Ratio-dependent fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a ratio type fluorescent probe FITC-Lip-Au NCs and a preparation method and application thereof.The preparation method comprises the steps that lipase serves as a template, chloroauric acid serves as a metal precursor, a reaction is conducted under the alkaline condition to generate a red fluorescent gold nanocluster, the red fluorescent gold nanocluster is further coupled with fluorescein isothiocyanate, and the ratio type biological fluorescent probe with double emission peaks is obtained. The probe shows high selectivity and high sensitivity to paraquat and the like, the detection limit of paraquat is as low as 15 nM, good linear relations are shown in the ranges of 0.025-10 [mu] M and 10-45 [mu] M respectively, and the probe shows excellent quantitative detection capacity; based on the sensitivity response of the probe to paraquat, an application device of test paper and cotton swabs is constructed in combination with artificial intelligence, the fluorescence intensity ratio (R / G) of a red channel to a green channel of the test paper and the paraquat concentration are in a good linear relation in a specific range, and the paraquat is visually, sensitively and quantitatively detected. The defects of complex process, high cost, long time consumption and the like of the existing detection method are overcome.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fluorescent probes, and particularly relates to a ratio-type fluorescent probe FITC-Lip-Au NCs and a preparation method and application thereof. BACKGROUND

[0002] Paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride, PQ) is a highly effective herbicide, but it causes serious problems due to its high toxicity and environmental persistence. In soil and water, paraquat can remain for many years (up to about 20 years), and it is easy to enter the human body through the skin, digestive tract and respiratory tract. Acute poisoning mainly damages the lungs and can cause progressive pulmonary fibrosis and respiratory failure; long-term exposure can also increase the risk of neurodegenerative diseases (such as Parkinson's disease). Given its threat to the environment and health, it is necessary to monitor and control paraquat residues in agricultural products and the environment.

[0003] At present, a variety of methods have been used for PQ detection, such as spectrophotometry (SP), high-performance liquid chromatography (HPLC), gas chromatography (GC) and surface-enhanced Raman spectroscopy (SERS). However, these methods, although having high selectivity and accuracy, have limitations such as long time-consuming, need for professional operation skills, complex equipment and tedious pretreatment, which are not convenient for on-site application. In addition, electrochemical sensors and enzyme-based biosensors have also been explored for PQ detection, which have the advantages of fast response, strong specificity and high sensitivity, and are suitable for real-time monitoring. However, the selectivity of such sensors is easily affected by interfering substances or environmental conditions such as temperature and ionic strength in complex matrix, which may lead to misjudgment. Fluorescence analysis has become an important means of on-site environmental detection due to its high detection sensitivity, easy miniaturization of probes, and remote signal transmission. In recent years, as a fluorescent probe, nanoclusters have unique optical, electrical and catalytic properties, and have become a prominent material for building fluorescent sensing platforms.

[0004] Metal nanoclusters (MNCs) are composed of several to dozens of metal atoms, with a size usually <2 nm, between single atoms and nanoparticles, and exhibit tunable photoluminescence, quantumized electronic transition, redox activity and catalysis, etc. due to the quantum confinement effect near Fermi wavelength. Gold nanoclusters (Au NCs) are widely used due to their clear composition, strong and light-stable luminescence. Surface ligands are crucial for the structural stability and functional regulation of Au NCs. With these tunable structures and surface chemistries, MNCs can specifically interact with paraquat, and have been used to construct a highly sensitive and selective paraquat detection system, showing potential advantages over traditional methods. However, current detection methods mostly rely on a single signal output mode, are easily disturbed by environmental background noise, have limited resistance to matrix effects, and lack self-calibration functions. In recent years, ratio fluorescent probes based on nanoclusters have effectively overcome the above problems through a dual-emission ratio mechanism, and have attracted widespread attention due to their higher sensitivity, stronger anti-interference ability and built-in self-calibration function. Therefore, it is urgent to develop a ratio fluorescent probe based on MNCs for highly sensitive on-site detection of paraquat. The present application innovatively synthesizes a ratio fluorescent gold nanocluster with lipase as a template, and further develops its application in the field of pesticide detection. SUMMARY

[0005] The present application relates to a ratio fluorescent probe FITC-Lip-Au NCs and a preparation method and application thereof. The fluorescent probe takes lipase (Lipase, Lip) as a template and tetrachloroauric acid as a metal precursor, and a one-pot method is used to synthesize gold nanoclusters (Lip-Au NCs) with red fluorescence. Further, under alkaline conditions, fluorescein isothiocyanate (FITC) reacts with the amino groups on the surface of Lip-Au NCs to prepare a FITC-Lip-Au NCs fluorescent probe with dual-emission characteristics. The probe can be used for the detection of paraquat residues in food and water samples, effectively overcoming the problems of existing detection methods such as complicated operation, expensive equipment, long time-consuming, and lack of internal reference. Based on the quenching effect of paraquat on the fluorescence of the probe, a fluorescence colorimetric determination method is established, and corresponding fluorescent visualization test paper and cotton swabs are developed. When used, only the sample to be tested needs to be added to the nanocluster-coated part, and the change in fluorescence intensity can be observed under a UV lamp to quickly determine the concentration of paraquat.

[0006] To achieve the above object, the technical scheme adopted by the present application is as follows: a preparation method of a ratio fluorescent probe FITC-Lip-Au NCs, characterized in that it comprises the following steps: S1. Dissolve lipase in deionized water to obtain a 1-5 mM lipase solution, and dissolve tetrachloroauric acid in deionized water to obtain a 5-45 mM tetrachloroauric acid aqueous solution. Then, mix the lipase solution and the tetrachloroauric acid aqueous solution at a molar ratio of (0.5-3.5):1 to obtain a mixture a, and place it on a vortex mixer and shake for 5-15 min. S2. Add 1M sodium hydroxide solution to the mixture a from step S1 to adjust the pH of mixture a to 8-14. Place it at 25-70°C in the dark for 1-24 hours to obtain mixture b. Centrifuge mixture b in a centrifuge at 10,000 rpm for 5-8 minutes and take the supernatant for later use, which is the Lip-Au NCs solution. S3. Take the supernatant Lip-Au NCs solution obtained in step S2 and dialyze it in NaOH buffer solution with pH 9.0~14 for 1-24 h to obtain mixture c. Mix the mixture c with 10-1000 μg / mL fluorescein isothiocyanate solution and incubate for 1-24 h. After incubation, pour it into a dialysis bag and dialyze it in phosphate buffer solution with pH 7.4 for 48-50 h to remove the solvent and free FITC in the system, thus obtaining the ratiometric fluorescent probe FITC-Lip-Au NCs.

[0007] Further, in step S3, the fluorescein isothiocyanate solution is obtained by dissolving fluorescein isothiocyanate in dimethyl sulfoxide to obtain a fluorescein isothiocyanate solution of 10-1000 μg / mL; the ratio of the supernatant Lip-Au NCs solution to the fluorescein isothiocyanate solution is: an initial mass concentration ratio of (50~4):1 and a volume ratio of (10~1):1.

[0008] The ratiometric fluorescent probe FITC-Lip-Au NCs prepared by the above method are FITC-Lip-Au NCs.

[0009] Furthermore, the ratiometric fluorescent probe FITC-Lip-Au NCs exhibits bright orange-red fluorescence under ultraviolet light.

[0010] Furthermore, the ratiometric fluorescent probe FITC-Lip-Au NCs exhibits dual emission peaks at 520 nm and 650 nm under 365 nm excitation light.

[0011] The above-mentioned ratiometric fluorescent probes FITC-Lip-Au NCs are applied in the field of pesticide detection.

[0012] Further, the pesticide detection field is pesticide residues in food and water samples, and the pesticide includes one of paraquat and dipyridyl pesticides; wherein the food and water samples include but are not limited to fruits, vegetables and various water samples.

[0013] Further, the ratio type fluorescent probe FITC-Lip-Au NCs realizes the detection of pesticides by using a fluorescence colorimetric method and a visualization method, and the standard addition recovery rate of the detection sample is 95.0% to 107.2%.

[0014] Further, in the fluorescence colorimetric method, the linear detection equation of the ratio type fluorescent probe FITC-Lip-Au NCs on paraquat is 0.025-10 μM (y1=-0.04982x+0.8454, R2=0.996) and 10-45 μM (y2=-0.00682x+0.37854, R2=0.992), and the LOD of the detection of paraquat is 15 nM.

[0015] Further, the visualization method device used by the ratio type fluorescent probe FITC-Lip-Au NCs includes but is not limited to test paper and a cotton swab.

[0016] Further, in combination with artificial intelligence, in the test paper determination of the visualization method, the ratio (R / G) of the red channel and the green channel of the test paper and the paraquat concentration present a good linear relationship in the range of 2.5-75 μM (y=-0.00433x+1.35431, R1²=0.9967) and 75-1000 μM (y=-0.000165x+1.01649, R2²=0.9995).

[0017] Compared with the prior art, the present application has the following beneficial effects: The ratio type fluorescent probe FITC-Lip-Au NCs has the advantages of simple and green preparation process, obvious orange-red fluorescence, high selectivity and sensitivity to paraquat, and dual functions of fluorescence colorimetric method and visualization detection. The ratio type fluorescent probe FITC-Lip-Au NCs can be applied to the detection of paraquat concentration in fruits, vegetables and various water samples. The detection method is simple, specific, has low sample consumption and low detection cost, can bring certain economic benefits, and promotes the development of the detection method. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is a physical picture of Lip-Au NCs and fluorescent probe FITC-Lip-Au NCs; Figure 2 It is a fluorescence excitation-emission spectrum of Lip-Au NCs; Figure 3 The emission spectrum of the fluorescent probe FITC-Lip-Au NCs; Figure 4 The ultraviolet absorption spectrum of the Lipase solution, the Lip-Au NCs solution, the FITC solution and the FITC-Lip-Au NCs fluorescent probe solution; Figure 5 The infrared spectrum of the Lipase powder, the Lip-Au NCs powder, the FITC powder and the FITC-Lip-Au NCs fluorescent probe powder; Figure 6 The selective response of the fluorescent probe FITC-Lip-Au NCs to different pesticides; Figure 7 The anti-interference performance of the fluorescent probe FITC-Lip-Au NCs in the detection of paraquat; Figure 8 The fluorescence change of the fluorescent probe FITC-Lip-Au NCs under different concentrations of paraquat; Figure 9 The fluorescence spectrum change of the fluorescent probe FITC-Lip-Au NCs under different concentrations of paraquat; Figure 10 The sensitivity and linear relationship fitting of the fluorescent probe FITC-Lip-Au NCs in the detection of paraquat; Figure 11 The schematic diagram of the visual detection test paper of paraquat based on the fluorescent probe FITC-Lip-Au NCs; Figure 12 The schematic diagram of the visual detection of cotton fibers of paraquat based on the fluorescent probe FITC-Lip-Au NCs. DETAILED DESCRIPTION

[0019] The specific embodiments of the present application will be described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present application, and are not used to limit the present application.

[0020] In the examples, the experimental methods are conventional methods unless otherwise specified.

[0021] The lipase (derived from Aspergillus oryzae) and other raw materials, reagents and equipment used are commercially available unless otherwise specified.

[0022] The present application will be described in detail below with reference to the examples. EXAMPLE

[0023] A preparation method of a ratio type fluorescent gold nanocluster FITC-Lip-Au NCs fluorescent probe, comprising the following steps: S1, dissolve lipase in deionized water to obtain a 4 mM lipase solution, dissolve tetrachloroauric acid in deionized water to obtain a 25 mM tetrachloroauric acid aqueous solution, then mix the lipase solution and the tetrachloroauric acid aqueous solution uniformly at a molar ratio of 2:1 to obtain a mixed solution a, and shake on a vortexer for 5 min; S2, add 1 mM sodium hydroxide solution to the mixed solution a of step S1 to adjust the pH value of the mixed solution a to 13, and react at 37°C in the dark for 12 h to obtain a mixed solution b, centrifuge the mixed solution b in a centrifuge at a speed of 10,000 rpm for 5 min, and take the supernatant for standby, which is the Lip-Au NCs solution; S3, dialyze the supernatant Lip-Au NCs solution obtained in step S2 in a NaOH buffer solution with a pH of 9.5 for 24 h to obtain a mixed solution c, mix the mixed solution c with a 100 μg / mL fluorescein isothiocyanate ester solution and incubate uniformly for 12 h; after the incubation is completed, pour it into a dialysis bag and dialyze in a phosphate buffer solution with a pH of 7.4 for 48 h to remove the solvent and free FITC in the system, and a ratiometric fluorescent probe FITC-Lip-Au NCs is obtained; wherein the fluorescein isothiocyanate ester solution is obtained by dissolving fluorescein isothiocyanate ester in dimethyl sulfoxide to obtain a 10-1000 μg / mL fluorescein isothiocyanate ester solution; the initial mass concentration ratio of the supernatant Lip-Au NCs solution to the fluorescein isothiocyanate ester solution is 40:1, and the volume ratio is 9:1. Example

[0024] According to the preparation method of Example 1, the reaction molar ratio of the lipase solution to the tetrachloroauric acid aqueous solution in step S1 is adjusted to 1:2 to obtain a mixed solution a1, the pH value of the mixed solution a1 is adjusted to 8 in step S2, and the mixed solution b1 is obtained by reacting at 25°C in the dark for 1 h, and the rest of the conditions are unchanged, and a ratiometric fluorescent probe FITC-Lip-Au NCs A1 is obtained. Example

[0025] According to the preparation method of Example 1, the reaction molar ratio of the lipase solution to the tetrachloroauric acid aqueous solution in step S1 is adjusted to 3.5:1 to obtain a mixed solution a2, the pH value of the mixed solution a2 is adjusted to 14 in step S2, and the mixed solution b2 is obtained by reacting at 70°C in the dark for 24 h, and the rest of the conditions are unchanged, and a ratiometric fluorescent probe FITC-Lip-Au NCs A2 is obtained. Example

[0026] According to the preparation method of Example 1, the initial mass concentration ratio of supernatant Lip-Au NCs solution to fluorescein isothiocyanate solution in step S3 is adjusted to 50:1, the volume ratio is 10:1, and the remaining conditions are unchanged, to obtain the ratio type fluorescent probe FITC-Lip-Au NCs A3. Example

[0027] According to the preparation method of Example 1, the initial mass concentration ratio of supernatant Lip-Au NCs solution to fluorescein isothiocyanate solution in step S3 is adjusted to 4:1, the volume ratio is 1:1, and the remaining conditions are unchanged, to obtain the ratio type fluorescent probe FITC-Lip-Au NCs A4. Example

[0028] Fluorescence spectral characterization of Lipase solution, Lip-Au NCs solution and FITC-Lip-Au NCs solution Take the Lip-Au NCs solution prepared in step S2 of Example 1 and the FITC-Lip-Au NCs solution prepared in step S3 of Example 1, respectively, and place them in centrifuge tubes. The fluorescence characteristics of the two are observed using a dark box ultraviolet analyzer.

[0029] The results show that under the irradiation of a 365 nm ultraviolet lamp, the Lipase solution has no fluorescence phenomenon; the Lip-Au NCs solution prepared in step S2 of Example 1 emits strong red fluorescence; the FITC-Lip-Au NCs solution prepared in step S3 of Example 1 presents strong orange-red fluorescence; as shown in Figure 1 .

[0030] Take 200 μL of the Lip-Au NCs solution prepared in step S2 of Example 1 in a micro quartz cuvette, and obtain its fluorescence excitation-emission light spectrum under a fluorescence spectrophotometer, as shown in Figure 2 , where Ex is the excitation wavelength and Em is the emission wavelength. The maximum excitation wavelength of Lip-Au NCs is at 470 nm, and the maximum emission wavelength is at 650 nm. Similarly, take 200 μL of the FITC-Lip-Au NCs solution prepared in step S3 of Example 1 in a micro quartz cuvette, and there are double emission peaks at 520 nm and 650 nm, as shown in Figure 3 . Example

[0031] UV absorption spectral characterization of Lipase, Lip-Au NCs, FITC and FITC-Lip-Au NCs The Lipase solution of S1 step of Example 1, the Lip-Au NCs solution prepared in S2 step of Example 1, the FITC solution of S3 step of Example 1 and the FITC-Lip-Au NCs solution prepared in S3 step of Example 1 were diluted to the same concentration and then put into a micro quartz cuvette, and the ultraviolet spectrum was detected by using a UV-1700 ultraviolet spectrophotometer, and the results are shown in Figure 4 .

[0032] The lipase solution prepared in Example 1 has a typical protein absorption peak (from aromatic amino acids) at 280 nm, while the Lip-Au NCs of the same concentration still retains the peak but the absorbance is higher than that of pure lipase, indicating that the enzyme still exists after synthesis, and the absorption of gold nanoclusters at this waveband is superimposed with the protein peak, which shows that they are not the same substance. FITC has a significant absorption peak at 490 nm, and FITC-Lip-Au NCs retains the protein peak at 280 nm and also has a new peak at 490 nm, which directly proves that FITC has been covalently connected to the surface of Lip-Au NCs. Example

[0033] Infrared spectrum characterization of Lipase, Lip-Au NCs, FITC and FITC-Lip-Au NCs The Lip-Au NCs solution prepared in S2 step of Example 1 and the FITC-Lip-Au NCs solution prepared in S3 step of Example 1 were first dried to constant weight in a 60°C constant temperature drying oven to obtain Lip-Au NCs powder and FITC-Lip-Au NCs powder, and then the Lip-Au NCs powder, the FITC-Lip-Au NCs powder, the lipase powder, the FITC powder and the potassium bromide were mixed in the order of 1:100 and then ground thoroughly, and the infrared spectrum was detected by using a Fourier transform infrared spectrometer, and the results are shown in Figure 5 .

[0034] In the infrared spectrum, the characteristic absorption peaks of lipase include N-H / O-H stretching vibration (about 3300 cm⁻¹), amide I band (C=O, about 1640 cm⁻¹) and amide II band (C-N+H, about 1540 cm⁻¹), which are shifted or changed in intensity after the formation of Lip-Au NCs, indicating that the structure of lipase still exists and its conformation and chemical environment are affected due to the stable gold nanoclusters. The characteristic peak of isothiocyanate group (-N=C=S) in FITC at 2100 cm⁻¹ disappears in the spectrum of FITC-Lip-Au NCs, which proves that FITC has been successfully covalently connected to Lip-Au NCs. Example

[0035] Selectivity of the ratiometric fluorescent probe FITC-Lip-Au NCs to different pesticides The standard samples of paraquat, methomyl, chlorantraniliprole, isoprocarb, dichlorvos, carbaryl, chlorpyrifos, phoxim, dimethoate and fenitrothion with a concentration of 1 mM, 20 μL were prepared respectively, 20 μL of the ratiometric fluorescent gold nanoclusters FITC-Lip-Au NCs fluorescent probe prepared in step S3 of Example 1 and 160 μL of ultrapure water (total 200 μL of mixed solution) were added to each standard sample; after the above-mentioned mixed solutions to be detected were placed in a 25 °C water bath for 5 min, the fluorescence intensity of each mixed solution at 520 nm and 680 nm was detected under the condition of excitation wavelength of 365 nm using a Spark 10M multifunctional microplate reader, and compared with the blank control group (20 μL of FITC-Lip-Au NCs described in Example 1 and 180 μL of ultrapure water). Among them, (F 650 / F 520 )0is the fluorescence intensity ratio of the blank control group, (F 650 / F 520 ) is the fluorescence intensity ratio after each pesticide reacts with FITC-Lip-Au NCs, and (F 650 / F 520 ) / (F 650 / F 520 )0is the evaluation index.

[0036] The results are shown in Figure 6 , only paraquat has obvious quenching effect on FITC-Lip-Au NCs in Example 1, and the rest of the pesticides have no quenching effect on the nanoclusters. It shows that the response of FITC-Lip-Au NCs to paraquat is specific. Example

[0037] Anti-interference of the ratiometric fluorescent probe FITC-Lip-Au NCs in detecting paraquat The standard solutions of paraquat, methomyl, chlorantraniliprole, isoprocarb, dichlorvos, carbosulfan, chlorpyrifos, phoxim, dimethoate and fenitrothion were prepared with a concentration of 1 mM, 20 μL, respectively. 20 μL of the ratio-type fluorescent probe FITC-Lip-Au NCs prepared in step S3 of Example 1 was added to each standard solution, and 20 μL of paraquat and 140 μL of ultrapure water were added (total of 200 μL of mixed solution). After the above-mentioned mixed solutions to be detected were placed in a 25 °C water bath for 5 min, the fluorescence intensity of each mixed solution at 520 nm and 680 nm was detected under the condition of excitation wavelength of 365 nm using a Spark 10M multifunctional microplate reader, and compared with the blank control group (40 μL of FITC-Lip-Au NCs described in Example 1 and 180 μL of ultrapure water). Among them, (F 650 / F 520 )0is the fluorescence intensity ratio of the blank control group, (F 650 / F 520 ) is the fluorescence intensity ratio after each pesticide reacts with FITC-Lip-Au NCs, and (F 650 / F 520 ) / (F 650 / F 520 )0is the evaluation index.

[0038] The results are shown in Figure 7 . No pesticide has an impact on the quenching effect of paraquat on FITC-Lip-Au NCs in Example 1, indicating that the response of FITC-Lip-Au NCs to paraquat is specific and anti-interference. Example

[0039] Sensitivity of the ratio-type fluorescent probe FITC-Lip-Au NCs for detecting paraquat Paraquat standard solution was prepared with a concentration of 0.75, 1, 2.5, 5, 7.5, 10, 25, 40, 50, 60, 75, 100, 250, 500, 750 and 1000 μM. 20 μL of the above standard solution was taken, 20 μL of the ratio-type fluorescent probe FITC-Lip-Au NCs prepared in step S3 of Example 1 was added to each standard solution, and 160 μL of ultrapure water was added (total of 200 μL of mixed solution), and the detection steps were the same as in Example 9.

[0040] The results show that under the irradiation of 365 nm ultraviolet lamp, the reaction system changes from orange-red fluorescence to green fluorescence. The emission peak of the ratio-type fluorescent probe FITC-Lip-Au NCs at 520 nm remains basically unchanged, while the emission peak at 650 nm decreases with the increase of the concentration of paraquat in the mixed solution, as shown in Figure 9The relative fluorescence intensity linear detection equation is 0.025-10 μM (y1=-0.04982x+0.8454, R2=0.996) and 10-45 μM (y2=-0.00682x+0.37854, R2=0.992), and the LOD of paraquat is 15 nM, as shown in Figure 10 Example

[0041] Visual detection of paraquat by the ratiometric fluorescent probe FITC-Lip-Au NCs (detection device: test paper) After cutting the filter paper into a circle, it was immersed in the ratiometric fluorescent probe FITC-Lip-Au NCs solution prepared in step S3 of Example 1, and placed at 4 ℃ for 3 hours; then it was naturally dried at room temperature to obtain a portable test paper for detecting paraquat. After different concentrations of paraquat solution were applied to the test paper, the fluorescence color change was observed under a 365 nm ultraviolet lamp, and the image was recorded using a smart phone. The color recognition function in the phone was used to scan and read the fluorescence RGB values of the test paper under different concentrations of paraquat, and the ratio of the fluorescence intensity of the red channel to the green channel (R / G) was calculated, and then a linear relationship between the ratio and the concentration of paraquat was established by fitting the function.

[0042] As shown in Figure 11 , with the increase of the concentration of paraquat, the fluorescence color gradually changed from red to green. The ratio of the fluorescence intensity of the red channel to the green channel (R / G) and the concentration of paraquat showed a good linear relationship in the range of 2.5-75 μM (y=-0.00433x+1.35431, R1²=0.9967) and 75-1000 μM (y=-0.000165x+1.01649, R2²=0.9995). Example

[0043] Visual detection of paraquat by the ratiometric fluorescent probe FITC-Lip-Au NCs (detection device: cotton swab) The cotton swab was immersed in 100 μL of the ratiometric fluorescent probe FITC-Lip-Au NCs solution prepared in step S3 of Example 1, and taken out after complete absorption; the prepared cotton swab was used to wipe the surface of the food sample, and the change of fluorescence color was observed. Then, 1 mM and 2 mM of paraquat standard solution were sprayed on the surface of the sample, and the cotton swab was used to wipe it, and the change of fluorescence color was observed.

[0044] As shown in Figure 12 ​As shown, under the condition of ultraviolet light irradiation, the first cotton swab as a control showed obvious and stable orange-red fluorescence. The fluorescence performance of the second cotton swab (wiping the surface of the food sample without spraying paraquat) was similar to that of the control cotton swab, still orange-red fluorescence, indicating that the surface of the food sample without spraying paraquat did not interfere with the fluorescence of the cotton swab. The third cotton swab (wiping the surface of the food sample sprayed with 500 μM paraquat) showed a significant decrease in the intensity of orange-red fluorescence compared to the control cotton swab, and green fluorescence appeared. The fourth cotton swab (wiping the surface of the food sample sprayed with 1 mM paraquat) showed further weakening of the orange-red fluorescence, even close to disappearing, showing green fluorescence. Embodiment

[0045] Detection of paraquat in real environmental samples by the ratio-type fluorescent probe FITC-Lip-Au NCs To verify the practical applicability of the method, two water samples (taken from the tap water of Huajin Campus of Anhui Normal University and the mirror lake water of Wuhu City) and four fruit and vegetable samples (market-purchased pears, apples, lettuce and rape) were selected as real samples for paraquat detection. The water samples were filtered through a 0.22 μm filter membrane before detection, and the clear liquid was obtained for detection; the fruit and vegetable samples were ground and centrifuged at 12000 r / min for 10 minutes, and the supernatant was diluted 10 times as the fruit and vegetable sample stock solution. Different concentrations of paraquat standard solution (final concentration of 0, 2.5, 5.0 μM) were added to the water sample and fruit and vegetable sample stock solution respectively, and the ratio-type fluorescent gold nanocluster FITC-Lip-Au NCs fluorescent probe solution prepared in step S3 in Example 1 was incubated at 25°C for 5 min, and then the fluorescence intensity was measured. The recovery rate of paraquat was calculated according to the formula "recovery rate (%) = (C1 / C2) x 100%", wherein C1 is the measured concentration of paraquat, and C2 is the added concentration of paraquat standard. The results show that the recovery rate of the ratio-type fluorescent probe FITC-Lip-Au NCs for pesticide detection in food and water samples is 95.0% to 107.2%, and the results are shown in Table 1.

[0046] Table 1 Amount added (μM) Amount detected (μM) Recovery (%) Tap water 0 ND - Tap water 2.5 2.43±0.08 97.2 Tap water 5.0 5.03±0.04 100.6 Mirror lake water 0 ND - Mirror lake water 2.5 2.53±0.01 101.2 Mirror lake water 5.0 4.98±0.03 99.7 Pear 0 ND - Pear 2.5 2.55±0.01 102.0 Pear 5.0 4.98±0.05 99.6 Apple 0 ND - Apple 2.5 2.50±0.12 100.1 Apple 5.0 4.90±0.12 98.1 Lettuce 0 ND - Lettuce 2.5 2.48±0.04 99.2 Lettuce 5.0 5.36±0.007 107.2 Rape 0 ND - Rape 2.5 2.51±0.02 100.4 Rape 5.0 4.98±0.01 98.0 It should be understood that although the present specification describes each embodiment separately, it is not intended that each embodiment only involves one independent technical solution. Such a description is for the sake of clarity, and those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that those skilled in the art can understand.

Claims

1. A method for preparing a ratiometric fluorescent probe FITC-Lip-Au NCs, characterized in that, Includes the following steps: S1. Dissolve lipase in deionized water to obtain a 1-5 mM lipase solution, and dissolve tetrachloroauric acid in deionized water to obtain a 5-45 mM tetrachloroauric acid aqueous solution. Then, mix the lipase solution and the tetrachloroauric acid aqueous solution at a molar ratio of (0.5-3.5):1 to obtain a mixture a, and place it on a vortex mixer and shake for 5-15 min. S2. Add 1M sodium hydroxide solution to the mixture a from step S1 to adjust the pH of mixture a to 8-14. Place it at 25-70°C in the dark for 1-24 hours to obtain mixture b. Centrifuge mixture b in a centrifuge at 10,000 rpm for 5-8 minutes and take the supernatant for later use, which is the Lip-Au NCs solution. S3. Take the supernatant Lip-Au NCs solution obtained in step S2 and dialyze it in NaOH buffer solution with pH 9.0~14 for 1-24 h to obtain mixture c. Mix the mixture c with 10-1000 μg / mL fluorescein isothiocyanate solution and incubate for 1-24 h. After incubation, pour it into a dialysis bag and dialyze it in phosphate buffer solution with pH 7.4 for 48-50 h to remove the solvent and free FITC in the system, thus obtaining the ratiometric fluorescent probe FITC-Lip-Au NCs.

2. The method for preparing a ratiometric fluorescent probe FITC-Lip-Au NCs according to claim 1, characterized in that: In step S3, the fluorescein isothiocyanate solution is prepared by dissolving fluorescein isothiocyanate in dimethyl sulfoxide to obtain a fluorescein isothiocyanate solution of 10-1000 μg / mL; the ratio of the supernatant Lip-Au NCs solution to the fluorescein isothiocyanate solution is: an initial mass concentration ratio of (50~4):1 and a volume ratio of (10~1):

1.

3. A ratiometric fluorescent probe FITC-Lip-Au NCs prepared by a method according to claim 1 or 2.

4. The ratiometric fluorescent probe FITC-Lip-Au NCs according to claim 3, characterized in that, The ratiometric fluorescent probe FITC-Lip-Au NCs exhibits orange-red fluorescence under ultraviolet light.

5. The application of the ratiometric fluorescent probe FITC-Lip-Au NCs as described in claim 3 or 4 in the field of pesticide detection.

6. The application of the ratiometric fluorescent probe FITC-Lip-Au NCs according to claim 5 in the field of pesticide detection, characterized in that: The pesticides mentioned include paraquat and one of the bipyridine pesticides.

7. The application of the ratiometric fluorescent probe FITC-Lip-Au NCs according to claim 5 in the field of pesticide detection, characterized in that: The detection methods for the pesticides are fluorescence colorimetry and visualization.

8. The application of the ratiometric fluorescent probe FITC-Lip-Au NCs according to any one of claims 5-7 in the field of pesticide detection, characterized in that: In the aforementioned fluorescence colorimetric method, the linear detection equations for paraquat using FITC-Lip-Au NCs of the ratiometric fluorescent probes were 0.025–10 μM (y1 = -0.04982x + 0.8454, R0). 2 =0.996) and 10-45 μM (y2=-0.00682x+0.37854, R 2 =0.992), and its detection LOD for paraquat was 15 nM.

9. The application of the ratiometric fluorescent probe FITC-Lip-Au NCs according to any one of claims 5-8 in the field of pesticide detection, characterized in that: The visualization method uses test strips or cotton swabs.

10. The application of the ratiometric fluorescent probe FITC-Lip-Au NCs according to any one of claims 5-9 in the field of pesticide detection, characterized in that: In the visualization test strip assay, the ratio of the red channel to the green channel (R / G) of the test strip showed a good linear relationship with the paraquat concentration in the ranges of 2.5-75 μM (y=-0.00433x+1.35431, R1²=0.9967) and 75-1000 μM (y=-0.000165x+1.01649, R2²=0.9995).

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