Lysozyme-mediated core-shell nanocomposite sensor and its application in detecting copper and chromium ions

Abstract

The present invention discloses a sensor based on lysozyme-mediated core-shell nanocomposite material and an application for detecting copper ions and chromium ions. After lysozyme is used as a coupling agent to connect silanized carbon quantum dots and metal nanoclusters, lysozyme is modified on the surface, and then encapsulated by ZIF-8 to obtain a sensor based on lysozyme-mediated core-shell nanocomposite material, or the composite material is uniformly spray-printed on cellulose paper as a sensor; wherein the metal nanoclusters are glutathione-stabilized gold nanoclusters or bovine serum albumin-stabilized copper nanoclusters. The coupling agent used in the present invention is non-toxic, has strong biocompatibility, and is short in time consumption. The constructed sensor can be used for ratio fluorescence detection of copper ions and chromium ions, and has higher selectivity and better stability.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescence detection, and particularly relates to a sensor based on a lysozyme-mediated core-shell nanocomposite and its application in the detection of Cu 2+ and Cr 6+ detection. Background Art

[0002] Heavy metal ions have a huge impact on the environment and human health and have attracted extensive attention. Copper ions are one of the three most abundant essential trace elements in the human body after iron and zinc, and it plays a key role as a catalytic cofactor for various metalloenzymes. However, abnormal levels of Cu 2+ in the human body can have toxic effects on human cells and tissues, leading to symptoms such as oxidative stress and logical nerve disorders, including Alzheimer's disease, Parkinson's disease, Menkes syndrome, etc. According to the World Health Organization's drinking water quality guidelines, the allowable concentration of Cu 2+ in drinking water is less than 2 mg / L (32 μM). Therefore, many analytical techniques, such as atomic absorption spectrometry, atomic emission spectrometry, inductively coupled plasma mass spectrometry, and electrochemical methods, are used to detect Cu 2+ in environmental water samples, but most of these analytical methods require expensive dedicated equipment and complex and time-consuming analytical procedures. Therefore, it is very necessary to develop a highly sensitive and selective Cu 2+ detection method.

[0003] Gold nanoclusters (AuNCs) have rapidly developed as a new type of luminescent material, which are usually composed of an inorganic gold core and an organic ligand shell. The photophysical properties of AuNCs are affected by the surrounding environment, the inner core, and the ligand shell, and can be adjusted through a series of pathways, providing great feasibility for constructing multifunctional chemical and biosensors. However, when using AuNCs alone to detect Cu 2+ there is only one emission peak, and changes in the probe concentration or the excitation light source will affect the detection accuracy. Recently, ratiometric fluorescence probes have attracted extensive attention because they can provide an internal reference and have the effect of single light excitation and dual fluorescence emission. When the target is added, one fluorescence does not change, and the other fluorescence changes with the addition of the target. The self-calibration of the two emission bands avoids the influence of the probe concentration, instrument state, and environmental state. In recent years, due to the obvious advantages of carbon quantum dots and AuNCs compared with traditional organic dyes, they are often used for ratiometric detection of heavy metal ions.

[0004] The environmental stability, optical properties, and photophysical properties of noble metal clusters largely depend on their cores, surface ligands, and dispersion media. Therefore, efforts have been made to improve the water stability and luminescence properties of these nanomaterials. Zeolitic imidazolate frameworks (ZIFs) have some outstanding advantages, such as easy preparation, large specific surface area, ultra-high porosity, and water dispersibility, and have been widely used in fields such as biosensing. Through the encapsulation of ZIF-8, not only can the emission activity of the loaded fluorescent substance be effectively enhanced, the entry of macromolecules be restricted, and analytes be selectively enriched to amplify the sensing signal, but the nanocomposites encapsulated with MOF are also expected to improve the thermodynamic stability with minimal aggregation. However, recent studies have shown that 2+ and Hg 2+ can both quench the fluorescence of AuNCs, but the quenching principles of the two are different. The former causes fluorescence quenching according to the coordination of Cu 2+ with glutathione, and the latter acts through the metallophilic interaction of Hg 2+ with Au + . Currently, researchers generally use reducing agents such as Sn 2+ or NaBH 4 to eliminate the influence of Hg 2+ , or add Ag + , and utilize the metallophilic interaction of Ag + with Au + to eliminate the influence of Hg 2+ . However, these methods all require harsh experimental conditions and complex detection steps. Therefore, developing a simple and feasible method to eliminate the influence of Hg 2+ has always been a challenging task for researchers. Summary of the Invention

[0005] Aiming at the above deficiencies, the purpose of the present invention is to provide a sensor based on lysozyme-mediated core-shell nanocomposites with simple preparation, stable performance, good sensitivity, and high selectivity for detecting copper ions and chromium ions, and to provide a new application for this sensor.

[0006] For the above purpose, the sensor based on lysozyme-mediated core-shell nanocomposites provided by the present invention is as follows: First, use lysozyme as a coupling agent to connect silanized carbon quantum dots and metal nanoclusters to obtain hybrid nanoparticles, then modify the surface of the hybrid nanoparticles with lysozyme, and finally encapsulate the lysozyme-modified hybrid nanoparticles with ZIF-8 to obtain lysozyme-mediated core-shell nanocomposites; use this composite material directly as a sensor, or uniformly spray-print this composite material on cellulose paper as a sensor. Among them, the metal nanoclusters are glutathione-stabilized gold nanoclusters or bovine serum albumin-stabilized copper nanoclusters.

[0007] The diameter of the above-mentioned silylated carbon quantum dots is 30-40 nm, and the preparation method thereof is as follows: Citric acid and ethylenediamine are ultrasonically dispersed in deionized water, and reacted at 180-220 °C for 4-6 h under closed conditions to obtain carbon quantum dots; the carbon quantum dots are ultrasonically dispersed in absolute ethanol, ammonia water and tetraethyl orthosilicate are added, and stirred for 18-24 h to obtain silylated carbon quantum dots. Among them, the molar ratio of the citric acid to the ethylenediamine is 1:0.9-1.1, and the volume ratio of the carbon quantum dots to the ammonia water and the tetraethyl orthosilicate is 1:1-3:1-3. The silylated carbon quantum dots have blue fluorescence emission and can be used as a reference fluorescence signal for detecting metal ions.

[0008] The preparation method of the above-mentioned glutathione-stabilized gold nanoclusters is as follows: Glutathione and chloroauric acid are uniformly dispersed in deionized water, heated and stirred at 65-75 °C for 20-24 h to obtain glutathione-stabilized gold nanoclusters. Among them, the molar ratio of the chloroauric acid to the glutathione is 1:1-2. The glutathione-stabilized gold nanoclusters have orange fluorescence emission and can be used as a sensing fluorescence signal for detecting copper ions.

[0009] The preparation method of the above-mentioned bovine serum albumin-stabilized copper nanoclusters is as follows: Under stirring conditions, copper chloride and bovine serum albumin are added to deionized water, the pH is adjusted to 11-12 with NaOH, stirred at room temperature for 10-15 min, then dithiothreitol is added, and the pH is adjusted to neutral with hydrochloric acid, and stirred continuously for 40-60 min to obtain bovine serum albumin-stabilized copper nanoclusters. Among them, the mass ratio of the copper chloride, the bovine serum albumin and the dithiothreitol is 1:6-12:2-6. The bovine serum albumin-stabilized copper nanoclusters have orange fluorescence emission and can be used as a sensing fluorescence signal for detecting chromium ions.

[0010] The preparation method of the above-mentioned lysozyme-modified hybrid nanoparticles consists of the following steps:

[0011] (1) The silylated carbon quantum dots are uniformly dispersed in a HEPES buffer solution containing lysozyme and tris(2-carboxyethyl)phosphine hydrochloride, and incubated at 37 °C for 20-60 min to obtain amino-functionalized silylated carbon quantum dots; the mass ratio of the silylated carbon quantum dots to the lysozyme and the tris(2-carboxyethyl)phosphine hydrochloride is 1:0.8-1.2:6-8;

[0012] (2) Mix glutathione-stabilized gold nanoclusters with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and mix and shake them for 20 - 40 min under dark conditions. Then add amino-functionalized silanized carbon quantum dots, and continue to shake for 2 - 4 h under dark conditions to obtain hybrid nanoparticles. Mix the hybrid nanoparticles with an aqueous solution of lysozyme, incubate them statically at 37 °C for 2 - 3 h, and centrifuge to obtain lysozyme-modified hybrid nanoparticles. Alternatively, mix bovine serum albumin-stabilized copper nanoclusters with amino-functionalized silanized carbon quantum dots, and shake them for 2 - 4 h under dark conditions to obtain hybrid nanoparticles. Mix the hybrid nanoparticles with an aqueous solution of lysozyme, incubate them statically at 37 °C for 30 - 50 min, and centrifuge to obtain lysozyme-modified hybrid nanoparticles. Among them, the mass ratio of the glutathione-stabilized gold nanoclusters to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 1:0.02 - 0.04:0.01 - 0.03, the mass ratio of the amino-functionalized silanized carbon quantum dots to the glutathione-stabilized gold nanoclusters or the bovine serum albumin-stabilized copper nanoclusters is 1:50 - 150, and the mass ratio of lysozyme to the hybrid nanoparticles is 1:1 - 3.

[0013] The diameter of the above-mentioned lysozyme-mediated core-shell nanocomposite material is 200 - 250 nm, and its preparation method is as follows: Disperse the lysozyme-modified hybrid nanoparticles in methanol, add a 2-methylimidazole methanol solution, mix well, and then add a zinc nitrate methanol solution. Let it stand at room temperature for 20 - 24 h to encapsulate the lysozyme-modified hybrid nanoparticles with ZIF-8 to obtain the lysozyme-mediated core-shell nanocomposite material. Among them, the mass ratio of the lysozyme-modified hybrid nanoparticles to 2-methylimidazole and zinc nitrate is 1:5 - 10:20 - 30. The excitation wavelength of the lysozyme-mediated core-shell nanocomposite material is 300 - 360 nm, and the strongest emission wavelengths are around 470 nm and around 620 nm, showing ratiometric fluorescence characteristics.

[0014] The sensor based on the lysozyme-mediated core-shell nanocomposite material of the present invention can be used for ratiometric fluorescence detection of Cu 2+ , where the metal nanocluster is glutathione-stabilized gold nanoclusters.

[0015] The sensor based on the lysozyme-mediated core-shell nanocomposite material of the present invention can also be used for ratiometric fluorescence detection of Cr 6+ , where the metal nanocluster is bovine serum albumin-stabilized copper nanoclusters.

[0016] The above-mentioned ratiometric fluorescence detection of Cu 2+ and Cr 6+ The specific detection method is as follows: Add the sensor based on the lysozyme-mediated core-shell nanocomposite material to different concentrations of Cu 2+or Cr 6+ In the standard solution, react for 1 min in the dark. Use a fluorescence spectrophotometer to measure the fluorescence intensities of the reaction solution at the wavelengths of the metal nanoclusters and carbon quantum dots. According to the fluorescence intensity ratios of different concentrations of Cu 2+ or Cr 6+ standard solutions, plot the standard curve of the fluorescence intensity ratio of C Cu 2+ -gold nanoclusters to carbon quantum dots or LogC Cr 6+ -copper nanoclusters to carbon quantum dots and calculate the regression equation; then use the same method to detect the fluorescence intensity ratio of metal nanoclusters to carbon quantum dots in the water sample to be tested containing Cu 2+ or Cr 6+ and calculate the concentration of Cu 2+ or Cr 6+ in the water sample to be tested according to the regression equation of the standard curve.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] 1. The lysozyme-mediated core-shell nanocomposite material of the present invention is prepared by encapsulating hybrid nanoparticles modified with lysozyme through the "bottle outside the boat" method using ZIF-8, wherein the hybrid nanoparticles are formed by connecting silanized blue carbon quantum dots and orange metal nanoclusters with lysozyme as a coupling agent. The present invention can directly use the lysozyme-mediated core-shell nanocomposite material as a sensor, or use the composite material as a ratio fluorescence probe. Using cellulose paper as a solid-phase carrier, the composite material is evenly sprayed on the cellulose paper through a membrane scribing and gold spraying instrument, and the obtained test strip is used as a sensor. The coupling agent used in this sensor is non-toxic and has strong biocompatibility. Using blue fluorescent carbon quantum dots as a reference fluorophore and orange fluorescent metal nanoclusters as a detection fluorophore, the constructed ratio fluorescence probe can measure two fluorescence peaks simultaneously. Through the fluorescence intensity ratio at two well-resolved emission peaks, it has an internal calibration self-calibration effect on the influence of the environment, can effectively eliminate external interferences such as light source fluctuations and background absorption, and has better stability and higher detection accuracy.

[0019] 2. In the sensor of the present invention, when the metal nanocluster used is a gold nanocluster, lysozyme plays a role in protecting the gold nanoclusters, blocking the metallophilic interaction between Hg 2+ and Au + , can eliminate the fluorescence quenching effect of Hg 2+ on the gold nanoclusters, avoid the influence of Hg 2+ on the detection, and improve the detection of Cu by the ratio fluorescence probe 2+Specificity; when the metal nanoclusters used are copper nanoclusters, lysozyme acts as a surfactant to control the nucleation and growth of ZIF-8 on the surface of hybrid nanoparticles. Moreover, through the encapsulation of the hybrid nanoparticles by ZIF-8, the stability of the metal nanoclusters is enhanced, the entry of macromolecules is restricted, and the analyte can be selectively enriched to amplify the sensing signal, realizing highly sensitive and highly selective ratio fluorescence detection of Cu 2+ and Cr 6+ .

[0020] 3. The lysozyme-mediated core-shell nanocomposite of the present invention is a solution, which can be directly printed on cellulose paper to make a test strip. By observing the fluorescence color change of the test strip after adding the analyte under ultraviolet light, it is possible to microscopically visualize and instantaneously and rapidly detect Cu 2+ and Cr 6+ , and it is applied to detect Cu 2+ and Cr 6+ in environmental water samples, with the characteristics of small dosage, stable structure, short time consumption, environmental friendliness, and convenient operation, etc. Description of the Drawings

[0021] Figure 1 is the transmission electron microscope images of CDs@SiO 2 (a), HNPs(b), and HNPs@ZIF-8(c) in Example 1.

[0022] Figure 2 is the ultraviolet-visible absorption spectra of CDs, AuNCs, ZIF-8, and HNPs@ZIF-8 in Example 1.

[0023] Figure 3 is the fluorescence spectra of CDs, AuNCs, HNPs, and HNPs@ZIF-8 in Example 1.

[0024] Figure 4 is the Fourier transform infrared spectra of AuNCs and GSH(A), and CDs, CDs@SiO 2 , CDs@SiO 2 @lyz, HNPs, HNPs@ZIF-8, and ZIF-8(B) in Example 1.

[0025] Figure 5 is the X-ray photoelectron spectra of AuNCs in Example 1.

[0026] Figure 6 is the quenching efficiency diagrams of HNPs@ZIF-8 and AuNCs after adding Cu 2+ and Hg 2+ respectively in Example 1.

[0027] Figure 7It is the fluorescence spectra (A) of HNPs@ZIF-8 in Example 1 at different concentrations of Cu in the presence of an excitation wavelength of 320 nm and the calibration curve (B) of F for Cu. 2+ in the presence of and F AuNCs / CDs for Cu 2+ .

[0028] Figure 8 It is the selectivity result diagram of HNPs@ZIF-8 for detecting Cu in Example 1. 2+ .

[0029] Figure 9 It is the fluorescence spectra (A) of HNPs@ZIF-8 in Example 2 at different concentrations of Cr in the presence of an excitation wavelength of 300 nm and the calibration curve (B) of F for Cr. 6+ in the presence of and F CuNCs / CDs for Cr 6+ .

[0030] Figure 10 It is the selectivity result diagram of HNPs@ZIF-8 for detecting Cr in Example 2. 6+ .

[0031] Figure 11 It is the visualization picture of the test strip for detecting Cu in an aqueous solution in Example 3. 2+ . Detailed implementation manners

[0032] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the protection scope of the present invention is not limited to these embodiments.

[0033] Example 1

[0034] I. Preparation of a sensor for detecting Cu 2+

[0035] 1. Synthesis of silanized carbon quantum dots (CDs@SiO 2 )

[0036] Disperse 1.0507 g (5 mmol) of citric acid and 335 μL (5 mmol) of ethylenediamine in 10 mL of deionized water and mix well. Then transfer the solution to a reaction kettle lined with polytetrafluoroethylene and heat it at 200 °C for 5 h under closed conditions. After the reaction, cool it to room temperature and obtain carbon quantum dots (CDs) through dialysis. Take 300 μL of CDs, dilute it with deionized water to 5 mL, add 15 mL of absolute ethanol, add 600 μL of ammonia water and 800 μL of tetraethyl orthosilicate, mix and stir for 18 h, centrifuge, and wash alternately with water and absolute ethanol until the supernatant has no fluorescence to obtain CDs@SiO 2 .

[0037] 2. Preparation of Glutathione-Stabilized Gold Nanoclusters (AuNCs)

[0038] Mix 10 mL of 4 mM HAuCl 4 aqueous solution and 10 mL of 6 mM glutathione (GSH) aqueous solution thoroughly. The mixture is continuously heated and stirred at 70 °C for 24 h. After the reaction ends, it is cooled to room temperature, and pure AuNCs are obtained through dialysis.

[0039] 3. Preparation of Lysozyme-Modified Hybrid Nanoparticles (HNPs)

[0040] (1) Disperse CDs@SiO 2 in deionized water to make the concentration of CDs@SiO 2 in the dispersion 10 mg / mL. Take 1 mL of 10 mg / mL CDs@SiO 2 dispersion in a centrifuge tube and disperse it by ultrasonic treatment. Add 5 mL of 2 mg / mL lysozyme (lyz) solution (prepared by dissolving 10 mg of lyz in 10 mM HEPES buffer with pH = 7.4) and 5 mL of 14 mg / mL tris(2-carboxyethyl)phosphine hydrochloride (TCEP) solution (prepared by dissolving 70 mg of TCEP in 10 mM HEPES buffer with pH = 5). Incubate the mixture at 37 °C for 40 min, then take it out and wash it several times with deionized water to obtain amino-functionalized silanized carbon quantum dots (CDs@SiO 2 @lyz).

[0041] (2) Take 1 g of AuNCs and add 2.5 mL of 8 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution (prepared by dissolving 20 mg of EDC in 0.1 mM MES buffer with pH = 6) and 2.5 mL of 6 mg / mL N-hydroxysuccinimide (NHS) solution (prepared by dissolving 15 mg of NHS in 0.1 mM MES buffer with pH = 6). Mix and shake at 25 °C in the dark for 30 min, then add 10 mg of CDs@SiO 2 @lyz, and continue to shake in the dark for 3 h. Wash it several times with deionized water to obtain hybrid nanoparticles; redisperse 5 mg of the hybrid nanoparticles in 1 mL of deionized water again, and add 5 mL of 0.5 mg / mL lysozyme aqueous solution. Incubate it statically at 37 °C for 2 h, centrifuge, and wash with deionized water to obtain HNPs.

[0042] 4. Preparation of Lysozyme-Mediated Core-Shell Nanocomposites (HNPs@ZIF-8)

[0043] Disperse 3 mg of HNPs in 10 mL of methanol. Take 5 mL of the HNPs methanol solution and add it to 10 mL of a 25 mM 2-methylimidazole methanol solution (containing 21 mg of 2-methylimidazole), and mix and shake for 5 min. Then add 10 mL of a 25 mM zinc nitrate hexahydrate methanol solution (containing 74 mg of zinc nitrate hexahydrate), and let it stand at room temperature for 24 h to encapsulate the lysozyme-modified hybrid nanoparticles with ZIF-8. Then centrifuge and wash several times with methanol to obtain HNPs@ZIF-8.

[0044] Figure 1 For the TEM images of CDs@SiO 2 , HNPs, and HNPs@ZIF-8, as shown by Figure 1 (a), the diameter of CDs@SiO 2 is 30 - 40 nm. When AuNCs are coupled to the surface of CDs@SiO 2 , it can be seen from 1(b) that some AuNCs surround the surface of the hybrid nanoparticles. It can be seen from 1(c) that HNPs are successfully encapsulated into ZIF-8, with a diameter of approximately 200 - 250 nm, indicating the successful synthesis of HNPs@ZIF-8.

[0045] Figure 2 For the UV-visible absorption spectra of CDs, AuNCs, HNPs@ZIF-8, and ZIF-8, it can be seen from the figure that CDs have an obvious absorption at 340 nm, and AuNCs have a weak absorption around 400 nm, proving the successful synthesis of CDs and AuNCs. The absorption band of pure ZIF-8 is weak. By encapsulating HNPs with ZIF-8, the obtained HNPs@ZIF-8 has a strong absorption band in the UV-visible absorption spectrum, proving the successful synthesis of HNPs@ZIF-8.

[0046] Figure 3 For the fluorescence spectra of CDs, AuNCs, HNPs, and HNPs@ZIF-8, when excited at a wavelength of 320 nm, the emission peak of CDs is around 470 nm, and the emission peak of AuNCs is around 620 nm. The emission peaks of HNPs obtained by lysozyme modification and HNPs@ZIF-8 obtained by encapsulation into ZIF-8 do not show obvious shifts, proving the successful synthesis of HNPs@ZIF-8.

[0047] Figure 4 For the Fourier transform infrared spectra of AuNCs, GSH, and CDs, CDs@SiO 2 , CDs@SiO 2 @lyz, HNPs, HNPs@ZIF-8, and ZIF-8, in AuNCs, at 2520 cm -1The absorption peak of -SH disappeared, indicating the formation of S-Au bonds, suggesting the successful synthesis of AuNCs, 1091 cm -1 corresponds to the absorption peak of O-Si-O, indicating the successful synthesis of CDs@SiO 2 , 1308 cm -1 corresponds to the characteristic absorption peak of C-N, 417 cm -1 corresponds to the characteristic absorption peak of Zn-N, indicating the successful synthesis of HNPs@ZIF-8.

[0048] Figure 5 is the X-ray photoelectron spectroscopy of AuNCs. Two strong peaks are located at 87.4 eV and 83.8 eV, corresponding to the Au 4f of Au(0) 5 / 2 and Au 4f 7 / 2 electron binding energies, which proves the successful synthesis of AuNCs.

[0049] Figure 6 is the quenching efficiency diagram of HNPs@ZIF-8 and AuNCs added with Cu 2+ and Hg 2+ respectively. It can be seen from the figure that AuNCs can quench both Cu 2+ and Hg 2+ , while HNPs@ZIF-8 only quenches Cu 2+ , eliminating the influence of Hg 2+ and improving the selectivity of Cu 2+ detection.

[0050] II. Detection of the sensor for Cu 2+

[0051] 1. Sensitivity and detection range

[0052] Mix 160 μL of HNPs@ZIF-8 with 40 μL of Cu 2+ standard solutions with different concentrations, react for 1 min in the dark, and then measure the fluorescence spectra of the reaction solutions using a fluorescence spectrophotometer. As Figure 7 (A) shows, under 320 nm excitation, as the concentration of Cu 2+ increases, the fluorescence intensity of carbon quantum dots remains basically unchanged, and the fluorescence intensity of gold nanoclusters gradually weakens. According to the ratio of the fluorescence intensity of gold nanoclusters at a wavelength of 620 nm and the fluorescence intensity of carbon quantum dots at a wavelength of 470 nm corresponding to the reaction solutions of different concentrations of Cu 2+ standard solutions, make a standard curve of the concentration of Cu 2+ - the ratio of the fluorescence intensities of gold nanoclusters and carbon quantum dots and calculate the regression equation. As Figure 7 (B) shows, when the concentration range of Cu 2+ is 0.04 - 4 μM, the linear equation is:​

[0053] F AuNCs / CDs = -0.2294C Cu 2+ +1.8877

[0054] In the formula, F AuNCs / CDs is the ratio of the fluorescence intensity of gold nanoclusters to that of carbon quantum dots, and C Cu 2+ is the concentration of Cu 2+ The correlation coefficient is R 2 = 0.9972. From the correlation coefficient, it can be seen that the linear relationship between the fluorescence intensity ratio and C Cu 2+ is good. After testing, the detection limit of Cu 2+ is 35 nmol / L.

[0055] 2. Selectivity

[0056] The selectivity of the sensor for the detection of Cu 2+ is as follows Figure 8 As can be seen, other metal ions including Pb 2+ , Zn 2+ , Cd 2+ , Fe 3+ , Mg 2 + , Na + , K + , Ca 2+ and Hg 2+ etc. do not interfere with the detection of Cu 2+ , indicating that the sensor has good selectivity for the detection of Cu 2+ .

[0057] Example 2

[0058] I. Preparation of a sensor for detecting Cr 6+

[0059] 1. Synthesis of silanized carbon quantum dots (CDs@SiO 2 )

[0060] This step is the same as step 1 of Example 1.

[0061] 2. Preparation of bovine serum albumin-stabilized copper nanoclusters (CuNCs)

[0062] Add 5 mL of 2 mg / mL CuCl 2 ​The aqueous solution was added to 4 mL of 25 mg / mL bovine serum albumin (BSA) aqueous solution under vigorous stirring. The pH was adjusted to 11 - 12 with 1 M NaOH aqueous solution, and stirring was continued at room temperature for 10 min. Then, 1 mL of 40 mg / mL dithiothreitol aqueous solution was added, and the pH was adjusted to neutral with hydrochloric acid. Stirring was continued for 50 min, and pure CuNCs were obtained through dialysis.

[0063] 3. Preparation of lysozyme - modified hybrid nanoparticles (HNPs)

[0064] (1) The preparation method of CDs@SiO 2 @lyz was the same as that in Step 3 of Example 1.

[0065] (2) 1 g of CuNCs and 10 mg of CDs@SiO 2 @lyz were mixed evenly, shaken at 25 °C for 3 h under dark conditions, washed several times with deionized water to obtain hybrid nanoparticles; 5 mg of the hybrid nanoparticles were redispersed in 1 mL of deionized water, and 5 mL of 0.5 mg / mL lysozyme aqueous solution was added. The mixture was incubated statically at 37 °C for 40 min, centrifuged, and washed with deionized water to obtain HNPs.

[0066] 4. Preparation of lysozyme - mediated core - shell nanocomposites (HNPs@ZIF - 8)

[0067] This step was the same as that in Step 4 of Example 1.

[0068] II. Detection of Cr 6+ by the sensor

[0069] 1. Sensitivity and detection range

[0070] 100 μL of HNPs@ZIF - 8 was mixed with 100 μL of Cr 6+ standard solutions with different concentrations, reacted for 1 min in the dark, and then the fluorescence spectrum of the reaction solution was measured using a fluorescence spectrophotometer. As Figure 9 (A) shows, under 300 nm excitation, as the Cr 6+ concentration increased, the fluorescence intensity of the carbon quantum dots remained basically unchanged, while the fluorescence intensity of the copper nanoclusters gradually decreased. According to the ratio of the fluorescence intensity of the copper nanoclusters at 610 nm and the fluorescence intensity of the carbon quantum dots at 470 nm in the reaction solutions corresponding to different concentrations of Cr 6+ standard solutions, a standard curve of Log C Cr 6+ - the ratio of the fluorescence intensities of the copper nanoclusters and the carbon quantum dots was plotted and the regression equation was calculated. As Figure 9 (B) shows, when the Cr 6+ concentration range was 0.15 - 100 μM, the linear equation was:

[0071] F CuNCs / CDs =-0.2360Log C Cr 6+ +2.1279

[0072] Where F CuNCs / CDs is the ratio of the fluorescence intensity of copper nanoclusters to carbon quantum dots, C Cr 6+ is Cr 6+ Concentration, correlation coefficient is R 2 =0.9915, as can be seen from the correlation coefficient, the fluorescence intensity ratio and Log C Cr 6+ has a good linear relationship. After testing, Cr 6+ The detection limit is 2nmol / L.

[0073] 2. Selectivity

[0074] Sensor to Cr 6+ Detection selectivity such as Figure 10 It can be seen that other metal ions including Pb 2+ 、Zn 2+ 、Cd 2+ 、Fe 3+ 、Mg 2+ 、Na + 、K + and Ca 2+ etc. do not interfere with Cr 6+ Detection shows that the sensor is sensitive to Cr 6+ The detection of has good selectivity.

[0075] Example 3

[0076] The HNPs@ZIF-8 obtained in Example 1 was dispersed in 2 mL of HEPES buffer, and the resulting dispersion was evenly sprayed onto a test paper using a film-spraying gold sprayer and dried in a dark place. Then, 0, 0.1 μM, 0.2 μM, 0.4 μM, 0.8 μM, 1 μM, 5 μM, 15 μM, and 20 μM of Cu were added to the test paper respectively 2+ , place in a dark place for 1 minute, and observe the fluorescence color change of the test paper under a 365nm ultraviolet lamp. Figure 11 It can be seen that with Cu 2+ As the concentration increases, the color of the test strip changes from pink to blue.​​​​​​​

Claims

1. A sensor based on lysozyme-mediated core-shell nanocomposite material, characterized in that: First, lysozyme is used as a coupling agent to connect silanized carbon quantum dots and metal nanoclusters to obtain hybrid nanoparticles, then lysozyme is modified on the surface of the hybrid nanoparticles, and finally the lysozyme-modified hybrid nanoparticles are encapsulated by ZIF-8 to obtain a lysozyme-mediated core-shell nanocomposite material; the composite material is directly used as a sensor, or the composite material is uniformly spray-printed on cellulose paper as a sensor; wherein the metal nanoclusters are glutathione-stabilized gold nanoclusters or bovine serum albumin-stabilized copper nanoclusters; the diameter of the silanized carbon quantum dots is 30-40 nm, and the diameter of the lysozyme-mediated core-shell nanocomposite material is 200-250 nm.

2. The sensor based on lysozyme-mediated core-shell nanocomposite material according to claim 1, characterized in that: The preparation method of the silanized carbon quantum dots is as follows: ultrasonically dispersing citric acid and ethylenediamine in deionized water, reacting at 180-220° C. for 4-6 hours under closed conditions to obtain carbon quantum dots; ultrasonically dispersing the carbon quantum dots in anhydrous ethanol, adding ammonia water and tetraethyl orthosilicate, stirring for 18-24 hours to obtain silanized carbon quantum dots; wherein the molar ratio of the citric acid to ethylenediamine is 1:0.9-1.1, and the volume ratio of the carbon quantum dots to ammonia water and tetraethyl orthosilicate is 1:1-3:1-3.

3. The sensor based on lysozyme-mediated core-shell nanocomposite material according to claim 1, characterized in that: The preparation method of the glutathione-stabilized gold nanoclusters is as follows: glutathione and chloroauric acid are uniformly dispersed in deionized water, and heated and stirred at 65-75° C. for 20-24 h to obtain glutathione-stabilized gold nanoclusters; wherein the molar ratio of the chloroauric acid to the glutathione is 1:1-2.

4. The sensor based on lysozyme-mediated core-shell nanocomposite material according to claim 1, characterized in that: The preparation method of the bovine serum albumin-stabilized copper nanoclusters is as follows: under stirring conditions, copper chloride and bovine serum albumin are added to deionized water, the pH is adjusted to 11-12 with NaOH, stirring at room temperature for 10-15 minutes, and then dithiothreitol is added, the pH is adjusted to neutral with hydrochloric acid, and stirring is continued for 40-60 minutes to obtain bovine serum albumin-stabilized copper nanoclusters; wherein the mass ratio of the copper chloride, bovine serum albumin, and dithiothreitol is 1:6-12:2-6.

5. The sensor based on lysozyme-mediated core-shell nanocomposite material according to claim 1, characterized in that: The preparation method of the lysozyme-modified hybrid nanoparticles is as follows: (1) uniformly dispersing silanized carbon quantum dots in a HEPES buffer solution containing lysozyme and tri(2-carboxyethyl)phosphine hydrochloride, and incubating at 37°C for 20 to 60 min to obtain amino-modified silanized carbon quantum dots; the mass ratio of the silanized carbon quantum dots to lysozyme and tri(2-carboxyethyl)phosphine hydrochloride is 1:0.8 to 1.2:6 to 8; (2) Mixing glutathione-stabilized gold nanoclusters with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, shaking the mixture in the dark for 20 to 40 min, then adding amino-silanized carbon quantum dots, and continuing to shake the mixture in the dark for 2 to 4 h to obtain hybrid nanoparticles; mixing the hybrid nanoparticles with a lysozyme aqueous solution, incubating the mixture at 37 °C for 2 to 3 h, and centrifuging the mixture to obtain lysozyme-modified hybrid nanoparticles; Alternatively, copper nanoclusters stabilized by bovine serum albumin are mixed with amino-silanized carbon quantum dots, and the mixture is shaken for 2 to 4 hours in the dark to obtain hybrid nanoparticles; the hybrid nanoparticles are mixed with a lysozyme aqueous solution, incubated at 37°C for 30 to 50 minutes, and centrifuged to obtain lysozyme-modified hybrid nanoparticles; The mass ratio of the glutathione-stabilized gold nanoclusters to 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide is 1:0.02-0.04:0.01-0.03, the mass ratio of the amino-silanized carbon quantum dots to the glutathione-stabilized gold nanoclusters or bovine serum albumin-stabilized copper nanoclusters is 1:50-150, and the mass ratio of lysozyme to the hybrid nanoparticles is 1:1-3.

6. The sensor based on lysozyme-mediated core-shell nanocomposite material according to claim 1, characterized in that: The preparation method of the lysozyme-mediated core-shell nanocomposite material is as follows: dispersing lysozyme-modified hybrid nanoparticles in methanol, adding 2-methylimidazole methanol solution, mixing, and then adding zinc nitrate methanol solution, standing at room temperature for 20 to 24 hours, so that ZIF-8 encapsulates the lysozyme-modified hybrid nanoparticles to obtain the lysozyme-mediated core-shell nanocomposite material; wherein the mass ratio of the lysozyme-modified hybrid nanoparticles to 2-methylimidazole and zinc nitrate is 1:5 to 10:20 to 30.

7. The sensor based on lysozyme-mediated core-shell nanocomposite material according to claim 1 is used for ratiometric fluorescence detection of Cu 2+ Applications in which The metal nanoclusters are glutathione-stabilized gold nanoclusters.

8. The sensor based on lysozyme-mediated core-shell nanocomposite material according to claim 1 is used for ratiometric fluorescence detection of Cr 6+ Applications in which The metal nanoclusters are copper nanoclusters stabilized by bovine serum albumin.

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