Preparation method of ABTS-HRP-based liposome bionic composite material

Abstract

The invention discloses a preparation method of an ABTS-HRP-based liposome bionic composite material, which comprises the following steps: encapsulating ABTS in a liposome through the method, and crosslinking catalase on the outer side of the liposome by using glutaraldehyde to form a bionic composite structure with efficient electron transfer capability and stability. Therefore, the hydrogen peroxide colorimetric-electrochemical dual-detection-mode sensor is constructed. The method comprises the specific steps of phospholipid membrane preparation, ABTS hydration packaging, ultrasonic dispersion, ultrafiltration purification and enzyme modification. The composite material solves the problems of complex preparation, poor stability, low response sensitivity and the like of a nanoprobe in the prior art, remarkably improves the detection efficiency and environmental adaptability of a biosensor, and can be widely applied to the fields of high-sensitivity detection of hydrogen peroxide and biomedicine.

Description

Technical Field

[0001] The present invention relates to the technical field of new energy materials, in particular to a method for preparing an ABTS-HRP-based liposome biomimetic composite material. Background Art

[0002] With the development of modern biosensors, improving their sensitivity, stability, and response speed has become a key research topic. Among various biosensors, hydrogen peroxide (H2O2) is an important biomarker and is widely used in medical diagnosis, environmental monitoring, and food safety testing. However, existing hydrogen peroxide sensors often face the following problems:

[0003] Complexity of nanoprobe preparation: Traditional nanoprobes involve multiple steps in the preparation process and require strict control of reaction conditions, which increases the complexity and cost of preparation.

[0004] Poor stability: Many nanoprobes lose stability during use due to environmental changes (such as temperature and pH changes), resulting in reduced reliability in practical applications.

[0005] Low response sensitivity: Many existing sensors exhibit poor sensitivity when detecting low concentrations of hydrogen peroxide, which limits their scope of practical applications, especially in areas requiring high-precision detection.

[0006] To overcome these problems, researchers have explored a variety of new materials and technologies to improve the performance of sensors. As an important biomaterial, liposomes have been widely used in drug delivery, gene therapy and biosensing due to their excellent biocompatibility, degradability and surface modifiability. The basic structure of liposomes is a phospholipid bilayer wrapped around an aqueous core, which can effectively encapsulate small molecules and biomacromolecules, enhancing their stability and biological activity. Liposome-based biomimetic composites have gradually become a research hotspot in biosensors in recent years due to their unique structural advantages. Liposomes can not only effectively encapsulate small molecule electron mediators such as ABTS, but also modify catalytic enzymes such as catalase (HRP) on the outside to build a more efficient and stable sensing system. These composite materials can greatly improve the stability of the catalyst while improving the efficiency of electron transfer, thereby improving the response speed and sensitivity of the sensor.

[0007] However, in existing studies, the application of ABTS encapsulation and HRP cross-linking in liposomes has not been fully explored, and how to optimize its structure to improve its performance in hydrogen peroxide detection remains an urgent problem to be solved. Summary of the Invention

[0008] The present invention aims to provide a method for preparing an ABTS-HRP-based liposome biomimetic composite material. By encapsulating ABTS within the liposomes and cross-linking catalase to the liposomes with glutaraldehyde, the resulting biomimetic composite structure exhibits efficient electron transfer and stability. This innovative design addresses existing issues such as the complex preparation, poor stability, and low response sensitivity of nanoprobes, significantly improving the detection efficiency and environmental adaptability of the biosensor, thus addressing the aforementioned background art issues.

[0009] To achieve the above object, the present invention provides the following technical solution: a method for preparing an ABTS-HRP-based liposome biomimetic composite material, comprising the following steps:

[0010] Step 1: Dissolve soybean lecithin and cholesterol in ethanol and sonicate until a transparent solution is formed;

[0011] Step 2: Pour the above solution into a round-bottom flask and perform thin-film rotary evaporation until a transparent film is formed on the flask;

[0012] Step 3: vacuum drying the round-bottom flask with the film;

[0013] Step 4: Add the diammonium salt (ABTS) solution to the round-bottom flask dried in step 3, place it in a rotary evaporator again for hydration, and collect the emulsion in the flask;

[0014] Step 5: The emulsion collected in step 4 is crushed in an ultrasonic crusher, and then ultrafiltered to obtain an ABTS-based liposome composite material;

[0015] Step 6: The ABTS-based liposome composite material obtained in step 5 is mixed with horseradish peroxidase (HRP) solution, and then drop-coated on the electrode and placed in glutaraldehyde vapor for cross-linking to obtain an ABTS-HRP liposome composite electrochemical biosensor.

[0016] Based on the above, in step 1, the phospholipid membrane is composed of soybean lecithin and cholesterol in a mass ratio of 4:1, 5 ml of ethanol is used, and ultrasonication is performed for 30 minutes.

[0017] Based on the above, in step 2, a thin film rotary evaporation is performed using a rotary evaporator, and the temperature of the rotary evaporator is controlled at 37°C.

[0018] Based on the above, in step 3, the vacuum drying temperature is 45° C. and the time is 2 h.

[0019] Based on the above, in step 4, the concentration of the diammonium salt (ABTS) solution is 6 mg / ml, the hydration temperature is 45° C., and the hydration time is 2 h.

[0020] Based on the above, in step 5, ultrasonic treatment is performed in an ice bath, with an ultrasonic crusher power of 60 W, 4 s pulse / 2 s interval, and lasting for 10 min.

[0021] Based on the above, in step six, the ABTS-based liposome composite material is mixed with the HRP solution and then drop-coated on the glassy carbon electrode and cross-linked with glutaraldehyde for 15 minutes.

[0022] Based on the above, liposomes are introduced to encapsulate the small molecule ABTS to enhance its stability in the electrode and electrolyte while maintaining its electrochemical activity.

[0023] Based on the above, the ABTS-based liposome composite material is prepared by the preparation method according to any one of claims 1-6.

[0024] Based on the above, the ABTS-based liposome composite material is cross-linked with HRP and used for detecting hydrogen peroxide in a neutral electrolyte.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) In the present invention, ABTS is encapsulated inside the liposome and cross-linked to the outside of the liposome using catalase (HRP) through glutaraldehyde. This design can effectively promote the transfer of electrons inside and outside the liposome. ABTS acts as an electron transfer medium, enhancing the response speed and sensitivity of the sensor.

[0027] (2) The present invention provides multiple signal outputs by combining the dual detection modes of colorimetry and electrochemical methods. Colorimetric detection can provide intuitive reaction progress information, while electrochemical detection can provide more accurate quantitative data, making the sensor detection more comprehensive and diversified. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 TEM images of blank liposomes and ABTS liposomes in the application examples of the present invention;

[0029] Figure 2 The particle size distribution diagrams of blank liposomes and ABTS liposomes in the application examples of the present invention are shown;

[0030] Figure 3 This is a DPV diagram of the ABTS-Lip-HRP electrochemical colorimetric dual-mode sensor in an application example of the present invention detecting different concentrations of hydrogen peroxide;

[0031] Figure 4 This is a daylight image of the ABTS-Lip-HRP electrochemical colorimetric dual-mode sensor in an application example of the present invention detecting different concentrations of hydrogen peroxide;

[0032] Figure 5 This is a UV-visible spectrum diagram of the ABTS-Lip-HRP electrochemical colorimetric dual-mode sensor in an application example of the present invention detecting different concentrations of hydrogen peroxide;

[0033] Figure 6 is a colorimetric detection curve diagram of the electrochemical colorimetric dual-mode hydrogen peroxide sensor solution at different hydrogen peroxide concentrations in an application example of the present invention;

[0034] Figure 7 This is an analysis diagram of the selectivity and anti-interference performance of the colorimetric mode of the electrochemical colorimetric dual-mode hydrogen peroxide sensor in an application example of the present invention;

[0035] Figure 8 This is the catalytic cyclic voltammetry curve of ABTS-Lip-HRP to 1mM H2O2 in the application example of the present invention. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0037] Example 1

[0038] This embodiment first provides a liposome material, the synthesis method of which includes the following steps: dissolving 75.8 mg of soybean lecithin and 19 mg of cholesterol in 5 ml of methanol, and performing ultrasonic treatment until completely dissolved.

[0039] The solution was poured into a 250 ml round-bottom flask and placed in a rotary evaporator to remove methanol under vacuum at 37°C, and then placed in a vacuum drying oven at 45°C for 2 h.

[0040] 5 ml of PBS solution was added to the dried round-bottom flask and placed in a rotary evaporator again for hydration at 45° C. for 2 h to obtain blank liposome material.

[0041] To characterize the blank liposomes, Figure 1 a is a TEM image of blank liposomes. It can be seen from the figure that the synthesized blank liposomes are uniform in size. Figure 2 a is the particle size distribution diagram of blank liposomes.

[0042] Example 2

[0043] This embodiment provides an ABTS liposome material, and the synthesis method thereof comprises the following steps: dissolving 75.8 mg of soybean lecithin and 19 mg of cholesterol in 5 ml of methanol, and performing ultrasonic treatment until completely dissolved.

[0044] The solution was poured into a 250 ml round-bottom flask and placed in a rotary evaporator to remove methanol under vacuum at 37°C, and then placed in a vacuum drying oven at 45°C for 2 h.

[0045] 30 mg of ABTS was dissolved in PBS solution and added to the dried round-bottom flask, which was then placed in a rotary evaporator again and hydrated at 45° C. for 2 h to obtain ABTS liposome material.

[0046] To characterize the blank liposomes, Figure 1 b is a TEM image of ABTS liposomes. It can be seen from the figure that the synthesized ABTS liposomes are uniform in size. Figure 2 b is the particle size distribution of ABTS liposomes.

[0047] Verification Example 1

[0048] To verify that ABTS-Lip still retains the electrochemical activity of ABTS itself, the ABTS-Lip solution obtained in Example 2 was drop-coated on a glassy carbon electrode and placed in 20 ml of PBS electrolyte at pH 7. An electrochemical scan was performed in the potential window of 0 V to 0.7 V to observe the changes in the electrochemical signal compared with ABTS alone.

[0049] At the same time, a 100-fold diluted ABTS-Lip solution was taken and placed in a cuvette, and its absorption intensity at 340 nm was measured in a UV spectrophotometer.

[0050] Example 3

[0051] This embodiment further provides a method for preparing an electrochemical colorimetric dual-mode hydrogen peroxide sensor, which specifically includes the following steps:

[0052] 20 μL of the mixed solution of ABTS-Lip and 3 mg / ml HRP obtained in Example 2 was drop-coated on a glassy carbon electrode and dried in a 37°C oven for 20 min. The sample on the electrode surface was removed before being completely dried and placed in glutaraldehyde vapor for cross-linking for 15 min. 1 ml of the ABTS-Lip obtained in Example 2 was mixed with 10 μL of 25% glutaraldehyde solution, reacted at 37°C for 20 min, dialyzed for 2 h, and then 3 mg of HRP solution was added. The mixture was incubated at 4°C overnight to obtain an electrochemical colorimetric dual-mode hydrogen peroxide sensor.

[0053] Example 4

[0054] This embodiment provides an electrochemical colorimetric dual-mode detection method for detecting hydrogen peroxide, which specifically includes the following steps:

[0055] The electrochemical sensing performance of the ABTS-LIP-HRP electrochemical sensor was tested using a three-electrode system with pH 7 PBS as the working solution. The three electrodes were a working electrode, a reference electrode, and a counter electrode. The working electrode was a glassy carbon electrode; the reference electrode was a saturated mercuric chloride electrode; and the counter electrode was a platinum electrode.

[0056] For the test of Example 4 Figure 3 As shown, the following results are obtained:

[0057] (1) The optimal operating voltage of the ABTS-LIP-HRP electrochemical sensor is 0.48 V, and it has specific selectivity for hydrogen peroxide;

[0058] (2) The ABTS-LIP-HRP electrochemical sensor can detect changes in hydrogen peroxide concentration within three seconds;

[0059] 10 μL of hydrogen peroxide solutions of different concentrations to be tested were added to the electrochemical colorimetric dual-mode hydrogen peroxide sensor prepared in Example 3 to prepare a 1 ml reaction system, and the color change of the solution was observed under sunlight.

[0060] Figure 4 Daylight images of the electrochemical colorimetric dual-mode hydrogen peroxide sensor solution at different hydrogen peroxide concentrations. As the hydrogen peroxide concentration increases, the solution gradually changes color from colorless to blue. UV-visible absorption spectra were measured using a UV-visible spectrophotometer, with the UV-visible spectrum observed in the 400-500 nm range.

[0061] Figure 5 The UV-visible spectra of the colorimetric electrochemical dual-mode hydrogen peroxide sensor solution at different hydrogen peroxide concentrations are shown. The functional relationship between the different hydrogen peroxide concentrations of the solution and the UV-visible absorption peak values is used to obtain the corresponding hydrogen peroxide concentration values between the UV-visible absorption peak values in the solution.

[0062] like Figure 5 As shown, the functional relationship between the hydrogen peroxide values of different concentrations of the solution and the UV-visible absorption peak values is used to obtain the hydrogen peroxide concentration value corresponding to the UV-visible absorption peak value in the solution.

[0063] Figure 6 This is a colorimetric detection curve of the electrochemical colorimetric dual-mode hydrogen peroxide sensor solution under different hydrogen peroxide concentrations, where the x-axis is the hydrogen peroxide concentration value and the y-axis is the UV-visible absorption peak value;

[0064] like Figure 6 As shown, the functional relationship between the UV-visible absorption peak value and the hydrogen peroxide concentration is: y = 0.00142x + 0.04194 (R 2=0.99), where y represents the UV-visible absorption peak at 420 nm and x represents the hydrogen peroxide concentration.

[0065] Verification Example 2

[0066] The electrochemical colorimetric dual-mode hydrogen peroxide sensor was prepared according to Example 3, and Na + Mg 2+ 、K+、Fe 2+ , His, UA, Glu, etc. were used as interfering ions to explore the selectivity and anti-interference ability of the electrochemical colorimetric dual-mode hydrogen peroxide sensor in detecting hydrogen peroxide. After reacting at 25-30℃ for 25min, spectral detection was performed using a UV-visible spectrophotometer, and the ultraviolet absorption changes at 420nm were observed by the electrochemical colorimetric dual-mode nanozyme sensor.

[0067] Figure 7 This is an analysis diagram of the selectivity and anti-interference of the colorimetric mode. It can be seen from the figure that the ultraviolet absorption value of the colorimetric-fluorescence dual-mode nanozyme sensor changes significantly only in the presence of hydrogen peroxide, indicating that the sensor has good selectivity and anti-interference for hydrogen peroxide detection.

Claims

1. A method for preparing an ABTS-HRP-based liposome biomimetic composite material, characterized in that: The method comprises the following steps: Step 1: Dissolve soybean lecithin and cholesterol in ethanol and sonicate until a transparent solution is formed; Step 2: Pour the above solution into a round-bottom flask and perform thin-film rotary evaporation until a transparent film is formed on the flask; Step 3: vacuum drying the round-bottom flask with the film; Step 4: Add the diammonium salt (ABTS) solution to the round-bottom flask dried in step 3, place it in a rotary evaporator again for hydration, and collect the emulsion in the flask; Step 5: The emulsion collected in step 4 is crushed in an ultrasonic crusher, and then ultrafiltered to obtain an ABTS-based liposome composite material; Step 6: The ABTS-based liposome composite material obtained in step 5 is mixed with horseradish peroxidase (HRP) solution, and then drop-coated on the electrode and placed in glutaraldehyde vapor for cross-linking to obtain an ABTS-HRP liposome composite electrochemical biosensor.

2. The method for preparing an ABTS-HRP-based liposome biomimetic composite material according to claim 1, wherein: In the step 1, the phospholipid membrane is composed of soybean lecithin and cholesterol in a mass ratio of 4:1, 5 ml of ethanol is used, and ultrasonication is performed for 30 minutes.

3. The method for preparing an ABTS-HRP-based liposome biomimetic composite material according to claim 1, wherein: In the step 2, a rotary evaporator is used to perform thin film rotary evaporation, and the temperature of the rotary evaporator is controlled at 37°C.

4. The method for preparing an ABTS-HRP-based liposome biomimetic composite material according to claim 1, wherein: In the step 3, the vacuum drying temperature is 45° C. and the time is 2 h.

5. The method for preparing an ABTS-HRP-based liposome biomimetic composite material according to claim 1, wherein: In the step 4, the concentration of the diammonium salt (ABTS) solution is 6 mg / ml, the hydration temperature is 45° C., and the hydration time is 2 h.

6. The method for preparing an ABTS-HRP-based liposome biomimetic composite material according to claim 1, characterized in that: In the step 5, ultrasonic treatment is performed in an ice bath with an ultrasonic disruptor power of 60 W, 4 s pulse / 2 s interval, and the duration is 10 min.

7. The method for preparing an ABTS-HRP-based liposome biomimetic composite material according to claim 1, characterized in that: In the step six, the ABTS-based liposome composite material is mixed with the HRP solution and then drop-coated on the glassy carbon electrode and cross-linked with glutaraldehyde for 15 minutes.

8. The method for preparing an ABTS-HRP-based liposome biomimetic composite material according to claim 1, characterized in that: The ABTS-based liposome composite material obtained in step five is prepared by encapsulating the small molecule ABTS in liposomes to enhance its stability in the electrode and electrolyte, while maintaining its electrochemical activity.

9. The method for preparing an ABTS-HRP-based liposome biomimetic composite material according to claim 8, characterized in that: The ABTS-based liposome composite material is prepared by the preparation method according to any one of claims 1 to 6.

10. Use of an ABTS-based liposome composite material prepared by the preparation method according to any one of claims 1 to 7, characterized in that: The ABTS-based liposome composite material is cross-linked with HRP and used for detecting hydrogen peroxide in a neutral electrolyte.

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