Sensor for regulating fluorescence based on nanogold size

Abstract

The invention discloses a sensor for regulating fluorescence based on nanogold size. The sensor regulates and controls the generation amount of thiocholine through an enzyme inhibition reaction dependent on organophosphorus concentration so as to control the synthesis size of AuNPs, and high-sensitivity response of a fluorescence signal is realized by combining the efficiency difference of fluorescence resonance energy transfer (FRET) between carbon quantum dots (CDs) and AuNPs with different sizes. According to the method, the detection limit reaches 3.2 nM, the linear range is wide and is 0-100 [mu] M, the anti-interference capability is high, the actual sample detection recovery rate is 87.0%-114.6%, the method is suitable for accurate analysis of OPs residues in agricultural products such as fruits and vegetables, and an efficient and reliable technical means is provided for food safety monitoring.

Description

Technical Field

[0001] The present invention belongs to the field of nano-optical sensors, and in particular relates to a fluorescent nano-sensor for detecting organophosphorus pesticides and an application method thereof. Background Art

[0002] Organophosphorus pesticides are widely used in modern agriculture due to their high efficacy and broad-spectrum insecticides. Their significance lies in three key aspects: First, they can rapidly kill a wide range of pests, significantly reducing crop losses caused by pests and diseases, and safeguarding food production; second, compared to traditional pesticides, they have a shorter lifespan and lower costs, enabling farmers to achieve cost-effective field management; and finally, their rational use can support agricultural intensification and alleviate the conflict between population growth and limited arable land. However, their drawbacks should not be overlooked: first, their high toxicity can cause damage to non-target organisms (such as pollinators and aquatic life) and ecosystems; second, long-term excessive use can easily lead to the development of pesticide resistance in pests, forcing escalating pesticide use and creating a vicious cycle; and third, residues can accumulate in the food chain, posing a threat to human health, particularly with potential damage to the nervous system and liver. Detecting organophosphorus residues in crops is crucial. On one hand, rigorous residue testing is a key component of ensuring food safety. Accurately measuring residue levels through techniques such as chromatography and mass spectrometry can effectively prevent agricultural products exceeding standards from entering the market, reducing the risk of chronic poisoning to consumers from long-term, low-dose organophosphorus ingestion. Furthermore, testing data provides a basis for scientific pesticide use, guiding farmers to adjust application schedules and dosages to reduce environmental impact. Furthermore, organophosphorus residue limits are widely set in international agricultural trade, and strengthening testing capabilities can help overcome technical barriers. Therefore, when weighing the pros and cons of organophosphorus pesticides, establishing a systematic residue monitoring system and promoting green alternative technologies are crucial paths to sustainable agricultural development.

[0003] Gold nanoparticles (AuNPs) have shown broad application prospects in the field of fluorescence sensors due to their unique optical properties and surface modifiability. Their surface plasmon resonance effect (SPR) and fluorescence enhancement / quenching properties can achieve signal amplification and selectivity improvement through size, morphology regulation and surface ligand design. In recent years, researchers have focused on the development of composite structures, such as combining with quantum dots, carbon dots or organic dyes to construct dual-mode sensing systems, which significantly improves detection sensitivity and anti-interference ability. For example, after AuNPs are surface-modified with aptamers or molecularly imprinted polymers, they can specifically recognize organophosphorus pesticide residues, cause changes in their dispersion state, and output sensing signals. In addition, AuNPs-based fluorescence sensors have outstanding potential in point-of-care testing (POCT). Their portability, rapid response and visual signal output (such as color / fluorescence changes) provide an efficient sensing system for food safety.

[0004] However, AuNPs have poor stability in complex sample matrices, and the anti-interference ability of sensing systems that rely on the dispersion state of AuNPs needs to be improved.

[0005] The optical properties of AuNPs are highly dependent on their size, with larger AuNPs exhibiting a stronger SPR effect. Therefore, designing a more robust organophosphorus detection system by manipulating the size of AuNPs is a novel approach. This paper proposes a controllable synthesis strategy for organophosphorus concentration-dependent AuNPs, enabling high-precision analysis of organophosphorus residues in crops. Summary of the Invention

[0006] To address the current problems, the present invention provides a novel fluorescence sensor. Its working principle relies on the size of AuNPs regulated by organic phosphorus content, as well as the efficiency difference of fluorescence resonance energy transfer (FRET) between fluorescent nanomaterials, carbon quantum dots (CDs), and AuNPs of different sizes, ultimately achieving the regulation of fluorescence signal intensity. This nano-fluorescence sensor has the advantages of high sensitivity and good reliability.

[0007] The first object of the present invention is to provide a method for preparing a sensor based on nanogold size-regulated fluorescence, comprising the following preparation steps:

[0008] a. The acetylcholinesterase (AChE) solution and the thioacetylcholine (ATCh) solution were mixed in a volume ratio of 1:1. The solvents of the acetylcholinesterase (AChE) solution and the thioacetylcholine (ATCh) solution were both PBS buffer. The mixture was allowed to stand in a constant temperature water bath at 37°C for 1 hour to hydrolyze the thioacetylcholine to form thiocholine (TCh).

[0009] b. To the reaction system of step a, chloroauric acid (HAuCl4) stock solution and sodium borohydride (NaBH4) stock solution were sequentially added, the mixture was shaken and allowed to stand for 10 minutes to synthesize gold nanoparticles (AuNPs) in situ;

[0010] c. Add 0.5 mL of carbon quantum dots (CDs) solution to the AuNPs dispersion in step b, mix well, scan the fluorescence spectrum in the range of 380-650 nm, and record the fluorescence intensity.

[0011] Furthermore, in step a, the concentration of the acetylcholinesterase (AChE) solution is 3 mU / mL, and the concentration of the thioacetylcholine (ATCh) solution is 500 μM.

[0012] Furthermore, the pH range of the PBS buffer in step a is 7.5-8.5, and the concentration is 8-12 mM; preferably, the pH of the PBS buffer is 8.0, and the concentration is 10 mM.

[0013] Furthermore, in step b, the concentration of the chloroauric acid (HAuCl4) stock solution is 0.05-0.2wt%, and the concentration of the sodium borohydride (NaBH4) stock solution is 50-200μM; preferably, the concentration of the chloroauric acid (HAuCl4) stock solution is 0.1wt%, and the concentration of the sodium borohydride (NaBH4) stock solution is 100μM.

[0014] Furthermore, the volume ratio of the chloroauric acid (HAuCl4) stock solution and the sodium borohydride (NaBH4) stock solution in step b to the acetylcholinesterase (AChE) solution in step a is 1:5.

[0015] Furthermore, the concentration of the carbon quantum dot (CDs) solution in step c is 15-25 μg / mL; preferably, the concentration of the carbon quantum dot (CDs) solution is 20 μg / mL; the volume ratio of the carbon quantum dot (CDs) solution to the acetylcholinesterase (AChE) solution in step a is 1:1.

[0016] Furthermore, in step c, the excitation wavelength of carbon quantum dots (CDs) is 350-370 nm; preferably, the excitation wavelength is 360 nm.

[0017] The second object of the present invention is to provide a sensor based on size-regulated fluorescence of gold nanoparticles, wherein the sensor is prepared using the aforementioned preparation method.

[0018] The third object of the present invention is to provide a method for detecting organophosphorus pesticides (OPs) based on the aforementioned sensor, comprising the following steps:

[0019] d. Prepare organophosphorus pesticide (OPs) concentration gradient solutions, which were added to the AChE and ATCh mixed solutions in step a to inhibit AChE activity to reduce TCh generation; repeat steps b and c to measure the fluorescence intensity of CDs at different OPs concentrations;

[0020] e. Draw a standard curve with OPs concentration as the horizontal axis and fluorescence intensity as the vertical axis and establish a linear regression equation;

[0021] f. Centrifuge and filter the sample to be tested, then dilute it. Measure the fluorescence intensity according to step d. Substitute the fluorescence intensity into the standard curve to calculate the actual concentration of organophosphorus pesticides (OPs) in the sample.

[0022] Furthermore, the concentration gradient of the organophosphorus pesticide (OPs) solution in step d is at least one of 0.001-100 μM.

[0023] Furthermore, the sample to be tested in step f is an extract of agricultural products, including a solution obtained by extracting crushed vegetables, fruits or grains using PBS buffer.

[0024] Beneficial effects of the present invention:

[0025] (1) The present invention utilizes the sensitivity of surface ligands to size regulation during the synthesis of AuNPs to establish a nanosensor based on the efficient optical coupling mechanism between CDs and AuNPs, which has the advantage of high sensitivity.

[0026] (2) The detection signal is based on the size change of AuNPs rather than the dispersion state, avoiding the interference of complex sample matrix on the dispersion stability and significantly improving the applicability and accuracy of the nanosensor in real samples.

[0027] (3) Combining the specificity of the enzyme inhibition reaction with the controllable synthesis strategy of AuNPs ensures that the detection results are stable and reliable, suitable for precise quantitative analysis, and the operation steps are simple and easy to standardize.

[0028] (4) The sensor regulates the amount of thiocholine (TCh) generated through an organophosphorus concentration-dependent enzyme inhibition reaction, thereby controlling the synthesis size of AuNPs. It also combines the difference in fluorescence resonance energy transfer (FRET) efficiency between carbon quantum dots (CDs) and AuNPs of different sizes to achieve a highly sensitive response to the fluorescence signal. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a transmission electron microscope photograph of the AuNPs prepared in Example 1.

[0030] Figure 2 This is a statistical diagram of the particle size of AuNPs prepared in Example 1.

[0031] Figure 3 This is a diagram of the fluorescence signal conversion mechanism disclosed in the present invention.

[0032] Figure 4 (a) The fluorescence response spectra of the fluorescence sensor to different concentrations of OPs and the standard curve (b) in Example 3.

[0033] Figure 5 This is a fluorescence intensity diagram of the fluorescent sensor in Example 4 in response to different types of pesticides.

[0034] Figure 6 This is a graph showing the fluorescence response intensity of the fluorescence sensor in Example 4 when interferents and OPs coexist. DETAILED DESCRIPTION

[0035] The present invention is further explained below with reference to the following examples, but the examples do not limit the present invention in any form.

[0036] Technical solution: The present invention involves a variety of correlation technologies.

[0037] Technical Solution 1. Preparation of the Fluorescent Sensor—Construction of the Catalytic Reaction System: Prepare PBS buffer, dissolve AChE and ATCh separately, and mix 0.5 mL of AChE solution with 0.5 mL of ATCh solution in a centrifuge tube. Shake thoroughly. Place the mixed solution in a water bath to allow ATCh to hydrolyze to form TCh under the catalysis of AChE.

[0038] Preferably, the concentration of PBS is 10 mM and the pH is 8.0; the concentrations of AChE and ATCh are 3 mU / mL and 500 μM, respectively; the water bath temperature is 37° C., and the incubation time is 1 hour.

[0039] Technical Solution 2. Preparation of Fluorescent Sensor—Synthesis of AuNPs: HAuCl4 and NaBH4 stock solutions were added sequentially to the reaction system from Technical Solution 1, and the mixture was rapidly shaken. After standing at room temperature, the NaBH4 reduced the HAuCl4 to form AuNPs, whose size was controlled by the TCh concentration.

[0040] Preferably, the volumes of the HAuCl4 stock solution and the NaBH4 stock solution are both 0.1 mL, and the concentrations are 0.1 wt% and 100 μM, respectively; the AuNPs synthesis time is 10 min, and the reaction is carried out at room temperature.

[0041] Technical Solution 3. Preparation of Fluorescence Sensor—CDs Fluorescence Response: Add CDs solution to the AuNPs dispersion and mix thoroughly. Measure and record the fluorescence intensity of the CDs using a fluorescence spectrometer.

[0042] Preferably, the concentration of the CDs solution is 20 μg / mL and the volume is 0.5 mL; after CDs and AuNPs are mixed, spectral scanning can be started without waiting; the fluorescence spectrum scanning range is 380-650 nm, and the excitation wavelength is 360 nm.

[0043] Technical Solution 4. OPs Detection Method - Standard Curve Drawing: Dilute the OPs stock solution with PBS buffer to prepare a series of OPs solutions with a final concentration of 0-100 μM. Replace the pure PBS buffer with PBS solutions of OPs at various concentrations to prepare mixed solutions of AChE (3 mU / mL) and ATCh (500 μM), and repeat the above sensor preparation technical solutions (Schemes 1-3). With the OPs concentration as the horizontal axis and the corresponding fluorescence intensity as the vertical axis, draw a standard curve and establish a linear regression equation Y=aX+b. Where Y is the dependent variable (fluorescence intensity), X is the independent variable (OPs concentration), and a and b are constants.

[0044] Preferably, the concentrations of the diluted OPs are: 0, 0.001, 0.1, 1, 10, 50, 100 μM.

[0045] Technical Solution 5. OPs Detection Method—Quantitative Analysis of Unknown OPs Concentrations: Centrifuge and filter the agricultural product sample (e.g., vegetable extract). Dilute the supernatant with PBS buffer. Prepare the diluted sample solution, construct a sensor according to Technical Solutions 1-3, and measure its fluorescence intensity. Substitute the measured fluorescence intensity into the standard curve equation to calculate the actual OPs concentration in the sample.

[0046] Example 1

[0047] In PBS buffer, AChE was used as the catalytic enzyme and ATCh as the substrate. After the catalytic reaction, NaBH₄ was added to the system to reduce HAuCl₄. AuNPs were prepared using the reduction effect of NaBH₄, using the same conditions as described in Technical Scheme 1-2. To simulate the inhibitory effect of OPs on AChE, AChE was removed from the catalytic reaction and the size of the AuNPs was compared. Figure 1 Transmission electron microscopy photos of AuNPs under two conditions. Among them, the system with AChE produces TCh tightly complexed with AuNPs, and its size is significantly smaller than that of AuNPs synthesized in the system without AChE. Figure 2 Figure 2 is the size distribution diagram of AuNPs in the two systems. Corresponding to the results of transmission electron microscopy, the average particle size of smaller-sized AuNPs is 1.6 nm, while the average particle size of larger-sized AuNPs is 4.9 nm.

[0048] Example 2

[0049] In this example, large-sized AuNPs were synthesized according to the method mentioned in Example 1, and their absorption spectrum was measured. A dilute solution of CDs was prepared with a concentration of 20 μg / mL, and its fluorescence spectrum was measured. The spectral measurement conditions were the same as those in Technical Solution 3. The relationship between the absorption spectrum of AuNPs and the fluorescence spectrum of CDs was compared to verify the optical coupling between the two and explore the fluorescence quenching mechanism of CDs. Figure 3 As shown, in the range of 450-680 nm, the fluorescence spectrum of CDs overlaps with the absorption spectrum of AuNPs over a large area, which meets the conditions for the occurrence of the FRET optical mechanism, resulting in the fluorescence quenching of CDs.

[0050] Example 3

[0051] In this example, AuNPs were synthesized in the presence of OPs at different concentrations in an AChE catalytic reaction system, and the synthesis conditions were the same as in Example 1. By regulating the rate of the enzyme-catalyzed reaction through OPs, the amount of TCh generated was regulated, and AuNPs of different particle sizes were further controllably synthesized. CDs were added to the AuNPs dispersion, and the fluorescence of the fluorescence sensor was regulated based on the strength of the FRET effect between CDs and AuNPs of different sizes. The fluorescence intensity of the fluorescence sensor was measured ( Figure 4As shown in the figure, the nanobiosensor exhibited an OPs concentration-dependent fluorescence intensity response, with a linear detection range of 0-100 μM and a detection limit of 3.2 nM, which is lower than the upper limit of organic phosphorus content in fruits and vegetables specified in my country's standard GB 2763-2021 (0.01 mg / kg).

[0052] Example 4

[0053] In this example, OPs was replaced with other types of insecticides, and fluorescence sensors were prepared according to the same process as in Example 3. The concentration of the insecticides was 100 μM, and the selectivity of the fluorescence sensors was studied. At the same time, various types of metal ions, glucose and other nutrients coexisted with OPs, and fluorescence sensors were prepared according to the same process as in Example 3 to evaluate the anti-interference ability of this method. + , K + , Ca 2+ , Mg 2+ , Fe 2+ , glucose, amino acid concentration is 100mmol / L, which is much higher than the concentration of OPs 100μM, and protein concentration is 1mg / mL. Figure 5 As shown, the fluorescent sensor exhibited significant fluorescence response only to OPs type insecticides (chlorpyrifos, omethoate, trthion), but had no response to organochlorine (quintozene, lindane, endosulfan) and pyrethroid (permethrin, cypermethrin, fenvalerate) insecticides, showing good specificity. Figure 6 The relative fluorescence intensity graph shown in Figure 2 demonstrates that the fluorescence sensor's response to OPs is unaffected by potential interferents, demonstrating strong anti-interference capabilities. Therefore, this fluorescence sensor can eliminate interference from other components in fruit and vegetable samples and can be used for the analysis of OPs residues in real samples.

[0054] Example 5

[0055] In this example, the OPs concentration in fruit and vegetable sample extracts was detected using the same process steps as described in Example 3. The fruit and vegetable samples used were extracts of crushed carrots and pakchoi spiked with varying concentrations of OPs pesticides. The fluorescence intensity measured by the fluorescence sensor was substituted into a standard curve to calculate the measured OPs concentration. Calculated recoveries of the spiked OPs pesticides in the fruit and vegetable samples ranged from 87.0% to 114.6%. Therefore, the present invention is suitable for detecting OPs pesticides in real fruit and vegetable samples, offering advantages such as simple operation, low cost, and high accuracy.

[0056] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a sensor based on size-regulated fluorescence of gold nanoparticles, characterized in that: The method comprises the following preparation steps: a. The acetylcholinesterase (AChE) solution and the thioacetylcholine (ATCh) solution were mixed in a volume ratio of 1:

1. The solvents of the acetylcholinesterase (AChE) solution and the thioacetylcholine (ATCh) solution were both PBS buffer. The mixture was allowed to stand in a constant temperature water bath at 37°C for 1 hour to hydrolyze the thioacetylcholine to form thiocholine (TCh). b. To the reaction system of step a, chloroauric acid (HAuCl4) stock solution and sodium borohydride (NaBH4) stock solution were sequentially added, the mixture was shaken and allowed to stand for 10 minutes to synthesize gold nanoparticles (AuNPs) in situ; c. Add 0.5 mL of carbon quantum dots (CDs) solution to the AuNPs dispersion in step b, mix well, scan the fluorescence spectrum in the range of 380-650 nm, and record the fluorescence intensity.

2. The preparation method according to claim 1, characterized in that The concentration of the acetylcholinesterase (AChE) solution in step a is 3 mU / mL, and the concentration of the thioacetylcholine (ATCh) solution is 500 μM; the PBS in step a The pH range of the buffer solution is 7.5-8.5, and the concentration is 8-12 mM; preferably, the pH of the PBS buffer solution is 8.0, and the concentration is 10 mM.

3. The preparation method according to claim 1, characterized in that In step b, the concentration of the chloroauric acid (HAuCl4) stock solution is 0.05-0.2wt%, and the concentration of the sodium borohydride (NaBH4) stock solution is 50-200μM; preferably, the concentration of the chloroauric acid (HAuCl4) stock solution is 0.1wt%, and the concentration of the sodium borohydride (NaBH4) stock solution is 100μM.

4. The preparation method according to claim 1, characterized in that The volume ratio of the chloroauric acid (HAuCl4) stock solution and the sodium borohydride (NaBH4) stock solution in step b to the acetylcholinesterase (AChE) solution in step a is 1:

5.

5. The preparation method according to claim 1, characterized in that The concentration of the carbon quantum dot (CDs) solution in step c is 15-25 μg / mL; preferably, the concentration of the carbon quantum dot (CDs) solution is 20 μg / mL; the volume ratio of the carbon quantum dot (CDs) solution to the acetylcholinesterase (AChE) solution in step a is 1:

1.

6. The preparation method according to claim 1, characterized in that In step c, the excitation wavelength of carbon quantum dots (CDs) is 350-370 nm; preferably, the excitation wavelength is 360 nm.

7. A sensor based on size-regulated fluorescence of gold nanoparticles, characterized in that: The compound is prepared by the preparation method according to any one of claims 1 to 6.

8. A method for detecting organophosphorus pesticides (OPs) based on the sensor according to claim 7, characterized in that: The following steps are involved: d. Prepare gradient concentration solutions of organophosphorus pesticides (OPs) and add them to the mixed solution of AChE and ATCh in step a to inhibit AChE activity to reduce TCh generation; repeat steps b and c to measure the fluorescence intensity of CDs at different OPs concentrations; e. Draw a standard curve with OPs concentration as the horizontal axis and fluorescence intensity as the vertical axis and establish a linear regression equation; f. Centrifuge and filter the sample to be tested, then dilute it. Measure the fluorescence intensity according to step d. Substitute the fluorescence intensity into the standard curve to calculate the actual concentration of organophosphorus pesticides (OPs) in the sample.

9. The detection method according to claim 8, characterized in that In the step d, the gradient concentration of the organophosphorus pesticide (OPs) is at least one of 0.001-100 μM.

10. The detection method according to claim 8, characterized in that The sample to be tested in step f is an extract of agricultural products, including a solution obtained by extracting crushed vegetables, fruits or grains using PBS buffer.

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