated onto the electrode surface and dried to obtain an af-GQD modified electrode.

[0042] Step 3: Modification of hollow gold nanospheres (HGNs)

[0043] In a 250 mL three-necked round-bottom flask, 100 mL of pure water was mixed with 100 μL of 0.4 M cobalt chloride solution and 400 μL of 0.1 M sodium citrate solution. Simultaneously, 100 μL of a 1 wt% polyvinylpyrrolidone (PVP) solution with an average molecular weight of 58,000 was added. The solution was bubbled with argon gas for 40 min to remove oxygen. Then, 100 μL of 1.0 M sodium borohydride solution was added to the solution. After a few seconds, the solution changed from a pale pink to a darker color, indicating the formation of cobalt nanoparticles.

[0044] Subsequently, 30 mL of the cobalt nanoparticle solution was transferred to a 100 mL beaker, and 10 mL of pure water and 62 μL of 1 wt% chloroauric acid solution were immediately added to reduce Au by cobalt. 3+ The solution was stirred for another five minutes under aerobic conditions to allow the oxidation and displacement reactions of cobalt to proceed simultaneously. The color slowly changing from black to dark green indicated that HGNs formation was complete and the cobalt had reacted completely. The cobalt oxide generated during the reaction, due to the addition of sodium citrate, decomposed to form cobalt ions. Finally, the solution was centrifuged at 10,000 rpm at room temperature for 15 minutes. The supernatant was removed and the solution dispersed in 10 mL of pure water to obtain a pure HGNs solution.

[0045] In step 2, a pure HGNs solution is drop-coated onto the surface of the af-GQD modified electrode, and after drying, an af-GQD-HGNs composite material modified electrode is obtained.

[0046] Step 4: Disperse glucose oxidase in a 0.2% (pH=5) chitosan solution and ultrasonically disperse until homogeneous. Then, drop-coat the resulting mixed solution onto the electrode surface prepared in Step 3 and allow it to stand for 12 hours to allow glucose oxidase to deposit on the electrode surface, thus obtaining the modified working electrode. The concentration of glucose oxidase in the mixed solution is 30 mg / mL. The prepared electrode is stored at 4°C.

[0047] Example 3

[0048] The flexible enzyme glucose sensor provided in this embodiment has the same structure as that in Embodiment 1, the difference being the specific modification method of the working electrode as follows:

[0049] Step 1: Activation of screen-printed electrode: Soak the electrode in 0.05 mol / mL sulfuric acid solution for 2 hours, then use sulfuric acid solution as electrolyte to scan 20 times by cyclic voltammetry. After the peak value of the CV graph is basically stable, take it out, rinse it with pure water, air dry it at room temperature, and store it at 4℃.

[0050] Step 2: Modification of Aminographene Quantum Dots (af-GQD)

[0051] Weigh 7.5g of citric acid into an Erlenmeyer flask and heat it in a homogeneous reactor at 200℃ for 40 minutes. After the reaction is complete, remove the flask; a pale yellow oily liquid will appear inside. After cooling, add 105mL of ammonium hydroxide in a fume hood. The liquid gradually turns orange-red. Due to the decrease in temperature, some of the oily liquid solidifies and needs to be sonicated until completely dissolved. Then seal and place in the dark for 1-2 days until the liquid turns dark yellow. Then place it in a 300D dialysis bag for dialyzing, changing the water every hour until the pH of the liquid in the dialysis bag reaches 7. Freeze-dry the resulting pale yellow liquid to obtain af-GQD powder.

[0052] af-GQD powder was added to deionized water to prepare a 6 mg / mL solution, which was then drop-coated onto the electrode surface and dried to obtain an af-GQD modified electrode.

[0053] Step 3: Modification of hollow gold nanospheres (HGNs)

[0054] In a 250 mL three-necked round-bottom flask, 100 mL of pure water was mixed with 100 μL of 0.4 M cobalt chloride solution and 400 μL of 0.1 M sodium citrate solution. Simultaneously, 100 μL of a 1 wt% polyvinylpyrrolidone (PVP) solution with an average molecular weight of 58,000 was added. The solution was bubbled with argon gas for 40 min to remove oxygen. Then, 100 μL of 1.0 M sodium borohydride solution was added to the solution. After a few seconds, the solution changed from a pale pink to a darker color, indicating the formation of cobalt nanoparticles.

[0055] Subsequently, 30 mL of the cobalt nanoparticle solution was transferred to a 100 mL beaker, and 10 mL of pure water and 62 μL of 1 wt% chloroauric acid solution were immediately added to reduce Au by cobalt. 3+ The solution was stirred for another five minutes under aerobic conditions to allow the oxidation and displacement reactions of cobalt to proceed simultaneously. The color slowly changing from black to dark green indicated that HGNs formation was complete and the cobalt had reacted completely. The cobalt oxide generated during the reaction, due to the addition of sodium citrate, decomposed to form cobalt ions. Finally, the solution was centrifuged at 10,000 rpm at room temperature for 15 minutes. The supernatant was removed and dispersed in 20 mL of pure water to obtain a pure HGNs solution.

[0056] In step 2, a pure HGNs solution is drop-coated onto the surface of the af-GQD modified electrode, and after drying, an af-GQD-HGNs composite material modified electrode is obtained.

[0057] Step 4: Disperse glucose oxidase in a 0.2% (w / v) chitosan solution at pH 5 and ultrasonically disperse until homogeneous. Then, drop-coat the resulting mixed solution onto the electrode surface prepared in Step 3 and allow it to stand for 12 hours to allow glucose oxidase to deposit on the electrode surface, thus obtaining the working electrode modified with the af-GQD-HGNs-CS-GOD composite material. The concentration of glucose oxidase in the mixed solution is 30 mg / mL. The prepared electrode is stored at 4°C.

[0058] I. Morphological and structural characterization of af-GQD

[0059] Figure 1 This is a transmission electron microscope image of the af-GQD prepared in Example 1. The image shows that the af-GQD particles are uniform in size, with an average particle size of approximately 5 nm. Figure 2The FTIR spectra further characterized the functional groups of af-GQD: at 3415 cm⁻¹ -1 There is a distinct FT-IR absorption peak at 1562 cm⁻¹, which should be attributed to the stretching vibration of the NH bond; while at 1562 cm⁻¹... -1 The absorption peak at 1400 cm⁻¹ is attributed to the bending vibration of the NH bond; the stretching vibrations of the CN and C=O bonds are respectively attributed to the bending vibrations of the NH bond. -1 and 1677cm -1 The absorption peak at a certain point indicates the presence of an amide in the af-GQD structure. These results fully demonstrate the successful preparation of af-GQD.

[0060] II. Morphological Characterization of Hollow Gold Nanospheres

[0061] Figure 3 This is a transmission electron microscope image of hollow gold nanospheres. The image shows that the material has a hollow structure and a particle size of about 25 nm.

[0062] III. Electrochemical Characterization of Gradually Modified Electrodes

[0063] The electrodes modified in steps 2-4 of Example 1 were subjected to cyclic voltammetry tests on a CHI660E electrochemical workstation. The electrolyte was a mixture of 0.1 M KCl and 5 mM potassium ferricyanide / potassium ferrocyanide (volume ratio 1:1). The results are as follows. Figure 4 As shown, the af-GQD modified electrode exhibits a faster electron transfer rate in the electrolyte than the bare electrode, resulting in an increased current response. The af-GQD-HGNs composite material modified electrode shows an even faster electron transfer rate in the electrolyte than the af-GQD modified electrode, further increasing the current response. However, the af-GQD-HGNs-CS-GOD composite material modified electrode shows a decreased electron transfer rate in the electrolyte; this decrease in current response is due to the chitosan film blocking electron flow at the electrode surface.

[0064] IV. Concentration Optimization of Graphene Quantum Dots

[0065] The aminated graphene quantum dots from step 2 of Example 1 were prepared into three concentrations of 2, 4, and 6 mg / mL. 30 μL of each aminated graphene quantum dot solution was dropped onto the electrode surface to obtain the af-GQD modified electrode.

[0066] The modified electrode was tested by cyclic voltammetry on a CHI660E electrochemical workstation. The electrolyte was a PBS solution with pH=8.5 containing 1mM glucose. Figure 5 The image shows a comparison of the current response of the working electrode modified with af-GQD obtained in step 2 under different concentrations of aminated graphene quantum dots. It can be seen that the sensor with an aminated graphene quantum dot concentration of 4 mg / mL has a higher current response for glucose detection.

[0067] V. pH Optimization of the Prepared Glucose Sensor

[0068] The electrode modified with the af-GQD-HGNs-CS-GOD composite material in Example 1 was subjected to cyclic voltammetry testing on a CHI660E electrochemical workstation. The electrolyte was a 0.1M PBS solution containing 1mM glucose at different pH values. Figure 6 Comparison of CV values ​​for glucose detection using modified electrodes at different pH values ​​(e.g.) Figure 6 (a) shows) and the linear relationship between oxidation current and pH value is obtained (as shown in Figure 1). Figure 6 As shown in (b), it can be seen that the oxidation current of glucose is highest at pH = 8.5.

[0069] VI. Response of the sensor obtained in Example 1 to glucose

[0070] Figure 7 The image shows the cyclic voltammetry curves of the glucose sensor prepared in Example 1 within a glucose concentration range of 50 μM to 1.4 mM. From bottom to top, the glucose concentrations are 0.05, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, and 1.6 mM. It can be seen that the oxidation current increases with increasing glucose concentration. Simultaneously, it can be obtained... Figure 8 The linear relationship.

[0071] Figure 8 The linear relationship between the oxidation peak value and glucose concentration of the glucose sensor prepared in Example 1 within the glucose concentration range of 50 μM-1.4 mM was determined based on y = 0.473x + 2.385, R... 2 =0.994 indicates that the sensor exhibits good linearity within the 50μM-1.4mM range. This range can also monitor glucose levels in urine, yielding a calculated sensitivity of 19.204 μA / mM / cm. 2 .

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A flexible enzyme glucose sensor capable of in vitro detection, characterized in that: The flexible enzyme glucose sensor includes a flexible substrate and an electrode system disposed on the flexible substrate; the flexible substrate is made of polyethylene terephthalate (PET); the electrode system includes a working electrode, a counter electrode, and a reference electrode; a functional layer is modified on the working electrode, the functional layer being composed of amino-based graphene quantum dots and hollow gold nanospheres; and chitosan-immobilized glucose oxidase is also modified on the functional layer. The functional layer is obtained by first drop-coating an amino-graphene quantum dot solution onto the surface of the working electrode, and then drop-coating a hollow gold nanosphere solution. The chitosan-immobilized glucose oxidase is formed by drop-coating a mixed solution of glucose oxidase and chitosan onto a functional layer. The counter electrode and the working electrode are formed by printing conductive carbon paste on a flexible substrate, and the reference electrode is formed by printing conductive silver paste on a flexible substrate; The hollow gold nanospheres are prepared as follows: In a 250 mL three-necked round-bottom flask, 100 mL of pure water is mixed with 100 μL of 0.4 M cobalt chloride solution and 400 μL of 0.1 M sodium citrate solution. At the same time, 100 μL of 1 wt% polyvinylpyrrolidone solution with an average molecular weight of 58000 is injected. The solution is bubbled with argon gas for 40 min to remove oxygen. 100 μL of 1.0 M sodium borohydride solution was injected into the solution to form cobalt nanoparticles; subsequently, 30 mL of the cobalt nanoparticle solution was transferred to a 100 mL beaker, and 10 mL of pure water and 62 μL of 1 wt% chloroauric acid solution were immediately added to reduce Au by cobalt. 3+ The solution was stirred for another five minutes under an aerobic environment to allow the oxidation and displacement reactions of cobalt to proceed simultaneously. Finally, the solution was centrifuged at 10,000 rpm for 15 minutes at room temperature to remove the supernatant and disperse it in 5 mL of pure water to obtain a hollow gold nanosphere solution.

2. The in vitro detectable flexible enzyme glucose sensor according to claim 1, characterized in that: The concentration of the amino-based graphene quantum dot solution is 1–6 mg / mL.

3. The in vitro detectable flexible enzyme glucose sensor according to claim 1, characterized in that: The glucose oxidase and chitosan mixed solution is obtained by adding glucose oxidase to a chitosan solution and dispersing it evenly by ultrasonication; the pH value of the chitosan solution is between 4.2 and 6.3, the mass concentration is between 0.1 and 1.0%, and the concentration of glucose oxidase in the resulting mixed solution is 15 to 30 mg / mL.

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