Rapid visual detection of hg 2+ fluorescent dual-emission ssdna-silver cluster probe for hg and preparation method thereof

Abstract

The application discloses a fluorescent double-emission ssDNA-silver cluster probe for rapidly and visually detecting Hg 2+ and a preparation method thereof. The application discloses a fluorescent double-emission ssDNA-silver cluster probe for rapidly and visually detecting Hg 2+ , which contains a template ssDNA capable of simultaneously emitting strong red fluorescence and weak green fluorescence. The sequence of the ssDNA is 5'-AACAAAGCCCCCCCCCCCCCTTTTTTTTTT-3'. The fluorescent double-emission ssDNA-silver cluster probe can rapidly and visually detect Hg 2+ . The ssDNA-silver cluster probe has excellent selectivity for Hg 2+ , and has been used for rapid detection of trace Hg 2+ in actual water samples, and the detection result is sensitive and accurate.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent material preparation, and is particularly concerned with a method for rapidly visually detecting Hg 2+ Fluorescent dual-emission ssDNA-silver cluster probe and preparation method. Background Art

[0002] Even low concentrations of mercury pollutants are highly toxic and can seriously harm the environment and human health. 2+ ) has good water solubility and exists stably, and is the most common form of mercury pollutants. 2+ After entering the soil and water, bacteria can convert it into organic mercury, which is then absorbed by humans through the food chain, causing chronic poisoning, damage to the brain, kidneys, stomach and intestines, and even death. Therefore, it is important to monitor Hg in soil and water. 2+ The content is very important.

[0003] Currently used to detect trace amounts of Hg 2+ Classical methods include atomic absorption spectroscopy, atomic emission spectroscopy, X-ray spectroscopy, inductively coupled plasma-mass spectrometry, etc. These methods require complex sample pretreatment, are time-consuming, and have high testing costs, and are difficult to achieve rapid on-site detection.

[0004] Currently reported visual detection of Hg 2+ The sensitivity of colorimetric and fluorescence methods is generally low (generally only able to detect trace amounts of Hg 2+ Visual detection), the detection time is long (usually requires 30 minutes of reaction before detection), and it is difficult to detect trace Hg 2+ Ratiometric fluorescence analysis is a rapid, on-site detection method. Due to its higher sensitivity, accuracy, and selectivity, it has been widely used in environmental safety monitoring and other fields in recent years. However, these reported ratiometric fluorescence methods typically require the preparation of two or more probes, and the probe preparation methods / steps are complex and time-consuming, making mercury ion detection time-consuming and costly. Summary of the Invention

[0005] In view of the above problems, the present invention aims to provide a rapid visual detection method for Hg 2+ Fluorescent dual-emission ssDNA-silver cluster probe and preparation method thereof, the probe is a method for rapid visual detection of trace Hg 2+ The fluorescent dual-emission ssDNA-AgNCs probe is simple, efficient, low-cost and environmentally friendly. 2+ The method is simple, rapid, sensitive, accurate and low cost.

[0006] In order to achieve the above object of the invention, the specific technical solutions of the present invention are as follows:

[0007] A rapid visual detection method for Hg 2+ Fluorescent dual-emission ssDNA-AgNCs probe contains template ssDNA that can simultaneously emit strong red fluorescence and weak green fluorescence, and the sequence of the ssDNA is 5'-AACAAAGCCCCCCCCCCCCCTTTTTTTTTT-3'.

[0008] The first invention objective of the present invention is to protect the above-mentioned fluorescent dual-emission ssDNA-AgNCs probe in rapid visual detection of Hg 2+ Application in.

[0009] The second invention object of the present invention is to protect the above-mentioned rapid visual detection of Hg 2+ The preparation method of the fluorescent dual-emission ssDNA-AgNCs probe comprises the following steps:

[0010] ssDNA aqueous solution and AgNO3 solution were added to Tris-HAc buffer, mixed and reacted at room temperature in the dark for a period of time; then, freshly prepared NaBH4 solution was added and incubated at room temperature in the dark overnight to obtain ssDNA-AgNCs probes, i.e., dual-emission ssDNA-AgNCs probes, which were then refrigerated for storage.

[0011] As a better embodiment of the present application, in the rapid visual detection of Hg 2+ In the preparation method of the fluorescent dual-emission ssDNA-AgNCs probe, the final molar ratio of ssDNA, AgNO3 and NaBH4 is 1-3:12-48:6-36.

[0012] As a better embodiment of the present application, in the rapid visual detection of Hg 2+ In the preparation method of the fluorescent dual-emission ssDNA-AgNCs probe, Tris-HAc buffer, ssDNA aqueous solution and AgNO3 solution are mixed and reacted at room temperature in the dark for 20 to 100 minutes.

[0013] As a better embodiment of the present application, in the rapid visual detection of Hg 2+ In the preparation method of the fluorescent dual-emission ssDNA-AgNCs probe, the Tris-HAc buffer solution is 5-30.0 mmol / L, the pH is 6.0-8.4; and the molar ratio of the ssDNA solution to the Tris-HAc buffer solution is 1-4:100-1000.

[0014] As a better embodiment of the present application, the dual-emission ssDNA-AgNCs probe detects Hg 2+ The specific application steps are as follows:

[0015] The ssDNA-AgNCs probe solution was mixed with Tris-HAc buffer at a volume ratio of 1-4:1-4, and then different concentrations of Hg 2+ Standard solution or Hg 2+ After the mixed solution was allowed to react at room temperature for 1 to 30 minutes, different Hg 2+ Fluorescence photos of the system under different concentrations; Simultaneously measure the dual fluorescence emission spectrum of the system; Then, according to the fluorescence color card of the standard solution, the Hg in the sample solution can be quickly semi-quantitatively / quantitatively detected by the naked eye or mobile phone software Image J 2+ concentration; finally, according to the red and green double emission peak fluorescence intensity ratio and Hg 2+ The concentration relationship is used to establish a standard curve, based on which the Hg content in the sample solution can be accurately quantified. 2+ content.

[0016] As a preferred embodiment of the present application, the ssDNA-AgNCs probe solution and the Tris-HAc buffer solution were mixed at a volume ratio of 1:1, and then allowed to react at room temperature for 5 minutes.

[0017] As a preferred embodiment of the present application, the fluorescence excitation wavelengths of the probes are 440 nm and 560 nm respectively.

[0018] As a preferred embodiment of the present application, the maximum fluorescence emission wavelengths of the probes are 530 nm and 630 nm, respectively.

[0019] Working principle:

[0020] The dual-emission fluorescent probe described in the present invention is a silver cluster (ssDNA-AgNCs) prepared using a single-stranded DNA of a specific sequence as a template in an aqueous solution system under mild conditions. The ssDNA-AgNCs fluorescent probe can emit weak green fluorescence and strong red fluorescence. The red fluorescence emitted by the probe solution can be seen by the naked eye under the irradiation of a portable ultraviolet lamp. The present invention uses the ssDNA-AgNCs solution as the only signal and target recognition probe. Different concentrations of Hg 2+ After reacting with the probe solution at room temperature for 5 minutes, the fluorescence of the probe solution gradually changes from red to yellow to green under ultraviolet light. Therefore, different concentrations of Hg can be quickly identified by the naked eye or mobile phone software based on the fluorescence signal. 2+ , which can achieve Hg 2+ On-site rapid visual detection of water samples with concentrations as low as 10.0nmol / L. By quickly identifying the RGB value of the fluorescent photo through mobile phone software, trace Hg can be accurately and quickly detected on-site. 2+ In addition, the fluorescence spectrophotometer was used to record the 2+Fluorescence emission spectra before and after the reaction, trace Hg 2+ The red fluorescence intensity decreases rapidly, while the green fluorescence gradually increases. 2+ The ratio change of fluorescence intensity of two different emission wavelengths can also quickly and accurately detect Hg 2+ Therefore, the present invention has developed a simple, rapid, accurate, efficient, low-cost and environmentally friendly method to prepare fluorescent dual-emission ssDNA-AgNCs. The probe is used as a unique signal and target recognition probe for trace Hg in water. 2+ Rapid visualization and ratiometric fluorescence detection.

[0021] Compared with the existing technology, the beneficial effects of the present invention are:

[0022] (1) The synthesis conditions of ratiometric fluorescent probes are mild (room temperature and refrigerated), and the aqueous system does not require organic solvents, resulting in low energy consumption, environmental protection, greenness, and low cost;

[0023] (2) Only one ssDNA strand is needed as a template and stabilizer to prepare the dual-emission fluorescent probe. The probe preparation process is simple, the conditions are mild, and the controllability is good. The fluorescent performance of the synthesized probe is reproducible and stable.

[0024] (3) The probe detects mercury ions at room temperature, with a short response time of only 5 minutes;

[0025] (4) Fluorescence signal detection can be performed by directly observing the fluorescence color and intensity of the solution with the naked eye. 2+ Rapid on-site visual detection;

[0026] (5) Fluorescence signal detection can be performed using mobile phone software to quickly quantify Hg based on the solution fluorescence RGB value. 2+ content;

[0027] (6) A fluorescence spectrophotometer can also be used to record the green and red fluorescence emission spectra of the solution at excitation wavelengths of 440 nm and 560 nm, respectively, and accurately detect Hg in the sample by ratiometric fluorescence method. 2+ content.

[0028] (7) The actual water sample does not require complex pre-treatment, only a syringe with a filter membrane is needed for processing; Hg 2+ The test results show that the test results of the present invention are consistent with those of the national standard method, and the test results are accurate and reliable.

[0029] (8) The prepared fluorescent probe can visually detect Hg at concentrations as low as 10.0 nmol / L 2+ . BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1Schematic diagram of the preparation process of dual-emission ssDNA-silver nanocluster probes;

[0031] Figure 2 Visual detection of Hg using dual-emission fluorescent probes 2+ Schematic diagram of;

[0032] Figure 3 is a two-dimensional fluorescence spectrum contour map of the dual-emission fluorescent probe of the present invention;

[0033] Figure 4 This is a high-resolution transmission electron microscopy image of the dual-emission ssDNA-AgNCs of the present invention;

[0034] Figure 5 The ssDNA-AgNCs probe (a) and ssDNA-AgNCs probe+Hg 2+ (b) Fluorescence emission curves (excitation wavelengths are 440 nm and 560 nm, respectively); the insets are ssDNA-AgNCs probe solution (a) and ssDNA-AgNCs probe + Hg 2+ Fluorescence photograph of solution (b) under UV light;

[0035] Figure 6 The fluorescent probe of the present invention responds to different concentrations of Hg 2+ Fluorescence dual emission curves;

[0036] Figure 7 It is a dual-emission fluorescent probe for ratiometric fluorescence detection of Hg 2+ The linear range of

[0037] Figure 8 The dual-emission fluorescent probe of the present invention responds to different concentrations of Hg 2+ Standard fluorescent color card;

[0038] Figure 9 This is the fluorescence response of the dual-emission fluorescent probe of the present invention to 15 different metal ions and its fluorescence response photos (interpolated photos). DETAILED DESCRIPTION

[0039] All features disclosed in this specification, or all steps in the disclosed methods or processes, except mutually exclusive features and / or steps, can be combined in any manner.

[0040] Any feature disclosed in this specification (including claims and abstract), unless otherwise stated, may be replaced by other equivalent or similar features. That is, unless otherwise stated, each feature is only an example of a series of equivalent or similar features.

[0041] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0042] Example 1

[0043] 1. By designing different ssDNA sequences as templates and stabilizers to synthesize silver clusters, the fluorescence emission of ssDNA templates-AgNCs with different sequences was investigated. As shown in Table 1, the fluorescence emission of silver clusters formed by ssDNA templates with different sequences is quite different: C 12 The SP-A5 and SP-G5 templates can only produce silver clusters with single fluorescence emission; the SP-A5 and SP-G5 templates can only produce silver clusters with weaker double fluorescence emission, and the double emission fluorescence peak difference of the SP-G5 template silver cluster is small, only 40nm; SP-T5, SP-T 10 、SP-T 15 and SP-T 20 The fluorescent dual-emission silver clusters prepared by the template have a peak difference of about 100nm between green fluorescence and red fluorescence, and the red fluorescence is enhanced to varying degrees. 10 The dual-emission silver clusters prepared by the template simultaneously emit weak green fluorescence and extremely strong red fluorescence.

[0044] Table 1 Fluorescence emission of AgNCs prepared with ssDNA templates of different sequences

[0045]

[0046] 2. According to the fluorescence emission of ssDNA template-silver clusters with different sequences, SP-T 10 For the optimal template, SP-T 10 The ssDNA sequence of the template is: 5'-AACAAAGCCCCCCCCCCCCCTTTTTTTTTT-3'.

[0047] The DNA involved in the present invention can be synthesized by conventional techniques in the art.

[0048] Example 2-1:

[0049] Preparation of fluorescent dual-emission ssDNA-AgNCs probes:

[0050] To 980.0 μL of Tris-HAc buffer (20 mmol / L) at pH 6.6, add 20.0 μL of ssDNA aqueous solution (100.0 μmol / L) and 48.0 μL of AgNO₃ solution (1.0 mmol / L). Mix on a shaker for 2 minutes and incubate at room temperature (25 ± 1°C) in the dark for 1 hour. Then, add 24.0 μL of freshly prepared NaBH₄ solution (1.0 mmol / L) and incubate overnight at room temperature in the dark to obtain the fluorescent dual-emission ssDNA-AgNCs probe. The probe solution was refrigerated at 4°C until use.

[0051] Based on Example 2-1, the effects of different pH values ​​and concentrations of Tris-HAc buffer on the dual-emission fluorescence intensity of the probe were investigated. The specific results are shown in Table 2.

[0052] Table 2 Effects of different pH and concentrations of Tris-HAc buffer on the dual-emission fluorescence intensity of the probe

[0053]

[0054] As shown in Table 2, the red fluorescence of the fluorescent dual-emission ssDNA-AgNCs probe prepared when the buffer solution was at pH 6.6 Tris-HAc (20.0 mmol / L) was the strongest.

[0055] Example 2-2:

[0056] Preparation of fluorescent dual-emission ssDNA-AgNCs probes:

[0057] Add 20.0 μL of ssDNA template solution (100.0 μmol / L) and 48.0 μL of AgNO₃ solution (1.0 mmol / L) to a 20.0 mmol / L Tris buffer solution (pH 6.6). Mix thoroughly on a shaker for 2 minutes and incubate in the dark at room temperature for 1 hour. Then, add 24.0 μL of freshly prepared NaBH₄ solution (1.0 mmol / L), mix thoroughly, and incubate at room temperature overnight in the dark to obtain a dual-emission fluorescent ssDNA-AgNCs probe. Refrigerate the probe solution at 4°C until use.

[0058] Based on Example 2-2, the influence of ssDNA, AgNO3 and NaBH4 at different molar ratios (final molar ratios) on the double-emission fluorescence intensity of the probe was investigated. The specific results are shown in Table 3.

[0059] Table 3 Effects of different molar ratios of ssDNA, AgNO3 and NaBH4 on the dual-emission fluorescence intensity of the probe

[0060]

[0061] As shown in Table 3, the red fluorescence of the fluorescent dual-emission ssDNA-AgNCs probe prepared when the molar ratio of ssDNA, AgNO3 and NaBH4 is 1:24:12 is the strongest.

[0062] Example 2-3:

[0063] Preparation of fluorescent dual-emission ssDNA-AgNCs probes:

[0064] To 980.0 μL of Tris-HAc buffer (20.0 mmol / L) at pH 6.6, add 20.0 μL of ssDNA aqueous solution (100.0 μmol / L) and 48.0 μL of AgNO₃ solution (1.0 mmol / L). Mix on a shaker for 2 minutes and allow to react at room temperature in the dark for 60 minutes. Then, add 24.0 μL of freshly prepared NaBH₄ solution (1.0 mmol / L). Incubate (stand) at room temperature in the dark overnight to obtain the fluorescent dual-emission ssDNA-AgNCs probe. The probe solution was refrigerated at 4°C until use.

[0065] Based on Example 2-3, the effect of mixing ssDNA and AgNO3 at room temperature in the dark for different reaction times on the probe dual-emission fluorescence intensity was investigated. The specific results are shown in Table 4.

[0066] Table 4 Effects of ssDNA+AgNO3 mixture at room temperature in the dark for different reaction times on the probe dual emission fluorescence intensity

[0067]

[0068] As shown in Table 4, the red fluorescence of the fluorescent dual-emission ssDNA-AgNCs probe prepared when ssDNA and AgNO3 were mixed and reacted at room temperature in the dark for 60 minutes was the strongest.

[0069] Example 2-4:

[0070] Preparation of fluorescent dual-emission ssDNA-AgNCs probes:

[0071] To 980.0 μL of Tris-HAc buffer (20.0 mmol / L) at pH 6.6, add 20.0 μL of ssDNA aqueous solution (100.0 μmol / L) and 48.0 μL of AgNO₃ solution (1.0 mmol / L). Mix on a shaker for 2 minutes and allow to react at room temperature in the dark for 60 minutes. Then, add 24.0 μL of freshly prepared NaBH₄ solution (1.0 mmol / L). Incubate (stand) at room temperature in the dark overnight to obtain the fluorescent dual-emission ssDNA-AgNCs probe. The probe solution was refrigerated at 4°C until use.

[0072] The fluorescence emission spectra of the dual-emission fluorescent probe prepared in Example 2-4 are as follows: Figure 3 As shown, the optimal fluorescence excitation wavelengths of the probes are 440 nm and 560 nm, and the maximum fluorescence emission wavelengths are 530 nm and 630 nm, respectively.

[0073] The morphology of the dual-emission fluorescent probe prepared in Example 2-4 is as follows: Figure 4 As shown, the average particle size of the spherical, monodispersed, and uniform silver nanoclusters is about 2.5 nm.

[0074] Example 3-1: Detection of Hg using fluorescent probe dilution 2+ The process:

[0075] The ssDNA-AgNCs fluorescent probe solution (80 μL, prepared in Example 2-4) was diluted with 80 μL Tris-HAc buffer solution (pH 6.6), and then 1.0 μL Hg 2+ The standard solution (20.0 μmol / L) was mixed and allowed to react at room temperature for 5 min. The fluorescence emission spectra of a series of solutions at excitation wavelengths of 440 nm and 560 nm were recorded using a fluorescence spectrophotometer.

[0076] Based on the premise of Example 3-1, the fluorescence probe solution and Hg 2+ The effects of different reaction times and different volume ratios of the fluorescent probe solution and Tris-HAc buffer solution on the probe dual-emission fluorescence intensity are shown in Table 5.

[0077] Table 5 Fluorescent probe solution and Hg 2+ Effects of different reaction times and different volume ratios of fluorescent probe solution and Tris-HAc buffer solution on the probe dual emission fluorescence intensity

[0078]

[0079] As shown in Table 5, the volume ratio of ssDNA-AgNCs probe solution to Tris-HAc buffer solution = 1:1, and the obtained probe solution is 2+ After the reaction was allowed to stand at room temperature for 5 minutes, the red fluorescence of the probe was quenched and the green fluorescence was enhanced to the greatest extent, and Hg was detected. 2+ Highest sensitivity.

[0080] Example 3-1 was mixed with a blank solution (0 μL Hg 2+ Compared with the standard solution), the dual-emission ssDNA-AgNCs fluorescent probe solution was added with 1.0 μL Hg 2+ After adding the standard solution (20.0 μmol / L), the red fluorescence was quenched and the green fluorescence was significantly enhanced. Figure 5As shown, the fluorescence color of the probe solution changes from bright red to bright green ( Figure 5 Interpolated fluorescence photograph). Accordingly, the dual-emission fluorescent probe of the present invention can be used for Hg 2+ Rapid ratiometric fluorescence and visualization detection.

[0081] Example 3-2: Probe ratio fluorescence detection of Hg 2+ The process of making the standard curve:

[0082] The ssDNA-AgNCs fluorescent probe solution (100.0 μL, prepared in Example 2-4) was diluted with 100.0 μL Tris-HAc buffer solution (pH 6.6), and then 1.0 μL of Hg 2+ The standard solution (10.0 nmol / L to 1.0 mmol / L) was mixed and allowed to react at room temperature for 5 min. The fluorescence emission spectra of a series of standard solutions at excitation wavelengths of 440 nm and 560 nm were recorded using a fluorescence spectrophotometer. Figure 6 As shown in the figure, 530nm is the green fluorescence emission peak, and 630nm is the red fluorescence emission peak. Figure 6 It can be seen that with the increase of Hg 2+ With the increase of Hg content, the fluorescence intensity at 530nm gradually increased, while the red fluorescence intensity at 630nm decreased rapidly. 2+ The concentration of the standard curve was drawn, and the linear range of the dual emission probe ratio fluorescence detection of mercury ions was 0.5-200.0nmol / L. Figure 7 shown.

[0083] Example 3-3: Visual detection of Hg by probe 2+ Production of standard fluorescent color card:

[0084] The ssDNA-AgNCs fluorescent probe solution (100.0 μL, prepared in Example 2-4) was diluted with 100.0 μL Tris-HAc (20.0 mmol / L) buffer at pH 6.6, and then 1.0 μL of Hg 2+ The standard solution (10.0 nmol / L to 1.0 mmol / L) was mixed and allowed to react at room temperature for 5 min. The color of the mixed solution was observed under a portable UV lamp, and a series of fluorescence photos of the standard solution were taken with a mobile phone to prepare Hg 2+ Standard fluorescent color cards, such as Figure 8 As shown. 2+With the increase of content, the fluorescence color of the detection system solution gradually changes from bright red to bright green, and the fluorescence color changes significantly. The concentration range of mercury ion detection by dual-emission probe fluorescence visualization is 10.0-300.0nmol / L; trace Hg can be detected according to the fluorescence color of the detection system solution. 2+ Visual detection, concentration as low as 10.0nmol / L Hg 2+ The residue can be quickly identified with the naked eye.

[0085] Example 3-4: Standard Curve Method Ratio Fluorescence Detection of Hg in Actual Water Samples 2+ Steps:

[0086] The ssDNA-AgNCs fluorescent probe solution (100.0 μL, prepared in Example 2-4) was diluted with 100.0 μL Tris-HAc buffer solution (pH 6.6), and then 0.1-10.0 μL of the actual water sample solution was added. After mixing, the reaction was allowed to stand at room temperature for 5 minutes. The fluorescence emission spectra of the sample solution at excitation wavelengths of 440 nm and 560 nm were recorded using a fluorescence spectrophotometer. The Hg in the water sample could be accurately quantified using the standard curve method. 2+ content.

[0087] Example 3-5: Standard addition method ratio fluorescence detection of Hg in actual water samples 2+ Steps:

[0088] The ssDNA-AgNCs fluorescent probe solution (100.0 μL, prepared in Example 2-4) was diluted with 100.0 μL Tris-HAc buffer solution (pH 6.6), and then 1.0 to 50.0 μL of the actual water sample solution was added, and then 1.0 μL of Hg 2+ The standard solution (10.0 nmol / L to 1.0 mmol / L) was mixed and allowed to react at room temperature for 5 minutes. The fluorescence emission spectra of a series of sample solutions at excitation wavelengths of 440 nm and 560 nm were recorded using a fluorescence spectrophotometer. The Hg content in the sample could be accurately quantified by the standard addition method. 2+ concentration.

[0089] Example 3-6: Detection of Hg in actual water samples by naked eye visualization 2+ Steps:

[0090] The ssDNA-AgNCs fluorescent probe solution (100.0 μL, prepared in Example 2-4) was diluted with 100.0 μL Tris-HAc (20.0 mmol / L) at pH 6.6, and then 1.0 to 50.0 μL of the actual water sample solution was added. After mixing, the mixture was allowed to react at room temperature for 5 minutes and the color of the mixed solution was observed under a portable UV lamp. The fluorescence color of the sample solution was compared with that of Hg2+ Standard fluorescent color card comparison, according to the solution fluorescence color and fluorescence intensity, Hg can be quickly semi-quantified by naked eyes 2+ content.

[0091] Example 3-7: Detection of Hg in actual water samples using mobile phone software visualization method 2+ Steps:

[0092] The ssDNA-AgNCs fluorescent probe solution (100.0 μL, prepared in Example 2-4) was diluted with 100.0 μL Tris-HAc (20.0 mmol / L) buffer at pH 6.6, and then 1.0-50.0 μL of the actual water sample solution was added. After mixing, the solution was allowed to react at room temperature for 5 min. The solution was placed under a portable UV lamp, and a fluorescent photo of the sample solution was taken with a mobile phone. The fluorescence was then compared with that of Hg 2+ Compare with the standard fluorescence color card photo and use the mobile phone software to identify the RGB value of the fluorescence photo, which can quickly quantify the Hg in the sample solution 2+ content.

[0093] Example 4: Selective detection of Hg by dual-emission fluorescent probe 2+ The process:

[0094] The ssDNA-AgNCs fluorescent probe solution (100.0 μL, prepared in Example 2-4) was diluted with 100.0 μL Tris-HAc (20.0 mmol / L) buffer at pH 6.6, and then 1.0 μL of a standard solution containing 15 metal ions (40.0 μmol / L) including mercury, europium, magnesium, calcium, cobalt, barium, lead, bismuth, cadmium, chromium, zinc, nickel, iron, potassium, and sodium was added. After mixing, the mixture was allowed to react at room temperature for 5 minutes. The mixture was placed under a portable UV lamp and a fluorescent photograph of the mixed solution was taken with a mobile phone, as shown in FIG. Figure 9 The fluorescence emission intensity of a series of mixed solutions at wavelengths of 530nm and 630nm was recorded using a fluorescence spectrophotometer. By comparing the fluorescence color and fluorescence emission intensity of each mixed solution, it was found that only mercury ions with a concentration of ≥10.0mmol / L caused a significant change in the fluorescence color of the probe solution; only mercury ions caused the red fluorescence intensity of the probe to decrease rapidly, while the green fluorescence intensity gradually increased. The experimental results show that the dual-emission fluorescent probe is effective for Hg 2+ It has a high fluorescence selectivity response.

[0095] Example 5: Detection of Hg in real water samples using a dual-emission fluorescent probe 2+

[0096] The dual-emission fluorescent probes prepared in Examples 2-4 were used to detect Hg in local tap water, river water, and sewage treatment plant effluent according to the preferred method and steps of Example 3. 2+The content was tested and the test results are shown in Table 6.

[0097] The national standard method (GB / T 37906-2019) for testing tap water, river water, and sewage treatment plant effluent uses the following process: collect and preserve water samples in accordance with GB / T 5750.2; process water samples and determine the mercury ion content in them in accordance with GB / T 37906-2019.

[0098] Table 6 Results of using dual-emission fluorescence probe and national standard method to detect mercury ion content in real water samples (the experimental results are the average of 3 parallel measurements)

[0099] sample Methods in Examples 3-6 Method in Example 3-4 Method in Example 5 Tap water (mercury ion content) Not detected Not detected Not detected River water (mercury ion content) Not detected 0.59±0.02nmol / L 0.63±0.02nmol / L Wastewater treatment plant effluent (mercury ion content) About 100 nmol / L 105.32±2.96nmol / L 109.64±3.38nmol / L <![CDATA[Water sample Hg 2+ Detection time]]> About 5 minutes About 10 minutes About 15 minutes

[0100] As shown in Table 6, the dual-emission fluorescence probe prepared by the present invention can be used for the visualization and ratiometric fluorescence detection of Hg in real water. 2+ The content is close to the test results of the national standard method (GB / T 37906-2019). The Bland-Altman method evaluation results (P>0.05) show that the quantitative detection results of the two methods (dual-emission fluorescent probe ratio fluorescence method and national standard method) have good consistency, indicating that the dual-emission fluorescent probe prepared by the present invention can be used to detect trace Hg in real water samples. 2+ Content detection.

[0101] The above-described embodiments merely represent specific implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of protection of the present application. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the technical concept of the present application, and all such variations and improvements fall within the scope of protection of the present application.

[0102] This background section is provided to generally present the context of the invention, and the work of the presently named inventors, the work to the extent described in this background section, and aspects of the description in this section that did not constitute prior art at the time of filing are neither explicitly nor implicitly admitted to be prior art to the present invention.

Claims

1. A rapid visual detection method for Hg 2+ Fluorescent dual-emission ssDNA-AgNCs probe, characterized by: The probe contains a template ssDNA that can simultaneously emit strong red fluorescence and weak green fluorescence, and the sequence of the ssDNA is 5'-AACAAAGCCCCCCCCCCCCCTTTTTTTTTT-3'.

2. The fluorescent dual-emission ssDNA-AgNCs probe as claimed in claim 1 is used for rapid visual detection of Hg 2+ Application in.

3. Rapid visual detection of Hg as claimed in claim 1 2+ The preparation method of the fluorescent dual-emission ssDNA-AgNCs probe is characterized in that The following steps are involved: ssDNA aqueous solution and AgNO3 solution were added to Tris-HAc buffer, mixed and reacted at room temperature in the dark for a period of time; then, freshly prepared NaBH4 solution was added and incubated at room temperature in the dark overnight to obtain ssDNA template-AgNCs probe, i.e., fluorescent dual-emission ssDNA-AgNCs probe, which was stored in a refrigerator.

4. The method for preparing the fluorescent dual-emission ssDNA-AgNCs probe according to claim 3, characterized in that: The final molar ratio of ssDNA, AgNO3 and NaBH4 is 1~3:12~48:6~36.

5. The method for preparing the fluorescent dual-emission ssDNA-AgNCs probe according to claim 3, wherein: The Tris-HAc buffer, ssDNA aqueous solution and AgNO3 solution are mixed evenly and reacted in the dark at room temperature for 20 to 100 minutes.

6. The method for preparing the fluorescent dual-emission ssDNA-AgNCs probe according to claim 3, wherein: The concentration of Tris-HAc buffer is 5-30.0 mmol / L, and the pH is 6.0-8.4; the molar ratio of ssDNA solution to Tris-HAc buffer is 1-4:100-1000.

7. The fluorescent dual-emission ssDNA-AgNCs probe as claimed in claim 2 is used for visual detection of Hg by naked eye or mobile phone software 2+ The application is characterized by The specific application steps are: The ssDNA-AgNCs probe solution was mixed with Tris-HAc buffer at a volume ratio of 1-4:1-4, and then different concentrations of Hg 2+ Standard solution or Hg 2+ After mixing, let it stand at room temperature for 1 to 30 minutes, and use a mobile phone to record the different Hg 2+ Fluorescence photos of the system under Hg content; then 2+ The fluorescence color card of the standard solution can be used to quickly semi-quantitatively / quantitatively analyze the Hg content in the sample solution by naked eyes or mobile phone software ImageJ. 2+ concentration.

8. The fluorescent dual-emission ssDNA-AgNCs probe as claimed in claim 2 is used to detect Hg by ratiometric fluorescence method 2+ The application is characterized by The specific application steps are as follows: ssDNA-AgNCs probe solution and Tris-HAc buffer were mixed at a volume ratio of 1-4:1-4, and then different concentrations of Hg 2+ Standard solution or Hg 2+ The sample solution was mixed and allowed to react at room temperature for 1 to 30 minutes. The dual fluorescence emission spectrum of the system was measured. The red and green dual emission peak fluorescence intensity ratio and Hg 2+ The concentration of Hg in the sample solution was accurately quantified by establishing a standard curve. 2+ concentration.

9. The dual-emission ssDNA-AgNCs probe as claimed in claim 8 is used for ratiometric detection of Hg 2+ The application is characterized by: The optimal fluorescence excitation wavelengths of the probes are 440nm and 560nm, respectively; the maximum fluorescence emission wavelengths of the probes are 530nm and 630nm, respectively.

10. Rapid visual detection of Hg by the fluorescent dual-emission probe according to claim 7 or claim 8 2+ The detection method is characterized in that If the sample solution Hg 2+ If the concentration is too high, dilute the sample appropriately with double-distilled water and retest.

Images