Molecularly imprinted ratiometric fluorescence sensor based on MXene quantum dots as well as preparation method and application of molecularly imprinted ratiometric fluorescence sensor

Abstract

The invention discloses a molecular imprinting ratiometric fluorescence sensor based on MXene quantum dots as well as a preparation method and application of the molecular imprinting ratiometric fluorescence sensor. The molecular imprinting ratio fluorescence sensor is a core-shell structure polymer obtained by polymerizing blue fluorescent MXene quantum dots (BMQDs), orange fluorescent MXene quantum dots (OMQDs), histamine, APTES and TEOS, OMQDs are coated with a silicon dioxide layer to obtain OMQDs (at) SiO2, histamine, APTES, TEOS and the like are synthesized into an imprinting layer to be grafted on the outer layer of OMQDs (at) SiO2, and BMQDs are embedded in the imprinting layer to obtain the molecular imprinting ratio fluorescence sensor. The blue / orange MXene quantum dots with high quantum yield are prepared, a molecular imprinting technology is combined, a molecular imprinting ratio fluorescence sensor only based on the MXene quantum dots is constructed, the synthesis method is simple, and specific recognition and rapid intelligent visual detection of histamine can be achieved by combining a smart phone.

Description

Technical Field

[0001] The present invention relates to the field of fluorescence sensing detection technology, and in particular to a molecular imprinting ratio fluorescence sensor based on MXene quantum dots and a preparation method and application thereof. Background Art

[0002] Histamine is one of the most toxic biogenic amines, and its content is often used as an important indicator to evaluate the freshness and degree of corruption of food. If the human body ingests more than 200 mg / kg, it will cause allergic poisoning, and in severe cases it will endanger life. All countries or regions have set limit standards for the content of histamine in food. my country stipulates that the content of histamine in high-histamine fish shall not exceed 40 mg / 100 g. At present, the detection of histamine mainly includes high-performance liquid chromatography (HPLC) and immunoassay. HPLC has the advantages of accuracy and sensitivity, but because histamine has no ultraviolet and fluorescent chromophores, it needs to be measured after complex and cumbersome derivatization, and the instrument is expensive and has high professional and technical requirements, which is difficult to meet the market detection needs of a wide range of large quantities. The immunoassay method is fast and simple, but due to the small molecular weight of histamine, it is difficult to prepare high-affinity antibodies directly against histamine, and derivatization is still required to achieve detection, and there are often problems such as false positives and cross-reactivity. Therefore, it is urgent to develop an efficient, sensitive, and easy-to-operate rapid detection method for on-site monitoring of the content of histamine in food.

[0003] Fluorescence sensing analysis is widely used in food safety, bioanalysis and other fields due to its high sensitivity, simple operation and high degree of visualization. Compared with single-wavelength fluorescence analysis, ratiometric fluorescence sensors can achieve self-calibration, overcome the influence of external environment, background noise and substrate concentration, and thus be more accurate and sensitive. In addition, the most attractive feature of ratiometric fluorescence sensors to researchers is that their color changes are easier to distinguish and the visual detection results are more reliable. In recent years, the popularity of smartphones, the continuous improvement of mobile phone camera functions and the widespread use of detection applications have provided new development opportunities for ratiometric fluorescence sensing to move towards on-site detection. The smartphone-based fluorescence sensing platform can not only give full play to the advantages of smartphones such as lightness, convenience, low cost and simple operation, but also make use of the principles and applicability of traditional sensing methods to make up for the shortcomings of traditional instruments in terms of detection timeliness.

[0004] The choice of fluorescent materials directly affects the interaction between the sensor and the target analyte, the optical conductivity of the sensing interface, and the service life of the sensor. As an emerging fluorescent nanomaterial, MXene quantum dots are known as the most anticipated two-dimensional materials in the 21st century due to their excellent water solubility, good biocompatibility, rich adjustable groups, and excellent luminescence ability. In recent years, MXene quantum dots have been gradually used to construct fluorescent sensors, but most of them are based on the single emission fluorescence method of blue MXene quantum dots. There are no reports on ratio fluorescence sensors and detection methods based on blue / orange MXene quantum dots.

[0005] In addition, the selectivity and response time of fluorescence sensing detection are closely related to the recognition element. Molecularly imprinted polymers (MIPs) are high molecular weight polymers that are prepared based on molecular imprinting technology by simulating the recognition of antibodies and antigens and completely match the template molecule in spatial structure and binding sites. They have excellent molecular recognition performance, excellent operational stability and low production cost, making them ideal recognition elements for constructing fluorescence sensors and are widely used. Molecularly imprinted fluorescence sensors combine the high selectivity of MIPs and the high sensitivity of fluorescence detection, and have unique advantages in the separation and detection of trace samples under complex matrix conditions. Summary of the invention

[0006] The purpose of the present invention is to provide a molecular imprinting ratio fluorescence sensor based on MXene quantum dots and a preparation method and application thereof.

[0007] Another object of the present invention is to provide an application of the MXene-based molecular imprinting ratio fluorescence sensor in the selective detection of histamine.

[0008] The present invention is conceived as follows: by synthesizing MXene quantum dots emitting two colors through different strategies, combining the dual-color emitting MXene quantum dots with molecular imprinting technology with specific recognition ability, a molecular imprinting ratio fluorescence sensor based on MXene quantum dots is constructed. When histamine is present, the fluorescence color of the sensor changes with the histamine concentration. At the same time, the red, green and blue (RGB) information of the fluorescence image is analyzed in combination with the image processing software of the smart phone, and the fluorescence data is visualized to achieve the purpose of rapid on-site detection of histamine.

[0009] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a method for preparing a molecular imprinting ratio fluorescence sensor based on MXene quantum dots, comprising the following steps:

[0010] A. Preparation of MXene nanosheets;

[0011] B. Preparation of blue fluorescent MXene quantum dots BMQDs;

[0012] C. Preparation of orange fluorescent MXene quantum dots OMQDs;

[0013] D. OMQDs were coated with a silica layer to obtain OMQDs@SiO2, and then OMQDs@SiO2, BMQDs, target molecules, APTES (3-aminopropyltriethoxysilane) and TEOS (tetraethyl orthosilicate) were mixed for polymerization reaction, and the target molecules were eluted from the reaction products to obtain a molecular imprinting ratio fluorescence sensor based on MXene quantum dots, denoted as OMQDs@BMQDs@MIPs.

[0014] The target molecules are biogenic amines, including but not limited to histamine, putrescine, cadaverine, tryptamine, and the like.

[0015] In the present invention, the MXene nanosheets are preferably Ti3C2 nanosheets.

[0016] Furthermore, the target molecule is histamine.

[0017] Step A uses LiF and concentrated hydrochloric acid as etchants and adopts a mild etching method to prepare Ti3C2 nanosheets. The preparation method includes:

[0018] Add 1.0-4.0g LiF to the lining of a polytetrafluoroethylene reactor, then add 10-50mL 6-10mol / L HCl, and cover the lid; then place the reactor in a constant temperature water bath at 30-50°C, with a rotation speed of 500-800rpm, and stir for 10-20min (preferably 15min) to allow the system to react fully. When the temperature is stable, add 1.0-3.0g Ti3AlC2 powder to the reactor lining in small amounts and multiple times, and wait for the bubbles to completely dissipate before each addition; then cover the lid and seal the reaction for 36-60h, then centrifuge at 3000-4000rpm for 1-2min; then wash the reaction mixture 3-5 times with 1-3mol / L HCl to eliminate the incompletely reacted LiF; the precipitate is then centrifuged and washed with deionized water until the pH value of the supernatant is about 5.8-6.2, and then filtered with a filter membrane to obtain Ti3C2 nanosheets.

[0019] Further, in step B, Ti3C2 nanosheets are treated by ultrasonic hydrothermal method and doped with ethylenediamine as a nitrogen source to prepare BMQDs, and the preparation method includes:

[0020] 0.1-0.5 g of synthesized Ti3C2 nanosheets are uniformly dispersed in 10-30 mL of deionized water, and ultrasonicated for 1-3 h to obtain a few-layer Ti3C2 nanosheet dispersion; the dispersion is transferred to a 50 mL Teflon reactor liner, 2-6 mL of ethylenediamine is added, and the reactor is placed in a reactor to react at 120-180 ° C for 4-10 h; the reaction mixture is centrifuged at 8000-10000 rpm for 20-30 min, the supernatant is taken, filtered with a filter membrane, and then transferred to a dialysis bag with a molecular weight cutoff of 500-2000 Da for 1-5 h. The reagent used for dialysis is ultrapure water, and the dialyzed liquid is collected to obtain blue fluorescent MXene quantum dots BMQDs.

[0021] Further, step C uses mixed acid ultrasonic treatment of Ti3C2 nanosheets, and then doping o-phenylenediamine solvothermal method to prepare OMQDs, the preparation method includes:

[0022] Take 0.5-2 mL of 98.3% (mass percentage) concentrated sulfuric acid and add it to 1.5-6 mL of 68% (mass percentage) concentrated nitric acid and mix them evenly. Then add 0.1-0.5 g of Ti3C2 powder to the mixed acid solution and ultrasonically treat it for 1-3 hours to obtain a Ti3C2 solution. Then, add 10-30 mL of deionized water to the Ti3C2 solution, cool it to 20-25°C in an ice bath, and then adjust the pH value to about 6.8-7.2 with 2-5 mol / L NaOH solution. Then transfer the solution to a centrifuge tube. , centrifuge at 6000-10000rpm for 3-10min, discard the supernatant after centrifugation, transfer the precipitate to a 50mL polytetrafluoroethylene reactor lining, add 15-30mL of deionized water and 5-15mL of methanol, and then add 0.1-0.5g of o-phenylenediamine. After the o-phenylenediamine is completely dissolved, react at 150-200℃ for 5-10h; after the reaction, transfer the solution in the reactor lining to a centrifuge tube, centrifuge at 8000-10000rpm for 5-20min, take the supernatant, and obtain orange fluorescent MXene quantum dots OMQDs.

[0023] Preferably, in steps B and C, the ultrasonic treatment time is 2 h, the ultrasonic power is 100 W, and the frequency is 40 KHz.

[0024] Furthermore, in step D, a molecular imprinting ratio fluorescence sensor is prepared by an improved sol-gel method, and the preparation method comprises:

[0025] 1) Adoption OMQDs@SiO2 was prepared by coating a layer of silicon dioxide on OMQDs.

[0026] Take 0.5-2 mL of OMQDs solution and add it to 5-15 mL of anhydrous ethanol, disperse the OMQDs by ultrasound, then add 0.5-2 mL of TEOS to the dispersed solution in a continuous droplet manner under stirring conditions, and stir for 15-30 minutes; then add 100-250 μL of 25% (mass percentage) concentrated ammonia water and stir continuously for 18-36 hours; after the reaction is completed, transfer the solution to a centrifuge tube, centrifuge at 6000-10000 rpm for 3-10 minutes, and then wash it with ethanol and deionized water once each in turn. After centrifugal washing, take out the precipitate and vacuum dry it (such as vacuum drying at 35-80°C for 4-10 hours, preferably at a vacuum drying temperature of 60°C) to obtain OMQDs@SiO2;

[0027] 2) Take 10-25 mg of OMQDs@SiO2 and 0.5-2 mL of BMQDs solution and disperse them in 10-25 mL of anhydrous ethanol, then add 10-25 mg of histamine standard as a template molecule and 50-200 μL of APTES as a functional monomer, stir for 0.5-1.5 h to prepolymerize the template molecule and the functional monomer; then add 100-300 μL of TEOS, and then add 50-200 μL of 25% concentrated ammonia water, stir at room temperature in the dark for 18-36 h; after the reaction is completed, use ethanol-acetic acid mixture as eluent to remove histamine by ultrasonic centrifugation; then vacuum dry the precipitate (such as vacuum drying at 35-60°C for 10-24 h, preferably vacuum drying temperature 60°C), grind with an agate mortar and sieve to obtain OMQDs@BMQDs@MIPs.

[0028] Preferably, the volume ratio of ethanol to acetic acid in the ethanol-acetic acid mixture is 9:1 (both are analytically pure).

[0029] Furthermore, in step D, the ultrasonic power is preferably 100 W and the frequency is preferably 40 KHz.

[0030] In a preferred embodiment of the present invention, the molecular imprinting ratio fluorescence sensor based on Mxene quantum dots is prepared as follows:

[0031] Step A uses LiF and concentrated hydrochloric acid as etchants and adopts a mild etching method to prepare Ti3C2 nanosheets. The preparation method includes: adding 3.2g LiF to the inner liner of a polytetrafluoroethylene reactor, and then adding 40mL 8mol / L HCl, and covering the lid. Then place the reactor liner in a 40°C constant temperature water bath, the speed is 600rpm, and stir for 15min to allow the system to fully react. When the temperature stabilizes at 40°C, add 2.0g Ti3A1C2 to the inner liner of the reactor in small amounts and multiple times, waiting for the bubbles to completely dissipate before each addition. Then cover the lid and seal the reaction for 48h, and then centrifuge at 3500rpm for 1min. Then wash the reacted mixture three times with 2mol / L HCl to eliminate the incompletely reacted LiF. The precipitate is then centrifuged and washed with deionized water until the pH value of the supernatant reaches about 6.0, and then filtered with a 0.22μm water filter membrane to obtain Ti3C2MXene nanosheets;

[0032] Step B uses ultrasonic hydrothermal method to treat Ti3C2 nanosheets and dopes them with ethylenediamine as nitrogen source to prepare BMQDs. The preparation method includes: uniformly dispersing 0.2g of synthesized Ti3C2 nanosheets in 20mL of deionized water, ultrasonicating for 2h, and obtaining a dispersion of few-layer Ti3C2 nanosheets. The dispersion is transferred to a 50mL Teflon reactor liner, 5mL of ethylenediamine is added, and the reactor is placed in a stainless steel reactor and reacted at 150°C for 6h. The reaction mixture is centrifuged at 10000rpm for 30min, the supernatant is taken, filtered with a 0.22μm water filter membrane, and then transferred to a dialysis bag with a retention capacity of 1000Da for 3h. The dialysis reagent is ultrapure water, and the dialyzed liquid is collected to obtain blue fluorescent BMQDs;

[0033] Step C uses mixed acid ultrasonic treatment of Ti3C2 nanosheets and then doping o-phenylenediamine solvent thermal method to prepare OMQDs. The preparation method includes: taking 1mL of concentrated sulfuric acid and adding it to 3mL of concentrated nitric acid to mix evenly, then adding 0.2g of Ti3C2 powder to the above mixed solution, and ultrasonic treatment for 2h. After that, 25mL of deionized water is added to the Ti3C2 solution, which is cooled to 25°C under ice bath conditions, and then the pH value is adjusted to about 7.0 using 3mol / L NaOH solution. Next, the solution is transferred to a centrifuge tube and centrifuged at 8000rpm for 5min. After centrifugation, the supernatant is discarded, and the precipitate is transferred to a 50mL polytetrafluoroethylene reactor liner, 20mL of deionized water and 10mL of methanol are added, and then 0.2g of o-phenylenediamine is added. After the o-phenylenediamine is completely dissolved, it is reacted at 180°C for 6h. After the reaction, the solution in the reactor liner was transferred to a centrifuge tube and centrifuged at 10000 rpm for 10 min. The supernatant was taken to obtain orange-yellow OMQDs.

[0034] Step D: using an improved sol-gel method to prepare a molecular imprinting ratio fluorescence sensor, the preparation method comprising: using The method was used to coat a layer of silica on OMQDs to obtain OMQDs@SiO2. 1 mL of OMQDs solution was added to 10 mL of anhydrous ethanol, and the OMQDs were dispersed by ultrasound. Then, 1 mL of tetraethyl orthosilicate was added to the dispersed solution in a continuous droplet manner under stirring conditions and stirred for 30 min. Then, 200 μL of 25% concentrated ammonia water was added and stirred continuously for 24 h. After 24 h of reaction, the solution was transferred to a centrifuge tube, centrifuged at 8000 rpm for 5 min, and then washed with ethanol and deionized water respectively. After centrifugal washing, the precipitate was taken out and vacuum dried at 40 ° C for 6 h to obtain OMQDs@SiO2. 20 mg of OMQDs@SiO2 and 1 mL of BMQDs solution were dispersed in 20 mL of anhydrous ethanol, and then 20 mg of histamine standard was added as a template molecule and 150 μL of APTES as a functional monomer. The mixture was stirred for 1 h to prepolymerize the template molecule and the functional monomer. Then, 260 μL of TEOS was added, followed by 150 μL of 25% concentrated ammonia water, and stirred at 25°C in the dark for 24 h. After the reaction, ethanol-acetic acid (v:v=9:1) was used as an eluent to remove the template molecules by ultrasonic centrifugation. The precipitate was then vacuum dried at 60°C for 12 h, ground with an agate mortar, and sieved to obtain the product.

[0035] In a second aspect, the present invention provides a molecular imprinting ratio fluorescence sensor based on MXene quantum dots prepared according to the method.

[0036] In a third aspect, the present invention provides any of the following applications of the fluorescent sensor:

[0037] 1) Used for separation and enrichment of target molecules;

[0038] 2) Used for qualitative and quantitative detection of target molecules;

[0039] 3) Used for preparing target molecule detection reagents or kits;

[0040] 4) Intelligent devices for preparing target molecule detection.

[0041] In a fourth aspect, the present invention provides a method for detecting histamine, characterized in that the method comprises: adding the fluorescent sensor prepared according to the above method to the sample solution to be tested, after oscillating reaction at room temperature, detecting using a fluorescence spectrometer, or using an ultraviolet dark box to obtain a fluorescent image of the sample solution to be tested, and then using color analysis software of a smart phone to obtain RGB information for visual detection.

[0042] Furthermore, the fluorescence detection conditions are as follows: the excitation and emission scanning slits are both set to 5-20 nm (preferably 10 nm), the excitation light wavelength is 310-410 nm, and the scanning emission wavelength is 400-800 nm.

[0043] The linear detection range of fluorescence detection for histamine in the sample solution is 1-60 μM, and the detection limit is 21.9 nM.

[0044] The linear detection range of smartphone visual detection for histamine in the sample solution is 10-60μM, and the detection limit is 92.2nM.

[0045] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0046] (i) The present invention synthesizes high quantum yield blue-emitting and orange-emitting MXene quantum dots using only one precursor material through clever processing and doping strategies, expanding the application of multicolor MXene quantum dots in the field of sensing analysis.

[0047] (ii) The present invention constructs a ratiometric fluorescence sensor using blue and orange MXene quantum dots. The two fluorescence signals can change dynamically and synchronously, which can eliminate the influence of interference factors in complex matrices. Compared with conventional single fluorescence emission fluorescence sensors, it has stronger anti-interference ability.

[0048] (III) The present invention combines molecular imprinting technology with ratiometric fluorescence technology based on MXene quantum dots to achieve specific adsorption and highly sensitive and rapid detection of histamine. The detection effect is stable and the application prospects are broad.

[0049] (IV) The present invention can visualize and analyze the histamine content in samples by combining with a smart phone, providing new ideas for the development of smart devices for on-site monitoring of histamine. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a scanning electron microscope image of Ti3C2 nanosheets in a preferred embodiment of the present invention.

[0051] Figure 2 Graph showing the fluorescence excitation and emission spectra of BMQDs in a preferred embodiment of the present invention.

[0052] Figure 3 Graph showing the fluorescence excitation and emission spectra of OMQDs in a preferred embodiment of the present invention.

[0053] Figure 4 This is a transmission electron microscope image of the molecular imprinting ratiometric fluorescence sensor OMQDs@BMQDs@MIPs in a preferred embodiment of the present invention.

[0054] Figure 5These are images related to the optimization of conditions for the molecular imprinting ratiometric fluorescence sensor OMQDs@BMQDs@MIPs in a preferred embodiment of the present invention.

[0055] Figure 6 This is the fluorescence response image of the molecular imprinting ratiometric fluorescence sensor OMQDs@BMQDs@MIPs to different concentrations of histamine in a preferred embodiment of the present invention.

[0056] Figure 7 Schematic diagram, standard curve and CIE chromaticity diagram of the molecular imprinting ratio fluorescence sensor OMQDs@BMQDs@MIPs combined with a smartphone for detecting histamine in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0057] The invention provides a molecular imprinting ratio fluorescence sensor for detecting histamine in fish food. The molecular imprinting ratio fluorescence sensor is a core-shell structure polymer obtained by polymerizing BMQDs, OMQDs, histamine, 3-aminopropyltriethoxysilane (APTES) and tetraethyl orthosilicate (TEOS); the OMQDs are coated with a silicon dioxide layer to obtain silicon-coated OMQDs; a synthetic imprinting layer of the histamine, 3-aminopropyltriethoxysilane and tetraethyl orthosilicate is grafted on the outer layer of the silicon-coated OMQDs; the BMQDs are embedded in the imprinting layer; and after eluting the histamine, the molecular imprinting ratio fluorescence sensor OMQDs@BMQDs@MIPs is obtained.

[0058] The present invention provides a method for preparing a molecular imprinting ratio fluorescence sensor for detecting histamine in fish food. The method comprises: coating OMQDs with silicon dioxide to obtain OMQDs@SiO2; then polymerizing a molecular imprinting layer on the surface of OMQDs@SiO2 using histamine as a template molecule, 3-aminopropyltriethoxysilane as a functional monomer, and ethyl orthosilicate as a crosslinking agent, and embedding BMQDs in the molecular imprinting layer to form a spherical material with a core-shell structure.

[0059] The preparation method of the molecular imprinting ratio fluorescence sensor OMQDs@BMQDs@MIPs mainly comprises the following steps:

[0060] Step 1: Preparation of Ti3C2 nanosheets;

[0061] Step 2: Preparation of blue fluorescent MXene quantum dots BMQDs;

[0062] Step 3: Preparation of orange fluorescent MXene quantum dots OMQDs;

[0063] Step 4: Mix OMQDs@SiO2, BMQDs, histamine, 3-aminopropyltriethoxysilane (polymerization monomer) and tetraethyl orthosilicate (cross-linking agent) to undergo polymerization reaction, and elute histamine from the reaction product to obtain a MQDs-based molecular imprinting ratio fluorescence sensor, denoted as OMQDs@BMQDs@MIPs.

[0064] The orange OMQDs are located in the inner layer as a fluorescent reference signal, and the blue BMQDs are located in the outer molecular imprinting layer as a fluorescent response signal.

[0065] Further, step 1 uses LiF and concentrated hydrochloric acid as etchants, and a mild etching method is used to prepare Ti3C2 nanosheets. The preparation method includes: adding 3.2g LiF to the inner liner of a polytetrafluoroethylene reactor, and then adding 40mL 8mol / L HCl, and covering the lid. Then place the reactor liner in a 40°C constant temperature water bath, the speed is 600rpm, and stir for 15min to allow the system to fully react. When the temperature stabilizes at 40°C, add 2.0g Ti3AlC2 to the inner liner of the reactor in small amounts and multiple times, waiting for the bubbles to completely dissipate before each addition. Then cover the lid and seal the reaction for 48h, and then centrifuge at 3500rpm for 1min. Then wash the reacted mixture three times with 2mol / L HCl to eliminate the incompletely reacted LiF. The precipitate was then washed by centrifugation with deionized water until the pH value of the supernatant reached about 6.0, and then filtered with a 0.22 μm water filter membrane. After vacuum drying at 40°C for 12 h, a black powder sample was obtained and stored at 4°C in the dark.

[0066] Furthermore, step 2 uses ultrasonic hydrothermal method to treat Ti3C2 nanosheets and dopes ethylenediamine as a nitrogen source to prepare BMQDs. The preparation method includes: 0.2g of synthesized Ti3C2 nanosheets are uniformly dispersed in 20mL of deionized water, ultrasonicated for 2h, and a few-layer Ti3C2 nanosheet dispersion is obtained. The dispersion is transferred to a 50mL Teflon reactor liner, 5mL of ethylenediamine is added, and a stainless steel reactor is placed, and the reaction is carried out at 150°C for 6h. The reaction mixture is centrifuged at 10000rpm for 30min, the supernatant is taken, filtered with a 0.22μm water filter membrane, and then transferred to a dialysis bag with a retention capacity of 1000Da for 3h, and the dialyzed liquid is collected and stored at 4°C in the dark.

[0067] Furthermore, step three uses mixed acid ultrasonic treatment of Ti3C2 nanosheets and then doping o-phenylenediamine solvent thermal method to prepare OMQDs, and the preparation method includes: taking 1mL of concentrated sulfuric acid and adding it to 3mL of concentrated nitric acid to mix evenly, then adding 0.2g of Ti3C2 powder to the above mixed solution, and ultrasonic treatment for 2h. After that, 25mL of deionized water is added to the Ti3C2 solution, which is cooled to 25°C under ice bath conditions, and then the pH value is adjusted to about 7.0 using 3mol / L NaOH solution. Next, the solution is transferred to a centrifuge tube and centrifuged at 8000rpm for 5min. After centrifugation, the supernatant is discarded, and the precipitate is transferred to a 50mL polytetrafluoroethylene reactor liner, 20mL of deionized water and 10mL of methanol are added, and then 0.2g of o-phenylenediamine is added. After the o-phenylenediamine is completely dissolved, it is reacted at 180°C for 6h. After the reaction was completed, the solution in the reactor liner was transferred to a centrifuge tube, centrifuged at 10,000 rpm for 10 min, and the supernatant was collected and stored at 4°C in the dark.

[0068] Furthermore, in step 4, a molecular imprinting ratio fluorescence sensor is prepared by an improved sol-gel method, and the preparation method comprises: using The method was used to coat a layer of silica on OMQDs to obtain OMQDs@SiO2. 1 mL of OMQDs solution was added to 10 mL of anhydrous ethanol, and the OMQDs were dispersed by ultrasound. Then, 1 mL of tetraethyl orthosilicate was added to the dispersed solution in a continuous droplet manner under stirring conditions and stirred for 30 min. Then, 200 μL of 25% concentrated ammonia water was added and stirred continuously for 24 h. After 24 h of reaction, the solution was transferred to a centrifuge tube, centrifuged at 8000 rpm for 5 min, and then washed with ethanol and deionized water respectively. After centrifugal washing, the precipitate was taken out and vacuum dried at 40 ° C for 6 h to obtain OMQDs@SiO2. 20 mg of OMQDs@SiO2 and 1 mL of BMQDs solution were dispersed in 20 mL of anhydrous ethanol, and then 20 mg of histamine standard was added as a template molecule and 150 μL of APTES as a functional monomer. The mixture was stirred for 1 h to prepolymerize the template molecule and the functional monomer. Then, 260 μL of TEOS was added, followed by 150 μL of 25% concentrated ammonia water, and stirred at 25°C in the dark for 24 h. After the reaction, ethanol-acetic acid (v:v=9:1) was used as an eluent to remove the template molecules by ultrasonic centrifugation. The precipitate was then vacuum dried at 60°C for 12 h, ground with an agate mortar, sieved, and stored at 4°C in the dark for later use. The prepared molecular imprinting ratio fluorescence sensor OMQDs@BMQDs@MIPs was used to detect histamine in fish food.

[0069] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0070] Example 1 Preparation of molecular imprinted ratiometric fluorescence sensor OMQDs@BMQDs@MIPs

[0071] The invention provides a molecular imprinting ratio fluorescence sensor for detecting histamine in fish products. The molecular imprinting ratio fluorescence sensor is a core-shell structure polymer obtained by polymerizing BMQDs, OMQDs, histamine, 3-aminopropyltriethoxysilane and tetraethyl orthosilicate. A synthetic molecular imprinting layer of histamine, 3-aminopropyltriethoxysilane and tetraethyl orthosilicate is grafted on an outer layer of OMQDs@SiO2, and the BMQDs are embedded in the imprinting layer. After ultrasonic centrifugation and elution, the molecular imprinting ratio fluorescence sensor OMQDs@BMQDs@MIPs is obtained.

[0072] The OMQDs are located in the inner core as a fluorescent reference signal, and the BMQDs are located in the outer layer as a fluorescent response signal. The synthesized polymer can timely, quickly and accurately identify and detect histamine in fish products.

[0073] This embodiment provides a method for preparing a molecular imprinting ratiometric fluorescence sensor for detecting histamine in fish products, which mainly includes the following steps:

[0074] Step 1: Preparation of Ti3C2 nanosheets;

[0075] Step 2: Preparation of blue fluorescent MXene quantum dots BMQDs;

[0076] Step 3: Preparation of orange fluorescent MXene quantum dots OMQDs;

[0077] Step 4: Use a silica layer to coat OMQDs to prepare OMQDs@SiO2, graft a molecular imprinting layer on the surface of OMQDs@SiO2, embed BMQDs in the molecular imprinting layer, and construct a fragment imprinted ratio fluorescence sensor OMQDs@BMQDs@MIPs.

[0078] Step 1: Use LiF and concentrated hydrochloric acid as etchants to prepare Ti3C2 nanosheets using a mild etching method, specifically: add 3.2g LiF to the inner liner of a polytetrafluoroethylene reactor, then add 40mL 8mol / L HCl, and cover the lid. Then place the reactor liner in a 40℃ constant temperature water bath and stir at 600rpm for 15min. When the temperature stabilizes at 40℃, add 2.0g Ti3AlC2 to the reactor liner in small amounts and multiple times, then cover the lid and seal the reaction for 48h, and centrifuge at 3500rpm for 1min. Then wash the reacted mixture three times with 2mol / L HCl to eliminate the incompletely reacted LiF. The precipitate is then centrifuged and washed with deionized water until the pH value of the supernatant reaches about 6.0, then filtered with a 0.22μm water filter membrane, and vacuum dried at 40℃ for 12h to obtain a black powder sample, which is stored at 4℃ in the dark. The diameter of the prepared Ti3C2 nanosheets is 5.0-10.0 μm, as shown in the electron microscope image. Figure 1 .

[0079] Step 2: Treat Ti3C2 nanosheets with ultrasonic hydrothermal method and dope them with ethylenediamine as nitrogen source to prepare BMQDs. Specifically, 0.2g of synthesized Ti3C2 nanosheets are evenly dispersed in 20mL of deionized water, ultrasonicated for 2h to obtain a few-layer Ti3C2 nanosheet dispersion, transferred to a 50mL Teflon reactor liner, added 5mL of ethylenediamine, placed in a stainless steel reactor, and reacted at 150°C for 6h. The reaction mixture was centrifuged at 10000rpm for 30min, the supernatant was taken, filtered with a 0.22μm water filter membrane, and then transferred to a dialysis bag with a retention capacity of 1000Da for 3h, the dialyzed liquid was collected, and stored at 4°C in the dark. The particle size of the prepared BMQDs is about 3.1nm, and it exhibits blue fluorescence under a 365nm ultraviolet lamp. The fluorescence spectrum is shown in Figure 2 .

[0080] Step 3: Prepare OMQDs by solvothermal method by doping o-phenylenediamine with mixed acid and ultrasonic treatment of Ti3C2 nanosheets. Specifically, 1 mL of concentrated sulfuric acid is added to 3 mL of concentrated nitric acid and mixed evenly. Then, 0.2 g of Ti3C2 powder is added to the mixed solution and ultrasonically treated for 2 h. After that, 25 mL of deionized water is added to the Ti3C2 solution, which is cooled to 25 ° C in an ice bath, and then the pH value is adjusted to about 7.0 using 3 mol / L NaOH solution. Next, the solution is transferred to a centrifuge tube and centrifuged at 8000 rpm for 5 min. After centrifugation, the supernatant is discarded, and the precipitate is transferred to a 50 mL polytetrafluoroethylene reactor liner, 20 mL of deionized water and 10 mL of methanol are added, and then 0.2 g of o-phenylenediamine is added. After the o-phenylenediamine is completely dissolved, it is reacted at 180 ° C for 6 h. After the reaction, the solution in the reactor liner was transferred to a centrifuge tube, centrifuged at 10000 rpm for 10 min, and the supernatant was taken and stored at 4°C in the dark. The particle size of the prepared OMQDs was about 7.4 nm, and it showed orange fluorescence under 365 nm ultraviolet light. The fluorescence spectrum is shown in Figure 3 .

[0081] Furthermore, in step 4, a molecular imprinting ratio fluorescence sensor is prepared by using an improved sol-gel method, specifically: using The method was used to coat a layer of silica on OMQDs to obtain OMQDs@SiO2. 1 mL of OMQDs solution was added to 10 mL of anhydrous ethanol, and the OMQDs were dispersed by ultrasound. Then, 1 mL of tetraethyl orthosilicate was added to the dispersed solution in a continuous droplet manner under stirring conditions and stirred for 30 min. Then, 200 μL of 25% concentrated ammonia water was added and stirred continuously for 24 h. After 24 h of reaction, the solution was transferred to a centrifuge tube, centrifuged at 8000 rpm for 5 min, and then washed with ethanol and deionized water respectively. After centrifugal washing, the precipitate was taken out and vacuum dried at 40 ° C for 6 h to obtain OMQDs@SiO2. 20 mg of OMQDs@SiO2 and 1 mL of BMQDs solution were dispersed in 20 mL of anhydrous ethanol, and then 20 mg of histamine standard was added as a template molecule and 150 μL of APTES as a functional monomer. The mixture was stirred for 1 h to prepolymerize the template molecule and the functional monomer. Then add 260 μL of TEOS, and then add 150 μL of 25% concentrated ammonia water, and stir at 25°C in the dark for 24 hours. After the reaction, use ethanol-acetic acid (v:v=9:1) as the eluent to remove the template molecules by ultrasonic centrifugation. Then, vacuum dry the precipitate at 60°C for 12 hours, grind it with an agate mortar and sieve it, and store it at 4°C in the dark for later use. The transmission electron microscopy image of the prepared molecular imprinting ratio fluorescence sensor OMQDs@BMQDs@MIPs is shown in Figure 4 .

[0082] Furthermore, the detection conditions of the ratio fluorescence sensor were optimized, specifically: the excitation wavelength, incubation time, pH value of the reaction system and concentration of OMQDs@BMQDs@MIPs during the fluorescence test were optimized. First, the excitation wavelength of BMQDs, which is the outer response signal, was optimized to ensure that the fluorescence emission ability of the sensor was maximized. We tested the fluorescence emission spectrum of BMQDs in the range of 310N410nm and recorded the excitation wavelength with the highest fluorescence intensity. The other three conditions of the molecular imprinting ratio fluorescence sensor were optimized by single-factor experiments. Among them, the incubation time was set to 0-30min, and the fluorescence spectrum was measured every 5min; the fluorescence spectrum of the sensor was tested in the pH range of 1N13; and the fluorescence spectrum of the sensor was tested at concentrations of 0.1, 0.2, 0.5, 1.0, and 2.0mg / mL. The above tests were all compared with blank samples, and the degree of quenching was used as an indicator to evaluate the response performance of the sensor. Related images of the optimization of the conditions for the molecular imprinting ratio fluorescence sensor OMQDs@BMQDs@MIPs are shown in Figure 5 (A~D).

[0083] When using, take 200 μL of 0.5 mg / mL MIPs@BCDs / RCDs@SiO2, add it to 200 μL of histamine standard solution and sample solution of different concentrations, react in a constant temperature oscillator at 25°C for 20 minutes, take it out, centrifuge it at 5000 rpm, take the supernatant after adsorption, and measure its fluorescence emission spectrum at an excitation wavelength of 380 nm using a fluorescence spectrometer. Qualitative and quantitative analysis is performed based on the relevant fluorescence spectrum and standard curve. Figure 6 The fluorescence response diagram of standard solutions of histamine at different concentrations shows that as the concentration of histamine increases, the fluorescence signal intensity of the inner OMQDs at 550 nm remains basically unchanged, while the fluorescence intensity of the outer BMQDs at 468 nm gradually decreases after the outer molecular imprinting layer binds to histamine. 468 / I 550 Establish a standard curve.

[0084] The excitation and emission scanning slits of the fluorescence spectrometer were both set to 10 nm, the excitation light wavelength range was set to 310-410 nm, and the scanning emission wavelength range was 400-800 nm.

[0085] The linear detection range of histamine in the sample solution is 1-60 μM, and the detection limit is 21.9 nM.

[0086] The visualization method used was consistent with the above conditions. After the sensor and the sample reacted completely, they were taken out of the UV dark box and fluorescent images were taken under 365nm UV light. The RGB color parameters of the image were read using a smartphone, and the R / B value was used as a parameter to establish a correlation with the histamine concentration. Figure 7 To visualize the implementation principle of the method, the fluorescence response standard curve and the CIE chromaticity diagram were shown. The linear range of the method was 10-60 μM, and the detection limit was 92.2 nM.

[0087] Example 2 Spiked recovery experiment and real sample detection

[0088] In order to verify the practicality of the molecular imprinting ratio fluorescence sensor prepared in Example 1, the method was verified using mackerel, frozen Atlantic cod meat and canned tuna as three different types of real samples. The real samples were subjected to spike recovery tests with three concentration gradients of histamine standards (0μM, 25μM, 50μM), and the accuracy was verified by HPLC. Take 2.0g of fish sample, add 10mL of 5% trichloroacetic acid, homogenize and ultrasonically treat for 10min, then centrifuge at 8000rpm for 3min, take the supernatant and add 5mL of n-hexane to remove fat to obtain a sample extract, which is directly used for fluorescence detection. Afterwards, the real sample extract and internal standard sample used for HPLC testing were derivatized, and the derivatization reagent was a 10mg / L dansyl chloride solution. The working conditions of HPLC detection are as follows: the chromatographic column is a C18 column (column length 250 mm, column inner diameter 4.6 mm, column filler particle size 5 μm), the ultraviolet detection wavelength is 254 nm, the injection volume is 20 μL, the column temperature is 35° C., the mobile phase A is 90% acetonitrile / 10% ammonium acetate solution containing 0.1% acetic acid at a concentration of 0.01 mol / L. The mobile phase B is 10% acetonitrile / 90% ammonium acetate solution containing 0.1% acetic acid at a concentration of 0.01 mol / L, and the flow rate is 0.8 mL / min. The fluorescence detection results and the liquid chromatography detection results are shown in Table 1.

[0089] Table 1 Determination results of histamine content in different fish samples by MIPs@BCDs / RCDs@SiO2

[0090]

[0091] ND: Not detected.

[0092] From the experimental results of spike recovery, the spike recovery rate of the ratio fluorescence sensor of the present invention for three samples ranges from 96.52% to 105.32%, and the relative standard deviation ranges from 1.57% to 5.57%. Moreover, the detection results of histamine by this method are highly consistent with the results verified by the HPLC method, and the t-test (P>5%) shows that there is no significant difference between the two methods at a confidence level of 95%. In addition, compared with instrumental analysis methods such as HPLC, the sample pretreatment of this method is simple, and only a simple extraction step is required, without complicated and time-consuming derivatization treatment.

[0093] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing a molecularly imprinted ratiometric fluorescence sensor based on MXene quantum dots, characterized in that: The following steps are involved: A. Preparation of MXene nanosheets; B. Preparation of blue fluorescent MXene quantum dots BMQDs; C. Preparation of orange fluorescent MXene quantum dots OMQDs; D. The OMQDs were coated with a silica layer to obtain OMQDs@SiO2, and then the OMQDs@SiO2, BMQDs, target molecules, APTES and TEOS were mixed to undergo a polymerization reaction, and the target molecules were eluted from the reaction products to obtain a molecular imprinting ratio fluorescence sensor based on MXene quantum dots, denoted as OMQDs@BMQDs@MIPs.

2. The method according to claim 1, characterized in that: The target molecules are biogenic amines, including histamine, putrescine, cadaverine, and tryptamine.

3. The method according to claim 2, characterized in that The MXene nanosheets are Ti3C2 nanosheets.

4. The method according to claim 3, characterized in that The target molecule is histamine; Step A uses LiF and concentrated hydrochloric acid as etchants to prepare Ti3C2 nanosheets by etching, and the preparation method includes: Add 1.0-4.0g LiF to the lining of a polytetrafluoroethylene reactor, then add 10-50mL 6-10mol / L HCl, and cover the lid; then place the lining of the reactor in a constant temperature water bath at 30-50°C, with a rotation speed of 500-800rpm, and stir for 10-20min to allow the system to react fully; when the temperature is stable, add 1.0-3.0g Ti3AlC2 powder to the lining of the reactor in small amounts and multiple times, and wait for the bubbles to completely dissipate before each addition; then cover the lid and seal the reaction for 36-60h, and then centrifuge at a speed of 3000-4000rpm for 1-2min; then wash the reaction mixture 3-5 times with 1-3mol / L HCl to eliminate the incompletely reacted LiF; the precipitate is then centrifuged and washed with deionized water until the pH value of the supernatant is 5.8-6.2, and then filtered with a filter membrane to obtain Ti3C2 nanosheets; Step B uses ultrasonic hydrothermal method to treat Ti3C2 nanosheets and dopes them with ethylenediamine as a nitrogen source to prepare BMQDs. The preparation method includes: Disperse 0.1-0.5 g of synthesized Ti3C2 nanosheets in 10-30 mL of deionized water, and ultrasonicate for 1-3 h to obtain a few-layer Ti3C2 nanosheet dispersion; transfer the dispersion to a Teflon reactor liner, add 2-6 mL of ethylenediamine, put it into the reactor, and react at 120-180 ° C for 4-10 h; centrifuge the reaction mixture at 8000-10000 rpm for 20-30 min, take the supernatant, filter it with a filter membrane, and then transfer it to a dialysis bag with a molecular weight cutoff of 500-2000 Da and dialyze it with ultrapure water for 1-5 h, collect the dialyzed liquid to obtain blue fluorescent MXene quantum dots BMQDs; Step C uses mixed acid ultrasonic treatment on Ti3C2 nanosheets, and then dopes them with o-phenylenediamine to prepare OMQDs by solvothermal method. The preparation method includes: Take 0.5-2 mL of 98.3% concentrated sulfuric acid and add it to 1.5-6 mL of 68% concentrated nitric acid and mix them evenly. Then add 0.1-0.5 g of Ti3C2 powder to the mixed acid solution and perform ultrasonic treatment for 1-3 hours to obtain a Ti3C2 solution. Then, add 10-30 mL of deionized water to the Ti3C2 solution, cool it to 20-25°C in an ice bath, and then adjust the pH value to 6.8-7.2 with 2-5 mol / L NaOH solution. Then transfer the solution to a centrifuge tube and simmer for 6000-7000 s.c. Centrifuge at 10000rpm for 3-10min, discard the supernatant after centrifugation, transfer the precipitate to the lining of a polytetrafluoroethylene reactor, add 15-30mL of deionized water and 5-15mL of methanol, and then add 0.1-0.5g of o-phenylenediamine. After the o-phenylenediamine is completely dissolved, react at 150-200°C for 5-10h; after the reaction is completed, transfer the solution in the reactor lining to a centrifuge tube, centrifuge at 8000-10000rpm for 5-20min, take the supernatant, and obtain orange fluorescent MXene quantum dots OMQDs; Step D: A molecular imprinting ratio fluorescence sensor is prepared by an improved sol-gel method. The preparation method comprises: 1) Adoption OMQDs@SiO2 was prepared by coating a layer of silicon dioxide on OMQDs. Take 0.5-2 mL of OMQDs solution and add it to 5-15 mL of anhydrous ethanol, disperse the OMQDs by ultrasound, then add 0.5-2 mL of TEOS to the dispersed solution in a continuous droplet manner under stirring conditions, and stir for 15-30 minutes; then add 100-250 μL of 25% concentrated ammonia water and stir continuously for 18-36 hours; after the reaction is completed, transfer the solution to a centrifuge tube, centrifuge at 6000-10000 rpm for 3-10 minutes, and then wash with ethanol and deionized water once each in turn. After centrifugal washing, take out the precipitate and vacuum dry it to obtain OMQDs@SiO2; 2) 10-25 mg of OMQDs@SiO2 and 0.5-2 mL of BMQDs solution were dispersed in 10-25 mL of anhydrous ethanol, followed by adding 10-25 mg of histamine standard as a template molecule and 50-200 μL of APTES as a functional monomer, stirring for 0.5-1.5 h to prepolymerize the template molecule and the functional monomer; then 100-300 μL of TEOS was added, followed by 50-200 μL of 25% concentrated ammonia water, stirring at room temperature in the dark for 18-36 h; after the reaction was completed, a mixture of ethanol and acetic acid was used as an eluent to remove the histamine by ultrasonic centrifugation; then the precipitate was vacuum dried, ground and sieved to obtain OMQDs@BMQDs@MIPs; The volume ratio of ethanol to acetic acid in the ethanol-acetic acid mixture is 9:

1.

5. A molecular imprinting ratio fluorescence sensor based on MXene quantum dots prepared according to the method of any one of claims 1 to 4.

6. Any of the following uses of the fluorescent sensor according to claim 5: 1) Used for separation and enrichment of target molecules; 2) Used for qualitative and quantitative detection of target molecules; 3) Used for preparing target molecule detection reagents or kits; 4) Intelligent devices for preparing target molecule detection.

7. A method for detecting histamine, characterized in that: The method comprises: adding the fluorescent sensor prepared according to the method of any one of claims 2 to 4 to a sample solution to be tested, performing an oscillation reaction at room temperature, and then performing detection using a fluorescence spectrometer, or obtaining a fluorescent image of the sample solution to be tested using an ultraviolet dark box, and then performing visual detection after obtaining RGB information using color analysis software of a smart phone.

8. The method according to claim 7, characterized in that The fluorescence detection conditions are as follows: the excitation and emission scanning slits are both set to 5-20 nm, the excitation light wavelength is 310-410 nm, and the scanning emission wavelength is 400-800 nm.

9. The method according to claim 8, characterized in that The fluorescence detection conditions were as follows: the excitation and emission scanning slits were both set to 10 nm.

10. The method according to claim 8 or 9, characterized in that: The linear detection range of fluorescence detection for histamine in the sample solution is 1-60 μM, and the detection limit is 21.9 nM; The linear detection range of smartphone visual detection for histamine in the sample solution is 10-60μM, and the detection limit is 92.2nM.

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