A cyanide on-site analysis method based on a ratiometric fluorescent probe

Abstract

The present application relates to a kind of cyanide on-site analysis method based on ratio fluorescent probe, it is related to cyanide detection analysis technical field, the on-site analysis method of cyanide includes the following steps: obtain the ratio fluorescent probe mixed with gold cluster and fluorescein;The ratio fluorescent probe is added to the cyanide-containing solution to be detected, utilizes portable fluorescence reading device and photographing equipment to carry out the steps such as fluorescence image photographing to the solution to be detected.The present application utilizes the etching effect of cyanide on gold cluster, introduces fluorescein to constitute ratio fluorescent probe, gold cluster synthesis process is simple and low in cost, this label-free ratio probe method does not need chemical modification, the rapid detection of cyanide can be realized by the simple mixing of both, combined with portable fluorescence reading device, the on-site real-time detection of cyanide can be realized.This technology can realize the on-site analysis of cyanide by simple mixing, provides a promising on-site detection strategy for cyanide poisoning scene.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cyanide detection and analysis, and particularly relates to a cyanide on-site analysis method based on a ratio fluorescent probe. BACKGROUND

[0002] As a highly toxic substance, cyanide is widely used in the fields of industry, metallurgy, electroplating, etc. Its leakage or accidental ingestion can lead to acute poisoning and even death. Therefore, developing a rapid, sensitive and reliable cyanide detection method is of great significance for environmental protection, food safety and clinical first aid. At present, the detection of cyanide mainly relies on large laboratory instruments (such as liquid chromatography, gas chromatography and their combined techniques). 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 period, which makes it difficult to meet the needs of on-site rapid detection.

[0003] To adapt to the scene of on-site detection, fast detection kits based on color development reactions have been gradually applied. Such kits usually use cyanide to react with specific color developers (such as pyridine-barbituric acid system) to generate colored products, and achieve qualitative or semi-quantitative analysis by visual colorimetry. However, common amino compounds (such as sulfides, thiosulfates) in actual samples are prone to competitive reactions with color developers, resulting in false positive results; in addition, background interference in complex matrices (such as blood, wastewater) can also significantly reduce the detection specificity. At the same time, existing kits mostly rely on visual colorimetry, lack of portable quantitative analysis devices, and are difficult to meet the urgent needs of rapid quantitative interpretation in poisoning incidents.

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

[0005] To solve the above problems, the present application provides a cyanide on-site analysis method based on a ratio fluorescent probe.

[0006] The present application provides a cyanide on-site analysis method based on a ratio fluorescent probe, which comprises the following steps:

[0007] obtain a ratio fluorescent probe mixed with gold clusters and fluorescein;

[0008] Add the ratio fluorescent probe to the cyanide-containing solution to be detected, take a fluorescent image of the solution to be detected by using a portable fluorescent reading device and a photographing device, and compare the fluorescent color chart to semi-quantitatively analyze the cyanide.

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

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

[0011] Further, the characteristic fluorescent wavelength of the gold clusters is 660 nm.

[0012] Further, the characteristic fluorescent wavelength of the fluorescein is 515 nm.

[0013] Further, the preparation method of the gold clusters comprises the following process:

[0014] Mix 5 mL of 10 mM HAuCl4 solution with 5 mL of 50 mg / mL BSA solution by vigorous stirring, add 0.5 mL of 1 M NaOH solution after 2 minutes, and obtain gold clusters after 12 hours of vigorous stirring at 37 DEG C.

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

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

[0017] The above technical solution provided by the embodiment of the present application has at least the following advantages compared with the prior art:

[0018] The embodiment of the present application provides a cyanide on-site analysis method based on a ratio fluorescent probe. BRIEF DESCRIPTION OF DRAWINGS

[0019] The drawings incorporated into the specification and forming a part thereof, illustrate embodiments consistent with the present application and together with the description, serve to explain the principles of the application.

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, for those skilled in the art, other drawings can also be obtained from these drawings without any creative effort.

[0021] Figure 1 Fig. 2 is a schematic diagram of the change of fluorescence intensity after the gold cluster reacts with cyanide of different concentrations in the test example 1;

[0022] Figure 2 Fig. 3 is a schematic diagram of the change of fluorescence intensity after the fluorescein reacts with cyanide of different concentrations in the test example 2;

[0023] Figure 3 Fig. 4 is a fluorescence diagram of the reaction of gold cluster and fluorescein with potassium cyanide in different proportions in the test example 3;

[0024] Figure 4 Fig. 5 is a fluorescence diagram of the reaction of the ratio fluorescent probe with potassium cyanide at different times in the test example 4;

[0025] Figure 5 Fig. 6 is a schematic diagram of the change of fluorescence intensity after the ratio fluorescent probe (gold cluster + fluorescein) reacts with potassium cyanide in the test example 5;

[0026] Figure 6 Fig. 7 is a fluorescence diagram of the reaction of the ratio fluorescent probe (gold cluster + fluorescein) with cyanide in the test example 6;

[0027] Figure 7 Fig. 8 is a photograph of a portable fluorescence reading device in the test example 7. DETAILED DESCRIPTION

[0028] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the following will combine the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without any creative effort fall within the scope of protection of the present application.

[0029] Unless otherwise specifically indicated, the various materials, reagents, instruments and equipment used in the present application can be purchased from the market or can be prepared by the existing methods.

[0030] The present application provides a cyanide on-site analysis method based on a ratio fluorescent probe. The cyanide on-site analysis method comprises the following steps:

[0031] The ratio fluorescent probe mixed with gold cluster and fluorescein is obtained.

[0032] The ratio fluorescent probe is added into the cyanide-containing solution to be detected, and a portable fluorescence reading device and a photographing device are used to take a fluorescence image of the solution to be detected, and a fluorescence color chart is compared to semi-quantitatively analyze the cyanide.

[0033] The embodiment of the present application provides a cyanide on-site analysis method based on a ratio fluorescent probe.

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

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

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

[0037] In some specific embodiments, the preparation method of the gold cluster comprises the following processes:

[0038] 5mL of 10mM HAuCl4 solution is mixed with 5mL of 50mg / mL BSA solution by vigorous stirring, 0.5mL of 1M NaOH solution is added after 2 minutes, and gold clusters are obtained after 12 hours of vigorous stirring at 37℃.

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

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

[0041] It should be noted that the components and raw materials involved in the cyanide on-site analysis method based on the ratio fluorescent probe provided by the embodiment of the present application can be directly used in the market or self-made by using the existing public preparation method if there is no special limitation or specific description; meanwhile, the steps and parameters involved can be performed according to the steps and parameters disclosed in the prior art or directly used by referring to the use instructions of the existing equipment if there is no special limitation or specific description, and the present application document will not be described one by one.

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

[0043] The technical solution of one specific embodiment of the application is:

[0044] A cyanide on-site analysis method based on a gold cluster ratio fluorescence strategy, the method comprising a sensor composed of gold clusters and fluorescein, the specific sensing steps are as follows: 20 μL of synthesized gold clusters and 10 μL of 0.1 mM fluorescein are respectively added to 200 μL of solution, after adding a certain concentration of cyanide, the portable fluorescence reading device is used to take pictures to analyze the fluorescence intensity changes of the gold clusters and the fluorescein; wherein, the preparation method of the gold clusters comprises the following process: 5 mL of 10 mM HAuCl4 solution and 5 mL of 50 mg / mL BSA solution are mixed by vigorous stirring, 0.5 mL of 1 M NaOH solution is added after 2 minutes, and the gold clusters are obtained after 12 hours of vigorous stirring at 37°C.

[0045] Screening and experimental verification:

[0046] Test Example 1: verification of the specific effect of cyanide on gold clusters

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

[0048] As shown in Figure 1 , the fluorescence intensity changes under the detection of a molecular fluorescence instrument and the images recorded by a portable fluorescence photographing device. After the gold clusters are added with cyanide, the fluorescence of the gold clusters at 660 nm is quenched to different degrees. With the increase of the cyanide concentration, the fluorescence intensity decreases more obviously.

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

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

[0051] As shown in Figure 2 , after different concentrations of cyanide are mixed with fluorescein, the fluorescence intensity changes of a molecular fluorescence instrument and the images recorded by a portable fluorescence photographing device.

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

[0053] Therefore, fluorescein can be used as a reference ratio signal for gold cluster sensing cyanide, and the gold cluster forms a ratio fluorescent probe.

[0054] Test example 3: explore the ratio of gold cluster and fluorescein

[0055] As shown in Figure 3 , the fluorescence change after adding cyanide after mixing different volume ratios of gold cluster and fluorescein;

[0056] The results show that when the volume ratio of gold cluster and fluorescein is between 5:1-2.5:1, the blank signal of ratio fluorescence is red to orange, the larger the ratio of gold cluster, the more the ratio signal tends to red, and vice versa. With the addition of potassium cyanide, the response signal in this ratio interval changes from red to dark red or brown. When the ratio of gold cluster and fluorescein is 2:1, the blank signal is orange, and the signal of the experimental group changes from dark brown to bright brown, showing a trend of green fluorescence. When the ratio of gold cluster and 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 fluorescent probe with significant fluorescence change and wide response concentration range, the volume ratio of gold cluster and fluorescein is finally selected as 2:1 as the best mixing ratio.

[0057] Test example 4: explore the reaction time of ratio fluorescent probe and potassium cyanide

[0058] As shown in Figure 4 , it is the fluorescence diagram of ratio fluorescent probe and potassium cyanide reacting for different times.

[0059] The results show that the target potassium cyanide can react immediately with the ratio probe. When the concentration of potassium cyanide is 20mg / L and 40mg / L, the fluorescence changes from orange red to dark orange immediately, and the fluorescence image changes little with the increase of reaction time to 5min. When the concentration of potassium cyanide is 80mg / L, the fluorescence of immediate reaction is dark brown, and the fluorescence of reaction time greater than 2min is dark green, indicating that the increase of potassium cyanide concentration needs more time to obtain green fluorescence; When the concentration of potassium cyanide is 100mg / L, the fluorescence of reaction time greater than 2min is bright green, which is brighter than the dark green fluorescence obtained by immediate reaction of potassium cyanide. Therefore, for low concentration of potassium cyanide (<40mg / L), it can react immediately after adding to the system; for high concentration of potassium cyanide (>40mg / L), the best response time of the system is 2min. In summary, the reaction time of the ratio probe is selected as 2min.

[0060] Test example 5: verify the ratio fluorescent probe (gold cluster + fluorescein) for detecting cyanide

[0061] AsFigure 5 The image shows the fluorescence intensity changes of the probe reacting with 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, and 50 mg / L cyanide, respectively, and the images captured by a portable fluorescence imaging device.

[0062] The results showed that the gold clusters exhibited significant fluorescence at 660 nm after binding with fluorescein. With the addition of potassium cyanide, the fluorescence of the gold clusters at 660 nm began to weaken, while the fluorescence intensity at 515 nm remained unchanged. Furthermore, the higher the concentration of potassium cyanide, the greater the quenching of fluorescence at 660 nm. Therefore, with the addition of potassium cyanide, the fluorescence of the gold clusters weakened while the fluorescence of fluorescein remained unchanged. Using this weak and unchanged signal (red fluorescence weakened, green fluorescence unchanged) to construct a ratiometric fluorescent probe, the fluorescence change of this probe from red to green indicates that this ratiometric fluorescence method can achieve the sensing analysis of potassium cyanide.

[0063] Experimental Example 6: Performance Analysis of Cyanide Detection

[0064] like Figure 6 The image shows fluorescence images (triple-parameters) of the reaction between the ratiometric fluorescent probe and the target analyte potassium cyanide (20-100 mg / L). As the potassium cyanide concentration increases, the fluorescence changes from bright orange to dark orange, brown, dark green, green, and finally bright green. Therefore, potassium cyanide can be semi-quantitatively detected based on the fluorescence variation range.

[0065] Figure 7 The image shown is a picture of the portable fluorescence reading device. The entire test kit includes pre-mixed gold clusters and fluorophore probes. The portable fluorescence reading device consists of an AA battery, a 365nm LED, a self-made quartz cuvette with an inner diameter of 1cm, and a 3D-printed fluorescence reading device measuring 6cm*8cm*10cm (length*width*height). Finally, it can be used with a mobile phone to read fluorescence signals.

[0066] In summary, this invention provides a method for on-site analysis of cyanide based on a ratiometric fluorescent probe. This method utilizes the etching effect of cyanide on gold clusters; the higher the cyanide concentration, the greater the fluorescence quenching. By introducing fluorescein and gold clusters to form a ratiometric fluorescent probe, the gold cluster synthesis process is simple and low-cost. This label-free ratiometric probe method requires no chemical modification; rapid detection of cyanide can be achieved through simple mixing of the two. Combined with a portable fluorescence reading device, real-time on-site detection of cyanide can be realized. This technology requires no chemical modification, only low-cost gold clusters and fluorescein reagents, and a portable fluorescence reading device costing approximately 50 yuan can achieve semi-quantitative analysis of cyanide. On-site analysis of cyanide can be achieved through simple mixing, providing a promising on-site detection strategy for cyanide poisoning scenarios.

[0067] 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 merely for convenience and brevity and should not be construed as a hard limitation on the scope of the invention; therefore, it should be considered that the range description has specifically disclosed all possible subranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed subranges 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 single numbers within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Furthermore, whenever a numerical range is referred to herein, it means including any referenced number (fraction or integer) within the range referred to.

[0068] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for on-site analysis of cyanide based on ratiometric fluorescent probes, characterized in that, The on-site analysis method for cyanide includes the following steps: A ratio fluorescent probe was obtained by mixing gold clusters and fluorescein; The ratiometric fluorescent probe is added to the cyanide-containing test solution. A portable fluorescence reading device and a camera are used to take a fluorescence image of the test solution. The fluorescence image is compared with the fluorescence colorimetric card to perform a semi-quantitative analysis of the detected cyanide. The volume ratio of the gold cluster to the fluorescein is (1~3):1; The characteristic fluorescence wavelength of the gold cluster is 660 nm; The characteristic fluorescence wavelength of the fluorescein is 515 nm. The preparation method of the gold clusters includes the following steps: 5 mL of 10 mM HAuCl4 solution and 5 mL of 50 mg / mL BSA solution are vigorously stirred and mixed. After 2 minutes, 0.5 mL of 1 M NaOH solution is added. After stirring vigorously at 37°C for 12 hours, gold clusters are obtained. The concentration of cyanide in the solution to be tested is 10-100 mg / L; and the cyanide is potassium cyanide.

2. The method for on-site analysis of cyanide based on ratiometric fluorescent probes according to claim 1, characterized in that, The volume ratio of the gold cluster to the fluorescein is 2:

1.

3. The method for on-site analysis of cyanide based on ratiometric fluorescent probes according to claim 1, characterized in that, The camera device includes a mobile phone.

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