Ochratoxin a detection method and application thereof

Abstract

The application discloses a perovskite paper chip for detecting ochratoxin A as well as a preparation method and application thereof, and relates to the technical field of food analysis. The perovskite paper chip comprises a paper-based carrier and a fluorescent signal unit and a specific recognition unit loaded on the paper-based carrier. The preparation method comprises the following steps: synthesizing silica-coated perovskite quantum dots and taking the silica-coated perovskite quantum dots as the fluorescent signal unit; preparing a molecular imprinting polymer embedding the perovskite quantum dots by means of a sol-gel molecular imprinting technology, and introducing amino groups into the molecular imprinting polymer by means of a post-modification method to obtain an amino-functionalized molecular imprinting polymer; and preparing the perovskite paper chip by sequentially loading the perovskite quantum dots and the amino-functionalized molecular imprinting polymer on the surface of the paper by means of covalent bonding. According to the fluorescence quenching effect caused by the combination of a target object and an imprinting site, the perovskite paper chip can realize sensitive and quantitative analysis of ochratoxin A, and has the advantages of simple operation, rapid response, low cost and the like.

Description

Technical Field

[0001] This invention relates to the field of food analysis technology, specifically to a perovskite paper chip for detecting ochratoxin A, its preparation method, and its application. Background Technology

[0002] Ochratoxin A (OTA) is a secondary metabolite produced by fungi of the genera Aspergillus and Penicillium. It widely contaminates grains such as corn, wheat, and rice, as well as their products and various agricultural products such as coffee and wine. OTA has strong nephrotoxicity, hepatotoxicity, immunosuppression, and teratogenicity, and has been classified as a Group 2B carcinogen by the International Agency for Research on Cancer.

[0003] Currently, the mainstream detection methods for OTA (over-occurring oxygen sulfide) include high-performance liquid chromatography-mass spectrometry (HPLC-MS) and enzyme-linked immunosorbent assay (ELISA). While HPLC-MS is sensitive and accurate, it requires expensive equipment, complex sample pretreatment, and is time-consuming. To improve detection convenience, ELISA is widely used, but its detection limit is relatively high and limited by antibody stability and batch-to-batch variability. In recent years, electrochemical sensors have made some progress in sensitivity; however, they are susceptible to interference in complex food matrices, affecting detection accuracy. Aptamer sensors have attracted attention due to their high specificity, but often require cumbersome sample pretreatment, and their long-term stability remains insufficient. Therefore, establishing a rapid, sensitive, and reliable OTA detection method is of paramount importance for ensuring food safety and public health.

[0004] Molecular imprinting is a technique that mimics natural molecular recognition systems (such as enzyme-substrate and antigen-antibody) to prepare "artificial antibodies," or molecularly imprinted polymers (MIPs), with predetermined selectivity. The basic principle of molecular imprinting is that template molecules and functional monomers are pre-assembled through covalent or non-covalent interactions, polymerized under the action of a cross-linking agent to form a rigid network, and after template elution, a three-dimensional cavity complementary to the target molecule in shape, size, and functional group arrangement is obtained. MIPs not only possess high specificity recognition capabilities but also have significant advantages such as simple preparation, low-cost raw materials, excellent physicochemical stability, and ease of functionalization modification, making them promising candidates for analytical sensing. Combining the specific recognition of molecular imprinting with the high sensitivity of fluorescence detection to construct molecularly imprinted fluorescent sensors is an important development direction in current rapid detection technologies. These sensors combine high selectivity, ease of operation, and rapid response. In recent years, all-inorganic perovskite quantum dots (CsPbX3, X=Cl, Br, I) have become highly promising high-performance fluorescent signal probes due to their excellent optical properties, such as high fluorescence quantum yield, tunable emission wavelength, and narrow emission peak. However, the ionic crystal structure of perovskite materials results in poor chemical stability. They are prone to fluorescence quenching or structural degradation under the influence of water, oxygen, light, heat, and polar solvents, which severely limits their application in practical sample matrices.

[0005] Paper, with its natural cellulose network as a framework, boasts advantages such as extremely low raw material cost, good biocompatibility, lightweight texture, and incineration. Paper chips, with their portability and low cost, have become an ideal carrier for building on-site real-time detection platforms. Meanwhile, the abundant hydroxyl groups on the surface of cellulose provide numerous active sites for chemical modification and functionalization, making paper substrates highly suitable as bases for immobilized signal probes and recognition elements to construct disposable sensing interfaces. Summary of the Invention

[0006] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a perovskite paper chip for detecting ochratoxin A, its preparation method, and its application. Specifically, the technical problem to be solved by this invention is that while mainstream OTA detection methods such as high-performance liquid chromatography-mass spectrometry (HPLC-MS) and enzyme-linked immunosorbent assay (ELISA) are highly accurate, they generally rely on large instruments, professional operators, and complex sample pretreatment, resulting in high costs, time-consuming and labor-intensive processes, and an inability to meet the needs of rapid on-site screening. In recent years, although some research has attempted to develop rapid detection methods based on fluorescent materials or molecular imprinting technology, they often face the following challenges: fluorescent probes (such as traditional organic dyes or quantum dots) suffer from poor photostability and are prone to quenching; recognition elements (such as antibodies or aptamers) are limited by high cost, poor stability, and easy inactivation; in addition, the stable and efficient integration of recognition elements and signal units into a portable detection platform remains a technical challenge. Therefore, developing a rapid on-site OTA detection technology that integrates high sensitivity, high stability, high selectivity, low cost, and ease of operation is a key problem that urgently needs to be solved in the field of food analysis.

[0007] This invention efficiently integrates fluorescent signal units, specific recognition units, and a paper-based carrier to prepare a perovskite paper chip. Specifically: First, silica-encapsulated perovskite quantum dots are synthesized and used as fluorescent signal units. Second, a molecularly imprinted polymer encapsulating the perovskite quantum dots is prepared using sol-gel molecular imprinting technology, and amino groups are introduced through post-modification to obtain an amino-functionalized molecularly imprinted polymer capable of specifically recognizing ochratoxin A. Finally, the molecularly imprinted perovskite paper chip is prepared by sequentially immobilizing perovskite quantum dots and covalently immobilizing the amino-functionalized molecularly imprinted polymer on the paper surface. During detection, the fluorescence quenching effect caused by the binding of the target analyte to the imprinted sites enables sensitive and quantitative analysis of ochratoxin A. This paper chip has the advantages of simple operation, rapid response, and low cost, and is suitable for rapid screening of ochratoxin A in food, providing a new technology and method for food safety supervision.

[0008] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a perovskite paper chip, the perovskite paper chip comprising a fluorescent signal unit, a specific recognition unit and a paper substrate, wherein the fluorescent signal unit and the specific recognition unit are both loaded on the paper substrate. The fluorescent signal unit is a perovskite quantum dot encapsulated in silica. The perovskite quantum dot encapsulated in silica is a CsPbBr3@SiO2 perovskite quantum dot, which is prepared by an in-situ coating method of methyl orthosilicate hydrolysis catalyzed by ammonia water. The specific recognition unit is a molecularly imprinted polymer, which is prepared from a pseudo-template molecule of the analyte, a functional monomer, and a crosslinking agent.

[0009] Optionally, the fluorescent signal unit is physically adsorbed and immobilized on the paper-based carrier, and the specific recognition unit is covalently immobilized on the paper-based carrier.

[0010] Optionally, the paper-based carrier is an aldehyde-based carrier, and the specific recognition unit is an aminated molecularly imprinted polymer. The specific recognition unit is immobilized on the paper-based carrier through a covalent bonding reaction between amino and aldehyde groups.

[0011] Optionally, the ratio of the fluorescent signal unit to the specific recognition unit is 0.5 mL: 4~6 mg; more preferably, it is 0.5 mL: 5 mg.

[0012] Secondly, the present invention provides a method for preparing the perovskite paper chip, comprising the following steps: S1. Prepare silica-encapsulated perovskite quantum dots by preparing a perovskite quantum dot solution from the silica-encapsulated perovskite quantum dots. A molecularly imprinted polymer is prepared, and the molecularly imprinted polymer is subjected to amination treatment to obtain an amination molecularly imprinted polymer. The amination molecularly imprinted polymer is then formulated into an amination molecularly imprinted polymer solution. S2. Immerse the filter paper in the perovskite quantum dot solution to obtain a paper base loaded with quantum dots; S3. The paper base loaded with quantum dots is subjected to surface amylation treatment and surface aldehyde-oxidation treatment to obtain an aldehyde-oxidized paper base. S4. Immerse the aldehyde-modified paper base in the aminated molecularly imprinted polymer solution to obtain the perovskite paper chip.

[0013] Optionally, in S1, the method for preparing the silica-encapsulated perovskite quantum dots includes: Lead bromide, cesium bromide, n-octylamine and oleic acid are added to the first solvent, heated and mixed evenly to obtain the precursor solution; Ammonia was added to the precursor solution and mixed thoroughly. Methyl orthosilicate was added while stirring, and the reaction was carried out under heating to obtain the product. The product was centrifuged and washed to obtain the silica-encapsulated perovskite quantum dots. The ratio of lead bromide, cesium bromide, n-octylamine, and oleic acid added is 0.3~0.5 mmol: 0.3~0.5 mmol: 0.5~0.7 mL: 1.6~2.0 mL; The volume ratio of the precursor solution to ammonia is 45-55:1, and the concentration of the ammonia is 2-3 wt.%.

[0014] Optionally, in S1, the method for preparing the aminated molecularly imprinted polymer includes: The second solvent is mixed evenly with the pseudo template molecule of the analyte, the functional monomer is added first for pre-assembly, and then the cross-linking agent is added for cross-linking polymerization to obtain the molecularly imprinted polymer. The molecularly imprinted polymer was added to an APTES ethanol solution for reaction to obtain an aminated molecularly imprinted polymer. The analyte to be detected includes ochratoxin A, and the pseudo-template molecule includes N-fluorenylmethoxycarbonyl-D-phenylalanine; The functional monomers are 3-aminopropyltriethoxysilane and phenyltrimethoxysilane; The crosslinking agent is tetraethoxysilane; The second solvent is a water / ethanol co-solvent, wherein the volume ratio of water to ethanol is 1.5~2.5:1; The molar ratio of the pseudo-template molecule to 3-aminopropyltriethoxysilane and phenyltrimethoxysilane is 1:1.5~2.5:0.5~1.5; The volume fraction of the APTES ethanol solution is 5-7%.

[0015] Optionally, in S2, filter paper is immersed in the perovskite quantum dot solution and shaken at room temperature in the dark for 10 to 14 hours. S3 includes the following specific steps: the quantum dot-loaded paper substrate is immersed in an 8-12% (v / v) APTES ethanol solution and reacted with shaking at room temperature for 6-10 hours. After the reaction is complete, it is washed to obtain an aminated paper substrate; the aminated paper substrate is immersed in an 8-12% (v / v) glutaraldehyde ethanol solution and reacted with shaking at room temperature for 0.5-1.5 hours. After the reaction is complete, it is washed to obtain an aldehyde-modified paper substrate; In step S4, the aldehyde-modified paper base is immersed in the aminated molecularly imprinted polymer solution and shaken at room temperature in the dark for 10 to 14 hours. The concentration of the aminated molecularly imprinted polymer solution is 0.1 to 1.0 mg / mL.

[0016] Optionally, the present invention provides a method for preparing a perovskite paper chip for detecting ochratoxin A, which efficiently integrates a fluorescent signal unit, a specific recognition unit, and a paper-based carrier through three distinct steps.

[0017] Step 1. Synthesis of perovskite quantum dots: Lead bromide, cesium bromide, oleylamine, and oleic acid were dissolved together in N,N-dimethylformamide, and the mixture was heated and stirred to obtain a clear precursor solution. A portion of this solution was taken, ammonia was added, and then the solution was rapidly injected into a toluene solution containing methyl orthosilicate, and the reaction was carried out under heating conditions. After the reaction was completed, the solution was centrifuged and washed to obtain a silica-encapsulated perovskite quantum dot dispersion.

[0018] Step 2. Preparation of Aminated Molecularly Imprinted Polymers: The pseudo-template molecule Fmoc-D-phenylalanine was dissolved in a mixed solvent of ethanol and water. Subsequently, 3-aminopropyltriethoxysilane and phenyltriethoxysilane, as functional monomers, and tetraethyl orthosilicate, as a crosslinking agent, were added sequentially to the solution to initiate a sol-gel polymerization reaction. After the reaction, the polymer was post-modified using APTES to introduce amino groups onto its surface. The template molecule was then eluted using a methanol / acetic acid mixture to finally obtain a molecularly imprinted polymer with an amino-rich surface that specifically recognizes ochratoxin A.

[0019] Step 3. Assembly of the perovskite molecularly imprinted paper chip: First, the paper substrate is immersed in the perovskite quantum dot dispersion prepared in step one, allowing the quantum dots to be immobilized on the paper substrate fibers through physical adsorption, forming a fluorescent layer. Then, the fluorescent paper substrate is sequentially immersed in an ethanol solution of 3-aminopropyltriethoxysilane and an ethanol solution of glutaraldehyde to react, sequentially attaching amino and active aldehyde groups to its surface. Finally, the aldehyde-modified paper substrate is immersed in the aminated molecularly imprinted polymer dispersion prepared in step two, and the recognition unit is firmly immobilized on the paper substrate surface through a covalent bonding reaction between the aldehyde and amino groups, thereby obtaining the perovskite molecularly imprinted paper chip.

[0020] As an improvement, the volume ratio of oleylamine to oleic acid in step one is approximately 1:3.

[0021] As an improvement, the concentration of ammonia water added in step one is approximately 2.8%.

[0022] As an improvement, the molar ratio of the functional monomers 3-aminopropyltriethoxysilane and phenyltriethoxysilane in step two is approximately 2:1.

[0023] As an improvement, the molar ratio of the pseudo-template molecule to the crosslinking agent tetraethyl orthosilicate in step two is approximately 1:10.

[0024] As an improvement, the volume ratio of the methanol-acetic acid mixture used to elute the template molecules in step two is approximately 9:1.

[0025] As an improvement, in step three, the concentration range of the aminated molecularly imprinted polymer dispersion used for immobilization is 0.1 mg / mL to 1.0 mg / mL.

[0026] Thirdly, the present invention provides the application of the perovskite paper chip in the detection of ochratoxin A.

[0027] Optionally, the application method includes: measuring the initial fluorescence intensity F0 of the paper chip using a fluorescence microarray scanner; adding the sample solution to be tested to the chip surface, reacting for a period of time, and measuring its fluorescence intensity; establishing a standard curve based on the relationship between the fluorescence quenching rate (F0-F) / F0 and the concentration of ochratoxin A, thereby achieving quantitative analysis of the target toxin in the sample.

[0028] Fourthly, the present invention provides a method for detecting ochratoxin A using the aforementioned perovskite paper chip, comprising the following steps: The initial fluorescence intensity F0 of the perovskite paper chip was measured using a fluorescence microarray scanner at an excitation wavelength of 532 nm. The sample solution to be tested was dropped onto the surface of the perovskite paper chip. After the reaction was completed, the fluorescence intensity F of the sample solution to be tested was measured using a fluorescence microarray scanner at an excitation wavelength of 532 nm. The fluorescence quenching rate (F0-F) / F0 of the sample solution to be tested was calculated. The concentration of the sample is obtained by substituting the fluorescence quenching rate (F0-F) / F0 of the test sample solution into the standard curve.

[0029] This invention has at least one of the following beneficial effects: Compared with existing technologies, the perovskite paper chip for detecting OTA, its preparation method, and its application provided by this invention have the following advantages: 1. This invention creatively combines the high fluorescence performance of perovskite quantum dots with the high specificity recognition capability of molecularly imprinted polymers and integrates them on a low-cost, easy-to-operate paper-based platform to construct a novel "signal-recognition-carrier" three-in-one sensing strategy, achieving high-performance and rapid detection of ochratoxin A.

[0030] 2. This invention employs an in-situ coating method using ammonia-catalyzed hydrolysis of methyl orthosilicate to construct a silica protective shell for perovskite quantum dots under mild conditions. This significantly enhances the chemical and photostability of the quantum dots in polar environments and during subsequent modification processes, solving the problem of easy degradation of perovskite materials in practical applications.

[0031] 3. This invention uses Fmoc-D-phenylalanine as a pseudotemplate to prepare silicon-based molecularly imprinted polymers via a sol-gel method. This pseudotemplate strategy avoids the safety and environmental risks associated with directly using highly toxic ochratoxin A. Furthermore, the prepared molecularly imprinted polymers are highly matched to ochratoxin A in terms of spatial structure and functional groups, exhibiting excellent selective recognition capabilities for the target analyte and effectively reducing the influence of interfering substances in complex sample matrices.

[0032] 4. This invention achieves a firm and uniform fixation of the recognition unit on the paper-based fluorescent layer through a layer-by-layer assembly process of "quantum dot immobilization → amination → aldehyde fixation → polymer covalent immobilization". This covalent bonding method prevents the recognition unit from falling off during use, ensuring good stability and reproducibility of the sensor.

[0033] 5. The perovskite paper chip prepared by this invention has the advantages of high sensitivity and rapid response. Combined with a fluorescence reading device, it can complete the quantitative analysis of ochratoxin A in a sample in a short time.

[0034] 6. The entire detection method of this invention is simple to operate, requiring no complex and expensive instruments or professional operators, and uses a small amount of reagents, resulting in low detection costs. The paper chip is easy to carry, store, and destroy, making it ideal for large-scale, rapid screening of ochratoxin A in on-site processes such as grain purchasing, storage, and processing, providing a practical solution for on-site food safety monitoring. Attached Figure Description

[0035] Figure 1 A schematic diagram illustrating the synthesis principle of a perovskite paper chip for detecting ochratoxin A shows the in-situ coating synthesis of CsPbBr3@SiO2 quantum dots, the preparation of NH2-OTA-MIP, and its layer-by-layer assembly process on a paper substrate. Figure 2 The fluorescence stability of CsPbBr3@SiO2 perovskite quantum dots under ambient light; Figure 3 The UV adsorption selectivity of the template Fmoc-D-phenylalanine for the aminated molecularly imprinted polymer (NH2-OTA-MIP) and the non-imprinted polymer (NH2-OTA-NIP) was studied. Figure 4 The fluorescence selectivity response of perovskite paper chip (P@CPB-OTA-MIP) and non-printed paper chip (P@CPB-OTA-NIP) to OTA was investigated. Figure 5 Experiments were conducted to demonstrate the fluorescence-specific response of the perovskite paper chip (P@CPB-OTA-MIP) to OTA and its structural analogs (OTB, AFB1, FB1, DON, ZEN). Figure 6 A fluorescence quenching rate-concentration standard curve was established for detecting different concentrations of OTA based on P@CPB-OTA-MIP paper chip; Figure 7 The results of spiked recovery experiments of P@CPB-OTA-MIP paper chips in actual samples.

[0036] Figure 8Images of perovskite quantum dots without silica coating and perovskite quantum dots coated with silica; among them, Figure 8 A represents the toluene solutions of Example 1 (left figure) and Comparative Example 1 (right figure). Figure 8 B represents the aqueous solutions of Example 1 (left figure) and Comparative Example 1 (right figure) after being left to stand for 4 hours. Detailed Implementation

[0037] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0038] Unless otherwise specified, the experimental methods described in the examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified. Quantitative filter paper (Whatman Grade 1) is used as the paper base.

[0039] Example 1: Synthesis of CsPbBr3@SiO2 perovskite quantum dots This embodiment provides a method for synthesizing perovskite quantum dots, including the following steps: 1. Preparation of precursor solution: 10 mL of N,N-dimethylformamide was added to a 25 mL round-bottom flask, followed by lead bromide (PbBr2, 0.1468 g, 0.4 mmol), cesium bromide (CsBr, 0.0851 g, 0.40 mmol), 0.6 mL of n-octylamine, and 1.8 mL of oleic acid. The mixture was placed in an oil bath at 90 °C and magnetically stirred for 2 hours to obtain a clear and transparent precursor solution.

[0040] 2. Silica Coating: Take 2 mL of the above precursor solution and add ammonia (2.8 wt.%, 40 μL), stir at room temperature for 5 minutes. Under vigorous stirring, rapidly inject 0.2 mL of this mixture into 10 mL of toluene containing methyl orthosilicate (TMOS, 5 μL). After 10 seconds, adjust the stirring speed to 500 rpm and transfer the reaction system to a 60°C water bath, continuing the reaction for 120 minutes.

[0041] 3. Purification and Dispersion: After the reaction, the mixed solution was centrifuged at 12000 rpm for 5 minutes, and the supernatant was discarded. The precipitate was washed three times with ethanol to remove unreacted raw materials and byproducts. Finally, the purified precipitate was redispersed in 4 mL of anhydrous ethanol to obtain a CsPbBr3@SiO2 perovskite quantum dot dispersion, which was stored at 4℃ in the dark. The synthesis principle is as follows. Figure 1 As shown.

[0042] Example 2: Preparation of Aminated Molecularly Imprinted Polymer (NH2-OTA-MIP) This embodiment provides a method for preparing an amino-imprinted polymer, the synthesis principle of which is as follows: Figure 1 As shown, it includes the following steps: 1. Pre-assembly: Weigh 0.0387 g (0.1 mmol) of Fmoc-D-phenylalanine and add it to a mixed solvent containing 4 mL of anhydrous ethanol and 8 mL of ultrapure water. Dissolve the solution by sonication. Place the solution in a 25°C water bath and slowly add the following solutions dropwise with stirring: 3-aminopropyltriethoxysilane (APTES, 48 μL, 0.20 mmol) and phenyltriethoxysilane (PTMOS, 18.7 μL, 0.10 mmol). After the addition is complete, continue stirring for 30 minutes.

[0043] 2. Crosslinking polymerization: Tetramethoxysilane (TEOS, 222 μL, 1.0 mmol) was slowly added dropwise to the above system. Polymerization was carried out with stirring at 25°C for 48 hours. After the reaction was complete, the polymer was collected by centrifugation and washed twice with ethanol.

[0044] 3. Amino Modification and Template Elution: The polymer precipitate was redispersed in a 6% (v / v) APTES ethanol solution and reacted with shaking at room temperature for 12 hours to modify the polymer surface with amino groups. After the reaction, the polymer was washed twice with ethanol. Subsequently, the polymer was eluted with a methanol / acetic acid (v / v 9:1) mixture, with the eluent changed every 2 hours, until template molecules were no longer detectable in the eluent at the characteristic absorption wavelength of the template using a UV spectrophotometer. After elution, the polymer was washed twice with ethanol to obtain the aminated molecularly imprinted polymer (NH2-OTA-MIP), which was then dispersed in ethanol for storage.

[0045] Comparative Example 1: Preparation of Aminated Non-Imprinted Polymer (NH2-OTA-NIP) NH2-OTA-NIP was prepared as a control. Except for omitting the template molecule Fmoc-D-phenylalanine, the preparation was carried out in accordance with Example 2. Aminated non-imprinted polymer (NH2-OTA-NIP) nanoparticles were generated under the same conditions as NH2-OTA-MIP.

[0046] Example 3: Assembly of perovskite molecularly imprinted paper chip (P@CPB-OTA-MIP) The CsPbBr3@SiO2 perovskite quantum dots synthesized in Example 1 and the aminated molecularly imprinted polymer prepared in Example 2 were assembled into a perovskite molecularly imprinted paper chip (P@CPB-OTA-MIP). The synthesis principle is as follows: Figure 1 As shown, the specific method is as follows: 1. Quantum Dot Immobilization (P@CPB): 2 mL of the CsPbBr3@SiO2 perovskite quantum dot dispersion prepared in Example 1 was diluted to 20 mL with ethanol. Fifty pre-cut circular filter paper sheets (5 mm in diameter) were immersed in this solution and placed in a constant-temperature shaker at room temperature in the dark for 12 hours. The paper sheets were then removed and thoroughly rinsed with ethanol to obtain the quantum dot-loaded paper substrate (P@CPB).

[0047] 2. Surface amination (P@CPB-NH2): P@CPB paper was immersed in APTES ethanol solution (10%, v / v, 30 mL) and reacted with shaking at room temperature for 8 hours. After the reaction, the paper was washed with ethanol to obtain the amination-modified paper base (P@CPB-NH2). The successful amination modification was verified by ninhydrin colorimetric assay.

[0048] 3. Surface aldehyde oxidation (P@CPB-NH2-CHO): Immerse the P@CPB-NH2 paper sheet in a glutaraldehyde ethanol solution (10%, v / v, 30 mL) and react with shaking at room temperature for 1 hour. After the reaction, wash the paper sheet with ethanol to obtain the aldehyde-oxidized paper base (P@CPB-NH2-CHO). A second ninhydrin test should show no obvious color development, indicating successful aldehyde oxidation.

[0049] 4. Covalent Immobilization of MIP (P@CPB-OTA-MIP): P@CPB-NH2-CHO paper discs were immersed in an ethanol dispersion (0.5 mg / mL, 10 mL) of NH2-OTA-MIP (prepared in Example 2) and shaken at room temperature in the dark for 12 hours. Covalent immobilization was achieved through a Schiff base reaction. After the reaction, the paper discs were washed with ethanol to obtain the final perovskite molecularly imprinted paper chip (P@CPB-OTA-MIP).

[0050] Comparative Example 2: Assembly of Perovskite Non-Paper Chip (P@CPB-OTA-NIP) The assembly process was the same as in Example 3, except that in step 4, the NH2-OTA-NIP dispersion prepared in Comparative Example 1 was used instead of NH2-OTA-MIP for the final immobilization step, resulting in a perovskite non-printed paper chip (P@CPB-OTA-NIP) as a control.

[0051] Comparative Example 3 The difference from Example 1 is that no silica protective shell is constructed, that is, the "silica coating" in step 2 is not performed. Everything else is the same as in Example 1, and a CsPbBr3 perovskite quantum dot dispersion is obtained.

[0052] The CsPbBr3@SiO2 perovskite quantum dots prepared in Example 1 and the CsPbBr3 perovskite quantum dots prepared in Comparative Example 3 were added to toluene and water, respectively, to obtain toluene solution and aqueous solution, and their fluorescence was observed.

[0053] The results are as follows Figure 8 As shown, Figure 8 A represents the toluene solutions of Example 1 (left figure) and Comparative Example 1 (right figure). Figure 8 After 4 hours of soaking in aqueous solutions of Example 1 (left) and Comparative Example 1 (right), it can be seen that the uncoated silica perovskite quantum dots exhibit weak fluorescence and are quickly quenched in water; while the CsPbBr3@SiO2 perovskite quantum dots prepared in Example 1 have brighter fluorescence and higher water stability.

[0054] Example 4: Fluorescence stability test of perovskite quantum dots The CsPbBr3@SiO2 quantum dots prepared in Example 1 were dispersed in aqueous ethanol solutions with different water contents of 0%, 25%, 50%, 75%, and 100%, and exposed to indoor light. Samples were taken at 0, 2, 4, 8, 24, and 48 hours to measure the fluorescence emission intensity under 470 nm excitation. The fluorescence retention rate at each time point was calculated with the initial fluorescence intensity as 100%, and the results were plotted. Figure 2 As shown, the fluorescence of CsPbBr3@SiO2 tends to be stable, proving that the SiO2 coating layer effectively improves the environmental stability of perovskite quantum dots.

[0055] Example 5: Adsorption Selectivity Test of Aminated Molecularly Imprinted Polymer (NH2-OTA-MIP) Prepare 2 mg / mL solutions of NH₂-OTA-MIP and NH₂-OTA-NIP polymers (ethanol / water, v / v, 1 / 3 solvent), add 70 μg / mL Fmoc-D-phenylalanine solution (ethanol / water, v / v, 1 / 3 solvent), and allow to adsorb at room temperature with shaking for 4 hours. After centrifugation, measure the absorbance of the supernatant and calculate the template adsorption amount. Figure 3 As shown, the adsorption capacity of NH2-OTA-MIP for Fmoc-D-phenylalanine is significantly higher than that of NH2-OTA-NIP, demonstrating that the prepared MIP has excellent selective recognition ability for the target analyte.

[0056] Example 6: Selectivity Testing of Perovskite Paper Chips The fluorescence response of P@CPB-OTA-MIP and P@CPB-OTA-NIP paper chips to the same concentration (100 ng / mL) of OTA standard solution was measured, and the fluorescence quenching rate was calculated. Results are as follows: Figure 4As shown, the fluorescence quenching rate of OTA by P@CPB-OTA-MIP is much higher than that of P@CPB-OTA-NIP. This difference is attributed to the presence of specific recognition sites in the MIP, thus demonstrating the selectivity of the paper chip of the present invention for OTA.

[0057] Example 7: Specificity Testing of Perovskite Paper Chips Standard solutions of OTA, OTB, AFB1, FB1, DON, and ZEN, each with a concentration of 100 ng / mL, were prepared. For each P@CPB-OTA-MIP paper chip, the initial fluorescence intensity F0 was measured. Different toxin solutions (20 μL) were then added to the surface. After a 10-minute reaction, the fluorescence intensity F was measured using a fluorescence microarray scanner (excitation wavelength 532 nm) to calculate the fluorescence quenching rate (F0-F) / F0. The results are as follows: Figure 5 As shown, the fluorescence quenching rate of P@CPB-OTA-MIP for OTA is significantly higher than that for several other toxins, demonstrating that the paper chip has a good selective response to OTA.

[0058] Example 8: Establishment of Standard Curve and Spike Recovery Experiment Standard curve establishment: A series of OTA standard solutions with concentrations of 0.1, 0.5, 1, 5, and 10 ng / mL were prepared (solvent: ethanol / water = 1 / 3, v / v). The fluorescence quenching rate at each concentration was determined using a P@CPB-OTA-MIP paper chip according to the method in Example 6. A linear regression was performed with OTA concentration (or its logarithm) on the x-axis and fluorescence quenching rate on the y-axis to establish a standard curve: y = 0.0504x + 0.0703 (R²). 2 =0.9820), such as Figure 6 As shown, the linear detection range is between 0.1 and 10 ng / mL, and the detection limit is 0.08 ng / mL.

[0059] Spiked recovery experiment: Blank rice, wheat, and corn samples were selected and pretreated by crushing, acetonitrile / water (70 / 30, v / v) extraction, and filtration. Low, medium, and high concentrations of OTA standard (OTA spiked concentrations of 0.1, 1, and 10 ng / mL, respectively) were then added. The paper chip of this invention was used to detect the spiked samples, and the OTA content was calculated based on the standard curve. The recovery rate and relative standard deviation (RSD) were also calculated. Figure 7 As shown in the figure, the experimental results indicate that the recovery rate of this method is between 82% and 103%, and the RSD is less than 5%, which meets the requirements of actual sample analysis and proves that the method is accurate and reliable.

[0060] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A perovskite paper chip, characterized in that, The perovskite paper chip includes a fluorescent signal unit, a specific recognition unit, and a paper substrate, wherein the fluorescent signal unit and the specific recognition unit are both loaded on the paper substrate. The fluorescent signal unit is a perovskite quantum dot encapsulated in silica. The perovskite quantum dot encapsulated in silica is a CsPbBr3@SiO2 perovskite quantum dot, which is prepared by an in-situ coating method of methyl orthosilicate hydrolysis catalyzed by ammonia water. The specific recognition unit is a molecularly imprinted polymer, which is prepared from a pseudo-template molecule of the analyte, a functional monomer, and a crosslinking agent.

2. The perovskite paper chip according to claim 1, characterized in that, The fluorescent signal unit is physically adsorbed and immobilized on the paper-based carrier, and the specific recognition unit is covalently immobilized on the paper-based carrier.

3. The perovskite paper chip according to claim 2, characterized in that, The paper-based carrier is an aldehyde-based carrier, and the specific recognition unit is an aminated molecularly imprinted polymer. The specific recognition unit is immobilized on the paper-based carrier through a covalent bonding reaction between amino and aldehyde groups.

4. The perovskite paper chip according to claim 1, characterized in that, The ratio of the fluorescent signal unit to the specific recognition unit is 0.5 mL: 4~6 mg.

5. The method for preparing the perovskite paper chip according to any one of claims 1 to 4, characterized in that, Includes the following steps: S1. Prepare silica-encapsulated perovskite quantum dots by preparing a perovskite quantum dot solution from the silica-encapsulated perovskite quantum dots. A molecularly imprinted polymer is prepared, and the molecularly imprinted polymer is subjected to amination treatment to obtain an amination molecularly imprinted polymer. The amination molecularly imprinted polymer is then formulated into an amination molecularly imprinted polymer solution. S2. Immerse the filter paper in the perovskite quantum dot solution to obtain a paper base loaded with quantum dots; S3. The paper base loaded with quantum dots is subjected to surface amylation treatment and surface aldehyde-oxidation treatment to obtain an aldehyde-oxidized paper base. S4. Immerse the aldehyde-modified paper base in the aminated molecularly imprinted polymer solution to obtain the perovskite paper chip.

6. The preparation method according to claim 5, characterized in that, In S1, the method for preparing the silica-encapsulated perovskite quantum dots includes: Lead bromide, cesium bromide, n-octylamine and oleic acid are added to the first solvent, heated and mixed evenly to obtain the precursor solution; Ammonia was added to the precursor solution and mixed thoroughly. Methyl orthosilicate was added while stirring, and the reaction was carried out under heating to obtain the product. The product was centrifuged and washed to obtain the silica-encapsulated perovskite quantum dots. The ratio of lead bromide, cesium bromide, n-octylamine, and oleic acid added is 0.3~0.5 mmol: 0.3~0.5 mmol: 0.5~0.7 mL: 1.6~2.0 mL; The volume ratio of the precursor solution to ammonia is 45-55:1, and the concentration of the ammonia is 2-3 wt.%.

7. The preparation method according to claim 5, characterized in that, In S1, the method for preparing the aminated molecularly imprinted polymer includes: The second solvent is mixed evenly with the pseudo template molecule of the analyte, the functional monomer is added first for pre-assembly, and then the cross-linking agent is added for cross-linking polymerization to obtain the molecularly imprinted polymer. The molecularly imprinted polymer was added to an APTES ethanol solution for reaction to obtain an aminated molecularly imprinted polymer. The analyte to be detected includes ochratoxin A, and the pseudo-template molecule includes N-fluorenylmethoxycarbonyl-D-phenylalanine; The functional monomers are 3-aminopropyltriethoxysilane and phenyltrimethoxysilane; The crosslinking agent is tetraethoxysilane; The second solvent is a water / ethanol co-solvent, wherein the volume ratio of water to ethanol is 1.5~2.5:1; The molar ratio of the pseudo-template molecule to 3-aminopropyltriethoxysilane and phenyltrimethoxysilane is 1:1.5~2.5:0.5~1.5; The volume fraction of the APTES ethanol solution is 5-7%.

8. The preparation method according to claim 5, characterized in that, Includes the following steps: In step S2, filter paper is immersed in the perovskite quantum dot solution and shaken at room temperature in the dark for 10-14 hours. S3 includes the following specific steps: the quantum dot-loaded paper substrate is immersed in an 8-12% (v / v) APTES ethanol solution and reacted with shaking at room temperature for 6-10 hours. After the reaction is complete, it is washed to obtain an aminated paper substrate; the aminated paper substrate is immersed in an 8-12% (v / v) glutaraldehyde ethanol solution and reacted with shaking at room temperature for 0.5-1.5 hours. After the reaction is complete, it is washed to obtain an aldehyde-modified paper substrate; In step S4, the aldehyde-modified paper base is immersed in the aminated molecularly imprinted polymer solution and shaken at room temperature in the dark for 10 to 14 hours. The concentration of the aminated molecularly imprinted polymer solution is 0.1 to 1.0 mg / mL.

9. The use of the perovskite paper chip according to any one of claims 1 to 4 in the detection of ochratoxin A.

10. A method for detecting ochratoxin A using the perovskite paper chip according to any one of claims 1 to 4, characterized in that, Includes the following steps: The initial fluorescence intensity F0 of the perovskite paper chip was measured using a fluorescence microarray scanner at an excitation wavelength of 532 nm. The sample solution to be tested was dropped onto the surface of the perovskite paper chip. After the reaction was completed, the fluorescence intensity F of the sample solution to be tested was measured using a fluorescence microarray scanner at an excitation wavelength of 532 nm. The fluorescence quenching rate (F0-F) / F0 of the sample solution to be tested was calculated. The concentration of the sample is obtained by substituting the fluorescence quenching rate (F0-F) / F0 of the test sample solution into the standard curve.

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