A paper-based molecularly imprinted sensor for detecting glucocorticoids and a preparation method thereof

Abstract

The application provides a paper-based molecular imprinting sensor for detecting glucocorticoids and a preparation method thereof, wherein the preparation method of the paper-based molecular imprinting sensor for detecting glucocorticoids comprises the following steps: using nitrogen-doped carbon / carbon nanotube composite material with a hierarchical structure as electrode modification material, and coating the electrode modification material on a screen-printed electrode; and assembling the obtained screen-printed electrode with a paper-based microfluidic device. The nitrogen-doped carbon and the carbon nanotube form a hierarchical porous structure, and high specific surface area and high conductivity make the sensor performance more excellent. The paper-based microfluidic process enables the sensor to simultaneously detect multiple glucocorticoids, and shows the application value of the sensor in the inspection field.

Description

Technical Field

[0001] This invention belongs to the field of biosensing and detection, and in particular relates to a paper-based molecular imprint sensor for detecting glucocorticoids and its preparation method. Background Technology

[0002] The adrenal glands produce a group of steroid hormones called glucocorticoids, including cortisol, cortisone, and corticosterone. Cortisol's main functions include raising blood sugar levels, suppressing the immune system, and aiding metabolism. Abnormal cortisol levels can lead to high blood pressure, hirsutism, and affect the cardiovascular system, as well as the gastrointestinal tract, metabolism, musculoskeletal system, immunity, and menstrual irregularities. Cortisone has the ability to bind to glucocorticoid receptors, which are commonly used in anti-inflammatory treatments; higher levels of cortisol may lead to Cushing's syndrome. Elevated corticosterone levels may increase the risk of aldosterone-secreting tumors, Cushing's syndrome, and adrenal cortical tumors. Therefore, effective, rapid, and reliable glucocorticoid testing is invaluable for dynamic health analysis, enabling comprehensive self-monitoring, health management, and personalized healthcare.

[0003] Molecularly imprinted polymers (MIPs) are polymer matrices obtained using molecular imprinting technology. This is a versatile synthetic technique used to design artificial receptors that selectively sense 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 typically require the addition of external redox signal probes, such as ferrocyanide, which limits their application. Untreated electrodes have limitations in sensitivity and detection range. Furthermore, the imprinted cavities in non-conductive molecularly imprinted polymers (MIPs) are independent, which significantly reduces conductivity and electrical signal response, thus affecting sensing performance. To improve electrical signal response, methods such as coating the electrode surface with conductive materials or nanocomposite materials can be employed.

[0004] Carbon materials possess numerous advantages, such as tunable pore structure, high specific surface area, and good thermal and chemical stability, making them one of the most widely used electrode materials for electrode modification. Nitrogen doping enhances the electrical conductivity, surface wettability, catalytic activity, and selectivity of carbon materials, thereby improving the electrochemical performance of modified electrodes. Carbon nanotubes possess unique mechanical and electronic properties, combined with chemical stability, and, depending on their structure, behave electrically as metals or semiconductors. For sensing applications, carbon nanotubes offer many advantages, such as small size, large surface area, and excellent electron transfer-promoting capabilities when used as electrode modifiers in electrochemical reactions. Paper-based microfluidics, characterized by fluid transport through the spontaneous capillary action of paper, show great promise in various applications, particularly in sensing. Furthermore, paper-based microfluidics enable the design of miniaturized electrochemical devices for the energy sector, which is particularly attractive for the rapidly growing market of small, disposable electronic products. Paper-based microfluidics also offer the possibility of simultaneously determining two or more analytes in different channels or at different points.

[0005] To date, researchers both domestically and internationally have explored various MIP-type sensors for glucocorticoid detection. Some researchers, such as Athira Mani et al. (Athira Mani, TS Anirudhan Electrochemicalsensing of cortisol by gold nanoparticle incorporated carboxylated graphene oxide based molecularly imprinted polymer, Chemical Engineering Journal, Volume 493, 2024, 152654), have achieved good results by incorporating carboxylated graphene oxide into allylated gold nanoparticles via graft copolymerization to improve electrode sensitivity and detection range. However, this method can only detect single components and cannot achieve simultaneous detection of multiple glucocorticoids. Therefore, there is a need to develop sensors with superior performance and multi-channel detection capabilities to meet practical needs. Summary of the Invention

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

[0007] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0008] The first aspect of the present invention provides a paper-based molecularly imprinted sensor for detecting glucocorticoids, comprising a screen-printed electrode modified with nitrogen-doped carbon / carbon nanotube composite material and a paper-based microfluidic device.

[0009] The second aspect of this invention provides a method for preparing a paper-based molecularly imprinted sensor for detecting glucocorticoids as described in the first aspect of this invention, comprising the following steps:

[0010] (1) A nitrogen-doped carbon / carbon nanotube composite material with a hierarchical structure was used as an electrode modification material and coated onto a screen-printed electrode;

[0011] (2) Assemble the screen-printed electrode obtained in step (1) with the paper-based microfluidic device.

[0012] Furthermore, in step (1), the nitrogen-doped carbon is a two-dimensional sheet structure with a size of 450-550µm, and the carbon nanotubes have a diameter of 45-55nm. The nitrogen-doped carbon and carbon nanotubes form a hierarchical structure.

[0013] Furthermore, the nitrogen-doped carbon in step (1) is a two-dimensional sheet structure with a size of 500µm and the carbon nanotubes have a diameter of 50nm.

[0014] Furthermore, the weight ratio of polyimide to carbon nanotubes in the nitrogen-doped carbon / carbon nanotube composite material is 10:1-5.

[0015] Preferably, the weight ratio of polyimide to carbon nanotubes in the nitrogen-doped carbon / carbon nanotube composite material is 10:3.

[0016] Furthermore, the preparation method of step (1) specifically includes the following steps:

[0017] 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 reaction vessel, continue polymerization, centrifuge, wash, and dry to obtain powder. Place the powder in a crucible and heat it to 300-400℃ in a nitrogen atmosphere at a rate of 5-10℃ / min and hold for 1 hour. Continue heating at the same rate to 700-900℃ and hold for 2 hours to obtain a nitrogen-doped carbon / carbon nanotube composite material with a hierarchical structure. Prepare a 1 mg / mL composite solution with deionized water. Take 7-13 μL of the composite solution and drop it onto a screen-printed electrode, then dry it in an oven at 30-50℃ for later use.

[0018] Furthermore, the stirring conditions are 12 hours, room temperature, and 800 rpm.

[0019] Furthermore, the polymerization time is 10 hours and the temperature is 180°C.

[0020] Furthermore, the centrifugation time is 10 min at 4500 rpm.

[0021] Furthermore, the washing step includes washing three times each with N,N-dimethylformamide, ethanol, and water.

[0022] Furthermore, the drying time is 12 hours.

[0023] Preferably, the pyrolysis temperature is 800℃.

[0024] Furthermore, the preparation method of step (2) specifically includes the following steps:

[0025] Cut the filter paper with a paper cutter, treat the cut filter paper with polydimethylsiloxane to make it hydrophobic, and stick it with double-sided tape to obtain a paper-based microfluidic device. Align the working area of ​​the screen-printed electrode obtained in step (1) with the working area of ​​the paper-based microfluidic device and fix it with double-sided tape.

[0026] Furthermore, the paper-based microfluidic device includes a circular test area in the center, and three working areas evenly distributed around the circular test area, with adjacent working areas connected by channels.

[0027] Furthermore, the filter paper has a diameter of 5 cm.

[0028] Furthermore, the diameter of the circular test area is 10-20mm, and the width of the channel is 0.5-1.5mm.

[0029] Preferably, the diameter of the circular test area is 15mm and the width of the channel is 1mm.

[0030] Compared with existing technologies, the paper-based molecularly imprinted sensor for detecting glucocorticoids and its preparation method described in this invention have the following advantages:

[0031] (1) The present invention uses nitrogen-doped carbon and carbon nanotubes to prepare a hierarchical porous composite material and uses it to modify screen-printed electrodes. The modified electrodes not only have a large specific surface area, high conductivity, and abundant element groups, but the hierarchical porous structure also increases the surface roughness of the material, which is beneficial to the adhesion of the imprinted molecular polymer during the electropolymerization process.

[0032] (2) The present invention prepares a multi-channel paper-based microfluidic chip through paper cutting process, which can realize the simultaneous detection of cortisol, cortisone and corticosterone. Attached Figure Description

[0033] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0034] Figure 1 This is a schematic diagram of the paper-based microfluidic device of the present invention;

[0035] Figure 2 This is a SEM image of the composite material prepared by polyimide and carbon nanotubes in a 10:3 ratio according to Example 1 of the present invention.

[0036] Figure 3 This is the DPV test curve for different cortisol concentrations (PBS solution) in Example 1 of the present invention;

[0037] Figure 4 This is the DPV test curve for different cortisone concentrations (PBS solution) in Example 1 of the present invention;

[0038] Figure 5 This is the DPV test curve for different corticosterone concentrations (PBS solution) in Example 1 of the present invention;

[0039] Figure 6 The CV test curves (SPCE is a screen-printed electrode) of the composite material prepared by polyimide and carbon nanotubes in different mass ratios in Example 2 of the present invention.

[0040] Figure 7 The CV test curves are for composite materials prepared from polyimide and carbon nanotubes at different pyrolysis temperatures in Example 3 of this invention. Detailed Implementation

[0041] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0042] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0043] Example 1

[0044] 1. Preparation of precursor materials:

[0045] (1) Add 3.11 g of 3,3ʹ,4,4ʹ-benzophenone tetracarboxylic dianhydride to a beaker and dissolve it with 60 mL of N,N-dimethylformamide;

[0046] (2) Add 1.04 g of p-phenylenediamine to the beaker in step (1) and stir for 12 h (room temperature, 800 rpm).

[0047] (3) Add 1.242g of carbon nanotubes to the reaction solution of step (2) and stir for 1h (room temperature, 800rpm).

[0048] (4) Transfer the solution obtained in step (3) to a 100 mL reactor and continue polymerization at 180 °C for 10 hours;

[0049] (5) Centrifuge (10 min, 4500 rpm), and wash three times each with N,N-dimethylformamide, ethanol and water;

[0050] (6) Dry at 60°C for 12 hours to obtain powder.

[0051] 2. Preparation of nitrogen-doped carbon / carbon nanotube composite materials:

[0052] The powder obtained in step 1 was placed in a crucible and heated to 350°C at a rate of 5°C / min in a nitrogen atmosphere and held for 1 hour. The temperature was then increased to 800°C at the same rate and held for 2 hours to obtain a nitrogen-doped carbon / carbon nanotube composite material with a hierarchical structure. The composite solution was prepared with deionized water to a concentration of 1 mg / mL.

[0053] 3. Electrode preparation:

[0054] Before modifying the screen-printed electrode with the composite solution, rinse the screen-printed electrode with deionized water and dry it with a rubber bulb. Take 10 μL of the composite solution obtained in step 2, drop it onto the screen-printed electrode, and dry it in an oven at 45°C.

[0055] 4. Prepare polymerization solution one: 0.027g o-phenylenediamine, 0.3g potassium ferricyanide, 0.1g ferric chloride, 0.42ml hydrochloric acid, 5ml cortisol, and 50ml of 0.1M (pH=4) acetate buffer solution (sodium acetate + acetic acid). Prepare polymerization solution two: 0.027g o-phenylenediamine, 0.3g potassium ferricyanide, 0.1g ferric chloride, 0.42ml hydrochloric acid, 5ml cortisone, and 50ml of 0.1M (pH=4) acetate buffer solution (sodium acetate + acetic acid). Prepare polymerization solution three: 0.027g o-phenylenediamine, 0.3g potassium ferricyanide, 0.1g ferric chloride, 0.42ml hydrochloric acid, 5ml cortisol, and 50ml of 0.1M (pH=4) acetate buffer solution (sodium acetate + acetic acid). Use polymerization solutions one, two, and three to prepare molecularly imprinted membranes using electropolymerization. Electropolymerization conditions: scan speed 25-100mV / s, potential range 0-1V, number of cycles 10-30, CV method polymerization.

[0056] 5. The polymerized electrode is immersed in anhydrous ethanol to remove the cortisol template from the polymer.

[0057] 6. Use a paper cutter to cut the filter paper according to the attached... Figure 1The paper is cut as shown, then hydrophobically treated with polydimethylsiloxane, and then glued with double-sided adhesive to form a paper-based microfluidic device, which is then assembled with the modified screen-printed electrode.

[0058] 7. Incubate in PBS solution for 10 min, rinse repeatedly with deionized water, and test SWV in PBS solution to test its sensing capability.

[0059] like Figures 2-5 As shown, nitrogen-doped carbon and carbon nanotubes form a hierarchical porous structure, which is excellent for fabricating high-performance biosensors. Paper-based microfluidic technology enables this sensor to simultaneously detect multiple glucocorticoids.

[0060] Example 2

[0061] 1. Preparation of precursor materials:

[0062] (1) Add 3.11 g of 3,3ʹ,4,4ʹ-benzophenone tetracarboxylic dianhydride to a beaker and dissolve it with 60 mL of N,N-dimethylformamide;

[0063] (2) Add 1.04 g of p-phenylenediamine to the beaker in step (1) and stir for 12 h (room temperature, 800 rpm).

[0064] (3) Add 0.414g, 0.828g, 1.242g, 1.656g and 2.07g of carbon nanotubes to the reaction solution in step (2) respectively, and stir for 1h (room temperature, 800rpm).

[0065] (4) Transfer the solution obtained in step (3) to a 100 mL reactor and continue polymerization at 180 °C for 10 hours;

[0066] (5) Centrifuge (10 min, 4500 rpm), and wash three times each with N,N-dimethylformamide, ethanol and water;

[0067] (6) Dry at 60°C for 12 hours to obtain powder.

[0068] 2. Preparation of nitrogen-doped carbon / carbon nanotube composite materials:

[0069] The powder obtained in step 1 was placed in a crucible and heated to 350°C at a rate of 5°C / min in a nitrogen atmosphere and held for 1 hour. The temperature was then increased to 800°C at the same rate and held for 2 hours to obtain a nitrogen-doped carbon / carbon nanotube composite material with a hierarchical structure. The composite solution was prepared with deionized water to a concentration of 1 mg / mL.

[0070] 3. Electrode preparation:

[0071] Before modifying the screen-printed electrode with the composite solution, rinse the screen-printed electrode with deionized water and dry it with a syringe. Take 10 μL of the composite solution obtained in step 2, drop it onto the screen-printed electrode, and dry it in an oven at 45°C.

[0072] 4. Test CV in PBS solution to test its sensing ability.

[0073] like Figure 6 As shown, the electrical signal response is greatest when the weight ratio of polyimide to carbon nanotubes is 10:3.

[0074] Example 3

[0075] 1. Preparation of precursor materials:

[0076] (1) Add 3.11 g of 3,3ʹ,4,4ʹ-benzophenone tetracarboxylic dianhydride to a beaker and dissolve it with 60 mL of N,N-dimethylformamide;

[0077] (2) Add 1.04 g of p-phenylenediamine to the beaker in step (1) and stir for 12 h (room temperature, 800 rpm).

[0078] (3) Add 1.242g of carbon nanotubes to the reaction solution of step (2) and stir for 1h (room temperature, 800rpm).

[0079] (4) Transfer the solution obtained in step (3) to a 100 mL reactor and continue polymerization at 180 °C for 10 hours;

[0080] (5) Centrifuge (10 min, 4500 rpm), and wash three times each with N,N-dimethylformamide, ethanol and water;

[0081] (6) Dry at 60°C for 12 hours to obtain powder.

[0082] 2. Preparation of nitrogen-doped carbon / carbon nanotube composite materials:

[0083] The powder obtained in step 1 was placed in a crucible and heated to 350°C at a rate of 5°C / min in a nitrogen atmosphere and held for 1 hour. The temperature was then increased to 700°C, 800°C, and 900°C at the same rate and held for 2 hours to obtain a nitrogen-doped carbon / carbon nanotube composite material with a hierarchical structure. The composite solution was prepared with deionized water to a concentration of 1 mg / mL.

[0084] 3. Electrode preparation:

[0085] Before modifying the screen-printed electrode with the composite solution, rinse the screen-printed electrode with deionized water and dry it with a syringe. Take 10 μL of the composite solution obtained in step 2, drop it onto the screen-printed electrode, and dry it in an oven at 45°C.

[0086] 4. Test CV in PBS solution to test its sensing ability.

[0087] like Figure 7 As shown, the electrical signal response is greatest at a pyrolysis temperature of 800℃.

[0088] 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a paper-based molecularly imprinted sensor for detecting glucocorticoids, characterized in that: Including the following steps: (1) A nitrogen-doped carbon / carbon nanotube composite material with a hierarchical structure was used as an electrode modification material and coated onto a screen-printed electrode. (2) Assemble the screen-printed electrode obtained in step (1) with the paper-based microfluidic device; The paper-based molecularly imprinted sensor for detecting glucocorticoids includes a screen-printed electrode modified with nitrogen-doped carbon / carbon nanotube composite material and a paper-based microfluidic device. The nitrogen-doped carbon in step (1) is a two-dimensional sheet structure with a size of 450-550 μm, and the carbon nanotubes have a diameter of 45-55 nm. The nitrogen-doped carbon and carbon nanotubes form a hierarchical structure. The weight ratio of polyimide to carbon nanotubes in the nitrogen-doped carbon / carbon nanotube composite material is 10:1-5. The paper-based molecular imprinting sensor for detecting glucocorticoids includes cortisol, cortisone, and corticosterone. The preparation method of step (2) specifically includes the following steps: Cut the filter paper with a paper cutter, treat the cut filter paper with polydimethylsiloxane to make it hydrophobic, and stick it with double-sided tape to obtain a paper-based microfluidic device. Align the working area of ​​the screen-printed electrode obtained in step (1) with the working area of ​​the paper-based microfluidic device and fix it with double-sided tape. The paper-based microfluidic device includes a circular test area in the middle, and three working areas evenly distributed around the circular test area, with adjacent working areas connected by channels; The filter paper has a diameter of 5 cm; The diameter of the circular test area is 10-20mm, and the width of the channel is 0.5-1.5mm; The preparation method of step (1) specifically includes 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 reaction vessel, continue polymerization, centrifuge, wash, and dry to obtain powder. Place the powder in a crucible and heat it to 300-400℃ in a nitrogen atmosphere at a rate of 5-10℃ / min and hold for 1 hour. Continue heating at the same rate to 700-900℃ and hold for 2 hours to obtain a nitrogen-doped carbon / carbon nanotube composite material with a hierarchical structure. Prepare a 1 mg / mL composite solution with deionized water. Take 7-13 μL of the composite solution and drop it onto a screen-printed electrode, then dry it in an oven at 30-50℃ for later use.

2. The method for preparing a paper-based molecularly imprinted sensor for detecting glucocorticoids according to claim 1, characterized in that: The washing steps include washing three times each with N,N-dimethylformamide, ethanol, and water.

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