On-site cyanide analysis method based on ratio fluorescent probe

Abstract

The invention relates to a cyanide field analysis method based on a ratiometric fluorescent probe, and relates to the technical field of cyanide detection and analysis, the cyanide field analysis method comprises the following steps: obtaining a ratiometric fluorescent probe formed by mixing gold clusters and fluorescein; adding the ratiometric fluorescent probe into a cyanide-containing solution to be detected, and performing fluorescence image photographing on the solution to be detected by using a portable fluorescence reading device and photographing equipment. According to the method, fluorescein is introduced to form the ratiometric fluorescent probe by utilizing the etching effect of cyanide on the gold cluster, the synthesis process of the gold cluster is simple and low in cost, chemical modification is not needed in the label-free ratiometric probe method, rapid detection of the cyanide can be realized through simple mixing of the fluorescein and the gold cluster, and a portable fluorescent reading device is combined to realize rapid detection of the cyanide. And on-site instant detection of cyanide can be realized. According to the technology, on-site analysis of cyanide can be realized through simple mixing, and a promising on-site detection strategy is provided for a cyanide poisoning scene.

Description

Technical Field

[0001] The present invention relates to the technical field of cyanide detection and analysis, and particularly to a field analysis method for cyanide based on a ratio fluorescence probe. Background Art

[0002] As a highly toxic substance, cyanide is widely used in industries such as metallurgy and electroplating. Its leakage or accidental ingestion can lead to acute poisoning and even death. Therefore, the development of rapid, sensitive, and reliable cyanide detection methods is of great significance for environmental protection, food safety, and clinical first aid. Currently, the detection of cyanide mainly relies on large laboratory instruments (such as liquid chromatography, gas chromatography, and their coupling technologies). Although these methods have high sensitivity and accuracy, they have limitations such as expensive equipment, complex operation, the need for professional technicians, and a long sample pretreatment cycle, making it difficult to meet the requirements of on-site rapid detection.

[0003] To adapt to on-site detection scenarios, rapid detection kits based on colorimetric reactions have gradually been applied. Such kits usually utilize the reaction of cyanide with specific colorimetric reagents (such as the pyridine-barbituric acid system) to generate colored products, and qualitative or semi-quantitative analysis is achieved through visual colorimetry. However, common amino compounds (such as sulfides and thiosulfates) in actual samples are prone to competitive reactions with colorimetric reagents, resulting in false positive results. In addition, background interference in complex matrices (such as blood and wastewater) will also significantly reduce the detection specificity. At the same time, existing kits mostly rely on visual colorimetry and lack portable quantitative analysis devices, making it difficult to meet the urgent need for rapid quantitative interpretation in poisoning incidents.

[0004] In recent years, fluorescence sensing technology has received attention due to its advantages such as high sensitivity, fast response, and real-time monitoring. However, traditional single-signal fluorescence probes are easily affected by probe concentration, environmental factors (such as pH and temperature), and instrument fluctuations, resulting in insufficient detection stability. In contrast, ratio fluorescence probes detect by simultaneously monitoring the ratio of the fluorescence signal intensities at two different wavelengths, which can effectively offset background interference and improve detection reliability. Currently, there is still a lack of research on ratio fluorescence detection of cyanide. Existing probes are mostly based on a single recognition mechanism (such as nucleophilic addition or coordination), have limited anti-interference ability in complex samples, and have not been effectively combined with portable detection devices, restricting their on-site application potential. Summary of the Invention

[0005] To solve the above problems, the present invention provides a field analysis method for cyanide based on a ratio fluorescence probe.

[0006] The present invention provides a field analysis method for cyanide based on a ratio fluorescence probe, and the field analysis method for cyanide includes the following steps:

[0007] A ratiometric fluorescence probe composed of gold clusters and fluorescein is obtained;

[0008] Add the ratiometric fluorescence probe into the solution to be detected containing cyanide, and use a portable fluorescence reading device and a photographing device to take fluorescence images of the solution to be detected. Compare with the fluorescence colorimetric card to conduct semi - quantitative analysis of the detected cyanide.

[0009] Furthermore, the volume ratio of the gold clusters to the fluorescein is (1 - 3):1.

[0010] Furthermore, the volume ratio of the gold clusters to the fluorescein is 2:1.

[0011] Furthermore, the characteristic fluorescence wavelength of the gold clusters is 660 nm.

[0012] Furthermore, the characteristic fluorescence wavelength of the fluorescein is 515 nm.

[0013] Furthermore, the preparation method of the gold clusters includes the following process:

[0014] Vigorously stir and mix 5 mL of 10 mM HAuCl 4 solution with 5 mL of 50 mg / mL BSA solution. After 2 minutes, add 0.5 mL of 1 M NaOH solution, and vigorously stir at 37 °C for 12 hours to obtain gold clusters.

[0015] Furthermore, the concentration of cyanide in the solution to be detected is 10 - 100 mg / L.

[0016] Furthermore, the cyanide includes potassium cyanide, and the photographing device includes a mobile phone.

[0017] The above - mentioned technical solution provided by the embodiments of the present invention has at least the following advantages compared with the prior art:

[0018] The embodiments of the present invention provide a method for on - site analysis of cyanide based on a ratiometric fluorescence probe. The present invention utilizes the etching effect of cyanide on gold clusters, introduces fluorescein to form a ratiometric fluorescence probe. The synthesis process of gold clusters is simple and low - cost. This label - free ratiometric probe method does not require chemical modification, and rapid detection of cyanide can be achieved through simple mixing of the two. Combined with a portable fluorescence reading device, on - site instant detection of cyanide can be realized. This technology can achieve on - site analysis of cyanide through simple mixing, providing a promising on - site detection strategy for cyanide poisoning scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings herein are incorporated into the specification and form a part of this specification, showing embodiments consistent with the present invention and used together with the specification to explain the principles of the present invention.

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the attached drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other attached drawings can also be obtained based on these attached drawings.

[0021] Figure 1 It is a schematic diagram of the change in fluorescence intensity after the reaction of gold clusters with cyanides of different concentrations in Test Example 1;

[0022] Figure 2 It is a schematic diagram of the change in fluorescence intensity after the reaction of fluorescein with cyanides of different concentrations in Test Example 2;

[0023] Figure 3 It is a fluorescence image for exploring the reaction of different ratios of gold clusters and fluorescein with potassium cyanide in Test Example 3;

[0024] Figure 4 It is a fluorescence image for exploring the reaction of the ratio fluorescence probe with potassium cyanide for different times in Test Example 4;

[0025] Figure 5 It is a schematic diagram of the change in fluorescence intensity for verifying the reaction of the ratio fluorescence probe (gold clusters + fluorescein) with potassium cyanide in Test Example 5;

[0026] Figure 6 It is a fluorescence image of the reaction of the ratio fluorescence probe (gold clusters + fluorescein) with cyanides in Test Example 6;

[0027] Figure 7 It is a physical image of the portable fluorescence reading device in Test Example 7. Detailed implementation manners

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the attached drawings in the embodiments of the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0029] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchases or can be prepared by existing methods.

[0030] The present invention provides a method for on-site analysis of cyanides based on a ratio fluorescence probe. The on-site analysis method of cyanides includes the following steps:

[0031] A ratiometric fluorescence probe composed of gold clusters and fluorescein is obtained;

[0032] Add the ratiometric fluorescence probe into the solution to be detected containing cyanide, take fluorescence images of the solution to be detected using a portable fluorescence reading device and a photographing device, compare with a fluorescence colorimetric card, and conduct semi - quantitative analysis of the detected cyanide.

[0033] The embodiment of the present invention provides a method for on - site analysis of cyanide based on a ratiometric fluorescence probe. The present invention utilizes the etching effect of cyanide on gold clusters, introduces fluorescein to form a ratiometric fluorescence probe. The synthesis process of gold clusters is simple and low - cost. This label - free ratiometric probe method does not require chemical modification, and rapid detection of cyanide can be achieved through simple mixing of the two. Combined with a portable fluorescence reading device, on - site instant detection of cyanide can be realized. This technology can achieve on - site analysis of cyanide through simple mixing, providing a promising on - site detection strategy for cyanide poisoning scenarios.

[0034] In some specific embodiments, the volume ratio of the gold clusters to the fluorescein is (1 - 3):1, preferably 2:1.

[0035] In some specific embodiments, the characteristic fluorescence wavelength of the gold clusters is 660 nm.

[0036] In some specific embodiments, the characteristic fluorescence wavelength of the fluorescein is 515 nm.

[0037] In some specific embodiments, the preparation method of the gold clusters includes the following process:

[0038] Vigorously stir and mix 5 mL of 10 mM HAuCl 4 solution with 5 mL of 50 mg / mL BSA solution, add 0.5 mL of 1 M NaOH solution after 2 minutes, and obtain gold clusters after vigorously stirring at 37 °C for 12 hours.

[0039] In some specific embodiments, the concentration of cyanide in the solution to be detected is 10 - 100 mg / L.

[0040] In some specific embodiments, the cyanide is potassium cyanide, and the photographing device includes a mobile phone.

[0041] It should be noted that for the component raw materials involved in the method for on - site analysis of cyanide based on a ratiometric fluorescence probe provided by the embodiment of the present invention, without special limitations or specific descriptions, commercially available products can be directly used or self - made using existing publicly disclosed preparation methods; meanwhile, for the steps and parameters involved, without special limitations or specific descriptions, they can be carried out according to the steps and parameters disclosed in the prior art or directly used with reference to the operating instructions of existing equipment. The present invention document will not elaborate one by one.

[0042] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following embodiments, they are generally determined according to national standards. If there is no corresponding national standard, they are carried out according to general international standards, conventional conditions, or the conditions recommended by the manufacturer.

[0043] The technical solution of a specific embodiment of the present invention is as follows:

[0044] A ratiometric fluorescence strategy based on gold clusters for on-site analysis of cyanide. The method uses a sensor composed of gold clusters and fluorescein. The specific sensing steps are as follows: Take 20 μL of the synthesized gold clusters and 10 μL of 0.1 mM fluorescein and dissolve them in 200 μL of solution. After adding a certain concentration of cyanide, immediately take a photo with a portable fluorescence reading device to analyze the fluorescence intensity changes of the gold clusters and fluorescein. Among them, the preparation method of the gold clusters includes the following process: Mix 5 mL of 10 mM HAuCl 4 solution with 5 mL of 50 mg / mL BSA solution by vigorous stirring. After 2 minutes, add 0.5 mL of 1 M NaOH solution and stir vigorously at 37 °C for 12 hours to obtain gold clusters.

[0045] Screening and experimental verification:

[0046] Experimental Example 1: Verifying the specific effect of cyanide on gold clusters

[0047] Cyanide can form a very stable Au(CN) 2 complex with Au through strong covalent bonds, which is called the Elsner reaction. Therefore, after adding cyanide to the gold cluster system, a gold cyanide complex is formed, resulting in fluorescence quenching of the gold clusters at 660 nm.

[0048] As Figure 1 shown, it is the fluorescence intensity change detected by a molecular fluorescence spectrometer and the image recorded by a portable fluorescence photography device. After adding cyanide to the gold clusters, the fluorescence of the gold clusters at 660 nm quenched to varying degrees. As the concentration of cyanide increased, the fluorescence intensity decreased more significantly.

[0049] The results show that the gold clusters have characteristic fluorescence at 660 nm, and as the concentration of cyanide increases, the fluorescence intensity weakens more strongly.

[0050] Experimental Example 2: Verifying that fluorescein can be used as a reference ratio signal

[0051] As Figure 2 shown, it is the fluorescence intensity change of a molecular fluorescence spectrometer and the image taken by a portable fluorescence photography device after mixing different concentrations of cyanide with fluorescein.

[0052] Results show that the fluorescence intensity of fluorescein does not change with the change in cyanide concentration.

[0053] Therefore, fluorescein can be used as a reference ratio signal for gold clusters to sense cyanide and form a ratio fluorescence probe with gold clusters.

[0054] Experimental Example 3: Exploring the ratio of gold clusters to fluorescein

[0055] As Figure 3 shown, the fluorescence changes after adding cyanide to the mixture of gold clusters and fluorescein with different volume ratios;

[0056] Results show that when the volume ratio of gold clusters to fluorescein is between 5:1 and 2.5:1, the blank signal of the ratio fluorescence is red to orange. The larger the proportion of gold clusters, the more the ratio signal tends to be red, and vice versa, it tends to be orange. With the addition of potassium cyanide, the response signal in this ratio range changes from red to dark red or brown. When the ratio of gold clusters to fluorescein is 2:1, the blank signal is orange, and the signal of the experimental group changes from dark brown to bright brown, and there is a tendency for green fluorescence to appear; when the ratio of gold clusters to fluorescein is 1:1, the blank signal is bright yellow, and the signal of the experimental group is bright green. In order to obtain a fluorescence probe with significant fluorescence changes and a wide response concentration range, the volume ratio of gold clusters to fluorescein of 2:1 is finally selected as the optimal mixing ratio.

[0057] Experimental Example 4: Exploring the reaction time of the ratio fluorescence probe with potassium cyanide

[0058] As Figure 4 shown, it is the fluorescence image of the ratio fluorescence probe reacting with potassium cyanide for different times.

[0059] Results show that the target potassium cyanide can react immediately with the ratio probe. When the concentration of potassium cyanide is 20 mg / L and 40 mg / L, the fluorescence immediately changes from orange-red to dark orange. As the reaction time increases to 5 min, the fluorescence image changes little. When the concentration of potassium cyanide is 80 mg / L, the fluorescence of the immediate reaction is dark brown, and the fluorescence after the reaction time is greater than 2 min is dark green, indicating that a higher concentration of potassium cyanide requires more time to obtain green fluorescence; when the concentration of potassium cyanide is 100 mg / L, the fluorescence after the reaction time is greater than 2 min is bright green, which is brighter than the dark green fluorescence obtained by the immediate reaction of potassium cyanide. Therefore, for low-concentration potassium cyanide (<40 mg / L), it can react immediately after being added to the system; for high-concentration potassium cyanide (>40 mg / L), the optimal reaction time for adding to the system is 2 min. In summary, the reaction time of 2 min is selected for this ratio probe.

[0060] Experimental Example 5: Verifying the detection of cyanide by the ratio fluorescence probe (gold clusters + fluorescein)

[0061] As shown Figure 5 in the figure, the fluorescence intensity changes of the probe reacting with cyanide at 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, and 50 mg / L, and the images taken by the portable fluorescence photography device are shown.

[0062] The results show that the fluorescence of the gold clusters is obvious at 660 nm after binding with fluorescein. After the addition of the target potassium cyanide, the fluorescence of the gold clusters at 660 nm begins to weaken, while the fluorescence intensity at 515 nm has no obvious change, and the greater the concentration of potassium cyanide, the higher the fluorescence quenching degree at 660 nm. Therefore, with the addition of potassium cyanide, the fluorescence of the gold clusters weakens while the fluorescence of fluorescein remains unchanged. Using a weak and unchanged (weakening red fluorescence and unchanged green fluorescence) signal to construct a ratio fluorescence probe, the fluorescence change from red to green of this probe indicates that this ratio fluorescence method can realize the sensing analysis of potassium cyanide.

[0063] Test Example 6: Performance Analysis of Detecting Cyanide

[0064] As shown Figure 6 in the figure, the fluorescence images (in triplicate) of the ratio fluorescence probe reacting with the target potassium cyanide (20 - 100 mg / L) are shown. As the concentration of potassium cyanide increases, the fluorescence changes from bright orange to dark orange, brown, dark green, green, and bright green. Therefore, semi - quantitative analysis and detection of potassium cyanide can be carried out according to the fluorescence change range.

[0065] Figure 7 As shown in the figure, the physical diagram of the portable fluorescence reading device is shown. The whole detection kit includes the pre - mixed gold clusters and fluorescein probe. The portable fluorescence reading device consists of a No. 5 battery, a 365 - nm lamp bead, a self - made quartz cuvette with an inner diameter of 1 cm, a 3D - printed fluorescence reading device with dimensions of 6 cm * 8 cm * 10 cm (length * width * height), and finally, fluorescence signal reading can be achieved by combining with a mobile phone.

[0066] In summary, the embodiment of the present invention provides a method for on - site analysis of cyanide based on a ratio fluorescence probe. This method utilizes the etching effect of cyanide on gold clusters. The greater the concentration of cyanide, the greater the fluorescence quenching degree. By introducing fluorescein and gold clusters to form a ratio fluorescence probe, the synthesis process of gold clusters is simple and the cost is low. This label - free ratio probe method does not require chemical modification, and rapid detection of cyanide can be achieved through the simple mixing of the two. Combined with a portable fluorescence reading device, on - site instant detection of cyanide can be realized. This technology does not require chemical modification, only low - cost gold cluster and fluorescein reagents, and combined with a portable fluorescence reading device with a cost of about 50 yuan, semi - quantitative analysis of cyanide can be achieved. On - site analysis of cyanide can be realized through simple mixing, providing a promising on - site detection strategy for cyanide poisoning scenarios.

[0067] The various embodiments of the present invention may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be construed as a rigid limitation on the scope of the present invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and individual values within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and individual numbers within the range, such as 1, 2, 3, 4, 5, and 6, and this applies regardless of the range. Additionally, whenever a numerical range is indicated herein, it means including any cited number (fraction or integer) within the indicated range.

[0068] The above are only specific embodiments of the present invention, enabling those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather will conform to the broadest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for on-site analysis of cyanide based on ratiometric fluorescent probe, characterized in that: The on-site analysis method of cyanide comprises the following steps: A ratiometric fluorescent probe composed of a mixture of gold clusters and fluorescein is obtained; The ratio fluorescent probe is added into a solution to be detected containing cyanide, and a fluorescent image of the solution to be detected is photographed using a portable fluorescent reading device and a photographing device, and compared with a fluorescent colorimetric card, a semi-quantitative analysis of the detected cyanide is performed.

2. The method for on-site analysis of cyanide based on ratiometric fluorescent probe according to claim 1, characterized in that: The volume ratio of the gold clusters to the fluorescent substance is (1-3):

1.

3. The on-site cyanide analysis method based on ratio fluorescence probe according to claim 1, characterized in that: The volume ratio of the gold clusters to the fluorescein is 2:

1.

4. The on-site cyanide analysis method based on ratio fluorescence probe according to claim 1, characterized in that: The characteristic fluorescence wavelength of the gold clusters is 660 nm.

5. The on-site cyanide analysis method based on ratio fluorescence probe according to claim 1, characterized in that: The characteristic fluorescence wavelength of the fluorescein is 515 nm.

6. The on-site cyanide analysis method based on ratio fluorescence probe according to claim 1, characterized in that: The preparation method of the gold clusters comprises the following steps: 5 mL of 10 mM HAuCl4 solution and 5 mL of 50 mg / mL BSA solution were vigorously stirred and mixed, and 0.5 mL of 1 M NaOH solution was added after 2 minutes. Gold clusters were obtained after vigorously stirring at 37 °C for 12 hours.

7. The on-site cyanide analysis method based on ratio fluorescence probe according to claim 1, characterized in that: The concentration of cyanide in the solution to be detected is 10-100 mg / L.

8. The method for on-site analysis of cyanide based on ratiometric fluorescent probe according to claim 1, characterized in that: The cyanide is potassium cyanide, and the photographing device includes a mobile phone.

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