CN114958361A

CN114958361A - Blue carbon dot/gold nanocluster-based ratiometric fluorescence sensor and application thereof in glyphosate detection

Info

Publication number: CN114958361A
Application number: CN202210649769.0A
Authority: CN
Inventors: 蒋长龙; 杨亮; 王振洋; 张淑东
Original assignee: Hefei Institutes of Physical Science of CAS
Current assignee: Hefei Institutes of Physical Science of CAS
Priority date: 2022-06-09
Filing date: 2022-06-09
Publication date: 2022-08-30
Anticipated expiration: 2042-06-09
Also published as: CN114958361B

Abstract

The invention discloses a fluorescent sensor based on blue carbon dot/gold nanocluster ratio and application thereof in glyphosate detection. The ratio fluorescence sensor is composed of a blue carbon dot and gold nanocluster composite system, wherein the fluorescence intensity ratio of the blue carbon dot to the gold nanoclusters is 9: 1. Carbon points containing abundant carboxyl and amino functional groups can be subjected to esterification and amide reaction with glyphosate to form a compound (CDs-Gly-CDs) so as to enable the compound to be aggregated, blue fluorescence is quenched, and orange fluorescence of the gold nanocluster serving as an internal standard is unchanged. Therefore, the ratio fluorescence sensor is prepared by taking the blue carbon dots as the signal probe and the gold nanocluster as the reference probe, an integrated intelligent mobile phone sensing platform is constructed by relying on a 3D printing technology, the ratio fluorescence sensor has the advantages of high sensitivity, visual detection, quick response and the like, the detection limit of glyphosate is as low as 4.19nM, and the ratio fluorescence sensor can be used for low-cost, quick and visual quantitative analysis of glyphosate residues in samples such as environmental water samples, fruit juice and the like.

Description

Blue carbon dot/gold nanocluster-based ratiometric fluorescence sensor and application thereof in glyphosate detection

Technical Field

The invention particularly relates to a blue carbon dot/gold nanocluster-based ratiometric fluorescence sensor and application thereof in glyphosate detection, and belongs to the fields of material synthesis, visual detection, food quality monitoring and the like.

Background

Glyphosate (glyphosate) is a systemic, broad-spectrum biocidal herbicide. It has wide herbicidal spectrum, complete herbicidal effect, high herbicidal effect and low cost, and is suitable for use in killing weeds. The glyphosate is found in a large number of crops as a herbicide with the largest international use amount, and can enter a water body ecological system in the modes of surface runoff, rain wash, irregular cleaning and spraying tools and the like. The glyphosate with high residue in the environment can enter the human body through diet and the like. The accumulation of glyphosate in vivo can cause health hazards such as hepatotoxicity, neurotoxicity and the like, and has certain carcinogenicity. In addition, the existing research results show that exposure of glyphosate with low concentration can affect the synthesis and secretion of sex hormone, destroy the functional structure of reproductive system, interfere the developmental maturity of gamete, cause the occurrence of poor pregnancy outcome and have certain reproductive toxicity. Therefore, the detection of the glyphosate, particularly the detection of the glyphosate in fruits, vegetables and drinking water has very important practical significance.

At present, biochemical sensors based on optical signals have proven to be a promising method for residue detection due to their simplicity, high sensitivity and good selectivity. Although these sensors have found potential applications in the field of residue detection, most sensors rely heavily on enzyme inhibition, which is prone to enzyme deactivation, affecting the sensitivity, accuracy and practicality of the sensor. In addition, the operation condition and the response speed are limited by internal/external factors, real-time/field application is not realized, the visual detection effect is poor, and accurate quantitative reading cannot be realized. Therefore, there is still an urgent need to design enzyme-free analytical sensors and further broaden their application fields.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a blue carbon dot/gold nanocluster-based ratiometric fluorescence sensor for high-sensitivity visual quantitative detection of glyphosate residues, and the enzyme-free sensing system can realize reliable, convenient and on-site detection of the glyphosate residues.

The novel enzyme-free rapid visual ratio fluorescence sensor serving as the core of the sensing system realizes selective quantitative detection of glyphosate by integrating designed blue carbon nanodots (CDs) and gold nanoclusters (Au NCs). After introduction of glyphosate, the blue fluorescence of CDs at 450nm can be rapidly quenched within 2 seconds by aggregation-induced quenching (ACQ) due to formation of CDs-Gly-CDs complex aggregates. While AuNCs served as a reference signal, there was no change in red fluorescence at 620nm, thus resulting in a distinct and immediate ratiometric fluorescence color change from blue to pink to orange. The detection limit of the glyphosate can be as low as 4.19 nm. In addition, the ratiometric fluorescent sensing system has been successfully applied to glyphosate detection in practical samples, and a new method is provided for constructing an enzyme-free visual quantitative system for trace hazard detection.

The invention discloses a blue carbon dot/gold nanocluster-based ratio fluorescence sensor which is composed of a composite system of blue carbon dots and a gold nanocluster solution, wherein the fluorescence intensity ratio of the blue carbon dots to the gold nanoclusters in the composite system is 9: 1.

The invention relates to a preparation method of a blue carbon dot/gold nanocluster-based ratio fluorescence sensor, which comprises the following steps of:

step 1: preparation of blue carbon dots

Dissolving 0.8-1.0 g of anhydrous sodium citrate and 0.45-0.52 g of polyacrylamide in 15-20 mL of ultrapure water, and performing ultrasonic treatment to obtain a clear transparent solution; then transferring the solution into a 50mL high-pressure reaction kettle with a polytetrafluoroethylene lining, carrying out heat preservation reaction in an oven at 180-200 ℃ for 3-5 hours, and naturally cooling to room temperature to obtain blue carbon dot liquid; dialyzing the blue carbon dot liquid for 4-6 hours by using a filter membrane dialysis bag with the molecular weight cutoff of 1000Da, drying the dialyzed blue carbon dot in an oven at 40-60 ℃ to obtain carbon dot powder, preparing into 0.8-1 mg/mL aqueous solution, and placing the aqueous solution in a refrigerator at 4 ℃ for later use.

Step 2: preparation of gold nanoclusters

Before the experiment, all glass instruments were soaked in aqua regia for two hours, then cleaned with pure water and dried. Dissolving 5.6-6.6 mg of 11-mercaptoundecanoic acid in 8-10 mL of deionized water, stirring, performing ultrasonic action for 30-50 min, adding NaOH (100 mu L,1M), and stirring to obtain a transparent solution; adding 350-400 mu L of HAuCl ₄ Reacting at room temperature for 5-8 h (mass ratio is 1%) to slowly turn the colorless solution to light yellow; dialyzing (1000Da) and purifying the gold-bearing nanocluster-containing solution for 4-6 h, and filtering by using a 0.22 mu m microporous membrane; the resulting colorless clear solution was stored at 4 ℃ for subsequent experiments.

And step 3: preparation of ratiometric fluorescent nano-sensing system

And (3) adding 20-30 mu L of the carbon dot solution obtained in the step (1) into 80-100 mu L of the gold nanocluster solution prepared in the step (2), diluting to 2-3 mL by using pure water, fully mixing, and reacting for 2-4 min to obtain the ratiometric fluorescent nano sensing system.

The invention discloses application of a blue carbon dot/gold nanocluster based ratiometric fluorescence sensor, which is used as a detection reagent in the process of detecting glyphosate residues.

The core of the invention for rapidly, visually and quantitatively detecting the glyphosate in the sample is a designed blue carbon dot/gold nanocluster ratio fluorescence sensing system. The ratio fluorescence sensing system of the glyphosate and the blue carbon dot/gold nanocluster reacts to generate a compound (CDs-Gly-CDs) so that the carbon dots are gathered to cause blue fluorescence quenching, orange red fluorescence of the gold nanocluster serving as an internal standard is not changed, and the color of the system continuously changes from blue to red, so that the glyphosate can be visually detected by the ratio fluorescence sensing system, and the content of the glyphosate in a sample can be detected through the fluorescence change degree in the ratio fluorescence system.

The detection method specifically comprises the following steps:

1. detection of glyphosate by ratiometric fluorescent probe solution

Dripping the ratiometric fluorescent nanoprobe solution into a quartz colorimetric tube with the length of 1-2 cm, sequentially adding 2-3 mu L of glyphosate solutions with different concentrations, and uniformly mixing. Obvious fluorescent color change can be observed under an ultraviolet lamp, and the glyphosate can be visually detected. And recording the fluorescence spectrum of the solution in the range of 400-800 nm by using 350nm exciting light, and realizing the quantitative detection of the glyphosate by establishing the relation between the change of the fluorescence peak intensity ratio and the concentration of the glyphosate.

2. Detection of glyphosate by fluorescent paper-based sensor

Adding the ratiometric fluorescent nanoprobe solution serving as ink into an empty ink box, repeatedly printing for 15-20 times on white A4 paper without fluorescent background interference to obtain fluorescent test paper (namely, a ratiometric fluorescent paper-based sensor), and cutting the fluorescent test paper into strips for further use.

And (3) dropwise adding 2-3 mu L of glyphosate solutions with different concentrations to the ratio fluorescent paper-based sensor, and after the paper-based sensor is dried for 0.5-1 minute, acquiring an image and a red-green-blue value (RGB value) by using software under a 365nm ultraviolet lamp light source. Quantitative detection is realized by establishing the relation between the ratio of R to B and the concentration of glyphosate.

The principle of detecting glyphosate by using a fluorescence sensor is based on a fluorescence quenching strategy, and particularly, the prepared active functional groups carboxyl and amino rich on the surface of a blue carbon dot react with glyphosate to form ester and phosphoric acid amide, so that a compound CDs-Gly-CDs is formed. The complex induces aggregation of blue carbon spots accompanied by fluorescence quenching, and the fluorescent signal response process can be completed within 2 seconds. Based on blue fluorescence quenching and red fluorescence invariance, the sensing system shows the conversion from blue fluorescence to red fluorescence under an ultraviolet lamp, and the visualized detection of the glyphosate is realized; the quantitative detection of the glyphosate is realized by establishing the relationship between the ratio fluorescence intensity and the glyphosate concentration.

Compared with the prior detection technology, the invention has the beneficial effects that:

1. compared with other monochromatic fluorescence detection, the ratio fluorescence sensing system disclosed by the invention has the advantages that the better visual detection effect is displayed, the instability problem of the monochromatic fluorescence intensity is effectively avoided, and the visual detection is realized.

2. The response time of the fluorescence sensor is finished within 2s, and the rapid detection is realized.

3. The detection limit of the fluorescence spectrometer for glyphosate is 4.19nM, which is lower than the allowable limit of glyphosate residue.

4. The fluorescence quenching system of the blue carbon dot and gold nanocluster ratio sensor prepared by the invention has good selectivity and sensitivity to glyphosate, can effectively avoid the interference of other impurities, and has quick response.

Drawings

FIG. 1 is a transmission electron micrograph of a ratiometric fluorescence sensing system.

FIG. 2 is a transmission electron micrograph of a ratiometric fluorescent sensor after addition of glyphosate.

FIG. 3 is an energy dispersive X-ray spectroscopy chart of a ratiometric fluorescence sensing system.

FIG. 4 is an infrared spectrum of a ratiometric fluorescence sensing system.

FIG. 5 is a comparison of x-ray photoelectron spectra of C1s (A, D), N1s (B, E) and O1s (C, F) before and after glyphosate addition in a ratiometric fluorescent sensing system.

Fig. 6 is a fluorescence excitation spectrum (a) and an emission spectrum (B) of a blue carbon dot in a ratiometric fluorescence sensing system, and a fluorescence excitation spectrum (C) and an emission spectrum (D) of gold nanoclusters.

FIG. 7(A) is a pH-optimized graph showing the effect of pH on fluorescence intensity in a ratiometric fluorescence sensing system. (B) Is a linear relationship between the ratio of change in fluorescence of the ratiometric probe and the pH.

FIG. 8 shows the fluorescence spectra of the probe solution after glyphosate addition when the fluorescence intensity ratios of the blue carbon dots/gold nanoclusters in the ratiometric fluorescence sensing system are 8:1(A),9:1(B) and 10:1 (C).

FIG. 9(A) is a graph showing the fluorescence spectrum and color change of a blue carbon dot/gold nanocluster mixed system with different concentrations of glyphosate. The fluorescence color of the solution gradually changes from blue to pink as the concentration of glyphosate increases. FIG. 9(B) is a graph of fluorescence intensity versus glyphosate concentration.

Fig. 10(A, B) is a schematic diagram of color recognition and visual detection effect of a blue carbon dot/gold nanocluster mixed system smart phone by different concentrations of glyphosate. FIG. 10(C) is a graph of the change in color of the fluorescent probe solution (red channel/blue channel) versus the concentration of glyphosate, with a linear relationship between the change in color of the fluorescent probe solution (red channel/blue channel) and the concentration of glyphosate.

FIG. 11(A) is a graph of the fluorescence spectrum and color change of different concentrations of glyphosate versus a fluorescent paper-based sensor. The fluorescence color of the solution gradually changes from blue to pink as the concentration of glyphosate increases. FIG. 11(B) is a graph of fluorescence intensity versus glyphosate concentration.

FIG. 12 is a graph showing selectivity and interference of a fluorescent probe, wherein (A) is a graph showing a change in fluorescence intensity of a solution system after addition of different interfering substances, (B) is a photograph of fluorescence under ultraviolet visible light, and (C) is a histogram showing fluorescence intensity.

Detailed Description

The technical scheme of the invention is further explained by combining the drawings and the specific embodiments as follows:

example 1: preparation of ratiometric fluorescent nanosensors

1. Preparation of blue carbon dots

0.8-1.0 g of anhydrous sodium citrate and 0.45-0.52 g of polyacrylamide are dissolved in 15-20 mL of ultrapure water, and a clear transparent solution is obtained by ultrasonic treatment. And then transferring the solution into a 50mL high-pressure reaction kettle with a polytetrafluoroethylene lining, carrying out heat preservation reaction in an oven at 180-200 ℃ for 3-5 hours, and naturally cooling to room temperature to obtain blue carbon dot liquid. Dialyzing the resultant for 4-6 hours by using a filter membrane dialysis bag with the molecular weight cutoff of 1000 Da. Drying the dialyzed blue carbon dots in an oven at 40-60 ℃ to obtain carbon dot powder, preparing into 0.8-1 mg/mL aqueous solution, and placing in a refrigerator at 4 ℃ for later use.

2. Preparation of gold nanoclusters

Before the experiment, all glass instruments were soaked in aqua regia for two hours, then cleaned with pure water and dried. Firstly, 5.6-6.6 mg of 11-sulfydryl decaDissolving the mono-alkanoic acid in 8-10 mL of deionized water, stirring, performing ultrasonic action for 30-50 min, adding NaOH (100 mu L,1M), and stirring to obtain a transparent solution. Adding 350-400 mu L of HAuCl ₄ And (1 percent of mass) reacting at room temperature for 5-8 hours to slowly turn the colorless solution into light yellow. And (3) purifying the gold-bearing nanocluster-containing solution for 4-6 hours by dialysis (1000Da), and filtering by using a 0.22 mu m microporous membrane. The resulting colorless clear solution was stored at 4 ℃ for subsequent experiments.

3. Preparation of ratiometric fluorescent nanosensors

And (3) adding 20-30 mu L of the carbon dot solution obtained in the step (1) into 80-100 mu L of the gold nanocluster solution prepared in the step (2), diluting to 2-3 mL by using pure water, fully mixing, and reacting for 2-4 min to prepare the ratio fluorescence sensing system.

Example 2: ratiometric fluorescence sensing mechanisms, component characterization, and optimization of detection conditions

1. The ratiometric fluorescence sensing system has obvious dual emission peaks under 365nm ultraviolet excitation. After introduction of glyphosate, the blue fluorescence of CDs at 450nm can be rapidly quenched within 2 seconds by aggregation-induced quenching (ACQ) due to the formation of CDs-Gly-CDs complex aggregates. And the Au NCs serve as a reference signal, the red fluorescence of the Au NCs does not change at 620nm, so that the obvious and instant ratio fluorescence color change from blue to pink to orange can be caused, the rapid visual detection of Gly by the ratio fluorescence sensing system can be realized, and the content of glyphosate in the sample can be detected through the fluorescence change degree in the ratio fluorescence system.

2. Ratiometric fluorescence sensing system component characterization

Taking into account that the detection sensitivity of the probe to the analyte is related to its own properties, the structural features and spectral characteristics of ratiometric fluorescent nanoprobes were studied using FT-IR, UV-vis and fluorescence spectroscopy, respectively. In addition, TEM, EDX and XPS were used to determine the morphology of ratiometric fluorescent nanoprobes and their elemental composition.

3. Influence of excitation wavelength, pH and ratio of fluorescence intensity of carbon dots/gold nanoclusters on ratiometric fluorescent probes

The excitation wavelength, the pH value and the fluorescence intensity of the carbon dots/gold nanoclusters have certain influence on the fluorescence intensity and the detection effect of the ratiometric fluorescent probe. As can be seen from FIG. 6, the blue fluorescence intensity of the carbon dots is different from the red fluorescence intensity of the gold nanoclusters at different excitation wavelengths, which indicates that the excitation wavelength has an influence on the fluorescence intensity of the fluorescence probe with the contrast ratio. Taken together, the probe of the present invention selects 350nm as the optimal excitation wavelength.

When the pH is less than 6.5, the blue fluorescence intensity of the ratiophor system is low around 450nm due to protonation and deprotonation of carboxyl groups and amino groups. The blue fluorescence intensity increases with increasing pH, reaches a maximum when pH reaches 6.5, and then decreases slightly. This is because the glyphosate molecule contains both phosphate and carboxyl groups. When the pH value is more than 7.0, glyphosate is easy to generate neutralization reaction, and the reaction of amide and CDs is not facilitated. After glyphosate is added, the pH value also affects the detection performance and the visualization effect. The ratiometric fluorescent sensing system exhibited the desired change in fluorescent color at a pH of 6.5. The combination of 6.5 is the optimum pH for detection.

The fluorescence intensity ratio of CDs and Au-NCs plays an important role in detecting the glyphosate. As shown in FIG. 8, the emission intensity ratio (I) at 365nm UV ₄₅₀ /I ₆₂₀ ) When adjusted to 9/1, a clear fluorescent color change from blue to pink was seen for the sensor solution after addition of glyphosate.

Example 3:

1. detection of glyphosate by ratiometric fluorescent probe solution

Dropping the ratiometric fluorescent nanoprobe solution prepared in example 1 into a quartz colorimetric tube with the length of 1-2 cm, sequentially adding 2-3 mu L of glyphosate solutions with different concentrations, and uniformly mixing. Obvious fluorescent color change can be observed under an ultraviolet lamp, and the glyphosate can be visually detected. And recording the fluorescence spectrum of the solution in the range of 400-800 nm by using 350nm exciting light, and realizing the quantitative detection of the glyphosate by establishing the relation between the change of the fluorescence peak intensity ratio and the concentration of the glyphosate.

2. Drawing of standard curve

Respectively adding the blue carbon dots/gold nanocluster ratiometric probes into a mixed systemAdding glyphosate solutions with different concentrations, mixing, testing fluorescence intensity, and establishing fluorescence intensity ratio (I) by showing that the blue fluorescence emission peak at 450nm is gradually weakened and the fluorescence emission peak at 620nm is almost unchanged ₄₅₀ /I ₆₂₀ ) And the quantitative detection of the glyphosate can be realized by the relationship between the concentration of the glyphosate and the concentration of the glyphosate. When the exciting light is 350nm, recording the fluorescence spectrum of the mixed system in the wavelength range of 400-800 nm. FIG. 9 shows the relationship between fluorescence intensity and glyphosate concentration, with the change in fluorescence intensity ratio being linear, where the abscissa is glyphosate concentration and the ordinate is the ratio of fluorescence intensity at 450nm and 620 nm.

Example 4:

1. preparation of blue carbon dots

The procedure for this step was the same as in example 1.

2. Preparation of gold nanoclusters

The procedure for this step was the same as in example 1.

3. Preparation of ratiometric fluorescent nanosensors

The procedure for this step was the same as in example 1.

4. Construction of detection platform of smart phone

This smart mobile phone testing platform includes two sample tanks (cell and paper strip groove), darkroom, small-size UV lamp support, filter (400) and smart mobile phone (HUAWEI nova 2).

5. Probe solution detection of glyphosate

And adding the fluorescent nano probe solution into a cuvette, then adding glyphosate solutions with different concentrations, and uniformly stirring. Fluorescent images are obtained through software of the smart phone in a dark environment, the fluorescent images are further decomposed into RGB values, and quantitative detection is completed through installation of a color recognition Application (APP).

6. Drawing of standard curve

The ratio fluorescence sensing cuvette dropwise added with glyphosate solutions of different concentrations is placed in a detection platform, and a fluorescence image is obtained through a smart phone by taking a 365nm ultraviolet lamp as a light source in a dark environment. Further decomposed into RGB values, and the inset 10 shows that the RGB values are linear with glyphosate concentration, where the abscissa is glyphosate concentration and the ordinate is the RGB ratio.

Example 5:

1. preparation of blue carbon dots

The procedure for this step was the same as in example 1.

2. Preparation of gold nanoclusters

The procedure for this step was the same as in example 1.

3. Preparation of ratiometric fluorescent nanosensors

The procedure for this step was the same as in example 1.

4. Preparation of ratiometric fluorescent paper-based sensors

The ratiometric probe solution prepared in 3 was added as ink to an empty cartridge, and printing was repeated multiple times on white a4 paper without fluorescent background interference to distribute the probes evenly, and a fluorescent test paper was printed and cut into strips for further use.

5. Construction of detection platform of smart phone

The procedure for this step was the same as in example 2.

6. Fluorescent paper-based sensor detection of glyphosate

And (3) dropwise adding 2-3 mu L of glyphosate solutions with different concentrations into the ratio fluorescent paper-based sensor, and after the paper-based sensor is dried for 0.5-1 minute, acquiring an image and a red-green-blue value (RGB value) by using a smart phone under a 365nm ultraviolet lamp light source. Quantitative detection is realized by establishing the relation between the ratio of R to B and the concentration of glyphosate.

7. Drawing of standard curve

The fluorescent paper-based sensor added with glyphosate solutions of different concentrations is placed in a detection platform, and a fluorescent image is obtained through a smart phone under a dark environment by taking a 365nm ultraviolet lamp as a light source, as shown in fig. 11. And further decomposing the glyphosate into RGB values, wherein the RGB values have a linear relation with the glyphosate values when the glyphosate concentration is 20-180 nM, the abscissa is the glyphosate concentration, and the ordinate is the RGB ratio.

Example 6:

1. preparation of blue carbon dots

The procedure for this step is the same as in example 1.

2. Preparation of gold nanoclusters

The procedure for this step was the same as in example 1.

3. Preparation of ratiometric fluorescent nanosensors

The procedure for this step was the same as in example 1.

4. Fluorescence nanoprobe selectivity and interference testing

Adding 150nM Trichlorfon (Trichlorofon), Dimethoate (Dimethoate), Acephate (Acephate), Paraoxon (Paraoxon), Carbendazim (Carbendazim), Chlorothalonil (Chlorothalonil) and inorganic ions (K) to the fluorescent nanoprobe system ⁺ ,Na ⁺ ,Ca ²⁺ ,Mg ²⁺ ,H ₂ PO ₄ ^- ,PO ₄ ^3- ) And the result shows that the fluorescence intensity ratio has no obvious change, and the blue fluorescence is quenched and the fluorescence intensity ratio is obviously changed by continuously adding 150nM glyphosate, as shown in FIG. 12. The results show that the system has good selectivity and anti-interference performance on glyphosate.

Claims

1. A blue carbon dot/gold nanocluster-based ratiometric fluorescence sensor, comprising:

the ratio fluorescence sensor is composed of a composite system of blue carbon dots and gold nanocluster solution, and the fluorescence intensity ratio of the blue carbon dots to the gold nanoclusters in the composite system is 9: 1.

2. A method for preparing a blue carbon dot/gold nanocluster-based ratiometric fluorescence sensor of claim 1, comprising the steps of:

step 1: preparation of blue carbon dots

Dissolving 0.8-1.0 g of anhydrous sodium citrate and 0.45-0.52 g of polyacrylamide in ultrapure water, and performing ultrasonic treatment to obtain a clear and transparent solution; then transferring the solution into a high-pressure reaction kettle with a polytetrafluoroethylene lining, carrying out heat preservation reaction for 3-5 hours at the temperature of 180-200 ℃, and naturally cooling to room temperature to obtain blue carbon dot liquid; dialyzing the blue carbon dot liquid, drying to obtain carbon dot powder, preparing into 0.8-1 mg/mL aqueous solution, and storing at 4 ℃ for later use;

step 2: preparation of gold nanoclusters

Dissolving 5.6-6.6 mg of 11-mercaptoundecanoic acid in deionized water, ultrasonically stirring, adding a NaOH solution, stirring until the solution is transparent, and then adding HAuCl ₄ Reacting at room temperature for 5-8 h to slowly turn the colorless solution into light yellow; filtering the solution containing the gold nanoclusters through a microporous membrane after dialysis; storing the obtained colorless transparent solution at 4 ℃ for later use;

and step 3: preparation of ratiometric fluorescent nano-sensing system

3. The method of claim 2, wherein:

in the step 1, the molecular weight of the filter membrane adopted for dialysis is 1000Da, and the dialysis time is 4-6 hours.

4. The method of claim 2, wherein:

in the step 2, the molecular weight of the filter membrane adopted for dialysis is cut off to be 1000Da, the dialysis time is 4-6 hours, and then the filtration is carried out by adopting a 0.22 mu m microporous membrane.

5. Use of a blue carbon dot/gold nanocluster based ratiometric fluorescence sensor according to claim 1, characterized in that:

the ratiometric fluorescent sensor is used as a detection reagent in the process of detecting glyphosate residues.

6. Use according to claim 5, characterized in that:

the ratio fluorescence sensor is used as a detection reagent, the detection reagent contains 16-20 mu g/mL of blue carbon dots, and the fluorescence intensity ratio of the blue carbon dots to the gold nanoclusters is 9: 1; the detection reagent solution is dripped into a quartz colorimetric tube with the length of 1-2 cm, 2-3 mu L of glyphosate solutions with different concentrations are sequentially added, the glyphosate solutions are uniformly mixed, and obvious fluorescent color change can be observed under an ultraviolet lamp, so that the glyphosate can be visually detected.

7. Use according to claim 5, characterized in that:

the ratio fluorescence sensor is used as a detection reagent, the detection reagent contains 16-20 mu g/mL of blue carbon dots, and the fluorescence intensity ratio of the blue carbon dots to the gold nanoclusters is 9: 1; dripping the detection reagent solution into a quartz colorimetric tube with the length of 1-2 cm, sequentially adding 2-3 mu L of glyphosate solutions with different concentrations, uniformly mixing, recording the fluorescence spectrum of the solution in the range of 400-800 nm by using excitation light with the length of 350nm, and realizing the quantitative detection of the glyphosate by establishing the relation between the change of the fluorescence peak intensity ratio and the glyphosate concentration.

8. Use according to claim 5, characterized in that:

printing the ratio fluorescent sensor solution on a white substrate as ink, and repeatedly printing for 15-20 times to obtain a ratio fluorescent paper-based sensor; and (3) dropwise adding 2-3 mu L of glyphosate solutions with different concentrations onto the ratio fluorescent paper-based sensor, after the paper-based sensor is dried for 0.5-1 minute, acquiring an image and RGB values by using software under an ultraviolet light source, and realizing quantitative detection by establishing a relation between the ratio of R to B and the glyphosate concentration.