A nanobiosensor and a method for directly detecting organophosphorus pesticides

Abstract

The present invention provides a method for directly detecting organophosphorus pesticides based on nano-biosensors, which realizes direct detection of organophosphorus pesticides OPs in one step by using a biosensor with carbon dots rich in phosphate groups on the surface, with high sensitivity and good specificity, and can avoid interference from components such as proteins, reducing sugars, amino acids, and metal ions, without the need for complex sample pre-treatment. The method comprises the following steps: using organic small molecules containing phosphate groups as raw materials, preparing carbon dots PCDs rich in phosphate groups on the surface by carbonization; mixing PCDs as fluorophores with dephosphorylase to construct a nano-biosensor; mixing organophosphorus pesticides OPs of known different concentrations with the nano-biosensor, measuring the fluorescence spectrum of the nano-biosensor after standing, and drawing a standard curve; mixing the OPs sample to be detected with the nano-biosensor, measuring the fluorescence spectrum of the nano-biosensor, and calculating the concentration of the OPs to be detected according to the standard curve.

Description

Technical Field

[0001] The invention belongs to the field of nano biosensors, and in particular relates to a carbon quantum dot nano biosensor for detecting organophosphorus pesticides and an application method thereof. Background Art

[0002] The use of pesticides in agricultural production is of great significance for crop protection and high yield and income increase. The rational use of pesticides can increase the yield of crops such as fruits, vegetables, and grains by 10% to 40%. Today, synthetic organic compound pesticides still account for the majority, and can be divided into organochlorine pesticides, organophosphorus pesticides (OPs), carbamate pesticides, pyrethroids, and pyrethroids according to their chemical composition. Organophosphorus pesticides have low cost, high insecticidal activity, and a wide range of control targets. They are the most widely used in my country and even in the world, especially in fruit and vegetable crops, accounting for about 80% of the total pesticide use in my country. However, illegal or excessive use of OPs pesticides will lead to excessive OPs content in fruit and vegetable products and processed fruit and vegetable foods, which greatly threatens human health and life safety. OPs can combine with the active site of acetylcholinesterase, causing nerves to be in a state of excitement for a long time, causing irreparable damage to the nervous system and respiratory system, and ultimately leading to death. The upper limit of OPs (chlorpyrifos, dimethoate, parathion, etc.) content in fruits and vegetables stipulated in my country's standard GB 2763-2021 is 0.01 mg / kg. In view of the frequent occurrence of OPs exceeding the standard in fruits and vegetables, the development of a new method for rapid, reliable and ultra-sensitive pesticide detection is of great significance for the detection of OPs residues in fruits and vegetables and the prevention and control of poisoning.

[0003] Traditional OPs detection technologies mainly include high performance liquid chromatography, liquid chromatography-mass spectrometry, gas chromatography, etc. Although the sensitivity of these detection methods can meet the requirements, they are time-consuming, the equipment is expensive, and the operation is cumbersome. They are not suitable for on-site detection, especially when sudden acute poisoning incidents require rapid detection. There are few reports on directly acting on OPs to achieve the purpose of detection, and the rapidly developing biosensor technology based on enzymatic reactions meets the needs of on-site rapid detection of OPs compounds. It is also very economical and reliable. Dephosphorylase is a type of phosphate monoester hydrolase widely distributed in human tissues. The performance of this type of enzyme in catalyzing the breakage of phosphate monoester bonds makes it have important application value in the construction of biosensors. Based on the inhibitory effect of OPs on dephosphorylase, the developed biosensor has the advantages of rapid response and environmental friendliness, and is expected to replace the above traditional methods for the rapid detection of OPs in food.

[0004] At present, the OPs detection strategies of biosensors using dephosphorylase as signal conversion units are all indirect detection based on multi-step cascade reactions. The enzyme catalyzes the hydrolysis of the substrate to generate products with special properties, which participate in downstream reactions and generate detection signals. On the contrary, if OPs are present in the system, the downstream reaction cannot occur due to the inhibition of enzyme activity. This multi-step cascade reaction system has made great progress in OPs detection, but it still faces the challenge of reliability (anti-interference ability) of the detection system when used for the analysis of OPs residues in fruit and vegetable samples.

[0005] Fluorescence analysis methods have the advantages of simple operation, high sensitivity, and easy-to-read signals, and are particularly suitable for the rapid analysis of OPs. As a zero-dimensional carbon-based material, carbon dots have many advantages, such as excellent optical properties, low toxicity, and easy preparation and modification. The nanobiosensors developed with carbon dots as fluorophores and hydrolases as signal conversion units have received widespread attention in the field of OPs detection. However, biosensors with carbon dots as fluorophores still output fluorescent signals through multi-step cascade reactions, which are indirect detection strategies and have no significant improvement in anti-interference ability.

[0006] At present, there is no report on the direct detection method of OPs using an optical biosensor that uses dephosphorylase as a signal conversion unit and carbon dots as both a fluorophore and a substrate of an enzymatic reaction to directly participate in the reaction and display a signal. Therefore, the present invention proposes a new method for directly detecting OPs in one step using carbon dots rich in phosphate groups and a nanobiosensor based on the carbon dots and an enzymatic reaction. Summary of the invention

[0007] In view of the existing problems, the present invention provides a nano biosensor, which is a nano biosensor based on fluorescent carbon dots (PCDs) rich in phosphate groups on the surface, which can be used for the direct detection of OPs. The nano biosensor has a simple and easy construction process, low cost and is environmentally friendly.

[0008] The nano biosensor of the present invention is prepared by the following method:

[0009] a. Using organic small molecules containing phosphate groups as raw materials, carbon dots PCDs with rich phosphate groups on the surface are prepared by carbonization;

[0010] b. PCDs are used as fluorophores and mixed with dephosphorylase to construct a nanobiosensor.

[0011] The specific process of step a of the above-mentioned nanobiosensor is as follows: dissolving the raw material in a solvent to prepare a solution, drying the solution at a temperature not exceeding 80°C to a crystalline state without free water, and then subjecting the crystalline raw material crystals to a solvent-free pyrolysis carbonization reaction to obtain a carbonization product, namely PCDs, with the reaction temperature not lower than 180°C and the reaction time not less than 4 hours.

[0012] In the above-mentioned nanobiosensor, in step a, PCDs are dissolved in ultrapure water, and unreacted raw materials and over-carbonized insoluble carbon residues are removed by filtration and dialysis to obtain a PCDs solution.

[0013] In the above-mentioned nano biosensor, in step a, the raw material is phytic acid PA, the solvent is pure water, the mass concentration of the raw material solution is 70%; the reaction temperature is 200° C., and the reaction time is 6 hours.

[0014] The specific process of step b of the above-mentioned nanobiosensor is as follows: a PCDs solution is prepared using a Tris-HCl buffer solution with a pH of 8.3 as a solvent, and alkaline phosphatase AP is added, and the solution is allowed to stand in a water bath at 37° C. for 2 hours to obtain a nanobiosensor.

[0015] In the above-mentioned nanobiosensor, in step b, the concentration of PCDs is 0.1 mg / mL, and the concentration of alkaline phosphatase AP is 1000 mU / mL.

[0016] The present invention also proposes a method for directly detecting organophosphorus pesticides based on nanobiosensors. The method realizes direct detection of organophosphorus pesticides OPs in one step based on nanobiosensors rich in carbon dots of phosphate groups and enzymatic reactions, without the need for step-by-step detection. The method is simple, rapid, reliable, highly sensitive, and has good specificity. It can avoid interference of other components such as proteins, reducing sugars, amino acids, and metal ions in the detection of OPs, and does not require complicated sample pretreatment.

[0017] The method for directly detecting organophosphorus pesticides based on nanobiosensors of the present invention comprises the following steps:

[0018] Using organic small molecules containing phosphate groups as raw materials, PCDs with rich surface phosphate groups were prepared by carbonization.

[0019] PCDs were used as fluorophores and mixed with dephosphorylase to construct nanobiosensors;

[0020] Organophosphorus pesticides OPs with known different concentrations were mixed with the nanobiosensor, and the fluorescence spectrum of the nanobiosensor was measured after standing to draw a standard curve;

[0021] The OPs sample to be detected is mixed with the nanobiosensor, the fluorescence spectrum of the nanobiosensor is measured, and the concentration of the OPs to be detected is calculated according to the standard curve.

[0022] The specific process of step a of the above-mentioned direct detection method of organophosphorus pesticides is as follows: dissolving the raw material in a solvent to prepare a solution, drying the solution at a temperature not exceeding 80°C to a crystalline state without free water, and then subjecting the crystalline raw material crystals to a solvent-free pyrolysis carbonization reaction to obtain a carbonization product, namely PCDs, with the reaction temperature not lower than 180°C and the reaction time not less than 4 hours.

[0023] In the above-mentioned direct detection method of organophosphorus pesticides, in step a, PCDs is dissolved in ultrapure water, and unreacted raw materials and over-carbonized insoluble carbon residues are removed by filtration and dialysis to obtain a PCDs solution.

[0024] In the above-mentioned direct detection method of organophosphorus insecticides, in step a, the raw material is phytic acid PA, the solvent is pure water, the mass concentration of the raw material solution is 70%; the reaction temperature is 200° C., and the reaction time is 6 hours.

[0025] The specific process of step b of the above-mentioned direct detection method of organophosphorus pesticides is as follows: a PCDs solution is prepared using a Tris-HCl buffer solution with a pH of 8.3 as a solvent, and alkaline phosphatase AP is added, and the solution is allowed to stand in a water bath at 37° C. for 2 hours to obtain a nanobiosensor.

[0026] In the above-mentioned direct detection method of organophosphorus pesticides, in step b, the PCDs concentration is 0.1 mg / mL and the alkaline phosphatase AP concentration is 1000 mU / mL.

[0027] The specific content of step c of the above-mentioned direct detection method of organophosphorus pesticides is as follows: adding 0-1000 μg / mL OPs of known different concentrations to the nanobiosensor solution and incubating at 37°C for not less than 30 minutes, measuring the fluorescence intensity of the nanobiosensor, and drawing a standard curve of the relationship between fluorescence intensity and OPs concentration.

[0028] In the above-mentioned method for direct detection of organophosphorus pesticides, in step c, the excitation wavelength of the nanobiosensor is 380 nm, and the wavelength scanning range is 510-600 nm.

[0029] In the above-mentioned direct detection method of organophosphorus pesticides, in step c, a regression equation is calculated based on the relationship between fluorescence intensity and OPs concentration.

[0030] In the above-mentioned direct detection method of organophosphorus insecticides, in step c, OPs is chlorpyrifos, and when the concentration of chlorpyrifos is 0.01-100 μg / mL, the regression equation is Y=863lgX+13204, where X is the concentration of chlorpyrifos and Y is the fluorescence intensity.

[0031] Beneficial effects of the present invention:

[0032] (1) The present invention uses organic small molecules rich in phosphate groups as carbonization raw materials and prepares PCDs by pyrolysis carbonization, which has the beneficial effects of low cost, environmental friendliness and good repeatability.

[0033] (2) The surface of PCDs retains the functional groups of the precursor, that is, it is rich in a large number of phosphate groups, so that PCDs produce fluorescence response to dephosphorylase. The enzymatic carbon dot fluorescence regulation involved in the present invention is the first in the world, with the beneficial effects of being simple, fast and rate-controllable, and at the same time laying a theoretical foundation for OPs detection.

[0034] (3) The OPs detection method disclosed in the present invention is a direct detection strategy, which avoids the interference of other components (such as proteins, reducing sugars, amino acids, metal ions, etc.) in real samples on OPs detection, has the beneficial effect of high accuracy, and avoids the complicated sample pretreatment process in the OPs detection process.

[0035] (4) The present invention is a method for directly detecting OPs. When OPs are added to the nanobiosensor, a concentration-dependent differential fluorescence signal output can be generated, without the need to detect OPs in separate steps. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a transmission electron microscope image of the PCDs prepared in Example 1.

[0037] Figure 2 The chemical composition of the PCDs prepared in Example 1 is characterized. a is a Fourier infrared spectrum; b is an X-ray photoelectron spectrum.

[0038] Figure 3 The fluorescence response of the PCDs prepared in Example 1 to the activity of dephosphorylase at different concentrations.

[0039] Figure 4 Schematic diagram of the mechanism of the nanobiosensor disclosed in the present invention for detecting OPs.

[0040] Figure 5 This is the standard curve of the fluorescence response of the nanobiosensor to different concentrations of OPs (chlorpyrifos).

[0041] Figure 6 This is the fluorescence response of the nanobiosensor to different interferents and when the interferents coexist with OPs. DETAILED DESCRIPTION

[0042] Technical solution: The present invention involves a variety of correlation technologies.

[0043] Technical solution 1: Preparation of carbon dots (PCDs) with rich surface phosphate groups. PCDs are prepared by pyrolysis and carbonization using organic compounds containing phosphate groups as raw materials. The raw material powder is dissolved in a solvent to prepare a solution of a certain concentration, and then the solution is dried to a crystalline state without free water, and the drying temperature does not exceed 80°C. The crystalline raw material crystals are then subjected to solvent-free pyrolysis and carbonization reaction, with the reaction temperature not lower than 180°C and the time not less than 4 hours. Finally, the carbonized product is dissolved in ultrapure water, and the unreacted raw materials and over-carbonized insoluble carbon residues are removed by filtration and dialysis to obtain a PCDs solution.

[0044] Preferably, the raw material selected is phytic acid (PA), the solvent is pure water, the raw material solution is a 70% (mass concentration) phytic acid aqueous solution, the reaction temperature is 200° C., and the reaction time is 6 hours.

[0045] Technical solution 2: Preparation of nanobiosensors. Using Tris-HCl buffer (pH 8.3) as the solvent system, the PCDs prepared in Technical Solution 1 as the fluorophore, and the dephosphorylase as the signal conversion unit, a certain concentration of PCDs and dephosphorylase were mixed to successfully construct a nanobiosensor for the detection of organophosphorus (OPs) pesticides.

[0046] Preferably, the concentration of PCDs in the nanobiosensor solution is 0.1 mg / mL, the type of dephosphorylating enzyme is alkaline phosphatase (AP), the enzyme concentration is 1000 mU / mL, and the standing time in the water bath is 2 hours.

[0047] Technical Solution 3: Quantitative relationship between the fluorescence intensity of the nanobiosensor and OPs. OPs of different concentrations were quickly added to the nanobiosensor prepared in Technical Solution 2, and the nanobiosensor mixed with OPs was placed in a 37°C water bath for a certain period of time, the fluorescence intensity of the nanobiosensor was measured, a standard curve was drawn, and the regression equation was calculated.

[0048] Preferably, in the above scheme, OPs uses chlorpyrifos as a representative insecticide, the standing time is 45 minutes, the excitation wavelength of the nanobiosensor is 380 nm, and the wavelength scanning range is 510-600 nm.

[0049] Technical Solution 4: Direct detection of unknown concentration of OPs. The OPs sample solution to be detected is mixed with the nanobiosensor constructed in Technical Solution 2, incubated and the fluorescence intensity of the nanosensor is detected according to the optimized conditions described in Technical Solution 3, and the fluorescence intensity value is substituted into the standard curve to calculate the concentration of OPs.

[0050] Example 1

[0051] PCDs were prepared by solvent-free pyrolysis carbonization method using phytic acid as raw material. A phytic acid aqueous solution with a mass concentration of 70% was prepared, and then the solution was transferred to an oven for low-temperature drying at 80°C to a crystalline state, and then a carbonization reaction occurred at a high temperature of 200°C for 6 hours. After cooling to room temperature, the carbonized product was dissolved in ultrapure water, the solution was ultrasonically treated and the supernatant was taken, filtered through a 0.22μm filter, and dialyzed for 6 hours to obtain a PCDs solution. Figure 1 This is a transmission electron microscope photo of PCDs. The PCDs are evenly distributed and the average size is 2.6nm. Figure 2 FTIR spectrum and XPS spectrum of the prepared carbon dots. Figure 2 As shown in a, PCDs are rich in phosphate groups, 1158 cm -1 The absorption peak at 1108 cm corresponds to the stretching vibration of P=O. -1 and 1008cm -1 Corresponding to the tensile vibration of POR and POH. And there are -OH and -COOH functional groups distributed on the surface of PCDs.

[0052] Example 2

[0053] In this example, the preparation process of PCDs is the same as that of Example 1. A PCDs solution with a concentration of 0.1 mg / mL is prepared, and the solvent is Tris-HCl buffer (pH 8.3). Different activity units of AP are added to the PCDs solution, incubated in a water bath at 37°C for 2 hours, and the fluorescence intensity of PCDs is detected. Figure 3 As shown, under the action of different active units, the phosphate groups on the surface of PCDs break to varying degrees, which is the passivation effect of the dephosphorylation group, and the green fluorescence of PCDs is quenched to varying degrees.

[0054] Example 3

[0055] In this example, the preparation process of PCDs is the same as that of Example 1. Construction of the nanobiosensor: The solvent is Tris-HCl buffer (pH 8.3), a PCDs solution with a concentration of 0.1 mg / mL is prepared, AP is added to the PCDs solution, and incubated in a water bath at 37°C for 2 hours. The active concentration of AP is 1000 mU / mL. The detection principle is as follows Figure 4 As shown, 0-1000 μg / mL of OPs was added to the nanobiosensor solution to inhibit enzyme activity, and the fluorescence intensity of the sensor was measured after incubation at 37°C ( Figure 5As shown in the figure, the nanobiosensor exhibited a fluorescence intensity response that was dependent on the OPs concentration, with a linear detection range of 0.01-100 μg / mL and a detection limit of 4.6 ng / mL, which is lower than the upper limit (0.01 mg / kg) of the content of organophosphorus (chlorpyrifos, dimethoate, parathion, etc.) in fruits and vegetables specified in the Chinese standard GB 2763-2021.

[0056] Example 4

[0057] In this embodiment, the preparation process of the carbon quantum dot fluorescence sensor is the same as that of embodiment 3, and the PCDs concentration is 0.1 mg / mL. The same volume of the interfering substance solutions present in the fruit and vegetable samples such as different ions, proteins, etc. are mixed with the sensor solution respectively, or OPs are added to the interfering substance solution and then mixed with the nanosensor solution. + ,Mg 2+ , Zn 2+ , K + , Glu, Cellulose, Pectin, Gly, BSA, UA, VC, etc. concentrations are 100mmol / L, which is much greater than the concentration of OPs 500μg / mL. Measure the fluorescence intensity of the sensor. Figure 6 The fluorescence response of the nanobiosensor to different interferents shown in (a) shows that the sensor has good selectivity for OPs. Only OPs can inhibit enzyme activity, while other interfering substances cannot. Changes in the functional groups on the PCDs surface lead to quenching of fluorescence. Figure 6 The fluorescence response of the nanobiosensor shown in b to the coexistence of interferents and OPs, simulating the Na + ,Mg 2+ , Zn 2+ , K + When interfering substances such as Glu, Cellulose, Pectin, Gly, BSA, UA, VC coexist with OPs, the results of the fluorescence sensor have no obvious effect. Therefore, by using fluorescence detection signals, the sensor can eliminate the interference of other components in fruit and vegetable samples and has good selectivity for organophosphorus pesticides OPs.

[0058] Example 5

[0059] In this embodiment, the concentration of OPs in the extract of fruit and vegetable samples was detected with reference to the process steps described in Example 3. The fruit and vegetable samples used were crushed extracts of cabbage and celery to which different concentrations of OPs pesticides were added. The measured fluorescence intensity of the nanobiosensor was substituted into the standard curve to calculate the measured concentration of OPs. After calculation, the recovery rate of the added OPs pesticides in the fruit and vegetable samples was 83.0%-115.1%. Therefore, the present invention is suitable for the detection of OPs pesticides in real fruit and vegetable samples, and has the advantages of simple operation, low cost and high accuracy.

[0060] The present invention uses organic small molecules containing phosphate groups as precursors, and prepares green fluorescent carbon dots (PCDs) rich in phosphate groups on the surface through carbonization. The phosphate groups of the precursors are retained on the surface of PCDs, and the rich phosphate monoester bonds provide a large number of hydrolysis sites for dephosphorylase. The enzyme catalyzes the cleavage of the phosphate groups on the surface of PCDs and converts them into hydroxyls, causing the fluorescence of PCDs to be quenched, and a nanobiosensor with PCDs as fluorophores and dephosphorylase as signal conversion units is successfully constructed. The activity of OPs is inhibited by enzymes to avoid the fluorescence quenching of PCDs, thereby realizing direct detection of OPs. The relationship between the fluorescence response of PCDs and the concentration of OPs is used to realize quantitative analysis of OPs. This direct detection strategy determines the good anti-interference ability of the nanobiosensor, has excellent reliability, and can be used for high-precision analysis of OPs in fruit and vegetable samples. The present invention is a new method for direct detection of OPs with high sensitivity and good specificity, has the significant advantages of simple use and high accuracy, and can be used for rapid and reliable analysis of OPs in real food samples.

Claims

1. A nanobiosensor, characterized in that: It is prepared by the following method: a. Using organic small molecules containing phosphate groups as raw materials, carbon dots PCDs with rich phosphate groups on the surface are prepared by carbonization; b. Mix PCDs as fluorophores and dephosphorylase to construct nanobiosensors; The specific process of step a is as follows: dissolving the raw material phytic acid PA in a solvent to prepare a solution, drying the solution at a temperature not exceeding 80° C. to a crystalline state without free water, and then subjecting the crystalline raw material crystals to a solvent-free pyrolysis carbonization reaction to obtain a carbonized product, namely PCDs, wherein the reaction temperature is not less than 180° C. and the reaction time is not less than 4 hours; The specific process of step b is as follows: a PCDs solution is prepared using a Tris-HCl buffer solution with a pH of 8.3 as a solvent, and alkaline phosphatase AP is added, and the solution is allowed to stand in a water bath at 37° C. for 2 hours to obtain a nanobiosensor.

2. The nanobiosensor according to claim 1, characterized in that: In step a, PCDs are dissolved in ultrapure water, and unreacted raw materials and over-carbonized insoluble carbon residues are removed by filtration and dialysis to obtain a PCDs solution.

3. The nanobiosensor according to claim 1, characterized in that: In step a, the solvent is pure water, the mass concentration of the raw material solution is 70%; the reaction temperature is 200° C., and the reaction time is 6 hours.

4. The nanobiosensor according to claim 1, characterized in that: In step b, the concentration of PCDs was 0.1 mg / mL and the concentration of alkaline phosphatase AP was 1000 mU / mL.

5. A method for direct detection of organophosphorus pesticides based on nanobiosensors, characterized in that: The following steps are involved: c. Mixing organophosphorus pesticides OPs of known different concentrations with the nanobiosensor according to claim 1, measuring the fluorescence spectrum of the nanobiosensor after standing, and drawing a standard curve; d. Mix the OPs sample to be detected with the nanobiosensor, measure the fluorescence spectrum of the nanobiosensor, and calculate the concentration of the OPs to be detected according to the standard curve.

6. The method for direct detection of organophosphorus pesticides according to claim 5, characterized in that: The specific content of step c is as follows: adding OPs of known different concentrations of 0-1000 μg / mL to the nanobiosensor solution and incubating at 37°C for not less than 30 minutes, measuring the fluorescence intensity of the nanobiosensor, and drawing a standard curve of the relationship between the fluorescence intensity and the OPs concentration.

7. The method for direct detection of organophosphorus pesticides according to claim 6, characterized in that: In step c, the excitation wavelength of the nanobiosensor is 380 nm, and the wavelength scanning range is 510-600 nm.

8. The method for direct detection of organophosphorus pesticides according to claim 6, characterized in that: In step c, OPs is chlorpyrifos, and the regression equation is calculated according to the relationship between the fluorescence intensity and the OPs concentration; when the chlorpyrifos concentration is 0.01-100 μg / mL, the regression equation is Y=863lgX+13204, X is the chlorpyrifos concentration, and Y is the fluorescence intensity.

Images