Enzyme cascade dual-mode sensor as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biosensing detection, and provides an enzyme cascade dual-mode sensor as well as a preparation method and application thereof. The sensor comprises a three-dimensional (3D) PCN / BS-rGO carrier, PtAuNCs (PtAuNCs) loaded on the 3D PCN / BS-rGO carrier, and Enzyme fixed on the carrier. The invention further discloses a preparation method of the sensor. According to the enzyme cascade dual-mode sensor, the electron transfer rate is increased by constructing the three-dimensional carrier and doping B, S and P, the platinum nanoclusters PtAuNCs are loaded on the three-dimensional carrier, and due to the aggregation-induced emission effect of the PtAuNCs and the carrier, the fluorescence intensity of the PtAuNCs is greatly improved; the natural enzyme is loaded on the nano composite material, so that the stability of the immobilized enzyme is improved, the loss of an intermediate product of an enzyme cascade reaction is reduced, the enzyme cascade reaction is accelerated, and the efficiency of the enzyme cascade reaction is improved.

Description

Technical Field

[0001] This invention relates to the field of biosensing and detection technology, and in particular to an enzyme cascade dual-mode sensor, its preparation method, and its application. Background Technology

[0002] In recent years, with improved living standards, people's diets have become increasingly high in purines. Purines are important base compounds that make up nucleic acids in the body, and their metabolic end product is uric acid. Uric acid is one of the main non-protein nitrogen (NPN) metabolites in the blood. Under normal physiological conditions, its content is relatively stable, but under pathological conditions, its content often fluctuates. Recent studies have shown that the concentration of serum uric acid is positively correlated with the concentrations of triglycerides and creatinine, and is associated with heart damage. Therefore, the detection and analysis of uric acid is of important reference value for clinical diagnosis and understanding disease progression.

[0003] Cholesterol is an important lipid in the human body, participating in physiological processes such as cell membrane formation, hormone synthesis, and bile acid production. It plays many vital functions in the body, including forming cell membranes and synthesizing hormones and vitamin D. However, abnormal cholesterol levels, especially excessively high levels, can trigger a series of health problems. High cholesterol can lead to atherosclerosis, which in turn can cause cardiovascular diseases such as heart disease and stroke, and may also cause biliary tract diseases such as gallstones.

[0004] Acetylcholine is an important neurotransmitter, primarily found in the central and peripheral nervous systems. It transmits information between neurons and participates in regulating various physiological functions, such as muscle contraction, heart rate regulation, and digestive system activity. Therefore, the detection of acetylcholine is of great significance in both basic research and clinical applications.

[0005] Immobilized enzymes are biocatalysts that confine free enzymes to the surface or interior of a specific support material through physical or chemical means, thereby retaining their catalytic activity while achieving spatial confinement. The advantages of immobilized enzymes are as follows: First, immobilization can address the problem of free enzymes' easy denaturation and inactivation in extreme pH, high temperature, or organic solvents through the protective effect of the support, enhancing stability. Second, they are reusable, reducing costs. Finally, by constructing cascade catalytic systems through multi-enzyme co-immobilization technology, complex reaction pathways can be completed, achieving multifunctional coordination. Enzyme immobilization can improve the production efficiency of biocatalysts, and the hydrophobicity of rGO can enhance the interaction between the enzyme and the support, improving the stability and catalytic efficiency of the immobilized enzyme. Existing enzyme cascade sensors suffer from poor support hydrophobicity, resulting in poor enzyme immobilization and low detection sensitivity. Therefore, there is an urgent need to develop an enzyme cascade sensor with strong enzyme-support interaction and high detection sensitivity. Summary of the Invention

[0006] In view of this, the present invention proposes an enzyme cascade dual-mode sensor, its preparation method and application, in which the interaction between the carrier and the enzyme is strong and the detection sensitivity is high.

[0007] The technical solution of the present invention is implemented as follows: In a first aspect, the present invention provides an enzyme cascade dual-mode sensor, the sensor comprising a three-dimensional 3D PCN / BS-rGO carrier, PtAuNCs loaded on the 3DPCN / BS-rGO carrier, and an enzyme immobilized on the carrier.

[0008] Specifically, reduced graphene oxide (rGO) is formed by the oxidation and reduction of graphene. The surface of graphene oxide (GO) has a large number of hydrophilic groups such as carboxyl, hydroxyl, and epoxy groups, exhibiting hydrophilicity. The co-doping of boron and sulfur helps to reduce GO more efficiently, removing hydrophilic oxygen-containing groups (such as –COOH, –OH) and restoring sp. 2 The structure makes the graphene matrix more hydrophobic. Simultaneously, the introduction of B and S reduces the content of polar groups on the surface and increases the number of nonpolar B–C and S–C structures, enhancing hydrophobic interactions.

[0009] Protein (enzyme) molecules themselves have "hydrophobic regions" and "hydrophilic regions" on the outside of their spatial structure. Suitable carrier hydrophobicity can enhance the hydrophobic interaction of proteins, allowing enzyme molecules to be more tightly adsorbed (non-covalently immobilized) on the material surface, thus improving immobilization efficiency. A moderate increase in hydrophobicity also helps protect the enzyme's native structure and activity, improving the sensitivity and detection limit of multifunctional sensors.

[0010] Based on the above technical solutions, preferably, the enzyme is any enzyme capable of catalyzing the production of hydrogen peroxide from disease biomarkers. In some preferred embodiments, the natural enzyme can be one of sarcosine oxidase, acetylcholinesterase, choline oxidase, uricase, glucose oxidase, cholesterol oxidase, β-galactosidase, and uricase. When the natural enzyme is sarcosine oxidase, the prepared fluorescence / colorimetric dual-mode enzyme cascade sensor can be used to detect sarcosine content; when the natural enzyme is acetylcholinesterase or choline oxidase, it can be used to detect acetylcholine content; when it is glucose oxidase, it can detect glucose content; when it is cholesterol oxidase, it can detect cholesterol content; when it is uricase, it can detect uric acid content; and when it is uricase, it can detect uric acid content.

[0011] Based on the above technical solutions, preferably, the preparation method of the three-dimensional 3D PCN / BS-rGO carrier includes the following steps:

[0012] S1, Preparation of phosphorus-doped graphitic carbon nitride (PCN): Urea was calcined and ground to obtain GCN, which was then dissolved in water and reacted with ammonium dihydrogen phosphate. The reactants were then rotary evaporated and calcined to obtain PCN.

[0013] S2, Preparation of boron-sulfur co-doped reduced graphene oxide BS-rGO: Graphite and sodium nitrate were mixed, and concentrated sulfuric acid and potassium permanganate were added sequentially. After mixing evenly, the mixture was heated to react. After the reaction was completed, the reactants were heated to reflux and the reaction was terminated with hydrogen peroxide. After the reaction was completed, the reactants were washed and freeze-dried to obtain GO. GO, sodium sulfide solid and boric acid were ultrasonically dispersed and subjected to hydrothermal reaction. After the reaction was completed, the mixture was dried to obtain BS-rGO.

[0014] S3, Preparation of 3D PCN / BS-rGO: The PCN prepared in step S1, the BS-rGO prepared in step S2, and the KOH solution are mixed and reacted. After the reaction is completed, the mixture is centrifuged, the precipitate is washed and dried to obtain the 3DPCN / BS-rGO carrier.

[0015] Based on the above technical solutions, preferably, in step S1, the mass ratio of GCN to ammonium dihydrogen phosphate is 9-11:1.

[0016] Based on the above technical solutions, preferably, in step S2, the mass-to-volume ratio of graphite, sodium nitrate, concentrated sulfuric acid, and potassium permanganate is 0.5-2g:0.5-3g:15-20mL:3-8g; the mass-to-volume ratio of GO, solid sodium sulfide, and boric acid is 2.5-3:80-120:80-120; the hydrothermal reaction temperature is 180-200℃; and the reaction time is 15-20h.

[0017] Based on the above technical solution, preferably, in step S3, the mass-to-volume ratio of PCN, BS-rGO, and KOH solution is 20 mg: 10-40 mg: 3-4 mL, the reaction temperature is 150-300℃, and the time is 4-6 h; the concentration of KOH solution is 0.754 mol / L.

[0018] Based on the above technical solutions, preferably, the preparation method of PtAu NCs is as follows: H2PtCl6, HAuCl4 and GMP are mixed and reacted, and the reaction product is dialyzed after the reaction is completed to obtain platinum nanoclusters PtAu NCs.

[0019] Based on the above technical solutions, preferably, the molar ratio of H2PtCl6, HAuCl4 and GMP is 5-9∶1∶2-4; the reaction temperature is 80-150℃, the time is 30-120min; and the particle size of platinum nanoclusters PtAu NCs is 2-3nm.

[0020] Secondly, the present invention provides a method for preparing the above-mentioned enzyme cascade dual-mode sensor, comprising the following steps:

[0021] 3D PCN / BS-rGO aqueous solution and Enzyme solution were prepared separately. Then, PtAu NCs solution was added to 3DPCN / BS-rGO aqueous solution, mixed, centrifuged, washed and dried to obtain 3D PtAuNCs / PCN / BS-rGO.

[0022] Then, 3D PtAuNCs / PCN / BS-rGO was dissolved in water, and enzyme solution was added for incubation. After incubation, the mixture was centrifuged and the product was washed to obtain enzyme@3D PtAuNCs / PCN / BS-rGO loaded with the natural enzyme.

[0023] Based on the above technical solutions, preferably, the mass-to-volume ratio of the 3D PCN / BS-rGO to PtAuNCs solution is 1 mg: 2.5-3.5 mL; the mass ratio of the 3D PtAuNCs / PCN / BS-rGO to Enzyme is 0.8-1.2: 4.5, and the incubation time is 30 min.

[0024] Thirdly, the present invention provides the application of the above-mentioned enzyme cascade dual-mode sensor in the preparation of reagents for in vitro detection of disease biomarkers, wherein the reagents for in vitro detection of disease biomarkers are reagents for in vitro detection of sarcosine, acetylcholine, choline, uric acid, glucose, cholesterol, β-galactosidase or uric acid.

[0025] Thirdly, the present invention provides a method for in vitro detection of disease biomarkers, comprising the following steps: preparing a series of disease biomarker standard solutions of different concentrations and adding them respectively to a buffer solution containing the enzyme cascade dual-mode sensor described in the first aspect of the present invention; detecting the fluorescence intensity of the reaction system; plotting a standard curve based on the change in fluorescence intensity with the concentration of the disease biomarker standard solution to obtain the fluorescence standard curve equation of the disease biomarker concentration; adding the disease biomarker sample to be tested to a buffer solution containing the enzyme cascade dual-mode sensor described in the first aspect of the present invention; detecting the fluorescence intensity of the reaction system; and calculating the concentration of the disease biomarker sample to be tested based on the fluorescence standard curve equation of the disease biomarker concentration.

[0026] Based on the above technical solutions, preferably, when the fluorescence method is used to detect disease biomarkers, the fluorescence intensity of the reaction system is detected at 430 nm; when the colorimetric method is used to detect disease biomarkers, the absorbance of the reaction system is detected at 652 nm.

[0027] Based on the above technical solutions, preferably, the disease marker is selected from one of sarcosine, acetylcholine, choline, uric acid, glucose, cholesterol, β-galactoside, or uric acid.

[0028] The principle of the enzyme cascade dual-mode sensor provided by this invention for detecting the content of disease markers is as follows: The weakening of the AIE effect of hydrogen peroxide platinum nanoclusters generated during the cascade process reduces the fluorescence intensity at 430 nm, resulting in a negative response between the concentration of the target marker and the fluorescence intensity. Furthermore, the nanoenzyme 3D PtAuNCs / PCN / BS-rGO with peroxidase-like activity catalyzes the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to a blue oxidation product (oxTMB) by hydrogen peroxide, increasing the absorbance at 652 nm. The concentration of the target marker shows a positive response to the absorbance, thereby quantifying the content of the added marker (see...). Figure 1 The fluorescence and colorimetric detection signals can respond independently and also verify each other, giving the enzyme cascade dual-mode sensor the characteristics of low detection limit, wide linear range, high sensitivity, simple operation, short response time, and high specificity.

[0029] The enzyme cascade dual-mode sensor, its preparation method, and its application, as described in this invention, have the following advantages over existing technologies:

[0030] (1) In this invention, boron and sulfur are co-doped onto reduced graphene oxide (BS-rGO). By promoting the removal of oxygen-containing polar groups on the surface and increasing the structure of non-polar heteroatoms (B–C, S–C, etc.), the surface polarity and surface energy of the material are reduced, resulting in a significant improvement in the hydrophobicity of the material. This is beneficial for adsorbing and immobilizing more enzyme molecules through hydrophobic interactions, thereby promoting the performance improvement of enzyme cascade sensors.

[0031] (2) The enzyme cascade dual-mode sensor of the present invention, by constructing a three-dimensional carrier, increases the electron transfer rate by doping with B, S and P, and loads platinum nanoclusters PtAu NCs on the three-dimensional carrier. Due to the aggregation-induced emission effect of PtAu NCs and the carrier, the fluorescence intensity of PtAu NCs is greatly improved. Loading natural enzymes on nanocomposite materials increases the stability of immobilized enzymes, reduces the loss of intermediate products in enzyme cascade reactions, accelerates the enzyme cascade reaction, and improves the efficiency of enzyme cascade reactions.

[0032] (3) The enzyme cascade dual-mode sensor provided by this invention can generate both fluorescence and colorimetric signals, enriching the detection methods. For fluorescence detection, the generation of H2O2 will reduce the fluorescence of the material at 430 nm; for colorimetric mode, the nanocomposite material with peroxidase-like activity can catalyze the oxidation of TMB by H2O2 to produce blue oxTMB, increasing the absorbance at 652 nm. This allows for quantitative detection of the added marker content. The two detection signals in the fluorescence and colorimetric dual modes can respond independently and verify each other, improving the reliability of the detection results. It features a low detection limit, a wide linear range, and high sensitivity. Simultaneously, the specific binding of the natural enzyme to the substrate enhances the specificity of the detection method. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 This is a schematic diagram illustrating the principle of the enzyme cascade dual-mode sensor of the present invention for detecting the content of disease markers.

[0035] Figure 2 Electron micrograph of 3D PtAu NCs / PCN / BS-rGO prepared in Example 1 of this invention; Figure 2 (A) is a 3D PCN / BS-rGO scanning electron microscope image; Figure 2 (B) is a transmission electron microscope image of PtAuNCs; Figure 2 (C) is a transmission electron microscope image of 3DPtAu NCs / PCN / BS-rGO.

[0036] Figure 3 The fluorescence spectrum of 3D PtAuNCs / PCN / BS-rGO prepared in Example 1 of this invention is shown.

[0037] Figure 4 XPS image of 3D PtAuNCs / PCN / BS-rGO prepared in Example 1 of this invention; Figure 4 (A) is the full spectrum of 3D PtAu / PCN / S-rGO; 4(B) is the fine spectrum of C1s; 4(C) is the fine spectrum of N1s; 4(D) is the fine spectrum of O1s; 4(E) is the fine spectrum of B1s; 4(F) is the fine spectrum of P 2p; 4(G) is the fine spectrum of S2p; 3(H) is the fine spectrum of Pt4f; 4(I) is the fine spectrum of Au 4f.

[0038] Figure 5 This is a schematic diagram illustrating the linear relationship between uric acid concentration and fluorescence intensity in Example 2 of the present invention.

[0039] Figure 6 This is a schematic diagram illustrating the linear relationship between uric acid concentration and absorbance in Example 2 of the present invention.

[0040] Figure 7 The diagram shows the contact angle measurements of rGO, S-rGO, and BS-rGO in Comparative Example 1 of this invention.

[0041] Figure 8 The fluorescence spectra of PtAuNCs under different reaction conditions in Comparative Example 2 of this invention are shown.

[0042] Figure 9 The fluorescence intensity comparison spectrum of PtAuNCs and 3D PtAuNCs / PCN / BS-rGO in Comparative Example 2 of this invention is shown. Detailed Implementation

[0043] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0044] Example 1

[0045] The enzyme cascade dual-mode sensor in this embodiment is prepared as follows:

[0046] (1) Preparation of graphene-like carbon nitride: A crucible containing 5g of urea was placed in a muffle furnace and calcined at a constant temperature of 550℃ for 2h. After the crucible spontaneously cooled to room temperature, the resulting pale yellow solid was thoroughly ground into powder for later use, yielding blocky GCN.

[0047] (2) Preparation of phosphorus-doped graphitic carbon nitride (PCN): 200 mg of GCN was dissolved in 10 mL of distilled water. The solution was magnetically stirred at room temperature for 30 min. 20 mg of ammonium dihydrogen phosphate was added to the reaction mixture, followed by ultrasonic treatment for 10 min. The prepared solution was rotary evaporated at 60 °C for 20 min to remove water. After cooling, the obtained precipitate was placed in a crucible and calcined in a muffle furnace at 300 °C for 1 h. After the reaction was completed, the muffle furnace was turned off, and the crucible was allowed to spontaneously cool to room temperature before being removed. The resulting pale yellow blocky PCN solid was ground into powder.

[0048] (3) Preparation of graphene oxide: 0.5 g of graphite and 0.25 g of sodium nitrate were mixed in a three-necked flask, and 15 mL of concentrated sulfuric acid was added. The mixture was stirred for 0.5 h under ice bath conditions. Then, 2 g of potassium permanganate was added to the mixture, and the mixture was stirred at room temperature for 1 h. 100 mL of deionized water was added, and the mixture was heated at 37 °C for 1 h. The reactants were then transferred to an oil bath and refluxed at 95 °C for 2 h. Finally, the reaction was terminated with 15 mL of 30% hydrogen peroxide. After the reactants cooled to room temperature, the mixture was washed several times with 5% hydrochloric acid and ultrapure water. The washed solution was then vacuum dried and lyophilized to obtain GO.

[0049] (4) Preparation of boron-sulfur co-doped reduced graphene oxide BS-rGO: 30 mg of GO was dispersed in 30 mL of pure water and sonicated for 0.5 h. Then, 1 g of boric acid and 1 g of sodium sulfide were added to the GO suspension and stirred for 30 min. The solution was transferred to an autoclave and placed in an oven at 180 °C for 20 h. After the reaction was complete, the solution was cooled to room temperature. Then, it was centrifuged at 8000 rpm, washed twice with ethanol, and washed several times with pure water. After vacuum drying at 60 °C for 12 h, dry BS-rGO powder was obtained.

[0050] (5) Preparation of 3D PCN / BS-rGO: A suspension containing 3 mL of distilled water (0.754 M KOH), 20 mg of BS-rGO, and 20 mg of PCN was ultrasonically mixed and placed in a sealed stainless steel autoclave lined with polytetrafluoroethylene. The temperature of the muffle furnace was maintained at 200 °C for 5 h. After the muffle furnace cooled to room temperature, the solution was washed several times with deionized water and then freeze-dried to obtain 3D PCN / BS-rGO solid.

[0051] (6) Preparation of platinum nanoclusters PtAuNCs: HAuCl4 (0.10 mL, 10 mM), H2PtCl6 (0.70 mL, 10 mM), and NaOH (0.4 mL, 100 mM) were mixed in 8 mL of ultrapure water and stirred at room temperature for 20 min. Then, GMP (0.3 mL, 10 mM) was added to the mixture. The resulting solution was placed in a polytetrafluoroethylene-lined autoclave and heated to 120 °C for 30 min. After the reaction was complete, the mixture was cooled at room temperature, the product was removed, and dialyzed against the final product using a dialysis bag (500 Da) for 48 h. The final product was then stored at 4 °C for later use.

[0052] (7) Preparation of 3D PtAuNCs / PCN / BS-rGO: 1 mg of 3D PCN / BS-rGO was dispersed in 4 mL of ultrapure water and sonicated for 20 min. Then, 3 mL of PtAuNCs was added to the 3D PCN / BS-rGO aqueous solution. After stirring for 3 h at room temperature in the dark, the mixture was centrifuged at 8000 rpm for 10 min and washed multiple times with deionized water to obtain the product 3D PtAuNCs / PCN / BS-rGO.

[0053] (8) Preparation of enzyme cascade dual-mode sensor UOx@3D PtAuNCs / PCN / BS-rGO: Dissolve 3 mg of uricase (UOx) in 2 mL of deionized water, dissolve 1 mg of 3D PtAuNCs / PCN / S-rGO powder in 1 mL of deionized water, and sonicate for 20 min to disperse it evenly. Then add 1 mL of UOx solution (containing 1.5 mg UOx), sonicate for 15 min, and incubate at room temperature for 30 min. Centrifuge the mixture at 8000 rpm for 10 min, wash three times with ultrapure water, and obtain the product UOx@3D PtAuNCs / PCN / BS-rGO.

[0054] Figure 2-4 The images shown are electron micrographs, fluorescence spectra, and XPS images of the 3D PtAuNCs / PCN / BS-rGO prepared in Example 1.

[0055] Figure 4 It can be seen that the composite nanomaterial contains C, N, O, B, S, P, Pt and Au elements simultaneously, indicating that the composite material has been successfully prepared.

[0056] Example 2

[0057] The enzyme cascade dual-mode sensor in this embodiment is prepared as follows:

[0058] (1) Preparation of graphene-like carbon nitride: A crucible containing 5g of urea was placed in a muffle furnace and calcined at a constant temperature of 550℃ for 2h. After the crucible spontaneously cooled to room temperature, the resulting pale yellow solid was thoroughly ground into powder for later use, yielding blocky GCN.

[0059] (2) Preparation of phosphorus-doped graphitic carbon nitride (PCN): 220 mg of GCN was dissolved in 10 mL of distilled water. The solution was magnetically stirred at room temperature for 30 min. 20 mg of ammonium dihydrogen phosphate was added to the reaction mixture, followed by ultrasonic treatment for 10 min. The prepared solution was rotary evaporated at 60 °C for 20 min to remove water. After cooling, the obtained precipitate was placed in a crucible and calcined in a muffle furnace at 300 °C for 1 h. After the reaction was completed, the muffle furnace was turned off, and the crucible was allowed to spontaneously cool to room temperature before being removed. The resulting pale yellow blocky PCN solid was ground into powder.

[0060] (3) Preparation of graphene oxide: 0.5 g of graphite and 0.5 g of sodium nitrate were mixed in a three-necked flask, and 15 mL of concentrated sulfuric acid was added. The mixture was stirred for 0.5 h under ice bath conditions. Then, 3 g of potassium permanganate was added to the mixture, and the mixture was stirred at room temperature for 1 h. 100 mL of deionized water was added, and the mixture was heated at 37 °C for 1 h. The reactants were then transferred to an oil bath and refluxed at 95 °C for 2 h. Finally, the reaction was terminated with 15 mL of 30% hydrogen peroxide. After the reactants cooled to room temperature, the mixture was washed several times with 5% hydrochloric acid and ultrapure water. The washed solution was then vacuum dried and lyophilized to obtain GO.

[0061] (4) Preparation of boron-sulfur co-doped reduced graphene oxide BS-rGO: 25 mg GO was dispersed in 30 mL of pure water and sonicated for 0.5 h. Then, 1.5 g boric acid and 1.5 g sodium sulfide were added to the GO suspension and stirred for 30 min. The solution was transferred to an autoclave and placed in an oven at 200 °C for 15 h. After the reaction was complete, the solution was cooled to room temperature. Then, it was centrifuged at 8000 rpm, washed twice with ethanol, and washed several times with pure water. After vacuum drying at 60 °C for 12 h, dry BS-rGO powder was obtained.

[0062] (5) Preparation of 3D PCN / BS-rGO: A suspension containing 3 mL of distilled water with 0.754 M KOH, 10 mg of BS-rGO, and 20 mg of PCN was ultrasonically mixed and placed in a sealed stainless steel autoclave lined with polytetrafluoroethylene. The temperature of the muffle furnace was maintained at 150 °C for 6 h. After the muffle furnace cooled to room temperature, the solution was washed several times with deionized water and then freeze-dried to obtain 3D PCN / BS-rGO solid.

[0063] (6) Preparation of platinum nanoclusters PtAuNCs: HAuCl4 (0.10 mL, 10 mM), H2PtCl6 (0.50 mL, 10 mM), and NaOH (0.4 mL, 100 mM) were mixed in 8 mL of ultrapure water and stirred at room temperature for 20 min. Then, GMP (0.2 mL, 10 mM) was added to the mixture. The resulting solution was placed in a polytetrafluoroethylene-lined autoclave and heated to 80 °C for 120 min. After the reaction was complete, the mixture was cooled at room temperature, the product was removed, and the final product was dialyzed through a dialysis bag (500 Da) for 48 h. The product was then stored at 4 °C for later use.

[0064] (7) Preparation of 3D PtAuNCs / PCN / BS-rGO: 1 mg of 3D PCN / BS-rGO was dispersed in 4 mL of ultrapure water and sonicated for 20 min. Then, 2.5 mL of PtAu NCs was added to the 3DPCN / BS-rGO aqueous solution. After stirring for 3 h at room temperature in the dark, the mixture was centrifuged at 8000 rpm for 10 min and washed multiple times with deionized water to obtain the product 3D PtAuNCs / PCN / BS-rGO.

[0065] (8) Preparation of enzyme cascade dual-mode sensor UOx@3D PtAuNCs / PCN / BS-rGO: Dissolve 3 mg of uricase (UOx) in 2 mL of deionized water, dissolve 0.8 mg of 3D PtAuNCs / PCN / S-rGO powder in 1 mL of deionized water, and sonicate for 20 min to disperse it evenly. Then add 1 mL of UOx solution (containing 1.5 mg UOx), sonicate for 15 min, and incubate at room temperature for 30 min. Centrifuge the mixture at 8000 rpm for 10 min, wash three times with ultrapure water, and obtain the product UOx@3D PtAuNCs / PCN / BS-rGO.

[0066] Example 3

[0067] The enzyme cascade dual-mode sensor in this embodiment is prepared as follows:

[0068] (1) Preparation of graphene-like carbon nitride: A crucible containing 5g of urea was placed in a muffle furnace and calcined at a constant temperature of 550℃ for 2h. After the crucible spontaneously cooled to room temperature, the resulting pale yellow solid was thoroughly ground into powder for later use, yielding blocky GCN.

[0069] (2) Preparation of phosphorus-doped graphitic carbon nitride (PCN): 180 mg of GCN was dissolved in 10 mL of distilled water. The solution was magnetically stirred at room temperature for 30 min. 20 mg of ammonium dihydrogen phosphate was added to the reaction mixture, followed by ultrasonic treatment for 10 min. The prepared solution was rotary evaporated at 60 °C for 20 min to remove water. After cooling, the obtained precipitate was placed in a crucible and calcined in a muffle furnace at 300 °C for 1 h. After the reaction was completed, the muffle furnace was turned off, and the crucible was allowed to spontaneously cool to room temperature before being removed. The resulting pale yellow blocky PCN solid was ground into powder.

[0070] (3) Preparation of graphene oxide: 2g of graphite and 3g of sodium nitrate were mixed in a three-necked flask, and 18mL of concentrated sulfuric acid was added. The mixture was stirred for 0.5h under ice bath conditions. Then, 8g of potassium permanganate was added to the mixture, and the mixture was stirred at room temperature for 1h. 100mL of deionized water was added, and the mixture was heated at 37℃ for 1h. The reactants were then transferred to an oil bath and refluxed at 95℃ for 2h. Finally, the reaction was terminated with 15mL of 30% hydrogen peroxide. After the reactants cooled to room temperature, the mixture was washed several times with 5% hydrochloric acid and ultrapure water. The washed solution was then vacuum dried and lyophilized to obtain GO.

[0071] (4) Preparation of boron-sulfur co-doped reduced graphene oxide BS-rGO: 28 mg of GO was dispersed in 30 mL of pure water and sonicated for 0.5 h. Then, 2 g of boric acid and 2 g of sodium sulfide were added to the GO suspension and stirred for 30 min. The solution was transferred to an autoclave and placed in an oven at 190 °C for 18 h. After the reaction was complete, the solution was cooled to room temperature. Then, it was centrifuged at 8000 rpm, washed twice with ethanol, and washed several times with pure water. After vacuum drying at 60 °C for 12 h, dry BS-rGO powder was obtained.

[0072] (5) Preparation of 3D PCN / BS-rGO: A suspension containing 4 mL of distilled water (0.754 M KOH), 40 mg of BS-rGO, and 20 mg of PCN was ultrasonically mixed and placed in a sealed stainless steel autoclave lined with polytetrafluoroethylene. The temperature of the muffle furnace was maintained at 300 °C for 4 h. After the muffle furnace cooled to room temperature, the solution was washed several times with deionized water and then freeze-dried to obtain 3D PCN / BS-rGO solid.

[0073] (6) Preparation of platinum nanoclusters PtAuNCs: HAuCl4 (0.10 mL, 10 mM), H2PtCl6 (0.90 mL, 10 mM), and NaOH (0.4 mL, 100 mM) were mixed in 8 mL of ultrapure water and stirred at room temperature for 20 min. Then, GMP (0.4 mL, 10 mM) was added to the mixture. The resulting solution was placed in a polytetrafluoroethylene-lined autoclave and heated to 150 °C for 30 min. After the reaction was complete, the mixture was cooled at room temperature, the product was removed, and the final product was dialyzed through a dialysis bag (500 Da) for 48 h. The product was then stored at 4 °C for later use.

[0074] (7) Preparation of 3D PtAuNCs / PCN / BS-rGO: 1 mg of 3D PCN / BS-rGO was dispersed in 4 mL of ultrapure water and sonicated for 20 min. Then, 3.5 mL of PtAu NCs was added to the 3DPCN / BS-rGO aqueous solution. After stirring for 3 h at room temperature in the dark, the mixture was centrifuged at 8000 rpm for 10 min and washed multiple times with deionized water to obtain the product 3D PtAuNCs / PCN / BS-rGO.

[0075] (8) Preparation of enzyme cascade dual-mode sensor UOx@3D PtAuNCs / PCN / BS-rGO: Dissolve 3 mg of uricase (UOx) in 2 mL of deionized water, dissolve 1.2 mg of 3D PtAuNCs / PCN / S-rGO powder in 1 mL of deionized water, and sonicate for 20 min to disperse it evenly. Then add 1 mL of UOx solution (containing 1.5 mg UOx), sonicate for 15 min, and incubate at room temperature for 30 min. Centrifuge the mixture at 8000 rpm for 10 min, wash three times with ultrapure water, and obtain the product UOx@3D PtAuNCs / PCN / BS-rGO.

[0076] Example 4

[0077] This embodiment discloses a method for detecting uric acid using an enzyme cascade dual-mode sensor, the specific steps of which are as follows:

[0078] (1) Detection of uric acid concentration by fluorescence method

[0079] Construction of the fluorescence standard curve: The UOx@3D PtAuNCs / PCN / BS-rGO prepared in Example 1 was dissolved in 1 mL of ultrapure water, and then diluted 10 times for later use. 100 μL of Tris buffer (pH 8.0), 50 μL of standard uric acid solutions of different concentrations, and 50 μL of UOx@3D PtAuNCs / PCN / BS-rGO solution were added to a test tube. After incubation in the dark for 20 min, the fluorescence value was detected using a multi-mode microplate reader. The parameters for the multi-mode microplate reader were set as follows: excitation wavelength 330 nm, emission wavelength 430 nm.

[0080] The concentrations of the prepared standard uric acid solutions were increased sequentially: 0.5, 5, 10, 30, 50, 80, 100, 130, and 150 μM. A standard curve was plotted with the standard uric acid concentration on the x-axis and fluorescence intensity on the y-axis. The concentration of the uric acid to be tested was determined based on the standard curve, and the detection limit was established. In this embodiment, the linear range for fluorescence detection of uric acid was 0.5–150 μM, and the detection limit was 0.27 μM. The linear relationship is shown in the figure. Figure 5 .

[0081] Add 50 μL of Tris buffer (pH 8.0), 50 μL of UOx@3D PtAuNCs / PCN / BS-rGO solution, and 50 μL of the uric acid solution to be tested to a test tube. Incubate in the dark for 20 min, then detect the fluorescence value using a multi-mode microplate reader. The parameters for the multi-mode microplate reader were set as follows: excitation wavelength 380 nm and emission wavelength 430 nm. Read the fluorescence intensity value and substitute it into... Figure 5 The concentration of uric acid is calculated from the standard curve.

[0082] (2) Colorimetric method for detecting uric acid concentration

[0083] Construction of colorimetric standard curves: The UOx@3D PtAuNCs / PCN / BS-rGO prepared in Example 1 was dissolved in 1 mL of ultrapure water and then diluted 10-fold for later use. 100 μL of Tris buffer (pH 8.0) and 50 μL of standard uric acid solutions of different concentrations (1, 5, 15, 30, 50, 80, 100, 130, 150, 180, 200, 230, and 250 μM) were added to test tubes, followed by 50 μL of UOx@3D PtAuNCs / PCN / BS-rGO solution. The solutions were incubated in the dark for 20 min. 150 μL of the solutions from each test tube were then added to 100 μL of HAC-NaAC buffer (pH 4.0) and 15 μL of 15 mM TMB solution, respectively. After incubation for 15 min, the absorbance at 652 nm was measured using a multi-mode microplate reader.

[0084] A standard curve was plotted with standard uric acid concentration on the x-axis and absorbance on the y-axis. The concentration of the uric acid to be tested was determined based on the standard curve, and the detection limit was established. In this embodiment, the linear range of the colorimetric method for detecting uric acid is 1-250 μM, and the detection limit is 0.65 μM. The linear relationship is shown in the figure. Figure 6 .

[0085] Add 100 μL of Tris buffer (pH 8.0), 50 μL of UOx@3D PtAu NCs / PCN / BS-rGO solution, and 50 μL of the uric acid solution to be tested to a test tube, and incubate in the dark for 20 min. Take 150 μL of the test solution, add 100 μL of HAC-NaAC buffer (pH 4.0), and 15 μL of 15 mM TMB solution, incubate for 15 min, and then use a multi-mode microplate reader to detect the absorbance at 652 nm. Read the absorbance value and substitute it into... Figure 6 The concentration of uric acid is calculated from the standard curve.

[0086] Example 5

[0087] This embodiment discloses a method for detecting uric acid levels in human blood using an enzyme cascade dual-mode sensor. The specific steps are as follows:

[0088] (1) Take a fresh human blood sample, centrifuge at 3000 rpm for 5 min and take the supernatant; centrifuge the supernatant at 10000 rpm for 10 min, take the supernatant and dilute it 5 times for later use.

[0089] (2) Dissolve the UOx@3D PtAuNCs / PCN / BS-rGO prepared in Example 1 in 1 mL of ultrapure water, and then dilute it 10 times for later use. Add 100 μL of Tris buffer (pH=8.0), 50 μL of UOx@3DPtAuNCs / PCN / BS-rGO solution, and 50 μL of human blood to be tested to a test tube. After incubation in the dark for 20 min, the fluorescence value is detected using a multi-functional microplate reader. The parameters for detection by the multi-functional microplate reader are set as follows: excitation wavelength 380 nm and emission wavelength 430 nm. Read the fluorescence intensity value and substitute it into... Figure 5 The standard curve is used to calculate the concentration of uric acid in human blood.

[0090] (3) Take 200 μL of the solution after the reaction in step (2), add 100 μL of HAC-NaAC buffer solution with pH=4.0, add 15 μL of 15 mM TMB solution, incubate for 15 min, and then use a multi-functional microplate reader to detect the absorbance at 652 nm. Read the absorbance value and substitute it into... Figure 6 The concentration of uric acid in human blood is calculated using the standard curve.

[0091] Example 6

[0092] The difference between the enzyme cascade dual-mode sensor in this embodiment and that in Example 1 is that uric acid oxidase is replaced with xanthine oxidase (XOD), resulting in XOD@3D PtAuNCs / PCN / BS-rGO. The enzyme cascade dual-mode sensor prepared in this embodiment detects xanthine using the same method as in Example 2.

[0093] Example 7

[0094] The difference between the enzyme cascade dual-mode sensor in this embodiment and that in Example 1 is that uric acid oxidase is replaced with glucose oxidase (GOD), resulting in GOD@3D PtAu NCs / PCN / BS-rGO. The enzyme cascade dual-mode sensor prepared in this embodiment is used to detect glucose content according to the same method as in Example 2.

[0095] Example 8

[0096] The difference between the enzyme cascade dual-mode sensor in this embodiment and that in Example 1 is that uricase is replaced with cholesterol oxidase (COD), resulting in COD@3D PtAuNCs / PCN / BS-rGO. The enzyme cascade dual-mode sensor prepared in this embodiment was used to detect cholesterol content using the same method as in Example 2.

[0097] Example 9

[0098] The difference between the enzyme cascade dual-mode sensor in this embodiment and that in Example 1 is that uricase is replaced with acetylcholinesterase (AChE), resulting in AChE@3D PtAuNCs / PCN / BS-rGO. The enzyme cascade dual-mode sensor prepared in this embodiment detects acetylcholine using the same method as in Example 2.

[0099] Comparative Example 1

[0100] This comparative analysis examines the static contact angles of reduced graphene oxide (rGO) without doping, with sulfur doping (S-rGO), and with boron-sulfur doping (BS-rGO).

[0101] Since UOx and BS-rGO are linked through hydrophobic interactions, the preparation method of enzyme immobilization using physical adsorption is simple. Figure 7 The static contact angles of rGO, S-rGO, and BS-rGO, measured by the seat drop method, were 98°, 132°, and 141°, respectively (contact angles greater than 90° are hydrophobic, and those less than 90° are hydrophilic). The results show that the hydrophobicity of rGO gradually increases with heteroatom doping, with BS-rGO exhibiting the strongest hydrophobicity and being able to immobilize more UOx.

[0102] Table 1. Results of uric acid detection using different methods

[0103]

[0104] As shown in Table 1, the enzyme cascade dual-mode sensor of this application is more sensitive than most existing methods for detecting uric acid, exhibiting a wider detection range and a lower detection limit. The comparative examples demonstrate that, compared to undoped or sulfur-doped methods, boron-sulfur doping results in a wider detection range and a lower detection limit.

[0105] Comparative Example 2

[0106] Based on Example 1, this comparative example discusses the effect of different reaction conditions on the fluorescence intensity of PtAuNCs, and the results are as follows: Figure 8 As shown.

[0107] Figure 8 (A) represents the fluorescence intensity at different reaction temperatures. Appropriate heating may promote the reaction between metal ions and ligands, thereby increasing the number of fluorescence emission centers. However, excessively high temperatures lead to the aggregation or structural changes of nanoclusters, thus reducing the number of effective fluorescence emission centers.

[0108] Figure 8(B) represents the fluorescence intensity of platinum nanoclusters at different reaction time periods. In the initial stage, as the reaction time increases, more metal ions are reduced, and nanoclusters gradually form and increase, thus increasing the fluorescence intensity. However, excessively long reaction times can lead to overgrowth or aggregation of nanoclusters, which reduces the efficiency of the fluorescence emission centers and causes a decrease in fluorescence intensity.

[0109] Figure 8 (C) illustrates different ratios of fluorescence intensity. As a ligand, GMP acts as a stabilizer during the formation of nanoclusters. It forms coordination bonds with metal ions, helping to control the size and morphology of nanoclusters while preventing aggregation and maintaining their dispersion. An inappropriate ratio of metal ions to ligands may lead to non-uniform size or irregular morphology of nanoclusters, affecting their fluorescence properties.

[0110] In summary, the optimal reaction conditions for synthesizing PtAuNCs are: a molar ratio of metal ions to GMP of 3:1, and a reaction at 120℃ for 0.5 h.

[0111] Comparative Example 3

[0112] This comparative example demonstrates the fluorescence properties of platinum nanoclusters PtAu NCs (Example 1) and 3D PtAu NCs / PCN / BS-rGO (Example 1).

[0113] Fluorescence properties of 3D PtAu NCs / PCN / BS-rGO, such as Figure 3 As shown, the maximum emission wavelength is 430 nm. The fluorescence intensity of 3D PtAuNCs / PCN / BS-rGO is significantly stronger than that of PtAuNCs (see...). Figure 9 This is because when PtAuNCs are loaded onto 3DPCN / BS-rGO, it induces aggregated emission (AIE); and by observing the position of the maximum emission peak, it can be found that the emission wavelength shifts to the right from 415nm to 430nm. This phenomenon is called redshift, and it is generally due to the formation of a new substance.

[0114] 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. An enzyme cascade dual-mode sensor, characterized in that: The sensor includes a three-dimensional 3D PCN / BS-rGO carrier, PtAuNCs loaded on the 3D PCN / BS-rGO carrier, and an enzyme fixed on the carrier.

2. The enzyme cascade dual-mode sensor as described in claim 1, characterized in that: The preparation method of the three-dimensional 3DPCN / BS-rGO carrier includes the following steps: S1, Preparation of phosphorus-doped graphitic carbon nitride (PCN): Urea was calcined and ground to obtain GCN, which was then dissolved in water and reacted with ammonium dihydrogen phosphate. The reactants were then rotary evaporated and calcined to obtain PCN. S2, Preparation of boron-sulfur co-doped reduced graphene oxide BS-rGO: Graphite and sodium nitrate were mixed, and concentrated sulfuric acid and potassium permanganate were added sequentially. After mixing evenly, the mixture was heated to react. After the reaction was completed, the reactants were heated to reflux and the reaction was terminated with hydrogen peroxide. After the reaction was completed, the reactants were washed and freeze-dried to obtain GO. GO, sodium sulfide solid and boric acid were ultrasonically dispersed and subjected to hydrothermal reaction. After the reaction was completed, the mixture was dried to obtain BS-rGO. S3, Preparation of 3D PCN / BS-rGO: The PCN prepared in step S1, the BS-rGO prepared in step S2, and the KOH solution are mixed and reacted. After the reaction is completed, the mixture is centrifuged, the precipitate is washed and dried to obtain the 3DPCN / BS-rGO carrier.

3. The enzyme cascade dual-mode sensor as described in claim 2, characterized in that: In step S1, the mass ratio of GCN to ammonium dihydrogen phosphate is 9-11:

1.

4. The enzyme cascade dual-mode sensor as described in claim 2, characterized in that: In step S2, the mass-to-volume ratio of graphite, sodium nitrate, concentrated sulfuric acid, and potassium permanganate is 0.5-2g:0.5-3g:15-20mL:3-8g; the mass ratio of GO, sodium sulfide solid, and boric acid is 2.5-3:80-120:80-120; the hydrothermal reaction temperature is 180-200℃; and the reaction time is 15-20h.

5. The enzyme cascade dual-mode sensor as described in claim 2, characterized in that: In step S3, the mass-to-volume ratio of PCN, BS-rGO, and KOH solution is 20 mg: 10-40 mg: 3-4 mL, the reaction temperature is 150-300℃, and the reaction time is 4-6 h; the concentration of KOH solution is 0.754 mol / L.

6. The enzyme cascade dual-mode sensor as described in claim 1, characterized in that: The preparation method of PtAu NCs is as follows: H2PtCl6, HAuCl4 and GMP are mixed and reacted, and the reaction product is dialyzed after the reaction is completed to obtain platinum nanoclusters PtAuNCs.

7. The enzyme cascade dual-mode sensor as described in claim 6, characterized in that: The molar ratio of H2PtCl6, HAuCl4 and GMP is 5-9:1:2-4; the reaction temperature is 80-150℃ and the time is 30-120 min; the particle size of platinum nanoclusters PtAuNCs is 2-3 nm.

8. The method for preparing the enzyme cascade dual-mode sensor according to any one of claims 1-7, characterized in that, Includes the following steps: 3D PCN / BS-rGO aqueous solution and Enzyme solution were prepared separately. Then, PtAu NCs solution was added to 3D PCN / BS-rGO aqueous solution, mixed, centrifuged, washed and dried to obtain 3D PtAuNCs / PCN / BS-rGO. Then, 3D PtAuNCs / PCN / BS-rGO was dissolved in water, and enzyme solution was added for incubation. After incubation, the mixture was centrifuged and the product was washed to obtain enzyme@3D PtAuNCs / PCN / BS-rGO loaded with the natural enzyme.

9. The method for preparing the enzyme cascade dual-mode sensor as described in claim 8, characterized in that, The mass-to-volume ratio of the 3DPCN / BS-rGO to PtAuNCs solution is 1 mg: 2.5-3.5 mL; the mass ratio of the 3D PtAuNCs / PCN / BS-rGO to Enzyme is 0.8-1.2: 4.5, and the incubation time is 20-30 min.

10. The application of the enzyme cascade dual-mode sensor as described in any one of claims 1-7 in the preparation of reagents for in vitro detection of disease biomarkers, characterized in that: The reagents used for in vitro detection of disease markers are reagents for in vitro detection of sarcosine, acetylcholine, choline, uric acid, glucose, cholesterol, β-galactosidase, or uric acid.

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