Paper-based molecularly imprinted sensor for detecting glucocorticoid and preparation method of paper-based molecularly imprinted sensor

Abstract

The invention provides a paper-based molecularly imprinted sensor for detecting glucocorticoid and a preparation method thereof, and the preparation method of the paper-based molecularly imprinted sensor for detecting glucocorticoid comprises the following steps: coating a screen-printed electrode with a nitrogen-doped carbon / carbon nanotube composite material with a hierarchical structure as an electrode modification material; and assembling the obtained screen-printed electrode and a paper-based microfluidic device. The nitrogen-doped carbon and the carbon nanotubes are utilized to form a hierarchical porous structure, and the sensor performance is more excellent due to high specific surface area and high conductivity. The paper-based microfluidic technology enables the sensor to realize simultaneous detection of various glucocorticoids, and shows the application value of the sensor in the field of inspection.

Description

Technical Field

[0001] The invention belongs to the field of biosensor detection, and in particular relates to a paper-based molecular imprinting sensor for detecting glucocorticoids and a preparation method thereof. Background Art

[0002] The adrenal glands produce a group of steroid hormones called glucocorticoids, including cortisol, cortisone, and corticosterone. Among them, the main effects of cortisol include raising blood sugar levels, suppressing the immune system, and helping metabolism. Abnormal cortisol can lead to high blood pressure, hirsutism, and affect cardiovascular as well as gastrointestinal, metabolic, musculoskeletal, immune, and menstrual disorders. Cortisone has the ability to bind to glucocorticoid receptors, which are often used in anti-inflammatory treatments, and higher levels of cortisone may lead to Cushing's syndrome. When corticosterone levels increase, there may be risks of aldosteroneoma, Cushing's syndrome, adrenal cortical tumors, etc. Therefore, effective, rapid, and reliable glucocorticoid testing is very valuable for dynamic health analysis to achieve comprehensive self-monitoring, health management, and personalized health care.

[0003] Molecularly imprinted polymers (MIPs) are polymer matrices obtained using molecular imprinting technology. This is a general synthetic technique used to design artificial receptors that are selectively sensitive to specific analytes. MIPs can mimic natural biological antibody-antigen receptors and use a "lock and key" mechanism to selectively and specifically bind to imprinted molecules. Currently commonly used MIP-based electrochemical detection methods for glucocorticoids usually require the addition of external redox signal probes, such as ferrocyanide, which limits their scope of use. Untreated electrodes have limitations in sensitivity and detection range. At the same time, the imprinted cavities in non-conductive molecularly imprinted polymers (MIPs) are independent of each other, which greatly reduces the conductivity and electrical signal response, thereby affecting the sensing effect. In order to improve the electrical signal response, the method of covering the electrode surface with conductive materials or nanocomposites can be used.

[0004] Carbon materials have multiple advantages, such as adjustable pore structure, high specific surface area, good thermal and chemical stability, and have become one of the most widely used electrode materials in electrode modification. Nitrogen doping is beneficial to improve the conductivity, surface wettability, catalytic activity and selectivity of carbon materials, thereby improving the electrochemical performance of modified electrodes. Carbon nanotubes have unique mechanical and electronic properties, combined with chemical stability, and behave electrically as metals or semiconductors depending on their structure. For sensing applications, carbon nanotubes have many advantages, such as small size, large surface area, and excellent electron transfer promotion ability when used as electrode modifiers in electrochemical reactions. Paper-based microfluidics, which is characterized by fluid transport through spontaneous capillary action of paper, shows great promise in various applications, especially in sensing. In addition, paper-based microfluidics enables the design of miniaturized electrochemical devices for applications in the energy field, which is particularly attractive for the rapidly growing market of small disposable electronics. Paper-based microfluidics also offers the possibility of simultaneous determination of two or more analytes in different channels or points.

[0005] So far, researchers at home and abroad have explored various MIP sensors for glucocorticoid detection. Some researchers, such as Athira Mani et al. (Athira Mani, TS Anirudhan Electrochemical sensing of cortisol by gold nanoparticle incorporated carboxylated graphene oxide based molecularly imprinted polymer, Chemical Engineering Journal, Volume 493, 2024, 152654), have used graft copolymerization to incorporate carboxylated graphene oxide into allylated gold nanoparticles to improve the sensitivity and detection range of the electrode, and have achieved good results. However, this can only detect a single component, and it is impossible to detect multiple glucocorticoids simultaneously. Therefore, it is necessary to develop sensors with better performance and multi-channel detection to meet practical needs. Summary of the invention

[0006] In view of this, the present invention aims to propose a paper-based molecular imprinting sensor for detecting glucocorticoids and a preparation method thereof, so as to solve the problem that the prior art can only detect a single component and cannot achieve simultaneous detection of multiple glucocorticoids.

[0007] To achieve the above object, the technical solution of the present invention is achieved as follows: A first aspect of the present invention provides a paper-based molecular imprinting sensor for detecting glucocorticoids, comprising a screen-printed electrode modified with a nitrogen-doped carbon / carbon nanotube composite material and a paper-based microfluidic device.

[0008] The second aspect of the present invention provides a method for preparing a paper-based molecular imprinting sensor for detecting glucocorticoids according to the first aspect of the present invention, comprising the following steps: (1) The nitrogen-doped carbon / carbon nanotube composite material with a hierarchical structure is used as an electrode modification material and coated on the screen-printed electrode; (2) Assembling the screen-printed electrode obtained in step (1) with the paper-based microfluidic device.

[0009] Furthermore, the nitrogen-doped carbon in step (1) is a two-dimensional sheet structure with a size of 450-550 μm, the diameter of the carbon nanotube is 45-55 nm, and the nitrogen-doped carbon and the carbon nanotube form a hierarchical structure.

[0010] Furthermore, the nitrogen-doped carbon in step (1) is a two-dimensional sheet structure with a size of 500 μm, and the diameter of the carbon nanotube is 50 nm.

[0011] Furthermore, the weight ratio of polyimide to carbon nanotubes in the nitrogen-doped carbon / carbon nanotube composite material is 10:1-5.

[0012] Preferably, the weight ratio of polyimide to carbon nanotubes in the nitrogen-doped carbon / carbon nanotube composite material is 10:3.

[0013] Furthermore, the preparation method of step (1) specifically comprises the following steps: Add p-phenylenediamine to a beaker and dissolve it with N,N-dimethylformamide to obtain a reaction solution, add carbon nanotubes to the above reaction solution, stir, transfer to a reactor, continue polymerization, centrifuge, wash, and dry to obtain powder; place the powder in a crucible, heat it to 300-400°C at 5-10°C / min in a nitrogen atmosphere and keep it warm for 1 hour, continue to heat it to 700-900°C at the same heating rate and keep it warm for 2 hours to obtain a nitrogen-doped carbon / carbon nanotube composite material with a hierarchical structure, and prepare it into a 1 mg / mL composite solution with deionized water; take 7-13 μL of the composite solution, drop it on the screen-printed electrode, and dry it in an oven at 30-50°C for standby use.

[0014] Furthermore, the stirring conditions are 12 h, room temperature, and 800 rpm.

[0015] Furthermore, the polymerization time is 10 hours and the temperature is 180°C.

[0016] Furthermore, the centrifugation time is 10 min, 4500 rpm.

[0017] Furthermore, the washing step comprises washing with N,N-dimethylformamide, ethanol and water three times respectively.

[0018] Furthermore, the drying time is 12 hours.

[0019] Preferably, the pyrolysis temperature is 800°C.

[0020] Furthermore, the preparation method of step (2) specifically comprises the following steps: The filter paper is cut with a paper cutter, the cut filter paper is subjected to hydrophobic treatment with polydimethylsiloxane, and the paper-based microfluidic device is obtained by bonding with double-sided tape. The working area of ​​the screen-printed electrode obtained in step (1) is aligned with the working area of ​​the paper-based microfluidic device, and the two are fixed with double-sided tape.

[0021] Furthermore, the paper-based microfluidic device includes a circular test area in the middle, three working areas evenly distributed around the circular test area, and adjacent working areas are connected by channels.

[0022] Furthermore, the filter paper has a diameter of 5 cm.

[0023] Furthermore, the diameter of the circular test area is 10-20 mm, and the width of the channel is 0.5-1.5 mm.

[0024] Preferably, the diameter of the circular test area is 15 mm, and the width of the channel is 1 mm.

[0025] Compared with the prior art, the paper-based molecular imprinting sensor for detecting glucocorticoids and the preparation method thereof described in the present invention have the following advantages: (1) The present invention uses nitrogen-doped carbon and carbon nanotubes to prepare a hierarchical porous composite material and uses it to modify a screen-printed electrode, so that the modified electrode not only has a large specific surface area, high conductivity, and abundant element groups, but also the hierarchical porous structure increases the roughness of the material surface, which is beneficial to the attachment of imprinted molecular polymers during the electropolymerization process.

[0026] (2) The present invention prepares a multi-channel paper-based microfluidic chip through a paper cutting process, which can realize the simultaneous detection of cortisol, cortisone, and corticosterone. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings: Figure 1 Schematic diagram of the paper-based microfluidic device of the present invention; Figure 2 This is a SEM image of a composite material prepared at a ratio of 10:3 of polyimide and carbon nanotubes in Example 1 of the present invention; Figure 3 This is the DPV test curve of different cortisol concentrations (PBS solution) in Example 1 of the present invention; Figure 4 The DPV test curves of different cortisone concentrations (PBS solution) in Example 1 of the present invention; Figure 5 This is the DPV test curve of different corticosterone concentrations (PBS solution) in Example 1 of the present invention; Figure 6 CV test curves of composite materials prepared from polyimide and carbon nanotubes in different mass ratios in Example 2 of the present invention (SPCE is a screen-printed electrode); Figure 7 This is a CV test curve of a composite material prepared from polyimide and carbon nanotubes prepared at different pyrolysis temperatures in Example 3 of the present invention. DETAILED DESCRIPTION

[0028] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0029] The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0030] Example 1 1. Preparation of precursor materials: (1) Add 3.11 g of 3,3ʹ,4,4ʹ-benzophenone tetracarboxylic dianhydride to a beaker and dissolve it in 60 mL of N,N-dimethylformamide; (2) Add 1.04 g of p-phenylenediamine to the beaker in step (1) and stir for 12 h (room temperature, 800 rpm); (3) Add 1.242 g of carbon nanotubes to the reaction solution of step (2) and stir for 1 h (room temperature, 800 rpm); (4) The solution obtained in step (3) was transferred to a 100 mL reactor and the polymerization was continued at 180° C. for 10 hours; (5) Centrifugation (10 min, 4500 rpm), washing three times with N,N-dimethylformamide, ethanol and water respectively; (6) Dry at 60°C for 12 hours to obtain a powder.

[0031] 2. Preparation of nitrogen-doped carbon / carbon nanotube composites: The powder obtained in step 1 was placed in a crucible, heated to 350°C at 5°C / min in a nitrogen atmosphere and kept warm for 1 hour, and then heated to 800°C at the same heating rate and kept warm for 2 hours to obtain a nitrogen-doped carbon / carbon nanotube composite material with a hierarchical structure, which was prepared into a 1 mg / mL composite solution with deionized water.

[0032] 3. Preparation of electrodes: Before modifying the screen-printed electrode with the composite solution, rinse the screen-printed electrode with deionized water and blow dry it with an ear bulb for later use. Take 10 μL of the composite solution obtained in step 2, drop it on the screen-printed electrode, and dry it in an oven at 45°C for later use.

[0033] 4. Prepare 0.027g o-phenylenediamine, 0.3g potassium ferrocyanide, 0.1g ferric chloride, 0.42ml hydrochloric acid, 5ml cortisol, and 50ml 0.1M (PH=4) acetate buffer solution (sodium acetate + acetic acid) as polymerization solution one; prepare 0.027g o-phenylenediamine, 0.3g potassium ferrocyanide, 0.1g ferric chloride, 0.42ml hydrochloric acid, 5ml cortisone, and 50ml 0.1M (PH=4) acetate buffer solution (sodium acetate + acetic acid) as polymerization solution two; prepare 0.027g o-phenylenediamine, 0.3g potassium ferrocyanide, 0.1g ferric chloride, 0.42ml hydrochloric acid, 5ml cortisone, and 50ml 0.1M (PH=4) acetate buffer solution (sodium acetate + acetic acid) as polymerization solution three; use the above polymerization solution one, polymerization solution two, and polymerization solution three to prepare molecular imprinting membranes respectively. Electropolymerization conditions: scanning speed 25-100 mV / s, potential range 0-1 V, cycle number 10-30 times, CV method polymerization.

[0034] 5. The polymerized electrode was immersed in anhydrous ethanol to remove the cortisol template in the polymer.

[0035] 6. Use a paper cutter to cut the filter paper into Figure 1 The cut filter paper is cut as shown, and then the cut filter paper is hydrophobically treated with polydimethylsiloxane and adhered with double-sided tape to make a paper-based microfluidic device, which is assembled with the modified screen-printed electrode.

[0036] 7. Incubate in PBS solution for 10 min, rinse repeatedly with deionized water, and test SWV in PBS solution to test its sensing ability.

[0037] like Figure 2-Figure 5 As shown, nitrogen-doped carbon and carbon nanotubes form a hierarchical porous structure, which is excellent for the preparation of high-performance biosensors. The paper-based microfluidic process enables the sensor to achieve simultaneous detection of multiple glucocorticoids.

[0038] Example 2 1. Preparation of precursor materials: (1) Add 3.11 g of 3,3ʹ,4,4ʹ-benzophenone tetracarboxylic dianhydride to a beaker and dissolve it in 60 mL of N,N-dimethylformamide; (2) Add 1.04 g of p-phenylenediamine to the beaker in step (1) and stir for 12 h (room temperature, 800 rpm); (3) Add 0.414 g, 0.828 g, 1.242 g, 1.656 g, and 2.07 g of carbon nanotubes to the reaction solution of step (2), respectively, and stir for 1 h (room temperature, 800 rpm); (4) The solution obtained in step (3) was transferred to a 100 mL reactor and the polymerization was continued at 180° C. for 10 hours; (5) Centrifugation (10 min, 4500 rpm), washing three times with N,N-dimethylformamide, ethanol and water respectively; (6) Dry at 60°C for 12 hours to obtain a powder.

[0039] 2. Preparation of nitrogen-doped carbon / carbon nanotube composites: The powder obtained in step 1 was placed in a crucible, heated to 350°C at 5°C / min in a nitrogen atmosphere and kept warm for 1 hour, and then heated to 800°C at the same heating rate and kept warm for 2 hours to obtain a nitrogen-doped carbon / carbon nanotube composite material with a hierarchical structure, which was prepared into a 1 mg / mL composite solution with deionized water.

[0040] 3. Preparation of electrodes: Before modifying the screen-printed electrode with the composite solution, rinse the screen-printed electrode with deionized water and blow dry it with an ear bulb for later use. Take 10 μL of the composite solution obtained in step 2, drop it on the screen-printed electrode, and dry it in an oven at 45°C for later use.

[0041] 4. Test CV in PBS solution to test its sensing ability.

[0042] like Figure 6 As shown, the electrical signal response is maximum when the weight ratio of polyimide to carbon nanotubes is 10:3.

[0043] Example 3 1. Preparation of precursor materials: (1) Add 3.11 g of 3,3ʹ,4,4ʹ-benzophenone tetracarboxylic dianhydride to a beaker and dissolve it in 60 mL of N,N-dimethylformamide; (2) Add 1.04 g of p-phenylenediamine to the beaker in step (1) and stir for 12 h (room temperature, 800 rpm); (3) Add 1.242 g of carbon nanotubes to the reaction solution of step (2) and stir for 1 h (room temperature, 800 rpm); (4) The solution obtained in step (3) was transferred to a 100 mL reactor and the polymerization was continued at 180° C. for 10 hours; (5) Centrifugation (10 min, 4500 rpm), washing three times with N,N-dimethylformamide, ethanol and water respectively; (6) Dry at 60°C for 12 hours to obtain a powder.

[0044] 2. Preparation of nitrogen-doped carbon / carbon nanotube composites: The powder obtained in step 1 was placed in a crucible, and the temperature was raised to 350°C at 5°C / min in a nitrogen atmosphere and kept for 1 hour. The temperature was then raised to 700°C, 800°C, and 900°C at the same heating rate and kept for 2 hours to obtain a nitrogen-doped carbon / carbon nanotube composite material with a hierarchical structure, which was prepared into a 1 mg / mL composite solution with deionized water.

[0045] 3. Preparation of electrodes: Before modifying the screen-printed electrode with the composite solution, rinse the screen-printed electrode with deionized water and blow dry it with an ear bulb for later use. Take 10 μL of the composite solution obtained in step 2, drop it on the screen-printed electrode, and dry it in an oven at 45°C for later use.

[0046] 4. Test CV in PBS solution to test its sensing ability.

[0047] like Figure 7 As shown, the electrical signal response is maximum when the pyrolysis temperature is 800℃.

[0048] 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, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A paper-based molecular imprinting sensor for detecting glucocorticoids, characterized in that: It includes screen-printed electrodes and paper-based microfluidic devices modified with nitrogen-doped carbon / carbon nanotube composites.

2. A method for preparing a paper-based molecular imprinting sensor for detecting glucocorticoids, characterized in that: The following steps are involved: (1) The nitrogen-doped carbon / carbon nanotube composite material with a hierarchical structure is used as an electrode modification material and coated on the screen-printed electrode; (2) Assembling the screen-printed electrode obtained in step (1) with the paper-based microfluidic device.

3. The method for preparing a paper-based molecular imprinting sensor for detecting glucocorticoids according to claim 2, characterized in that: The nitrogen-doped carbon in step (1) is a two-dimensional sheet structure with a size of 450-550 μm, the diameter of the carbon nanotube is 45-55 nm, and the nitrogen-doped carbon and the carbon nanotube form a hierarchical structure.

4. The method for preparing a paper-based molecular imprinting sensor for detecting glucocorticoids according to claim 2, characterized in that: The weight ratio of polyimide to carbon nanotubes in the nitrogen-doped carbon / carbon nanotube composite material is 10:1-5.

5. The method for preparing a paper-based molecular imprinting sensor for detecting glucocorticoids according to claim 2, characterized in that: The preparation method of step (1) specifically comprises the following steps: Add p-phenylenediamine to a beaker and dissolve it with N,N-dimethylformamide to obtain a reaction solution, add carbon nanotubes to the above reaction solution, stir, transfer to a reactor, continue polymerization, centrifuge, wash, and dry to obtain powder; place the powder in a crucible, heat it to 300-400°C at 5-10°C / min in a nitrogen atmosphere and keep it warm for 1 hour, continue to heat it to 700-900°C at the same heating rate and keep it warm for 2 hours to obtain a nitrogen-doped carbon / carbon nanotube composite material with a hierarchical structure, and prepare it into a 1 mg / mL composite solution with deionized water; take 7-13 μL of the composite solution, drop it on the screen-printed electrode, and dry it in an oven at 30-50°C for standby use.

6. The method for preparing a paper-based molecular imprinting sensor for detecting glucocorticoids according to claim 5, characterized in that: The washing step comprises washing with N,N-dimethylformamide, ethanol and water three times respectively.

7. The method for preparing a paper-based molecular imprinting sensor for detecting glucocorticoids according to claim 2, characterized in that: The preparation method of step (2) specifically comprises the following steps: The filter paper is cut with a paper cutter, the cut filter paper is subjected to hydrophobic treatment with polydimethylsiloxane, and the paper-based microfluidic device is obtained by bonding with double-sided tape. The working area of ​​the screen-printed electrode obtained in step (1) is aligned with the working area of ​​the paper-based microfluidic device, and the two are fixed with double-sided tape.

8. The method for preparing a paper-based molecular imprinting sensor for detecting glucocorticoids according to claim 7, characterized in that: The paper-based microfluidic device comprises a circular test area in the middle, three working areas evenly distributed around the circular test area, and adjacent working areas are connected by channels.

9. The method for preparing a paper-based molecular imprinting sensor for detecting glucocorticoids according to claim 7, characterized in that: The filter paper has a diameter of 5 cm.

10. The method for preparing a paper-based molecular imprinting sensor for detecting glucocorticoids according to claim 8, characterized in that: The diameter of the circular test area is 10-20 mm, and the width of the channel is 0.5-1.5 mm.

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