CN115356316A

CN115356316A - Sensor based on lysozyme-mediated core-shell nanocomposite and application of sensor to detection of copper ions and chromium ions

Info

Publication number: CN115356316A
Application number: CN202211029743.2A
Authority: CN
Inventors: 党福全; 范苗
Original assignee: Shaanxi Normal University
Current assignee: Shaanxi Normal University
Priority date: 2022-08-25
Filing date: 2022-08-25
Publication date: 2022-11-18

Abstract

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

Description

Sensor based on lysozyme-mediated core-shell nanocomposite and application of sensor in detection of copper ions and chromium ions

Technical Field

The invention belongs to the technical field of fluorescence detection, and particularly relates to a lysozyme-mediated core-shell nanocomposite-based sensor and application thereof in Cu ²⁺ And Cr ⁶⁺ Application in detection.

Background

Heavy metal ions have great influence on the environment and human health, and have attracted much attention. Copper ion is one of the third most abundant essential trace elements in the human body after iron and zinc, and plays a key role as a catalytic cofactor of a plurality of metalloenzymes. However, abnormal levels of Cu in the human body ²⁺ Can produce toxic effect on human cells and tissues, and cause symptoms of oxidative stress and logic nerve disorder, including Alzheimer's disease, parkinson disease, menkes' syndromeAnd the symptoms are combined. Cu in drinking water according to the Drinking Water quality guidelines of the world health organization ²⁺ Is less than 2mg/L (32. Mu.M). Thus, many analytical techniques, such as atomic absorption spectroscopy, atomic emission spectroscopy, inductively coupled plasma mass spectrometry, and electrochemical methods, are used to detect Cu in environmental water samples ²⁺ However, most of these analysis methods require expensive dedicated equipment and complicated and time-consuming analysis procedures. Therefore, cu with high sensitivity and strong selectivity was developed ²⁺ Detection methods are highly desirable.

Gold nanoclusters (AuNCs), which are rapidly developed as a new luminescent material, are generally composed of an inorganic gold core and an organic ligand. The photophysical properties of AuNCs are influenced by the surrounding environment, the inner core and the ligand shell, and can be adjusted through a series of ways, thereby providing great feasibility for constructing multifunctional chemical and biological sensors. However, cu was detected by AuNCs alone ²⁺ There is only one emission peak, and variations in probe concentration or excitation light source can affect the detection accuracy. Ratiometric fluorescent probes have recently attracted considerable attention because they can provide an intrinsic reference, with the effect of a single light exciting dual fluorescent emission. When the target is added, one fluorescence does not change, the other fluorescence changes along with the addition of the target, and the influence of the concentration of the probe, the state of the instrument and the state of the environment is avoided through the self calibration of the two emission bands. In recent years, carbon quantum dots and AuNCs have been used for the quantitative detection of heavy metal ions, because of their significant advantages over conventional organic dyes.

The environmental stability, optical properties and photophysical properties of the noble metal cluster depend to a large extent on its core, surface ligands and dispersive medium. Therefore, efforts are made to improve the water stability and the light emitting properties of these nanomaterials. Zeolite Imidazole Frameworks (ZIFs) have some outstanding advantages, such as ease of preparation, large specific surface area, ultra-high porosity and water dispersibility, and have wide applications in the fields of biosensing and the like. Through ZIF-8 encapsulation, the emission activity of a loaded fluorescent substance can be effectively enhanced, the entrance of macromolecules is limited, and analytes are selectively enriched to amplify sensing signals, and the sensing signals are encapsulated by MOFThe loaded nanocomposites are expected to improve thermodynamic stability with minimal agglomeration. However, recent studies have shown that Cu ²⁺ And Hg ²⁺ Both can quench the fluorescence of AuNCs, but the quenching principles are different, the former is based on Cu ²⁺ Coordination with glutathione leads to fluorescence quenching, the latter through Hg ²⁺ And Au ⁺ The metallophilic effect of (2). Currently, researchers commonly use Sn ²⁺ Or NaBH ₄ Hg elimination by reducing agent ²⁺ Or by addition of Ag ⁺ Using Ag ⁺ And Au ⁺ Eliminating Hg by metallophilic action ²⁺ However, these methods require severe experimental conditions and complicated detection steps, and therefore, a simple and easy method for eliminating Hg has been developed ²⁺ The impact of (c) has been a challenging task for researchers to face.

Disclosure of Invention

In view of the above disadvantages, the present invention aims to provide a sensor based on lysozyme-mediated core-shell nanocomposite, which has the advantages of simple preparation, stable performance, good sensitivity for detecting copper ions and chromium ions, and high selectivity, and provides a new application for the sensor.

In view of the above objects, the present invention provides a sensor based on lysozyme-mediated core-shell nanocomposites, comprising: firstly, lysozyme is taken as a coupling agent to connect silanized carbon quantum dots and metal nanoclusters to obtain hybrid nanoparticles, then lysozyme is modified on the surfaces of the hybrid nanoparticles, and finally the lysozyme-modified hybrid nanoparticles are packaged by ZIF-8 to obtain a lysozyme-mediated core-shell nanocomposite; the composite material is directly used as a sensor, or the composite material is uniformly sprayed and printed on cellulose paper to be used as a sensor. Wherein the metal nanocluster is a gold nanocluster stabilized by glutathione or a copper nanocluster stabilized by bovine serum albumin.

The diameter of the silanized carbon quantum dot is 30-40 nm, and the preparation method comprises the following steps: ultrasonically dispersing citric acid and ethylenediamine in deionized water, and reacting for 4-6 h at 180-220 ℃ under a closed condition to obtain carbon quantum dots; ultrasonically dispersing the carbon quantum dots in absolute ethyl alcohol, adding ammonia water and ethyl orthosilicate, and stirring for 18-24 hours to obtain the silanized carbon quantum dots. Wherein the molar ratio of the citric acid to the ethylenediamine is 1.9-1.1, and the volume ratio of the carbon quantum dots to the ammonia water and the tetraethoxysilane is 1-3. The silanized carbon quantum dot has blue fluorescence emission and can be used as a reference fluorescence signal for detecting metal ions.

The preparation method of the gold nanocluster with stable glutathione comprises the following steps: uniformly dispersing glutathione and chloroauric acid in deionized water, heating and stirring at 65-75 ℃ for 20-24 h to obtain the gold nanocluster with stable glutathione. Wherein the molar ratio of the chloroauric acid to the glutathione is 1-2. The gold nanocluster with stable glutathione has orange fluorescence emission and can be used as a sensing fluorescence signal for detecting copper ions.

The preparation method of the bovine serum albumin stable copper nanocluster comprises the following steps: under the condition of stirring, adding copper chloride and bovine serum albumin into deionized water, adjusting the pH value to 11-12 by NaOH, stirring at normal temperature for 10-15 min, adding dithiothreitol, adjusting the pH value to neutrality by hydrochloric acid, and continuously stirring for 40-60 min to obtain the bovine serum albumin stable copper nanoclusters. Wherein the mass ratio of the copper chloride to the bovine serum albumin to the dithiothreitol is (1-12). The copper nano cluster with the stable bovine serum albumin has orange fluorescence emission and can be used as a sensing fluorescence signal for detecting chromium ions.

The preparation method of the lysozyme modified hybrid nano-particle comprises the following steps:

(1) Uniformly dispersing the silanized carbon quantum dots in HEPES buffer solution containing lysozyme and tris (2-carboxyethyl) phosphine hydrochloride, and incubating for 20-60 min at 37 ℃ to obtain aminated silanized carbon quantum dots; the mass ratio of the silanized carbon quantum dot to lysozyme to tri (2-carboxyethyl) phosphine hydrochloride is (1);

(2) Mixing a gold nano cluster with stable glutathione with 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, mixing and oscillating for 20-40 min under a dark condition, then adding aminated silanized carbon quantum dots, and continuing oscillating for 2-4 h under the dark condition to obtain hybrid nano particles; and mixing the hybrid nano-particles with a lysozyme aqueous solution, standing and incubating for 2-3 h at 37 ℃, and centrifuging to obtain the lysozyme modified hybrid nano-particles. Or mixing copper nanoclusters stabilized by bovine serum albumin with aminated silanized carbon quantum dots, and oscillating for 2-4 h under a dark condition to obtain hybrid nanoparticles; and mixing the hybrid nano-particles with a lysozyme aqueous solution, standing and incubating for 30-50 min at 37 ℃, and centrifuging to obtain the lysozyme modified hybrid nano-particles. Wherein the mass ratio of the gold nanocluster stabilized by the glutathione to 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide is (1).

The diameter of the lysozyme mediated core-shell nano composite material is 200-250 nm, and the preparation method comprises the following steps: dispersing lysozyme-modified hybrid nanoparticles in methanol, adding a 2-methylimidazole methanol solution, uniformly mixing, adding a zinc nitrate methanol solution, standing at room temperature for 20-24 hours, and encapsulating the lysozyme-modified hybrid nanoparticles by ZIF-8 to obtain a lysozyme-mediated core-shell nano composite material; wherein the mass ratio of the lysozyme modified hybrid nano-particles to 2-methylimidazole to zinc nitrate is (1) - (10). The lysozyme-mediated core-shell nano composite material has the excitation wavelength of 300-360 nm, the strongest emission wavelengths of about 470nm and about 620nm, and has the ratio fluorescence characteristic.

The sensor based on the lysozyme-mediated core-shell nano composite material can be used for detecting Cu by ratio fluorescence ²⁺ Wherein the metal nanocluster is a gold nanocluster stabilized by glutathione.

The sensor based on the lysozyme-mediated core-shell nano composite material can also be used for detecting Cr by ratio fluorescence ⁶⁺ Wherein the metal nanoclusters are copper nanoclusters stabilized by bovine serum albumin.

Fluorescence detection of Cu at the above ratios ²⁺ And Cr ⁶⁺ The specific detection method comprises the following steps: will be based on lysozyme mediated core-shell sodiumThe sensor made of rice composite material is added with Cu with different concentrations ²⁺ Or Cr ⁶⁺ The reaction was carried out in a standard solution for 1min with the exclusion of light. And (3) measuring the fluorescence intensity of the reaction solution at the wavelengths of the metal nano-cluster and the carbon quantum dot by using a fluorescence spectrophotometer. According to different concentrations of Cu ²⁺ Or Cr ⁶⁺ Fluorescence intensity ratio of standard solution, as C _Cu ²⁺ Fluorescence intensity ratio of gold nanoclusters to carbon quantum dots or LogC _Cr ⁶⁺ -a standard curve of the ratio of the fluorescence intensity of the copper nanoclusters to the fluorescence intensity of the carbon quantum dots and calculating a regression equation; then detecting Cu content by the same method ²⁺ Or Cr ⁶⁺ The fluorescence intensity ratio of the metal nanoclusters and the carbon quantum dots in the water sample to be detected is calculated according to the regression equation of the standard curve ²⁺ Or Cr ⁶⁺ The concentration of (c).

Compared with the prior art, the invention has the following beneficial effects:

1. the lysozyme-mediated core-shell nano composite material is prepared by adopting an outboard bottle-making method and ZIF-8 packaging lysozyme-modified hybrid nano particles, wherein the hybrid nano particles are formed by connecting silanized blue carbon quantum dots and orange metal nano clusters by using lysozyme as a coupling agent. The lysozyme-mediated core-shell nano composite material can be directly used as a sensor, the composite material can also be used as a ratiometric fluorescent probe, cellulose paper is used as a solid phase carrier, the composite material is uniformly sprayed and printed on the cellulose paper by a film-scribing metal spraying instrument, and the obtained test strip is used as the sensor. The coupling agent used by the sensor is non-toxic and high in biocompatibility, blue fluorescent carbon quantum dots are used as reference fluorophores, orange fluorescent metal nanoclusters are used as detection fluorophores, the constructed ratiometric fluorescent probe can simultaneously measure two fluorescent peaks, the self-calibration effect of built-in correction is achieved on the influence of the environment through the fluorescent intensity ratio of two well-resolved emission peaks, external interferences such as light source fluctuation and background absorption can be effectively eliminated, and the sensor has high stability and high detection precision.

2. In the sensor, when the metal nanoclusters are gold nanoclusters, lysozyme plays a role in protecting the gold nanoclusters and blocks Hg ²⁺ And Au ⁺ Has metal affinity and can eliminate Hg ²⁺ The quenching effect on the fluorescence of the gold nanocluster is avoided, thereby avoiding Hg ²⁺ The influence on detection is improved by detecting Cu with a ratiometric fluorescent probe ²⁺ The specificity of (a); when the used metal nanocluster is a copper nanocluster, lysozyme is used as a surfactant to control the ZIF-8 to nucleate and grow on the surface of the hybrid nanoparticle, the ZIF-8 encapsulates the hybrid nanoparticle, so that the stability of the metal nanocluster is enhanced, the entrance of macromolecules is limited, analytes can be selectively enriched to amplify sensing signals, and the high-sensitivity and high-selectivity ratio fluorescence detection of Cu is realized ²⁺ And Cr ⁶⁺ 。

3. The lysozyme-mediated core-shell nano composite material is a solution, can be directly sprayed and printed on cellulose paper to prepare a test strip, changes the fluorescence color of the test strip after a test object is added on the test strip under an ultraviolet lamp, and can detect Cu in a micro-visual, instant and rapid manner ²⁺ And Cr ⁶⁺ Applied to detecting Cu in environmental water sample ²⁺ And Cr ⁶⁺ Has the characteristics of small using amount, stable structure, short time consumption, environmental friendliness, convenient operation and the like.

Drawings

FIG. 1 is CDs @ SiO in example 1 ₂ (a), HNPs (b), HNPs @ ZIF-8 (c).

FIG. 2 is a UV-visible absorption spectrum of CDs, auNCs, ZIF-8 and HNPs @ ZIF-8 in example 1.

FIG. 3 is a fluorescence spectrum of CDs, auNCs, HNPs and HNPs @ ZIF-8 in example 1.

FIG. 4 is the AuNCs and GSH (A) and CDs, CDs @ SiO in example 1 ₂ 、CDs@SiO ₂ Fourier transform infrared spectrogram of @ lyz, HNPs @ ZIF-8, ZIF-8 (B).

FIG. 5 is an X-ray photoelectron spectrum of AuNCs in example 1.

FIG. 6 shows that in example 1, cu was added to each of HNPs @ ZIF-8 and AuNCs ²⁺ And Hg ²⁺ Graph of quenching efficiency.

FIG. 7 is a graph of HNPs @ ZIF-8 in example 1 at different concentrations of Cu at an excitation wavelength of 320nm ²⁺ Fluorescence spectra in the Presence (A) and F _AuNCs/CDs For Cu ²⁺ Calibration curve (B) of (a).

FIG. 8 shows the detection of Cu by HNPs @ ZIF-8 pair in example 1 ²⁺ The selectivity results of (a).

FIG. 9 is a graph of HNPs @ ZIF-8 at different concentrations of Cr at 300nm excitation wavelength in example 2 ⁶⁺ Fluorescence spectra in the Presence (A) and F _CuNCs/CDs For Cr ⁶⁺ The calibration curve (B) of (1).

FIG. 10 shows the detection of Cr by HNPs @ ZIF-8 pair in example 2 ⁶⁺ Selective result diagram of (2).

FIG. 11 is a graph showing the test strips of example 3 for detecting Cu in an aqueous solution ²⁺ The visual picture of (2).

Detailed Description

The present invention will be described in further detail with reference to the drawings and examples, but the scope of the present invention is not limited to these examples.

Example 1

1. Preparation of test Cu ²⁺ Sensor (2)

1. Silanized carbon quantum dots (CDs @ SiO) ₂ ) Synthesis of (2)

1.0507g (5 mmol) of citric acid and 335 mu L (5 mmol) of ethylenediamine are dispersed in 10mL of deionized water and mixed well, then the solution is transferred to a reaction kettle lined with polytetrafluoroethylene and heated for reaction for 5h at 200 ℃ under closed conditions. After the reaction is finished, cooling to room temperature, and dialyzing to obtain the carbon quantum dots (CDs). Diluting 300 μ LCDs with deionized water to 5mL, adding 15mL anhydrous ethanol, adding 600 μ L ammonia water and 800 μ L ethyl orthosilicate, mixing and stirring for 18h, centrifuging, alternately washing with water and anhydrous ethanol until supernatant has no fluorescence to obtain CDs @ SiO ₂ 。

2. Preparation of glutathione-stabilized gold nanoclusters (AuNCs)

10mL4mM HAuCl ₄ The aqueous solution and 10mL of 6mM Glutathione (GSH) aqueous solution are fully mixed, the mixture is continuously heated and stirred for 24h at 70 ℃, the reaction is cooled to room temperature after the reaction is finished, and pure AuNCs are obtained by dialysis.

3. Preparation of Lysozyme-modified Hybrid Nanoparticles (HNPs)

(1) Mixing CDs @ SiO ₂ Dispersing in deionized water to make CDs @ SiO in the dispersion ₂ The concentration of (2) is 10mg/mL. 1mL of 10mg/mLCDs @ SiO ₂ The dispersion was ultrasonically dispersed in a centrifuge tube, 5mL of a 2mg/mL lysozyme (lyz) solution (prepared by dissolving 10mg of lyz in 10mM HEPES buffer solution having pH = 7.4) and 5mL of a 14mg/mL tris (2-carboxyethyl) phosphine hydrochloride (TCEP) solution (prepared by dissolving 70mg of TCEP in 10mM HEPES buffer solution having pH = 5) were added, the mixture was incubated at 37 ℃ for 40min and then taken out, and the mixture was washed several times with deionized water to obtain aminated silanized carbon quantum dots (CDs @ SiO quantum dots) ₂ @lyz)。

(2) 1g of AuNCs was added to a solution of 2.5mL8mg/mL of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (20 mg of EDC was dissolved in 0.1mM MES buffer pH = 6) and a solution of 2.5mL6mg/mLN-hydroxysuccinimide (NHS) (15 mg of NHS was dissolved in 0.1mM MES buffer pH = 6), mixed and shaken at 25 ℃ for 30min under dark conditions, and then added to 10mg of CDs @ SiO ₂ Continuing to vibrate for 3h under the dark condition at @ lyz, and washing for several times by using deionized water to obtain hybrid nanoparticles; and dispersing 5mg of hybrid nanoparticles into 1mL of deionized water again, adding 5mL of 0.5mg/mL lysozyme aqueous solution, standing and incubating at 37 ℃ for 2h, centrifuging, and washing with deionized water to obtain HNPs.

4. Preparation of lysozyme-mediated core-shell nano composite material (HNPs @ ZIF-8)

Dispersing 3mg of HNPs in 10mL of methanol, adding 5mL of methanol solution of HNPs into 10mL of 25mM methanol solution of 2-methylimidazole (containing 21mg of 2-methylimidazole) of 10mL of methanol solution of 25mM, mixing and shaking for 5min, adding 10mL of 25mM methanol solution of zinc nitrate hexahydrate (containing 74mg of zinc nitrate hexahydrate), standing at room temperature for 24h to enable ZIF-8 to encapsulate the lysozyme-modified hybrid nanoparticles, centrifuging, and washing with methanol for several times to obtain the @ ZIF-8 of HNPs.

FIG. 1 is CDs @ SiO ₂ TEM images of HNPs and HNPs @ ZIF-8, as seen from FIG. 1 (a), CDs @ SiO ₂ Diameter of 30-40 nm when AuNCs is coupled to CDs @ SiO ₂ After surface, 1 (b) shows that the surface of the hybrid nanoparticles surrounds some AuNCs, and 1 (c) shows that HNPs are successfully encapsulated in ZIF-8, the diameter is about 200-250 nm, which indicates that the HNPs @ ZIF-8 is successfully synthesized.

FIG. 2 is an ultraviolet-visible absorption spectrogram of CDs, auNCs, HNPs @ ZIF-8, from which it can be seen that CDs have an obvious absorption at 340nm, auNCs have a weak absorption at about 400nm, which proves successful synthesis of CDs and AuNCs, the absorption band of pure ZIF-8 is weaker, HNPs are packaged by ZIF-8, and the obtained HNPs @ ZIF-8 has a stronger absorption band in the ultraviolet-visible absorption spectrogram, which proves successful synthesis of HNPs @ ZIF-8.

FIG. 3 is a fluorescence spectrum of CDs, auNCs, HNPs @ ZIF-8, wherein the emission peak of CDs is about 470nm and the emission peak of AuNCs is about 620nm under the excitation of 320nm wavelength, the emission peak of HNPs obtained by lysozyme modification and the emission peak of HNPs @ ZIF-8 obtained by encapsulating into ZIF-8 do not move significantly, which proves that HNPs @ ZIF-8 are successfully synthesized.

FIG. 4 shows AuNCs, GSH, CDs @ SiO ₂ 、CDs@SiO ₂ Fourier transform infrared spectrogram of @ lyz, HNPs @ ZIF-8, 2520cm in AuNCs ^-1 The disappearance of the absorption peak at-SH, indicating the formation of S-Au bond, indicates the successful synthesis of AuNCs at 1091cm ^-1 The position corresponds to an O-Si-O absorption peak, which indicates that CDs @ SiO is successfully synthesized ₂ ，1308cm ^-1 Is corresponding to C-N characteristic absorption peak at 417cm ^-1 The position corresponds to a Zn-N characteristic absorption peak, which indicates that HNPs @ ZIF-8 is successfully synthesized.

FIG. 5 is an X-ray photoelectron spectrum of AuNCs, with two intensity peaks at 87.4eV and 83.8eV, corresponding to Au 4f of Au (0) _5/2 And Au 4f _7/2 This demonstrates the successful synthesis of AuNCs.

FIG. 6 shows the addition of Cu to HNPs @ ZIF-8 and AuNCs, respectively ²⁺ And Hg ²⁺ The quenching efficiency of AuNCs to Cu is shown in the figure ²⁺ And Hg ²⁺ All can be quenched, while HNPs @ ZIF-8 only for Cu ²⁺ Quench and eliminate Hg ²⁺ Has an effect on Cu ²⁺ Selectivity of detection.

2. Sensor pair Cu ²⁺ Detection of (2)

1. Sensitivity and detection Range

160 μ LHNPs @ ZIF-8 was mixed with Cu at a concentration of 40 μ L ²⁺ Mixing the standard solutions, and standing in dark placeThe reaction solution is subjected to fluorescence spectrometry for 1min, and then the fluorescence spectrum of the reaction solution is measured by using a fluorescence spectrophotometer. As shown in FIG. 7 (A), with Cu excitation at 320nm ²⁺ The concentration is increased, the fluorescence intensity of the carbon quantum dots is basically kept unchanged, and the fluorescence intensity of the gold nanoclusters is gradually weakened. According to different concentrations of Cu ²⁺ The ratio of the fluorescence intensity of the gold nanocluster of the reaction solution corresponding to the standard solution under the wavelength of 620nm to the fluorescence intensity of the carbon quantum dots under the wavelength of 470nm is used as the Cu ²⁺ Concentration-standard curve of ratio of fluorescence intensity of gold nanoclusters to carbon quantum dots and calculating regression equation, as shown in FIG. 7 (B), in Cu ²⁺ When the concentration ranges from 0.04 to 4 mu M, the linear equation is as follows:

F _AuNCs/CDs ＝-0.2294C _Cu ²⁺ +1.8877

in the formula F _AuNCs/CDs Is the ratio of the fluorescence intensity of the gold nanoclusters to that of the carbon quantum dots, C _Cu ²⁺ Is Cu ²⁺ Concentration, coefficient of correlation R ² =0.9972, as seen by correlation coefficient, fluorescence intensity ratio and C _Cu ²⁺ The linear relationship of (a) is good. Tested, cu ²⁺ The detection limit was 35nmol/L.

2. Selectivity is selected

Sensor pair Cu ²⁺ The selectivity of the detection can be seen in FIG. 8, with 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 Cu ²⁺ Detection shows that the sensor is paired with Cu ²⁺ Has good selectivity in detection.

Example 2

1. Preparation and detection of Cr ⁶⁺ Sensor (2)

1. Silanized carbon quantum dots (CDs @ SiO) ₂ ) Synthesis of (2)

This step was the same as in step 1 of example 1.

2. Preparation of bovine serum albumin stabilized copper nanoclusters (CuNCs)

5mL of 2mg/mLCuCl ₂ The aqueous solution was added to 4mL of a 25mg/mL aqueous solution of Bovine Serum Albumin (BSA) under vigorous stirringAdjusting the pH value to 11-12 with 1M NaOH aqueous solution, continuously stirring for 10min at normal temperature, adding 1mL of 40mg/mL dithiothreitol aqueous solution, adjusting the pH value to be neutral with hydrochloric acid, continuously stirring for 50min, and dialyzing to obtain pure CuNCs.

3. Preparation of Lysozyme-modified Hybrid Nanoparticles (HNPs)

(1)CDs@SiO ₂ The preparation method of @ lyz is the same as in step 3 of example 1.

(2) 1g of CuNCs and 10mg of CDs @ SiO were taken ₂ Mixing the @ lyz uniformly, shaking for 3h at 25 ℃ under a dark condition, and washing with deionized water for several times to obtain hybrid nanoparticles; and dispersing 5mg of hybrid nanoparticles into 1mL of deionized water again, adding 5mL of 0.5mg/mL lysozyme aqueous solution, standing and incubating at 37 ℃ for 40min, centrifuging, and washing with deionized water to obtain HNPs.

4. Preparation of lysozyme mediated core-shell nano composite material (HNPs @ ZIF-8)

This step was the same as in step 4 of example 1.

2. Sensor pair Cr ⁶⁺ Detection of (2)

1. Sensitivity and detection Range

Mixing 100 μ L of Cr with different concentrations of 100 μ L of Cr and 100 μ L of LHNPs @ ZIF-8 ⁶⁺ Mixing the standard solutions, reacting for 1min in a dark place, and then measuring the fluorescence spectrum of the reaction solution by using a fluorescence spectrophotometer. As shown in FIG. 9 (A), with Cr excitation at 300nm ⁶⁺ The concentration is increased, the fluorescence intensity of the carbon quantum dots is basically kept unchanged, and the fluorescence intensity of the copper nanoclusters is gradually weakened. According to different concentrations of Cr ⁶⁺ The ratio of the fluorescence intensity of the copper nanoclusters of the reaction solution corresponding to the standard solution under the wavelength of 610nm to the fluorescence intensity of the carbon quantum dots under the wavelength of 470nm is taken as Log C _Cr ⁶⁺ Standard curve of the ratio of fluorescence intensity of copper nanoclusters to that of carbon quantum dots and calculation of regression equation, as shown in FIG. 9 (B), at Cr ⁶⁺ When the concentration range is 0.15-100 mu M, the linear equation is as follows:

F _CuNCs/CDs ＝-0.2360Log C _Cr ⁶⁺ +2.1279

in the formula F _CuNCs/CDs Is the ratio of the fluorescence intensity of the copper nanoclusters to the carbon quantum dots, C _Cr ⁶⁺ Is Cr ⁶⁺ Concentration, coefficient of correlation R ² =0.9915, as seen by the correlation coefficient, the ratio of fluorescence intensity to Log C _Cr ⁶⁺ The linear relationship of (a) is good. Tested, cr ⁶⁺ The detection limit was 2nmol/L.

2. Selectivity is selected

Sensor pair Cr ⁶⁺ The selectivity of the detection can be seen in FIG. 10, and other metal ions include 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 in Cr pair ⁶⁺ Has good selectivity.

Example 3

The HNPs @ ZIF-8 obtained in example 1 was dispersed in 2mL of HEPES buffer solution, and the resulting dispersion was uniformly spray-printed on a test paper by a film-drawing gold spraying apparatus, and dried in the dark, after which 0, 0.1. Mu.M, 0.2. Mu.M, 0.4. Mu.M, 0.8. Mu.M, 1. Mu.M, 5. Mu.M, 15. Mu.M, and 20. Mu.M of Cu was dropped onto the test paper, respectively ²⁺ Standing in dark place for 1min, and observing the change of fluorescence color of the test paper under a 365nm ultraviolet lamp. As can be seen from FIG. 11, with Cu ²⁺ The test strip changed color from pink-purple to blue as the concentration increased.

Claims

1. A sensor based on lysozyme-mediated core-shell nano composite material is characterized in that lysozyme is used as a coupling agent to connect silanized carbon quantum dots and metal nanoclusters to obtain hybrid nanoparticles, lysozyme is modified on the surface of the hybrid nanoparticles, and finally the lysozyme-modified hybrid nanoparticles are packaged through ZIF-8 to obtain the lysozyme-mediated core-shell nano composite material; the composite material is directly used as a sensor, or the composite material is uniformly sprayed and printed on cellulose paper to be used as a sensor; wherein the metal nanocluster is a gold nanocluster stabilized by glutathione or a copper nanocluster stabilized by bovine serum albumin.

2. The lysozyme-mediated core-shell nanocomposite-based sensor according to claim 1, wherein: the diameter of the silanized carbon quantum dot is 30-40 nm, and the diameter of the lysozyme-mediated core-shell nano composite material is 200-250 nm.

3. The lysozyme-mediated core-shell nanocomposite-based sensor according to claim 1 or 2, wherein the silanized carbon quantum dots are prepared by a method comprising: ultrasonically dispersing citric acid and ethylenediamine in deionized water, and reacting for 4-6 h at 180-220 ℃ under a closed condition to obtain carbon quantum dots; ultrasonically dispersing the carbon quantum dots in absolute ethyl alcohol, adding ammonia water and ethyl orthosilicate, and stirring for 18-24 hours to obtain silanized carbon quantum dots; wherein the molar ratio of the citric acid to the ethylenediamine is 1.9-1.1, and the volume ratio of the carbon quantum dots to the ammonia water and the tetraethoxysilane is 1-3.

4. The lysozyme-mediated core-shell nanocomposite-based sensor according to claim 1, wherein the glutathione-stabilized gold nanoclusters are prepared by a method comprising: uniformly dispersing glutathione and chloroauric acid in deionized water, heating and stirring at 65-75 ℃ for 20-24 h to obtain a gold nanocluster with stable glutathione; wherein the molar ratio of the chloroauric acid to the glutathione is 1-2.

5. The lysozyme-mediated core-shell nanocomposite-based sensor according to claim 1, wherein the bovine serum albumin-stabilized copper nanoclusters are prepared by a method comprising: under the condition of stirring, adding copper chloride and bovine serum albumin into deionized water, adjusting the pH value to 11-12 by using NaOH, stirring for 10-15 min at normal temperature, adding dithiothreitol, adjusting the pH value to be neutral by using hydrochloric acid, and continuously stirring for 40-60 min to obtain a bovine serum albumin stable copper nano cluster; wherein the mass ratio of the copper chloride to the bovine serum albumin to the dithiothreitol is 1-6.

6. The lysozyme-mediated core-shell nanocomposite-based sensor according to claim 1, wherein the lysozyme-modified hybrid nanoparticles are prepared by a method comprising:

(1) Uniformly dispersing the silanized carbon quantum dots in HEPES buffer solution containing lysozyme and tris (2-carboxyethyl) phosphine hydrochloride, and incubating for 20-60 min at 37 ℃ to obtain aminated silanized carbon quantum dots; the mass ratio of the silanized carbon quantum dots to lysozyme and tris (2-carboxyethyl) phosphine hydrochloride is 1.8-1.2;

(2) Mixing a gold nano cluster with stable glutathione with 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, mixing and oscillating for 20-40 min under a dark condition, then adding aminated silanized carbon quantum dots, and continuing oscillating for 2-4 h under the dark condition to obtain hybrid nano particles; mixing the hybrid nano-particles with a lysozyme aqueous solution, standing and incubating for 2-3 h at 37 ℃, and centrifuging to obtain lysozyme-modified hybrid nano-particles;

or mixing the copper nanoclusters stabilized by the bovine serum albumin with the aminated silanized carbon quantum dots, and oscillating for 2-4 hours under the dark condition to obtain hybrid nanoparticles; mixing the hybrid nano-particles with a lysozyme aqueous solution, standing and incubating for 30-50 min at 37 ℃, and centrifuging to obtain lysozyme-modified hybrid nano-particles;

the mass ratio of the gold nanocluster stabilized by the glutathione to 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide is (1).

7. The lysozyme-mediated core-shell nanocomposite-based sensor according to claim 1, wherein the lysozyme-mediated core-shell nanocomposite is prepared by a method comprising: dispersing lysozyme-modified hybrid nanoparticles in methanol, adding a 2-methylimidazole methanol solution, uniformly mixing, adding a zinc nitrate methanol solution, standing at room temperature for 20-24 hours to enable ZIF-8 to encapsulate the lysozyme-modified hybrid nanoparticles, and thus obtaining a lysozyme-mediated core-shell nano composite material; wherein the mass ratio of the lysozyme modified hybrid nanoparticles to the 2-methylimidazole and the zinc nitrate is 1.

8. The lysozyme-mediated core-shell nanocomposite-based sensor of claim 1 for ratiometric fluorescence detection of Cu ²⁺ Wherein the metal nanoclusters are gold nanoclusters stabilized by glutathione.

9. The lysozyme-mediated core-shell nanocomposite-based sensor of claim 1 for the detection of Cr by ratiometric fluorescence ⁶⁺ Wherein the metal nanoclusters are copper nanoclusters stabilized with bovine serum albumin.