A single-atom active site-based znpc, dual-mode sensor and method

Abstract

The application belongs to the field of sports health, and provides a single-atom active site-based ZnPc, a dual-mode sensor and a method, which comprises the following steps: stirring and reacting an initial product obtained by solid-phase reaction of 4-nitrophthalonitrile, zinc acetate, urea and ammonium molybdate in HCl solution and NaOH solution; cooling to room temperature, washing with deionized water after filtration, and vacuum drying to obtain tetranitro ZnPc; reacting the tetranitro ZnPc with Na2S·9H2O in N,N-dimethylformamide; after cooling to room temperature, dispersing the reaction product in deionized water, washing with deionized water until neutral, and drying the obtained solid in a vacuum oven to obtain tetraamino ZnPc; and preparing a photoelectric / colorimetric dual-mode sensor by using the tetraamino ZnPc, wherein the preparation method is simple, and the post-treatment is relatively easy; and the dual-mode sensor method is used for PSA detection, and has high sensitivity, good reliability and good stability.

Description

Technical Field

[0001] This invention belongs to the field of sports and health, and relates to a ZnPc dual-mode sensor and method based on a single-atom active site. More specifically, it relates to a ZnPc photoelectric / colorimetric dual-mode sensor based on a Zn-N4 single-atom active site, and its preparation and detection methods. Background Technology

[0002] Cancer is a disease caused by the uncontrolled proliferation of mutated cells within the body. Appropriate exercise can help prevent cancer, but anaerobic exercise produces lactic acid. Lactic acid, an important tool for cancer cells to modify their microenvironment, provides an acidic environment that offers a proton gradient, driving proton-coupled proteins in cancer cells and inhibiting the growth of other normal cells. Therefore, developing sensitive detection technologies for cancer markers to effectively prevent cancer is crucial for the development of sports and health services. Prostate-specific antigen (PSA) is a specific biomarker for prostate cancer, which has a high mortality rate. PSA is found in both diseased and normal prostate cells. Generally, 4 ng / mL of PSA is considered the cutoff value in serum; PSA levels above 4 ng / mL are generally considered to indicate a risk of prostate cancer. Therefore, there is an urgent need to develop a method for monitoring PSA levels to achieve early and sensitive diagnosis of prostate cancer and prevent cancer progression. Existing detection methods mainly include fluorescence, electrochemical, and colorimetric methods. However, these methods are primarily based on a single-sensor model, which is susceptible to influences from operating conditions, the biological environment, and many other factors, leading to data errors and affecting the accuracy of the analysis. Therefore, the dual-mode strategy, which combines two detection methods to construct a sensor, not only provides multiple methods for actual detection needs, but also enables mutual verification of quantitative measurement results, thereby improving the reliability and accuracy of the data.

[0003] Metal single-atom materials are a new class of catalysts developed in recent years, possessing definite single-atom positions and unique catalytic properties. Among them, metal phthalocyanines (MPcs) are centered around a metal ion, with four nitrogen ligands coordinating with the metal center to form M-N4 units. The M-N4 single-atom active center can effectively enhance catalytic performance. Metal phthalocyanines have been widely used in photocatalysis due to their high stability, low cost, excellent catalytic performance, and photosensitivity to visible / near-infrared light. Zinc phthalocyanine (ZnPc), in particular, has a long excited-state lifetime, which is beneficial for charge separation and transfer in the excited state. Simultaneously, the reversible structure and oxidation state changes of ZnPc endow it with catalytic reduction capabilities, and the presence of the Zn-N4 active site further enhances the catalytic activity of the material. These advantages make it possible to construct highly efficient single-atom mimic enzymes based on ZnPc and apply them to analytical sensing. Summary of the Invention

[0004] To address at least one of the technical problems mentioned above, this invention provides a method for fabricating a dual-mode sensor based on a single-atom active site. The photoelectric / colorimetric dual-mode aptamer sensor constructed based on the Zn-N4 single-atom active site ZnPc is simple to build and can realize PSA detection. It exhibits high sensitivity, good reliability, and good stability when used for PSA detection.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a method for preparing Zn-N4 single-atom active site ZnPc, comprising: reacting 4-nitrophthalonitrile, zinc acetate, urea, and ammonium molybdate in a solid phase at 160-180℃ for 5-6 h. The resulting initial product is then reacted with 200-300 mL of 1M HCl solution and 200-300 mL of 1M NaOH solution at 80-90℃ for 1-2 h by stirring. After cooling to room temperature, the mixture is filtered, washed with deionized water, and vacuum dried at 60-70℃ for 7-8 h to obtain tetranitroZnPc; wherein the molar ratio of 4-nitrophthalonitrile, zinc acetate, urea, and ammonium molybdate is 1:0.4-0.5:8.0-9.0:0.01-0.02;

[0007] Zinc tetranitrophthalocyanine and Na₂S·9H₂O were reacted in 30-40 mL of N,N-dimethylformamide at 60-70 °C for 2-3 h. After cooling to room temperature, the reaction product was dispersed in 100-200 mL of deionized water, centrifuged and washed with deionized water until neutral, and the resulting solid was dried in a vacuum oven at 60-70 °C for 6-8 h to obtain tetraaminoZnPc, i.e., Zn-N₄ single-atom active site ZnPc; wherein the molar ratio of zinc tetranitrophthalocyanine to Na₂S·9H₂O was 1:12.0-13.0; the structural formula of Zn-N₄ single-atom active site, i.e., ZnPc, is as follows. Figure 1 As shown.

[0008] Secondly, this invention provides the application of Zn-N4 single-atom active site ZnPc in the preparation of a photoelectric / colorimetric dual-mode sensor. A photoelectric / colorimetric dual-mode sensor includes a three-electrode system: the sensor photoelectrode is the working electrode, the counter electrode is a platinum wire electrode, the reference electrode is an Ag / AgCl electrode, a xenon lamp is the light source, and the electrolyte is a phosphate buffer solution containing hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine.

[0009] Thirdly, the present invention provides a method for fabricating a photoelectric / colorimetric dual-mode sensor as described in the second aspect, comprising:

[0010] (1) Cleaning of FTO: Clean the FTO electrode three times with toluene, acetone, ethanol and water, and dry it for later use;

[0011] (2) Commercial P25 TiO2 was modified onto FTO using spin coating. 0.1-0.2 g of commercial P25 TiO2 was dispersed in ethanol. The spin coating rate was 1000-1500 rpm, and the time was 40-60 s. The spin coating was repeated 3-5 times to ensure the TiO2 thickness.

[0012] (3) Add 25-30 μL of chitosan solution (0.1 wt%) to the surface of the TiO2-modified FTO electrode, dry it, and rinse it with sodium hydroxide solution and ultrapure water; drop 30-50 μL of glutaraldehyde solution (5.0 wt%) onto the electrode and keep it for 60-80 minutes; drop 30-50 μL of PSA antibody 1 (Ab1) solution onto the activated electrode, incubate it at 4 ℃ for 6-8 h, and wash it thoroughly with ultrapure water; block the non-specific binding sites with 50-80 μL of 25% bovine serum albumin at 4 ℃ for 2-4 hours;

[0013] (4) Add 30-50 μL of glutaraldehyde solution (5.0 wt%) and 25-30 μL of chitosan solution (0.1 wt%) to a 5-10 wt% Zn-N4 aqueous solution. After reacting at room temperature for 60-80 minutes, centrifuge to separate the product. Subsequently, redisperse the product in water and add 30-50 μL of PSA antibody 2 (Ab2) solution. Incubate at 4 °C for 6-8 h to obtain an Ab2 solution labeled with Zn-N4.

[0014] (5) Add 20-40 μL of PSA solution of different concentrations, incubate for 35-60 minutes, and wash; then add 30-50 μL of Ab2 labeled with Zn-N4 single-atom active site ZnPc, incubate for 35-60 minutes, and wash for later use.

[0015] Fourthly, the present invention provides a performance testing method for a photoelectric / colorimetric dual-mode sensor, comprising:

[0016] A three-electrode system was used, with the sensor photoelectrode as the working electrode, a platinum wire electrode as the counter electrode, and an Ag / AgCl electrode as the reference electrode. The bias voltage was 0 V, and a xenon lamp was used as the light source. The electrolyte was a phosphate buffer solution (pH=7.4) containing hydrogen peroxide (H2O2) and 3,3',5,5'-tetramethylbenzidine (TMB). After waiting for 10 minutes, the photoelectric performance (It curve) was tested using the chronoamperometry method. Colorimetric tests were performed based on ultraviolet spectroscopy and color development. The concentration of H2O2 was 3.0-5.0 mM, and the concentration of TMB was 80-100 μM.

[0017] This invention discloses a photoelectric / colorimetric dual-mode sensor for PSA determination, using commercially available P25 TiO2, PSA antigen solution, PSA antibody solution, and ZnPc with Zn-N4 single-atom active sites. ZnPc exhibits high photosensitivity and catalytic oxidation capabilities. Simultaneously, the peroxidase-like activity of ZnPc catalyzes the decomposition of H2O2, leading to the color development of TMB. Therefore, the constructed photoelectric / colorimetric dual-mode sensor is simple, rapid, and enables sensitive detection of PSA.

[0018] The beneficial effects of this invention are:

[0019] (1) The method for preparing ZnPc single-atom active sites of Zn-N4 used in this invention is simple and the post-processing is relatively easy;

[0020] (2) The photoelectric / colorimetric dual-mode aptamer sensor based on the ZnPc single-atom active site of Zn-N4 is simple to construct and can realize the detection of PSA;

[0021] (3) The Zn-N4 single-atom active site ZnPc, which is fixed on the surface of the photoelectrode by the immune reaction triggered by PSA, acts as a highly efficient catalase-like catalyst to catalyze the colorimetric reaction between H2O2 and TMB. While obtaining the colorimetric signal, it consumes a large amount of electron donor H2O2 in the electrolyte, thereby significantly quenching the photocurrent signal and realizing dual-mode detection of PSA.

[0022] (4) The dual-mode sensing method constructed in this invention is used for PSA detection and has high sensitivity, good reliability and good stability.

[0023] Advantages of additional aspects of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0024] 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 improper limitation of the invention.

[0025] Figure 1 This is a structural diagram of the ZnPc single-atom active site in Zn-N4;

[0026] Figure 2 This is the ultraviolet spectrum of ZnPc of the present invention;

[0027] Figure 3 This is an infrared image of the ZnPc of the present invention;

[0028] Figure 4 This is a photoelectrochemical curve of the photoelectric / colorimetric dual-mode sensor of the present invention with and without the target analyte;

[0029] Figure 5 This is a graph showing the ultraviolet absorption curves of the photoelectric / colorimetric dual-mode sensor of the present invention with and without a target object. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0031] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0032] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0033] In this invention, terms such as "upper," "lower," "left," "right," "front," "back," "vertical," "horizontal," "side," and "bottom" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. These terms are used only to facilitate the description of the structural relationships of the various components or elements of this invention and do not specifically refer to any component or element in this invention. They should not be construed as limiting the invention.

[0034] In this invention, terms such as "fixed connection," "connected," and "linked" should be interpreted broadly, indicating a fixed connection, an integral connection, or a detachable connection; a direct connection or an indirect connection through an intermediate medium. Those skilled in the art can determine the specific meaning of these terms in this invention based on the specific circumstances, and they should not be construed as limitations on the invention.

[0035] Example 1

[0036] This embodiment provides a method for preparing ZnPc with single-atom active sites in Zn-N4, specifically providing the following two examples:

[0037] Case 1.1

[0038] (1) 3.8 g of 4-nitrophthalonitrile, 0.91 g of zinc acetate, 9.6 g of urea and 100 mg of ammonium molybdate were reacted in a solid-state reaction at 160 °C for 5 h. The resulting primary product was reacted in 300 mL of 1 M HCl solution and 300 mL of 1 M NaOH solution at 90 °C for 1 h with stirring. After cooling to room temperature, the mixture was filtered, washed with deionized water, and dried under vacuum at 60 °C for 7 h to obtain zinc tetranitrophthalocyanine.

[0039] (2) 1.5 g of tetranitrophthalocyanine zinc and 5.8 g of Na2S·9H2O were reacted in 30 mL of N,N-dimethylformamide at 60 °C for 2 h. After cooling to room temperature, the reaction product was dispersed in 100 mL of deionized water, centrifuged and washed with deionized water until neutral, and the obtained solid was dried in a vacuum oven at 60 °C for 6 h to obtain tetraaminoZnPc, i.e., Zn-N4 single-atom active site ZnPc;

[0040] (3) The obtained product was fully characterized by ultraviolet and infrared spectroscopy, which proved the successful preparation of ZnPc single-atom active site Zn-N4.

[0041] Case 1.2

[0042] (1) 1.9 g of 4-nitrophthalonitrile, 0.45 g of zinc acetate, 4.8 g of urea and 50 mg of ammonium molybdate were reacted in a solid-state reaction at 180 °C for 6 h. The resulting primary products were reacted in 100 mL of 1 M HCl solution and 200 mL of 1 M NaOH solution at 90 °C for 2 h with stirring. After cooling to room temperature, the mixture was filtered, washed with deionized water, and dried under vacuum at 70 °C for 7 h to obtain zinc tetranitrophthalocyanine.

[0043] (2) 1.5 g of tetranitrophthalocyanine zinc and 5.8 g of Na2S·9H2O were reacted in 40 mL of N,N-dimethylformamide at 65 °C for 3 h. After cooling to room temperature, the reaction product was dispersed in 200 mL of deionized water, centrifuged and washed with deionized water until neutral, and the obtained solid was dried in a vacuum oven at 70 °C for 8 h to obtain tetraaminophthalocyanine zinc, i.e., Zn-N4 single-atom active site ZnPc;

[0044] (3) The obtained solid product was fully characterized: the results were consistent with those in Case 1.1.

[0045] Example 2

[0046] This embodiment provides a method for fabricating a photoelectric / colorimetric dual-mode sensor, including:

[0047] (1) Cleaning of working electrode: Clean the FTO electrode three times each with toluene, acetone, ethanol and water, and dry it for later use;

[0048] (2) Commercial P25 TiO2 was modified onto FTO by spin coating. 0.15 g of commercial P25 TiO2 was dispersed in ethanol. The spin coating rate was 1200 rpm and the time was 60 s. The spin coating was repeated 4 times to ensure the TiO2 thickness.

[0049] (3) Add 30 μL of chitosan solution (0.1 wt%) to the surface of the TiO2-modified FTO electrode, dry it, and rinse it with sodium hydroxide solution and ultrapure water; drop 45 μL of glutaraldehyde solution (5.0 wt%) onto the electrode and keep it for 60 minutes; drop 30 μL of LPSA antibody 1 (Ab1) solution onto the activated electrode, incubate it at 4 ℃ for 6 h, and wash it thoroughly with ultrapure water; block the non-specific binding sites with 50 μL of 25% bovine serum albumin at 4 ℃ for 2 hours.

[0050] (4) Add 30-50 μL of glutaraldehyde solution (5.0 wt%) and 25-30 μL of chitosan solution (0.1 wt%) to a 5-10 wt% Zn-N4 aqueous solution. After reacting at room temperature for 60-80 minutes, centrifuge to separate the product. Subsequently, redisperse the product in water and add 30-50 μL of PSA antibody 2 (Ab2) solution. Incubate at 4 °C for 6-8 h to obtain an Ab2 solution labeled with Zn-N4.

[0051] (5) Add 25 μL of PSA solution of different concentrations, incubate for 45 minutes, and wash; then add 35 μL of Ab2 labeled with Zn-N4 single-atom active site ZnPc, incubate for 40 minutes, and wash for later use.

[0052] Example 3

[0053] This embodiment provides a method for measuring the performance of a photoelectric / colorimetric dual-mode sensor, including:

[0054] A three-electrode system was adopted, with the sensor photoelectrode as the working electrode, a platinum wire electrode as the counter electrode, and an Ag / AgCl electrode as the reference electrode. The bias voltage was 0 V, a xenon lamp was used as the light source, and the electrolyte was a phosphate buffer solution (pH=7.4) containing 4.0 mM H2O2 and 90 μM TMB. After waiting for 10 minutes, the photoelectric performance was tested using the It curve, and colorimetric tests were performed based on the ultraviolet spectrum and color development.

[0055] When the target analyte is present, the ZnPc single-atom active site of Zn-N4 is immobilized on the photoelectrode surface by the immune sandwich structure, catalyzing the colorimetric reaction between H2O2 and TMB in the electrolyte to generate a colorimetric signal. Simultaneously, due to the significant consumption of the electron donor H2O2, ZnPc can substantially quench the photocurrent signal, enabling dual-mode detection of PSA.

[0056] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for fabricating a photoelectric / colorimetric dual-mode sensor, characterized in that, include: Step 1, cleaning FTO: Clean the FTO electrode three times with toluene, acetone, ethanol and water, and dry it for later use. Step 2: Modify FTO with commercial P25 TiO2 using spin coating; disperse 0.1-0.2 g of commercial P25 TiO2 in ethanol; spin coating rate is 1000-1500 rpm, time is 40-60 s; repeat spin coating 3-5 times to ensure TiO2 thickness; Step 3: Add 25-30 μL of chitosan solution to the TiO2-modified FTO electrode surface, dry it, and then rinse it with sodium hydroxide solution and ultrapure water; drop 30-50 μL of glutaraldehyde solution onto the electrode and keep it for 60-80 minutes; drop 30-50 μL of PSA antibody 1 solution onto the activated electrode and incubate it at 4 ℃ for 6-8 h, then rinse it thoroughly with ultrapure water; block the non-specific binding sites with 50-80 μL of 25% bovine serum albumin at 4 ℃ for 2-4 hours. Step 4: Add 30-50 μL of glutaraldehyde solution and 25-30 μL of chitosan solution to a 5-10 wt% aqueous solution of Zn-N4 single-atom active site ZnPc; react at room temperature for 60-80 minutes and then centrifuge to separate the product; subsequently, redisperse the product in water and add 30-50 μL of PSA antibody 2 solution, and incubate at 4 ℃ for 6-8 h to obtain an Ab2 solution labeled with Zn-N4; Step 5: Add 20-40 μL of PSA solution of different concentrations, incubate for 35-60 minutes, and wash; then add 30-50 μL of Ab2 labeled with Zn-N4 single-atom active site ZnPc, incubate for 35-60 minutes, and wash for later use. The methods for preparing Zn-N4 single-atom active sites ZnPc include: The initial product was obtained by reacting 4-nitrophthalonitrile, zinc acetate, urea and ammonium molybdate in a solid phase at 160-180 °C for 5-6 h. The initial product was reacted in HCl solution and NaOH solution at 80-90 °C for 1-2 h with stirring. After cooling to room temperature, it was filtered, washed with deionized water, and dried under vacuum at 60-70 °C for 7-8 h to obtain tetranitroZnPc. TetranitroZnPc and Na2S·9H2O were reacted in a set amount of N,N-dimethylformamide at 60-70 °C for 2-3 h. After cooling to room temperature, the reaction product was dispersed in 100-200 mL of deionized water, centrifuged and washed with deionized water until neutral, and the obtained solid was dried in a vacuum oven at 60-70 °C for 6-8 h to obtain tetraaminoZnPc.

2. The method for fabricating a photoelectric / colorimetric dual-mode sensor as described in claim 1, characterized in that, The molar ratio of 4-nitrophthalonitrile, zinc acetate, urea and ammonium molybdate is 1:0.4-0.5:8.0-9.0:0.01-0.

02.

3. The method for fabricating a photoelectric / colorimetric dual-mode sensor as described in claim 1, characterized in that, The molar ratio of the zinc tetranitrophthalocyanine and Na2S·9H2O is 1:12.0-13.

0.

4. The method for fabricating a photoelectric / colorimetric dual-mode sensor as described in claim 1, characterized in that, The HCl solution is 200-300 mL of 1 M solution; the NaOH solution is 200-300 mL of 1 M solution.

5. A photoelectric / colorimetric dual-mode sensor, characterized in that, The sensor is prepared using the photoelectric / colorimetric dual-mode sensor preparation method as described in claim 1, comprising a three-electrode system: the sensor photoelectric electrode is the working electrode, the counter electrode is a platinum wire electrode, the reference electrode is an Ag / AgCl electrode, a xenon lamp is the light source, and the electrolyte is a phosphate buffer solution containing hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine.

6. A performance testing method for a photoelectric / colorimetric dual-mode sensor as described in claim 5, characterized in that, include: A three-electrode system was adopted, with the sensor photoelectrode as the working electrode, a platinum wire electrode as the counter electrode, and an Ag / AgCl electrode as the reference electrode. The bias voltage was 0 V, a xenon lamp was used as the light source, and the electrolyte was a phosphate buffer solution containing hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine. After waiting for 10 minutes, the photoelectric performance (It curve) was tested using the chronoamperometry method, and colorimetric tests were performed based on ultraviolet spectroscopy and color development. The concentration of H2O2 was 3.0-5.0 mM, and the concentration of TMB was 80-100 μM.

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