Phosphorescent silicon-dot nanoscale enzyme and its application in biochemical detection and triple anti-counterfeiting

Abstract

The application belongs to the technical field of nanomaterials and biological functional materials, and more particularly relates to a phosphorescent silicon dot nanoscale enzyme and application thereof in biochemical detection and triple anti-counterfeiting. The phosphorescent silicon dot nanoscale enzyme with fluorescence, room-temperature phosphorescence and photo-induced oxidase activity is synthesized by a one-step hydrothermal method using a silicon source, iodide, hematin and a phosphorus source as raw materials. The fluorescence / phosphorescence dual-mode detection based on the phosphorescent silicon dot nanoscale enzyme and the aqueous silicon dot nanoscale enzyme can realize sensitive detection of carmine, and can effectively avoid interference of non-target factors, and has high detection accuracy. The phosphorescent silicon dot nanoscale enzyme can also be used for detecting tiopronin (TPN) concentration, and has a detection lower limit of 9.8 nM and strong applicability in actual biological sample detection; and the hydrogel loaded with the phosphorescent silicon dot nanoscale enzyme can realize on-site rapid visual detection of TPN. In addition, the phosphorescent silicon dot nanoscale enzyme can also be used for preparing a fluorescence, phosphorescence and enzymatic color change triple anti-counterfeiting system.

Description

Technical Field

[0001] This application belongs to the field of nanomaterials and biofunctional materials technology, and more specifically, relates to phosphorescent silicon dot nanozymes, their preparation methods and applications. Background Technology

[0002] Silicon quantum dots (SiQDs) have attracted much attention from researchers as a candidate to replace existing semiconductor quantum dots. The design and development of nanomaterials with phosphorescence, fluorescence, and oxidase activities is a current emerging research hotspot.

[0003] Room-temperature phosphorescent (RTP) materials possess advantages such as long lifetime, visible emission, large Stokes shift, good stability, and high signal-to-noise ratio. Compared to conventional SiQDs that rely solely on changes in fluorescence signal, multi-parameter or multi-response phosphorescent SiQDs can effectively improve the selectivity and accuracy of detection. The RTP performance of nanomaterials is typically achieved by embedding them in a matrix; however, selecting a suitable matrix is ​​time-consuming and the preparation process is complex, severely hindering the further development of RTP materials. There is an urgent need to develop a matrix-free, ultra-long-life, non-matrix-assisted room-temperature phosphorescent SiQD with a simple, rapid, and environmentally friendly preparation method.

[0004] Carmine, as an artificial colorant, can produce aromatic amines, which may lead to nucleic acid mutations. Long-term or high-dose ingestion of carmine poses a significant health risk to organisms. Currently, its detection is mainly achieved through single-signal readout methods, which suffer from large measurement errors and low efficiency. Phosphorescent nanomaterials typically exhibit longer wavelength emission after the excitation source is removed. Compared to single fluorescence, they possess a larger Stokes shift, avoiding interference and self-absorption from excitation light. Furthermore, their long phosphorescence lifetime allows for appropriate time-gating for emission detection, thus avoiding spectral interference from the system and light scattering. Developing a rapid, sensitive, efficient, and accurate method for detecting carmine based on dual-signal readout of fluorescence and phosphorescence is of great significance.

[0005] Thiopronine (N-(2-mercaptopropionyl)glycine, TPN) is a drug used to treat heavy metal poisoning, cystinuria, and liver disease. Currently, TPN is mainly detected by fluorescence, colorimetry, chemiluminescence, and high-performance liquid chromatography (HPLC). However, these methods are highly dependent on laboratory equipment and cannot achieve rapid, on-site, and visual detection.

[0006] Anti-counterfeiting labels with a single anti-counterfeiting output mode have poor anti-counterfeiting performance, necessitating the development of labels with multiple anti-counterfeiting modes. Compared to fluorescent anti-counterfeiting materials, phosphorescent anti-counterfeiting materials have a longer lifespan, higher stability, and greater scarcity. Enzyme-catalyzed color-changing anti-counterfeiting is easily observed with the naked eye and is irreversible. Developing triple anti-counterfeiting labels combining fluorescence, phosphorescence, and enzyme-catalyzed color-changing, compared to traditional single-light information encryption static anti-counterfeiting labels, can encode optical information on spatial or temporal scales, making it difficult to copy, and providing a promising future for high-security anti-counterfeiting applications. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the purpose of this application is to provide phosphorescent silicon dot nanozymes and their applications in biochemical detection and triple anti-counterfeiting. The aim is to provide phosphorescent silicon dot nanozymes with phosphorescent, fluorescent, and photoinduced oxidase activities, which can detect carmine rapidly, sensitively, efficiently, and accurately through dual signal readout of fluorescence and phosphorescence, and can be used for rapid on-site visual detection of thiopronine, as well as for the preparation of triple anti-counterfeiting labels.

[0008] To achieve the above objectives, this application provides a method for preparing phosphorescent silicon dot nanozymes, comprising the following steps:

[0009] The first silicon source, iodide, heme and phosphorus source were mixed evenly and heated to react. After cooling, a solid product was obtained. The solid product was mixed evenly with water, centrifuged, dialyzed and freeze-dried to obtain phosphorescent silicon dot nanozyme.

[0010] Preferably, the first silicon source is at least one selected from N-[3-(trimethoxysilyl)propyl]ethylenediamine, dimethyloctadecyl[3-trimethoxysilyl]ammonium chloride, and 3-(2-aminoethylamino)propylmethyldimethoxysilane; and / or,

[0011] The iodide mentioned above is selected from at least one of potassium iodide, sodium iodide, and sodium periodate; and / or,

[0012] The phosphorus source mentioned above is selected from at least one of phosphoric acid, sodium dihydrogen phosphate, and sodium pyrophosphate.

[0013] Preferably, the mass ratio of the first silicon source, iodide, heme and phosphorus source is (100-150):(4-5):(0.1-0.5):(9-12).

[0014] Preferably, the temperature of the above heating reaction is 80℃~90℃, and the time is 2min~30min.

[0015] This application provides a phosphorescent silicon dot nanozyme prepared according to the above preparation method.

[0016] On the other hand, this application also provides an aqueous phosphorescent silicon dot nanozyme as described above.

[0017] Preferably, the preparation method of the above-mentioned aqueous phosphorescent silicon dot nanozyme includes the following steps:

[0018] The above-mentioned phosphorescent silicon dot nanozyme, the second silicon source, ammonia and water were mixed evenly, and the reaction solution was subjected to hydrolysis, condensation and polymerization reactions in sequence to obtain aqueous phosphorescent silicon dot nanozyme.

[0019] Preferably, the second silicon source is selected from ethyl silicate or tetraethoxysilane.

[0020] Preferably, the volume ratio of the second silicon source, ammonia, and water is (0.5-1):(0.5-1):(9-18).

[0021] Preferably, the concentration of phosphorescent silicon dot nanozyme in the above reaction solution is 100 μg / mL to 300 μg / mL.

[0022] On the other hand, this application also provides a hydrogel containing the above-mentioned phosphorescent silicon dot nanozyme.

[0023] This application also provides the application of the above-mentioned phosphorescent silicon dot nanozyme or the above-mentioned aqueous phosphorescent silicon dot nanozyme in the detection of carmine.

[0024] This application also provides the application of the above-mentioned phosphorescent silicon dot nanozyme or the above-mentioned hydrogel in the detection of thiopronine.

[0025] This application also provides the application of the above-mentioned phosphorescent silicon dot nanozyme in the preparation of fluorescent, phosphorescent and enzyme-catalyzed color-changing triple anti-counterfeiting labels.

[0026] In summary, compared with the prior art, the technical solutions conceived in this application have the following main technical advantages:

[0027] (1) The method for preparing phosphorescent silicon dot nanozymes provided in this application optimizes and screens halides and other nanozyme synthesis raw materials, using silicon sources, iodides, heme, and phosphorus sources as raw materials, and synthesizes phosphorescent silicon dot nanozymes in a one-step hydrothermal method. These nanozymes exhibit yellow-green room-temperature phosphorescence with a long phosphorescence lifetime, blue fluorescence, and excellent UV-induced oxidase activity. Compared with existing technologies, the method for preparing phosphorescent silicon dot nanozymes provided in this application uses environmentally friendly and harmless materials, requires no matrix, and features a simple, rapid synthesis process that does not require high temperatures or complex instruments, making it highly valuable for widespread application.

[0028] (2) The phosphorescent silicon dot nanozyme provided in this application has phosphorescent and fluorescence properties. The fluorescence mode detection based on phosphorescent silicon dot nanozyme and the phosphorescent mode detection based on aqueous silicon dot nanozyme have excellent selectivity and high responsiveness to carmine, enabling sensitive detection of carmine. They also have mutual verification functions, effectively avoiding interference from non-targeting factors, and have high detection accuracy in complex real samples.

[0029] (3) The phosphorescent silicon dot nanozyme provided in this application has UV-induced oxidase activity, enabling the detection of TPN. It exhibits excellent selective response to TPN and demonstrates good recovery rate, reproducibility, and strong applicability in practical biological sample detection. In a preferred embodiment, the detection limit for TPN reaches 9.8 nM. Furthermore, the hydrogel loaded with silicon dot nanozyme provided in this application enables visual detection of TPN concentration in samples and has low dependence on experimental instruments, achieving rapid on-site detection of TPN concentration in samples.

[0030] (4) The phosphorescent silicon dot nanozyme provided in this application can be used to prepare a triple anti-counterfeiting system. By combining fluorescence properties, matrix-free RTP properties, and photoinduced oxidase activity, a multi-layered information anti-counterfeiting encryption system has been successfully constructed, showing application potential in information anti-counterfeiting encryption. Compared with traditional anti-counterfeiting technologies, the multi-mode information encryption technology of fluorescence, RTP, and color development of the phosphorescent silicon dot nanozyme provided in this application can achieve a higher level of anti-counterfeiting information security. Attached Figure Description

[0031] Figure 1 The following is a characterization of the SiQDs@heme obtained in this application, wherein (a) is a TEM image and (b) is a particle size distribution histogram;

[0032] Figure 2 The images show the infrared spectrum and XPS spectrum of the SiQDs@heme obtained in this application, where (a) is the FT-IR spectrum and (b) is the XPS spectrum.

[0033] Figure 3 These are XPS images of SiQDs@heme obtained in this application, wherein content (a) is C1s, content (b) is N1s, content (c) is O1s, content (d) is P 2p, content (e) is Si 2p, and content (f) is I 3d;

[0034] Figure 4 These are the ultraviolet absorption spectrum and fluorescence / phosphorescence emission spectrum of SiQDs@heme obtained in this application, wherein (a) is the ultraviolet absorption spectrum and (b) is the fluorescence / phosphorescence emission spectrum under optimal excitation.

[0035] Figure 5The fluorescence and phosphorescence properties of SiQDs@heme prepared in this application are shown in (a) fluorescence / phosphorescence spectra at different excitation wavelengths, (b) CIE chromaticity diagram, (c) fluorescence decay diagram of SiQDs@heme solution, and (d) phosphorescence lifetime of SiQDs@heme solid powder.

[0036] Figure 6 The oxidase-like activity of SiQDs@heme prepared in this application is shown in (a) TMB UV absorption spectrum, (b) stepwise behavior of SiQDs@heme photoinduced oxidation activity under continuous on / off light source, (c) Michaelis-Menten curve of SiQDs@heme catalytic activity with TMB as substrate, and (d) Lineweaver-Burk curve of SiQDs@heme catalytic activity with TMB as substrate.

[0037] Figure 7 Phosphorescence duration diagrams for SiQDs synthesized with different halogen elements;

[0038] Figure 8 The photoinduced enzyme activity and phosphorescence of SiQDs synthesized from metal salts and the heme protein family, wherein (a) shows the difference in photoinduced enzyme activity and (b) shows the phosphorescence phenomenon.

[0039] Figure 9 This application shows the effect of SiQDs@heme on detecting carmine, wherein (a) is the fluorescence emission spectrum of SiQDs at different carmine concentrations, (b) is the linear calibration diagram of the fluorescence emission of carmine, (c) is the phosphorescence emission spectrum of SiQDs at different carmine concentrations, and (d) is the linear calibration diagram of the phosphorescence emission of carmine.

[0040] Figure 10 The UV absorption spectrum of SiQDs@heme mixed with carmine and the fluorescence / phosphorescence emission spectrum of SiQDs@heme prepared in this application are shown.

[0041] Figure 11 This application describes the selective response capabilities of the SiQDs@heme obtained in this application, including (a) the response capability of the fluorescence properties of SiQDs@heme to metal and dye-like interfering substances, (b) the response capability of the phosphorescence properties of SiQDs@heme to metal and dye-like interfering substances, (c) the response capability of the fluorescence properties of SiQDs@heme to sugar and amino acid-like interfering substances, and (d) the response capability of the phosphorescence properties of SiQDs@heme to sugar and amino acid-like interfering substances.

[0042] Figure 12The following are the results of SiQDs@heme in detecting TPN: (a) shows the absorption spectrum of TMB of SiQDs@heme at different TPN concentrations; (b) shows the linear calibration diagram of absorbance for different TPN concentrations; (c) shows the selective response capability to TPN; and (d) shows the linear calibration diagram of TPN detection by gel colorimetric sensing.

[0043] Figure 13 The SiQDs@heme-based multi-information anti-counterfeiting encryption system includes the following content: (a) shows that the areas coated with SiQDs@heme ink and regular SiQDs ink exhibit blue fluorescence under ultraviolet light; (b) shows that the areas coated with SiQDs@heme ink exhibit yellow-green phosphorescence after the ultraviolet light is turned off; and (c) shows that the areas coated with SiQDs@heme ink exhibit blue fluorescence after being coated with the color developer TMB and irradiated with ultraviolet light for 1 minute. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0045] This application provides a method for preparing phosphorescent silicon dot nanozymes, comprising the following steps:

[0046] The first silicon source, iodide, heme and phosphorus source are mixed evenly and heated to react. After cooling, a solid product is obtained. The solid product is mixed evenly with water, centrifuged, dialyzed and freeze-dried to obtain phosphorescent silicon dot nanozyme, namely SiQDs@heme.

[0047] In some embodiments, the first silicon source is at least one of N-[3-(trimethoxysilyl)propyl]ethylenediamine, dimethyloctadecyl[3-trimethoxysilyl]ammonium chloride, and 3-(2-aminoethylamino)propylmethyldimethoxysilane.

[0048] In some embodiments, the iodide is selected from at least one of potassium iodide, sodium iodide, and sodium periodate. In some embodiments, the phosphorus source is selected from at least one of phosphoric acid, sodium dihydrogen phosphate, and sodium pyrophosphate.

[0049] In some embodiments, the mass ratio of the first silicon source, iodide, heme and phosphorus source is (100-150):(4-5):(0.1-0.5):(9-12).

[0050] In some embodiments, the heating temperature is 80℃~90℃, and the heating time is 2min~30min. It is understood that this application does not limit the heating method described above, and it can be, but is not limited to, microwave or hydrothermal methods. In some embodiments, a microwave method is used to prepare phosphorescent silicon dot nanozymes. This application does not limit the instruments used in the microwave method, the microwave treatment time, etc. Those skilled in the art can select appropriate instruments for processing according to the actual situation, such as, but not limited to, using a microwave oven (750W) for 6min.

[0051] In this application, the solid product and water are mixed uniformly to disperse the solid product in the water. Large particulate impurities are then removed by subsequent centrifugation. This application does not limit the mass ratio of the solid product to water, as long as the solid product is uniformly dispersed in the water. This application also does not limit the method of uniform mixing; it can be, but is not limited to, ultrasonic treatment. In some embodiments, dialysis with a 500 Da molecular weight cutoff bag is used to remove minute impurities, followed by freeze-drying to obtain a pale yellow powder, namely, phosphorescent silicon dot nanozyme.

[0052] This application also provides a phosphorescent silicon dot nanozyme prepared using the above-described preparation method.

[0053] This application also provides an aqueous phosphorescent silicon dot nanozyme comprising the above-mentioned phosphorescent silicon dot nanozyme.

[0054] In some embodiments, the preparation method of the above-mentioned aqueous phosphorescent silicon dot nanozyme includes the following steps:

[0055] The above-mentioned phosphorescent silicon dot nanozyme, second silicon source, ammonia and water are mixed evenly, and the reaction solution is subjected to hydrolysis, condensation and polymerization reactions in sequence to obtain aqueous phosphorescent silicon dot nanozyme, namely SiQDs@heme-TEOS.

[0056] In the above reaction process of this application, the second silicon source is hydrolyzed to generate silicon hydroxide, which then forms covalent bonds (Si-OC) and hydrogen bonds with the above phosphorescent silicon dot nanozyme, so that the phosphorescent silicon dot nanozyme is embedded in the silica network and the rigidity of the cross-linked framework is enhanced, thereby effectively suppressing non-radiative dissipation, preventing the negative effects of dissolved oxygen, and realizing aqueous phase phosphorescence.

[0057] In some embodiments, the second silicon source is selected from ethyl silicate or tetraethoxysilane.

[0058] In some embodiments, the volume ratio of the second silicon source, ammonia, and water is (0.5-1):(0.5-1):(9-18).

[0059] In some embodiments, the concentration of phosphorescent silicon dot nanozymes in the aqueous phase phosphorescent silicon dot nanozyme is 100 μg / mL to 300 μg / mL. It is understood that those skilled in the art can adjust the concentration of phosphorescent silicon dot nanozymes in the aqueous phase according to actual needs, all within the scope of protection of this application.

[0060] The phosphorescent silicon dot nanozymes and aqueous phosphorescent silicon dot nanozymes prepared in this application can be used to detect carmine. The phosphorescent silicon dot nanozymes prepared in this application exhibit blue fluorescence, and the fluorescence emission intensity is quenched proportionally to the concentration of carmine. The specific steps for detecting carmine in a sample based on phosphorescent silicon dot nanozymes are as follows:

[0061] S1. Mix the above-mentioned SiQDs@heme aqueous solution, PBS solution, and carmine standard solutions of different concentrations, wherein the concentration of the carmine standard solution is greater than or equal to 0. Measure the fluorescence intensity of the mixture at the emission wavelength of 446 nm under the excitation wavelength of 370 nm. The fluorescence intensity of the mixture when the concentration of the carmine standard solution is 0 is F0, and the fluorescence intensity of the mixture of carmine standard solutions of different concentrations is F. Calculate the fluorescence intensity difference (Δfluorescence intensity) = F0 - F.

[0062] S2. Using carmine concentration as the independent variable and Δfluorescence intensity F0-F as the dependent variable, analyze and obtain the relationship between carmine concentration and Δfluorescence intensity F0-F;

[0063] S3. Extract carmine from the sample to be tested, then mix it evenly with the above-mentioned aqueous solution of carbon nanozyme and PBS, measure the fluorescence intensity Fn at the emission wavelength of 446nm under the excitation wavelength of 370nm, and calculate the fluorescence intensity difference F0-Fn.

[0064] S4. Substitute the fluorescence intensity difference F0-Fn of the sample to be tested into the relationship in S2 to obtain the carmine concentration of the sample to be tested.

[0065] In some embodiments, the detection limit of the above-mentioned phosphorescent silicon nanozyme for carmine is 0.33 μM.

[0066] The phosphorescent silicon dot nanozyme prepared in this application exhibits yellow-green phosphorescence, and the phosphorescence emission intensity is quenched proportionally to the carmine concentration. The specific steps for detecting carmine in the sample based on the aqueous phosphorescent silicon dot nanozyme are as follows:

[0067] S1. Mix the above SiQDs@heme-TEOS with PBS solution and carmine standard solutions of different concentrations, wherein the concentration of the carmine standard solution is greater than or equal to 0. Measure the phosphorescence intensity of the mixture at the emission wavelength of 530nm under the excitation wavelength of 350nm. The phosphorescence intensity of the mixture when the concentration of the carmine standard solution is 0 is P0, and the phosphorescence intensity of the mixture of carmine standard solutions of different concentrations is P. Calculate the fluorescence intensity difference (Δphosphorescence intensity) = P0 - P.

[0068] S2. Using carmine concentration as the independent variable and Δphosphorescence intensity P0-P as the dependent variable, analyze and obtain the relationship between carmine concentration and Δphosphorescence intensity P0-P.

[0069] S3. Extract carmine from the sample to be tested, then mix it evenly with the above SiQDs@heme-TEOS and PBS, measure the phosphorescence intensity Pn at the emission wavelength of 530nm under the excitation wavelength of 350nm, and calculate the phosphorescence intensity difference P0-Pn.

[0070] S4. Substitute the phosphorescence intensity difference P0-Pn of the sample to be tested into the relationship in S2 to obtain the carmine concentration of the sample to be tested.

[0071] In some embodiments, the detection limit of the above-mentioned aqueous phosphorescent silicon nanoparticle nanozyme for carmine is 51 nM.

[0072] In some embodiments, the test sample or sample may be, but is not limited to, food samples, cosmetics, pharmaceuticals, or combinations thereof.

[0073] It is understood that this application does not limit the methods of analysis described above, and may include, but is not limited to, linear analysis and regression analysis.

[0074] This application utilizes the fluorescence / phosphorescence dual-mode detection platform provided by SiQDs@heme and SiQDs@heme-TEOS to achieve sensitive detection of carmine, and has a mutual verification function, which can effectively avoid or reduce interference triggered by non-target factors, and has high detection accuracy in complex real samples.

[0075] This application also provides a hydrogel containing the above-mentioned phosphorescent silicon dot nanozyme.

[0076] In some embodiments, the preparation method of the above-mentioned hydrogel includes the following steps: after stirring and mixing polyvinyl alcohol and water, the above-mentioned phosphorescent silicon nanozyme, acrylamide, N,N'-methylenebisacrylamide and ammonium persulfate are added and mixed evenly to obtain the hydrogel.

[0077] In some embodiments, the mass ratio of the above-mentioned phosphorescent silicon nanoparticles, polyvinyl alcohol, acrylamide, N,N'-methylenebisacrylamide and ammonium persulfate is (1-2):(0.5-1):(2-3):(0.005-0.1):(0.05-0.1).

[0078] It is understood that those skilled in the art can choose other raw materials to replace the above-mentioned polyvinyl alcohol, acrylamide, N,N'-methylenebisacrylamide and ammonium persulfate to prepare hydrogels containing the above-mentioned phosphorescent silicon dot nanoenzymes, all of which are within the scope of protection of this application.

[0079] This application does not limit the size of the hydrogel spheres or the thickness of the hydrogel film. Those skilled in the art can choose the appropriate size and thickness according to the needs of the actual application scenario.

[0080] The phosphorescent silica dot nanozyme and the hydrogel loaded with the phosphorescent silica dot nanozyme prepared in this application both exhibit photoinduced oxidase activity and can be used to detect thiopronine. The specific steps for detecting thiopronine in the sample based on the phosphorescent silica dot nanozyme are as follows:

[0081] S1. Mix the above-mentioned SiQDs@heme aqueous solution with the chromogenic substrate solution, PBS solution, and TPN standard solutions of different concentrations. Measure the absorbance of the mixture at 652 nm. The absorbance of the mixture before adding TPN solution is A0, and the absorbance of the mixture after adding different concentrations of TPN standard solutions is A. TPN Calculate the absorbance difference (Δabsorbance) = A0 - A TPN ;

[0082] S2. Using TPN concentration as the independent variable, Δabsorbance A0-A TPN Using TPN concentration and Δabsorbance A0-A as the dependent variable, the analysis yielded the dependent variable. TPN Relationship;

[0083] S3. Extract the TPN from the sample to be tested, and then mix it with the above-mentioned aqueous solution of SiQDs@heme, chromogenic substrate solution and PBS. Measure the absorbance An of the mixture at 652nm and calculate the absorbance difference A0-An.

[0084] S4. Substitute the absorbance difference A0-An of the sample to be tested into the relationship in S2 to obtain the TPN concentration of the sample to be tested.

[0085] In some embodiments, the lower limit for TPN detection by the above-mentioned phosphorescent silicon nanozyme is 9.8 nM.

[0086] The specific steps for detecting thiopronine in the sample based on the above-mentioned hydrogel loaded with phosphorescent silicon dot nanozymes are as follows:

[0087] S1. Place the above hydrogel in a chromogenic substrate solution and TPN standard solutions of different concentrations, irradiate with ultraviolet light, and then observe the color change of the hydrogel. Use the corresponding CIE plugin Convert to perform RGB color sampling on the gels under different TPN concentrations, and convert the blanks to L* / a* / b* values.

[0088] S2. Using TPN concentration as the independent variable and L* value as the dependent variable, analyze and obtain the relationship between TPN concentration and L* value.

[0089] S3. Extract the TPN from the sample to be tested, repeat step S1, and obtain the L* value corresponding to the TPN concentration of the sample to be tested.

[0090] S4. Substitute the L* value of the sample to be tested into the relationship in S2 to obtain the TPN concentration of the sample to be tested.

[0091] This application also provides the application of the above-mentioned phosphorescent silicon dot nanozyme in the preparation of fluorescent, phosphorescent and enzyme-catalyzed color-changing triple anti-counterfeiting labels.

[0092] The above technical solutions are described in detail below with reference to specific embodiments. It should be understood that these are merely exemplary and not intended to limit this application. Materials of the same or similar type, model, quality, properties, or function as the reagents and instruments described below can be used to implement this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0093] The following is an example:

[0094] Example 1

[0095] Preparation of phosphorescent silicon dot nanozymes: 0.1 mL of N-[3-(trimethoxysilyl)propyl]ethylenediamine (DAMO) was dissolved in 5 mL of KI aqueous solution (KI concentration: 5 mM), and 0.147 mg of heme was added. Then, the mixture was stirred and 0.1 mL of phosphate solution (concentration: 1.02 mol / L) was added dropwise. The resulting transparent aqueous solution was transferred to a 50 mL beaker and heated in a microwave oven (750 W) for 6 min (heating temperature: 80–90 °C). After the reactor cooled naturally to room temperature, the obtained gel-like solid was dissolved in 30 mL of deionized water and sonicated for 10 min. The crude aqueous solution was centrifuged at 10,000 rpm for 10 min, and large particles or agglomerates were filtered through a 0.22 μm filter membrane. The collected solution was dialyzed (MWCO: 500 Da) for 24 h (with water changed every 6 h), and freeze-dried for 48 h to obtain a pale yellow powder, i.e., phosphorescent silicon dot nanozymes (SiQDs@heme).

[0096] To study the phosphorescence properties of the aforementioned phosphorescent silicon dot nanozymes, an aqueous phosphorescent silicon dot nanozyme was prepared. The specific method is as follows: 1.5 mg SiQDs@heme, 0.5 mL tetraethyl orthosilicate (TEOS), 0.5 mL NH4OH, and 14 mL water were mixed thoroughly to obtain 15 mL of a 100 μg / mL phosphorescent silicon dot nanozyme solution. This solution was then stirred at room temperature for 16 h to sequentially undergo hydrolysis, condensation, and polymerization reactions. The solution was then filtered multiple times using a 0.22 μm filter membrane, and the resulting transparent solution was collected, which is the aqueous phosphorescent silicon dot nanozyme (SiQDs@heme-TEOS). Specifically, in the initial stage of the reaction, TEOS hydrolyzes to generate silicon hydroxide, which then forms covalent bonds (Si-OC) and hydrogen bonds with SiQDs@heme. This allows SiQDs@heme to embed within the silica network, enhancing the rigidity of the cross-linked framework. This effectively suppresses non-radiative dissipation, prevents the negative effects of dissolved oxygen, and achieves aqueous phosphorescence.

[0097] Example 2

[0098] 2.1 Morphology and chemical structure of SiQDs@heme

[0099] The morphology and structure of SiQDs@heme were observed using transmission electron microscopy (TEM). Figure 1 Content (a), Figure 1 As shown in section (b), the SiQDs@heme are spherical and uniformly dispersed, with a particle size ranging from 4.29 to 9.92 nm and an average particle size of 7.38 nm. Their lattice spacing is 0.17 nm, corresponding to the (004) lattice plane of graphite 2H. The surface potential of SiQDs@heme is +6.29 mV, which is analyzed to be related to its particle surface structure.

[0100] The surface structure, elemental composition, and chemical composition of SiQDs@heme were detected using FT-IR and XPS. Figure 2 As shown in content (a), the FT-IR spectrum is displayed at 1075.55 cm⁻¹. -1 1469.43cm -1 and 527.48cm -1 The characteristic absorptions of CO / PO / Si-O-Si / Si-OC, heme, and CI are shown respectively. For example... Figure 2As shown in content (b), the XPS spectra indicate that 284.8 eV, 531.51 eV, 400.93 eV, 102.01 eV, 132.83 eV, 726.26 eV, and 618.24 eV correspond to C1s, O 1s, N1s, Si 2p, P 2p, Fe 2p, and I 3d, respectively, with corresponding contents of 41.16%, 33.16%, 10.89%, 5.84%, 6.95%, 0.32%, and 1.69%.

[0101] Further analysis of the XPS images of SiQDs@heme was conducted, such as... Figure 3 As shown in content (a), C 1s exhibits two characteristic peaks, CC and CO, at 284.48 eV and 285.95 eV; as Figure 3 As shown in content (b), N 1s exhibits two characteristic peaks, CNC, NN, and CN, at 398.88 eV, 400.20 eV, and 401.01 eV; Figure 3 As shown in content (c), the O 1s peaks at 530.24 eV and 531.79 eV are characteristic peaks of C=O; Figure 3 As shown in content (d), P 2p exhibits two characteristic peaks at 132.57 eV and 133.49 eV, representing PO and PC / PN, respectively; as... Figure 3 As shown in section (e), Si 2p exhibits two characteristic peaks at 101.90 eV and 102.62 eV: C-Si-O and Si-O. Figure 3 As shown in content (f), I3d exhibits I2 at 618.13 eV and 618.33 eV. + and I - Two characteristic peaks.

[0102] 2.2 Fluorescence and phosphorescence properties of SiQDs@heme

[0103] 1) The SiQDs@heme was dispersed and dissolved in deionized water to obtain an aqueous solution of SiQDs@heme. The absorbance at different wavelengths was then measured in the detection cell of a UV-Vis spectrophotometer to detect the fluorescence performance of SiQDs@heme. The SiQDs@heme-TEOS was then placed in the detection cell of a UV-Vis spectrophotometer to detect the absorbance at different wavelengths to detect the phosphorescence performance of SiQDs@heme.

[0104] like Figure 4 As shown in content (a), SiQDs@heme exhibits a major absorption peak at 341 nm, while SiQDs@heme-TEOS exhibits a major absorption peak in the range of 258–285 nm.

[0105] 2) Place the SiQDs@heme aqueous solution and SiQDs@heme-TEOS in the detection cell of a fluorescence spectrophotometer to measure the fluorescence intensity and phosphorescence intensity at the optimal emission wavelength, the changes in fluorescence intensity and phosphorescence intensity at different emission wavelengths, and plot the CIE colorimetric diagram.

[0106] like Figure 4 As shown in content (b), the maximum emission wavelength of SiQDs@heme aqueous solution at the optimal excitation wavelength of 370nm is 446nm (black line, fluorescence spectrum), and it exhibits bright blue fluorescence under 365nm ultraviolet light irradiation (the left inset shows the fluorescent and fluorescent images of SiQDs@heme aqueous solution, respectively); the maximum emission wavelength of SiQDs@heme-TEOS at the optimal excitation wavelength of 350nm is 530nm (red line, phosphorescence spectrum), and it exhibits yellow-green phosphorescence after irradiation with 365nm ultraviolet light (the right inset shows the fluorescent, fluorescent, and phosphorescent images of SiQDs@heme-TEOS, respectively). Figure 5 As shown in content (a), with increasing excitation wavelength, both fluorescence emission wavelength and phosphorescence emission wavelength exhibit a redshift, and the emission intensity gradually decreases. Figure 5 As shown in content (b), the fluorescence coordinates of the SiQDs@heme aqueous solution are (0.178, 0.266), and the phosphorescence coordinates of SiQDs@heme-TEOS are (0.172, 0.209).

[0107] 3) Detection of fluorescence lifetime, fluorescence quantum yield, phosphorescence lifetime, and phosphorescence quantum yield.

[0108] The fluorescence intensity of SiQDs@heme aqueous solution was tested over time at excitation / emission wavelengths of 370 nm and 446 nm, respectively, and a fluorescence decay plot was plotted. Figure 5 Content (c), fluorescence lifetime is 6 ns.

[0109] Using quinine sulfate (dissolved in 0.1 M H2SO4) as a reference, the fluorescence quantum yield of SiQDs@heme (dissolved in deionized water) was calculated according to equation (1):

[0110]

[0111] In this context, the subscripts "sam" and "ref" refer to the sample and standard substance, respectively. and φ ref The values ​​are QY for sample QY and quinine sulfate (QY is 54.6%), respectively; A is the absorbance value; I is the integral value of the emission intensity at the same excitation wavelength (Ex is 370 nm); n is the refractive index (n sam The value is 1.33, n refThe fluorescence quantum yield of SiQDs@heme is calculated to be 7.59%. The UV absorption intensity remains below 0.1.

[0112] The phosphorescence intensity of SiQDs@heme at excitation / emission wavelengths of 350 nm and 530 nm, respectively, was measured over time, and a phosphorescence decay diagram was plotted. Figure 5 Content (d), calculated, shows that the phosphorescence quantum yield of SiQDs@heme is 5.51%. The phosphorescence intensity variation data were fitted using the multi-exponential decay function of equation (2), and then the weighted average lifetime of the phosphorescence intensity was calculated according to equation (3).

[0113] <τ>=∑a i τ i Equation (3)

[0114] Where Ai is the total amplitude, ai is the amplitude fraction, A is the amplitude, and τi is the time constant. The calculated phosphorescence lifetime of SiQDs@heme is 126 ms.

[0115] 2.3 UV-induced oxidase activity of SiQDs@heme

[0116] The enzyme activity of SiQDs@heme was initially evaluated using TMB as a chromogenic substrate. 20 μL of a 2 mg / mL SiQDs@heme aqueous solution, 50 μL of a 3 mM TMB solution, and 430 μL of a 20 mM PBS solution (pH 4) were added to a cuvette and mixed thoroughly. The cuvette was then irradiated under a 365 nm UV lamp for 10 min (30 s on / off alternating). The photoinduced oxidase-like activity of SiQDs@heme was determined by measuring the absorbance at 652 nm.

[0117] like Figure 6 Content (a) shows that under 365 nm UV irradiation, SiQDs@heme catalyzes the oxidation of colorless TMB to a blue product (oxTMB), with a strong peak at 652 nm, indicating that SiQDs@heme possesses oxidase-like activity. However, without UV irradiation, the mixed solution of SiQDs@heme and TMB remains colorless, indicating that the oxidase-like activity of SiQDs@heme is induced by UV radiation. When the UV lamp is repeatedly turned on and off, as... Figure 6 Content (b) shows that the oxidase-like activity of SiQDs@heme increases in a stepwise manner, indicating that SiQDs@heme has optically controlled oxidase-like activity.

[0118] The photo-controlled oxidase activity of SiQDs@heme was further investigated using steady-state kinetic analysis. Specifically, 20 μL of a 2 mg / mL SiQDs@heme aqueous solution, 430 μL of a 20 mM PBS solution (pH 4), and 50 μL of a 0.05–0.5 mM TMB solution were mixed. The absorbance was recorded at 652 nm at 30-second intervals using a UV spectrophotometer. Michaelis-Menten curves were obtained by plotting the initial reaction rate of SiQDs@heme and the TMB concentration. The Michaelis-Menten curve data were then processed using a double reciprocal method to obtain a Lineweaver-Burk plot.

[0119] like Figure 6 Content (c) Figure 6 As shown in content (d), SiQDs@heme's V max The maximum reaction rate reached 3.4 × 10⁻⁶. -9 Ms -1 K m The Michaelis constant is 0.29 mM. Compared to some natural and artificial enzymes, SiQDs@heme has a higher Km. m Lower and V max The larger value indicates that SiQDs@heme, as an oxidase mimic, has a higher affinity and catalytic activity for TMB.

[0120] Example 3

[0121] Following the method provided in Example 1, 0.15 mL of 3-(2-aminoethylamino)propylmethyldimethoxysilane was dissolved in 6 mL of NaI solution (NaI concentration: 5 mM), and 0.5 mg of heme was added. Then, the mixture was stirred and 0.75 mL of sodium dihydrogen phosphate solution (concentration: 0.1 mol / L) was added dropwise. The resulting transparent aqueous solution was transferred to a 50 mL beaker and heated in a microwave oven (750 W) for 15 min (heating temperature: 80–90 °C). After centrifugation, dialysis, and freeze-drying, solid sample 1 was obtained.

[0122] 0.13 g of 3-(2-aminoethylamino)propylmethyldimethoxysilane was dissolved in 4.5 mL of sodium periodate solution (5 mM), and 0.3 mg of heme was added. Then, the mixture was stirred and 12 mg of sodium pyrophosphate was added. The resulting transparent aqueous solution was transferred to a 50 mL beaker and heated in a microwave oven (750 W) for 10 min (heating temperature 80–90 °C). After centrifugation, dialysis, and freeze-drying, solid sample 2 was obtained.

[0123] The samples were tested according to the method in Example 1. Experiments showed that the aqueous solutions of the solid samples all exhibited blue fluorescence under 365 nm ultraviolet light irradiation, and emitted yellow-green phosphorescence after being irradiated for a period of time and then the ultraviolet lamp was turned off. Furthermore, the samples could not catalyze the oxidation of colorless TMB to a blue product without ultraviolet light irradiation, but could catalyze the oxidation of colorless TMB to a blue product under 365 nm ultraviolet light irradiation, indicating that the oxidase activity of the solid samples was induced by ultraviolet light. In addition, the oxidase-like activity of the samples was comparable to that of the phosphorescent silicon dot nanozyme prepared in Example 1.

[0124] Following the method provided in Example 1, 1.0 mg of solid sample 1, 0.5 mL of ethyl silicate, 0.5 mL of NH4OH and 9 mL of water were mixed evenly to prepare 10 mL of solid sample 1 solution with a concentration of 100 μg / mL. The solution was placed at room temperature and stirred for 16 h to carry out hydrolysis, condensation and polymerization reactions in sequence. Then, it was filtered multiple times using a 0.22 μm filter membrane to collect transparent solution 1.

[0125] 6.0 mg of solid sample 2, 1 mL of tetraethoxysilane, 1 mL of NH4OH and 18 mL of water were mixed evenly to prepare 20 mL of solid sample 2 solution with a concentration of 300 μg / mL. The solution was placed at room temperature and stirred for 16 h to carry out hydrolysis, condensation and polymerization reactions in sequence. Then, it was filtered multiple times using a 0.22 μm filter membrane to collect transparent solution 2.

[0126] Tests showed that both transparent solution 1 and transparent solution 2 exhibited yellow-green phosphorescence, achieving aqueous phase phosphorescence.

[0127] Comparative Example 1

[0128] Phosphorescent silicon dot nanozymes were prepared by replacing KI with NaF, NaCl, and KBr, respectively, with the remaining operations the same as in Example 1. The prepared phosphorescent silicon dot nanozymes doped with different halogen atoms were arranged sequentially on black cardstock. In a dark environment, a portable ultraviolet lamp at a wavelength of 365 nm provided the excitation light source. After irradiation for 2 minutes, the phosphorescence emission duration of the prepared phosphorescent silicon dot nanozymes was recorded by video using a mobile phone. Subsequently, the recorded video was imported into Adobe Premiere Pro to capture phosphorescence images at different time points.

[0129] Depend on Figure 7 It can be seen that different halides have a significant impact on the room temperature phosphorescence performance of phosphorescent silicon dot nanozymes. Among them, the phosphorescence duration of SiQDs@heme prepared with KI as a halogen raw material is significantly better than that of NaF, NaCl, and KBr, and the phosphorescence duration is effectively extended.

[0130] Comparative Example 2

[0131] Phosphorescent silicon dot nanozymes were prepared by replacing heme with MnCl2, FeCl2, CoCl2, GdCl3, and HB, respectively, with the remaining operations being the same as in Example 1. The effects of changing the type of dopant element on the photoinduced oxidase activity and phosphorescence (RTP) of the phosphorescent silicon dot nanozymes were analyzed.

[0132] like Figure 8 As shown in (a), compared with SiQDs, the photoinduced oxidase activity of phosphorescent silicon dot nanozymes synthesized from HB, MnCl2, FeCl2, and GdCl2 was reduced. The phosphorescent silicon dot nanozymes prepared above were irradiated with a 365nm wavelength ultraviolet lamp for 2 min, and the changes before and after ultraviolet lamp irradiation were observed. Figure 8 As shown in section (b), compared with SiQDs, the phosphorescent silicon dot nanozymes synthesized from CoCl2, MnCl2, FeCl2, and GdCl3 do not exhibit RTP performance. In summary, the SiQDs@heme synthesized from heme exhibits the highest photoinduced oxidase activity and excellent RTP performance.

[0133] Example 4: Fluorescence / phosphorescence dual-mode detection system for carmine based on SiQDs@heme

[0134] 4.1 Relationship between fluorescence intensity of SiQDs@heme and carmine concentration

[0135] SiQDs@heme exhibits blue fluorescence, with an excitation wavelength / emission wavelength ratio (λ). Ex / λ Em The fluorescence emission spectra were measured at an excitation wavelength of 370 nm and an excitation wavelength of 446 nm. 40 μL of 2 mg / mL SiQDs@heme aqueous solution, 860 μL of PBS solution (pH 4) and 100 μL of carmine standard solutions of different concentrations were mixed. The fluorescence emission spectra were measured at an excitation wavelength of 370 nm and the fluorescence intensity at the emission wavelength of 446 nm was recorded. A linear relationship between fluorescence intensity and carmine concentration was constructed.

[0136] like Figure 9 As shown in content (a), when SiQDs@heme reacts with carmine, the fluorescence emission intensity of SiQDs@heme is quenched in a manner proportional to the concentration of carmine. Figure 9Content (b) shows the curve of the difference in fluorescence intensity at 446 nm emission wavelength under 370 nm excitation before and after the addition of carmine, as a function of carmine concentration. The fluorescence intensity of the mixture when the concentration of the carmine standard solution is 0 is denoted as F0, and the fluorescence intensity of the mixture of carmine standard solutions of different concentrations is denoted as F. It can be seen that when the carmine concentration is 0 μM to 55.6 μM, the difference in fluorescence intensity at 446 nm emission wavelength (Δfluorescence intensity = F0 - F) has a linear relationship with the carmine concentration, with a regression equation of y = 197.85x + 1512 and a correlation coefficient (R²). 2 The value of 0.9919 is given by x, where x is the concentration of carmine and y is the difference in fluorescence intensity at 446 nm before and after the addition of carmine (Δ fluorescence intensity). The detection limit (LOD) is 0.33 μM.

[0137] 4.2 Relationship between phosphorescence intensity and carmine concentration of SiQDs@heme-TEOS

[0138] SiQDs@heme-TEOS exhibits yellow-green phosphorescence, with an excitation wavelength / emission wavelength ratio (λ). Ex / λ Em The phosphorescence emission spectra were measured at 350 nm and 530 nm. 40 μL of 2 mg / mL SiQDs@heme-TEOS solution, 860 μL of PBS (pH 4) solution, and 100 μL of carmine standard solutions of different concentrations were mixed. The phosphorescence emission spectra were measured at 350 nm, and the phosphorescence intensity at the emission wavelength of 530 nm was recorded. A linear relationship between phosphorescence intensity and carmine concentration was established.

[0139] like Figure 9 As shown in content (c), when SiQDs@heme-TEOS is mixed with carmine standard solutions of different concentrations (0 μM to 34.8 μM), the phosphorescence emission intensity of SiQDs@heme-TEOS is proportionally quenched as the concentration of the carmine standard solution increases. Figure 9 Content (d) shows the curve of the difference in phosphorescence intensity at 530 nm emission wavelength under 350 nm excitation before and after the addition of carmine, as a function of carmine concentration. It can be seen that the difference in phosphorescence intensity at 530 nm emission wavelength (Δphosphorescence intensity) has a linear relationship with carmine concentration, with a regression equation of y = 123.79x + 830.01 and a correlation coefficient (R²). 2 The value is 0.9939, where x is the concentration of carmine, y is the difference in phosphorescence intensity at 530 nm before and after the addition of carmine (Δphosphorescence intensity), and the detection limit (LOD) is 51 nM.

[0140] The mechanism by which carmine quenches SiQDs@heme is likely the internal filtering effect (IFE). Carmine exhibits a broad UV-Vis absorption spectrum with a broad absorption peak (red line) in the 370–580 nm range. When the absorption peak of the absorber (quencher carmine) in the fluorescence / phosphorescence detection system overlaps with the fluorescence emission peak (446 nm, PL) and phosphorescence emission peak (530 nm, phos.) of SiQDs@heme, the IFE strategy converts the absorption signal into a significantly quenched fluorescence / phosphorescence signal. Figure 10 ).

[0141] 4.3 Selectivity of Fluorescence / Phosphorescence Detection Systems

[0142] The study investigated interfering substances including sugars (lactose, glucose), amino acids (L-lysine, L-glutamine, L-serine, glycine, D-malic acid, glutathione, cysteine), and metal ions (Fe). 2+ Al 3+ Ag + Pb 2+ Cu 2+ Zn 2+ Mn 2+ Mg 2+ Fe 3+ The fluorescence / phosphorescence signals in the presence of methyl red and dyes (methyl red, safranin, Congo red, neutral red) were used to evaluate the selectivity of the fluorescence / phosphorescence detection system for carmine.

[0143] Take 40 μL of 2 mg / mL SiQDs@heme aqueous solution, 860 μL of PBS (pH 4) solution, and 40 μL of different interfering substances with a concentration of 30 μM and mix them thoroughly. Record the fluorescence intensity of the mixed solution at an excitation wavelength of 370 nm and an emission wavelength of 446 nm before and after the addition of the interfering substances. F0 represents the initial fluorescence intensity without the addition of any interfering substances, and F is the fluorescence intensity measured after the addition of different interfering substances.

[0144] Take 40 μL of SiQDs@heme-TEOS solution with a concentration of 2 mg / mL, 860 μL of PBS solution (pH 4), and 40 μL of aqueous solutions of different interfering substances with a concentration of 30 μM. Mix them thoroughly and record the phosphorescence intensity of the mixed solution at an excitation wavelength of 350 nm and an emission wavelength of 530 nm before and after the addition of the interfering substances. Here, P0 represents the initial phosphorescence intensity without the addition of any interfering substances, and P is the phosphorescence intensity measured after the addition of different interfering substances.

[0145] like Figure 11 Content (a), Figure 11 Content (b) Figure 11 Content (c) Figure 11As shown in content (d), compared with the above-mentioned interfering substances, the fluorescence / phosphorescence detection system has the largest difference in absorbance value for carmine, and has a high responsiveness to carmine, which can be effectively applied to the detection of carmine.

[0146] In summary, the fluorescence / phosphorescence dual-mode detection platform provided by SiQDs@heme can achieve sensitive detection of carmine and has the function of mutual verification, which can effectively avoid or reduce interference triggered by non-target factors and has high detection accuracy in complex real samples.

[0147] Example 5

[0148] 5.1 TPN colorimetric sensor based on SiQDs@heme photoinduced oxidase activity

[0149] 40 μL of 2 mg / mL SiQDs@heme aqueous solution, 100 μL of 3 mM TMB chromogenic substrate solution, 760 μL of PBS (pH 4) solution, and 100 μL of TPN solutions of different concentrations were mixed and the UV absorption was measured at wavelengths of 400–800 nm. The characteristic absorption of TMB was at a wavelength of 652 nm.

[0150] Depend on Figure 12 As shown in content (a), with the increase of TPN concentration, the absorbance of the mixed solution at 652 nm gradually decreases, from... Figure 12 As shown in section (b), the color of the mixed solution gradually lightens with increasing TPN concentration (the inset shows the corresponding solution color), indicating that the interaction between TPN and SiQDs@heme inhibits the UV-induced oxidase-like activity of SiQDs@heme. The reason for this may be that TPN, as a thiol compound, has a strong binding affinity to the metal ions doped on the surface of SiQDs@heme, inhibiting the catalytically active sites of SiQDs@heme, thereby reducing the generation of the blue product oxTMB. This results in a reduced or no color change in TMB, and the reducing properties of TPN may also restore oxTMB to its colorless state.

[0151] Figure 12 Content (b) shows the curve of the absorbance difference before and after the addition of TPN as a function of TPN concentration, where the absorbance of the mixture before the addition of TPN solution is A0, and the absorbance of the mixture after the addition of TPN standard solutions of different concentrations is A0. TPN It can be seen that when the TPN concentration is between 0.015 μM and 25 μM, the absorbance difference (Δabsorbance = A0 - A) is significant. TPN There is a linear relationship between γ and TPN concentration, with the equation y = 0.0216x + 0.0714 and a correlation coefficient (R²). 2The value is 0.9913, where x is the TPN concentration and y is the difference in absorbance at 652nm wavelength before and after the addition of TPN (Δabsorbance = A0 - A). TPN The detection limit (LOD) of TPN is 9.8 nM.

[0152] 5.2 Selectivity of TPN colorimetric sensors

[0153] Following the method in 5.1, 100 μL of a 10 μM interfering substance, SiQDs@heme aqueous solution, TMB solution, and PBS solution were mixed. The ratio of the absorbance difference of the mixed solution at 652 nm before and after the addition of the interfering substance (ΔA / A0=(A0-A)) was measured. 干扰物质 The selectivity of the TPN colorimetric sensor to TPN was evaluated, with interfering substances including sugars (lactose, glucose, sucrose), amino acids (L-lysine, L-glutamine, L-serine, glycine, D-malic acid), and metal ions (Fe). 2+ Al 3+ Cr 3+ Pb 2+ Hg 2+ Mg 2+ Fe 3+ ).

[0154] Depend on Figure 12 As can be seen from content (c), different metal ions and most biochemical substances have no effect on the colorimetric detection of TPN, and the TPN colorimetric sensor has excellent selective response to TPN.

[0155] 5.3 Performance of TPN colorimetric sensor in detecting TPN concentration of actual drugs

[0156] 40 μL of 2 mg / mL SiQDs@heme aqueous solution, 100 μL of 3 mM TMB solution, 760 μL of PBS (pH 4) solution, and 0.1 g / tablet of enteric-coated TPN tablets were mixed, with final TPN concentrations of 0.05, 10, and 25 μM in the mixture. The absorbance difference (A0-A10) of the mixed solution at 652 nm was measured. TPN肠溶片 Substituting these values ​​into the equation in section 5.1 above, the TPN detection concentration is obtained, and the recovery rate is calculated as (TPN detection concentration / TPN addition concentration) × 100%. The results of detecting the actual drug TPN concentration using the TPN colorimetric sensor based on SiQDs@heme photoinduced oxidase activity are shown in Table 1.

[0157] Table 1. Recovery of TPN from TPN Enteric-coated Tablets

[0158] TPN concentration (μM) TPN detection concentration (μM) Recovery rate (%) RSD (%) 0.05 0.053 105.82 0.8 10 10.92 109.21 2.0 25 24.96 99.85 1.1

[0159] In Table 1, the recovery rate of TPN enteric-coated tablets was 99.85%–109.21%, and the relative standard deviation (RSD) was less than or equal to 2.0%, indicating that the TPN colorimetric sensor has good recovery rate and reproducibility, and is highly applicable to the detection of biological samples.

[0160] 5.4 TPN Smart Gel Colorimetric Sensor Based on SiQDs@heme Photoinduced Oxidase Activity

[0161] 0.5 g of polyvinyl alcohol (PVA) was added to 10 mL of water and dissolved by thorough stirring in an 80 °C water bath. Then, 2.5 g of acrylamide (AM), 5 mg of N,N'-methylenebisacrylamide (MBA), and 75 mg of ammonium persulfate (APS) were added. The mixture was sonicated for 1 min to remove air bubbles generated during stirring. Finally, 400 μL of a 2 mg / mL SiQDs@heme aqueous solution was added to obtain a gel solution. Using a 96-well plate as a mold, 80 μL of the gel solution was added to each well. The plate was irradiated with a 365 nm UV lamp (4 V, 52 W) at room temperature for 1 h to obtain the PVMA hydrogel, which is the TPN smart gel colorimetric sensor.

[0162] During the testing process, the gels were placed in TPN solutions of different concentrations and TMB solutions of a fixed concentration (3mM) for 3 minutes, and then irradiated with ultraviolet light for 1 minute. The color change of the gels was observed, and the corresponding CIE plugin Convert was used to perform RGB color sampling on the gels under different TPN concentrations. After subtracting the blank, the values ​​were converted to L* / a* / b* values.

[0163] like Figure 12 As shown in section (d), the gel color gradually faded with increasing TPN concentration (inset below). Analysis revealed that only the L* value (y) showed a good linear relationship with TPN concentration (x), with the linear equation y = -0.098x + 3.1022 and a correlation coefficient (R²). 2 The value is 0.9969.

[0164] Example 6: A Multi-layered Information Anti-counterfeiting Encryption System Based on SiQDs@heme

[0165] Ink 1 and Ink 2 were prepared by dissolving equal masses of SiQDs@heme and conventional SiQDs (with only blue fluorescence and no yellow-green phosphorescence) in water. Ink 1 was applied to cover the area of ​​the number "666", and Ink 2 was applied to the remaining area. Figure 13 As shown in content (a), under 365nm ultraviolet light irradiation, both ink 1 and ink 2 areas emit blue fluorescence, displaying the letter "888". After the ultraviolet light is turned off, the area coated with ink 1 emits yellow-green phosphorescence, displaying the code "666". Figure 13(As shown in content (b)). Next, TMB (pH=3) was used to prepare a color developer, which was then applied to the areas coated with ink 1 and ink 2. After irradiation with a UV lamp for 1 minute, the light was turned off. Due to the photoinduced oxidase activity of SiQDs@heme, the area coated with ink 1 showed a visible blue code "666" (see content (b)). Figure 13 (See content (c)).

[0166] It can be seen that the SiQDs@heme prepared in this application can be used as a high-quality anti-counterfeiting ink with advanced encryption. By combining fluorescence properties, matrix-free RTP properties, and photoinduced oxidase activity, a multi-mode anti-counterfeiting encryption system has been successfully constructed, showing application potential in information anti-counterfeiting encryption. Compared with traditional anti-counterfeiting technologies, the multi-mode information encryption technology of SiQDs@heme provided in this application, including fluorescence, RTP, and color development, can achieve a higher level of anti-counterfeiting information security.

[0167] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing phosphorescent silicon dot nanozymes, characterized in that, Includes the following steps: The first silicon source, iodide, heme and phosphorus source are mixed evenly and microwave heated for 2 min to 30 min. After cooling, a solid product is obtained. The solid product is mixed evenly with water, centrifuged, dialyzed and freeze-dried to obtain phosphorescent silicon dot nanozyme. The first silicon source is at least one of N-[3-(trimethoxysilyl)propyl]ethylenediamine, dimethyloctadecyl[3-trimethoxysilyl]ammonium chloride, and 3-(2-aminoethylamino)propylmethyldimethoxysilane; The iodide is selected from at least one of potassium iodide and sodium iodide; The phosphorus source is selected from at least one of phosphoric acid, sodium dihydrogen phosphate, and sodium pyrophosphate. The mass ratio of the first silicon source, iodide, heme and phosphorus source is (100~150):(4~5):(0.1~0.5):(9~12).

2. A phosphorescent silicon dot nanozyme prepared by the preparation method according to claim 1.

3. An aqueous phosphorescent silicon dot nanozyme comprising the phosphorescent silicon dot nanozyme of claim 2.

4. The aqueous phosphorescent silicon dot nanozyme according to claim 3, characterized in that, The preparation method of the aqueous phosphorescent silicon dot nanozyme includes the following steps: The phosphorescent silicon dot nanozyme, the second silicon source, ammonia, and water are mixed evenly, and the reaction solution is subjected to hydrolysis, condensation, and polymerization reactions in sequence to obtain aqueous phosphorescent silicon dot nanozyme.

5. The aqueous phosphorescent silicon dot nanozyme according to claim 4, characterized in that, The second silicon source is selected from ethyl silicate or tetraethoxysilane; and / or, The volume ratio of the second silicon source, ammonia, and water is (0.5~1):(0.5~1):(9~18); and / or, The concentration of phosphorescent silicon dot nanozyme in the reaction solution is 100 μg / mL to 300 μg / mL.

6. A hydrogel comprising the phosphorescent silicon dot nanozyme of claim 2.

7. The application of the phosphorescent silicon dot nanozyme of claim 2 or the aqueous phosphorescent silicon dot nanozyme of claim 3 in the detection of carmine.

8. The application of the phosphorescent silicon nanozyme of claim 2 or the hydrogel of claim 6 in the detection of thiopronine.

9. The application of the phosphorescent silicon dot nanozyme according to claim 2 in the preparation of a triple anti-counterfeiting label with fluorescence, phosphorescence and enzymatic color change.

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