Hairpin type DNA-copper cluster probe for rapid ratio fluorescence detection of Hg < 2 + > and preparation method of hairpin type DNA-copper cluster probe
By preparing hairpin-type DNA-copper cluster probes, a rapid, simple, and accurate Hg2+ detection method was achieved using the dual fluorescence emission ratio method. This method solves the problems of long time consumption and high cost in existing technologies and is suitable for rapid detection of Hg2+ in water.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-08
- Publication Date
- 2026-04-10
AI Technical Summary
Existing methods for detecting trace amounts of Hg2+ are time-consuming, costly, and difficult to implement rapid on-site detection. Traditional DNA-metal nanocluster probes are complex and time-consuming to prepare.
A hairpin-type DNA-copper cluster probe was used to prepare a dual-fluorescent emission probe that could respond rapidly at room temperature by adding copper sulfate and sodium ascorbate to an aqueous system with a specific sequence of hpDNA as a template. The fluorescence emission spectrum was recorded using a fluorescence spectrophotometer for ratiometric fluorescence detection of Hg2+.
It achieves rapid, simple, accurate, and low-cost Hg2+ detection with short response time, wide detection range, and accurate and reliable results. It is suitable for actual water samples without complex pretreatment.
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Figure CN121825530A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of fluorescent material preparation technology, and more specifically, this invention relates to a rapid ratio fluorescence detection method for Hg. 2+ Hairpin-type DNA-copper cluster probe and its preparation method. Background Technology
[0002] Even low concentrations of mercury pollutants are highly toxic, seriously endangering the environment and human health. Divalent mercury ions (Hg) 2+ It is highly water-soluble and stable, and is the most common form of mercury contaminant. (Hg) 2+ Once in soil and water, bacteria can convert mercury into organic mercury, which can then be absorbed by humans through the food chain, causing chronic poisoning and damaging the brain, kidneys, stomach, and intestines, even leading to death. Therefore, monitoring mercury levels in soil and water is crucial. 2+ The content of [something] is very important.
[0003] Currently used for detecting trace / ultra-scale Hg 2+ Classical methods include atomic absorption spectroscopy, atomic emission spectroscopy, X-ray spectroscopy, and inductively coupled plasma mass spectrometry. These methods require complex sample pretreatment, are very time-consuming, have high testing costs, and are difficult to implement for rapid on-site detection.
[0004] Currently reported DNA-metal nanocluster-based Hg detection 2+ Probe preparation time and detection of Hg 2+ The time required is relatively long (usually more than 30 minutes), making it difficult to achieve Hg. 2+ Rapid on-site detection is needed. Current technologies for synthesizing DNA-metal nanoclusters use sugars (such as sucrose, glucose, and fructose) as stabilizers to improve their stability. Ratio fluorescence analysis has been widely used in environmental safety monitoring and other fields in recent years due to its high sensitivity, accuracy, and selectivity. However, these reported ratio fluorescence methods typically require the preparation of two or more probes, and the probe preparation methods / steps are complex, time-consuming, and costly, resulting in long detection times and high costs for mercury ion detection. Summary of the Invention
[0005] One object of the present invention is to solve at least the above-mentioned problems and / or defects, and to provide at least the advantages described below.
[0006] To achieve these and other advantages according to the invention, the present invention provides a rapid ratio fluorescence detection method for Hg. 2+The hairpin DNA-copper cluster probe contains a hairpin DNA template (hpDNA) that can simultaneously emit strong red fluorescence and weak blue fluorescence, and the sequence of the hairpin DNA template (hpDNA) is 5'-TTTTTTTTTTTTTTTTTTTTTTTTTCGCGCGCGATATATCGCGCGCG-3'.
[0007] A rapid ratio fluorescence detection method for Hg 2+ The preparation method of the hairpin-type DNA-copper cluster probe includes the following steps: Step 1: Prepare a single-stranded DNA aqueous solution using 3-morpholinopropanesulfonic acid buffer (MOPS). Heat and keep the single-stranded DNA aqueous solution in a constant temperature water bath, then cool it to room temperature and store it in a refrigerator for a certain period of time to form an hpDNA template solution. Step 2: Add copper sulfate solution to the obtained hpDNA template solution and incubate at a constant temperature for a certain period of time to obtain a mixed solution; then, add sodium ascorbate (SA) solution to the mixed solution and react at a constant temperature for a certain period of time to allow the Cu molecules coordinated on the hpDNA template to react. 2+ Reduced by sodium ascorbate, a rapid ratio fluorescence detection of Hg was obtained. 2+ Hairpin-type DNA-copper cluster probe; the prepared rapid ratio fluorescence detection of Hg 2+ The hairpin-type DNA-copper cluster probes are stored in dark brown centrifuge tubes and kept at 4°C for later use.
[0008] Preferably, in step one, the concentration of the single-stranded DNA aqueous solution is 1.0~10.0 µM; The temperature of the single-stranded DNA aqueous solution was heated in a constant temperature water bath to 90℃~100℃ for 10~15 minutes. The temperature for refrigeration and insulation in the refrigerator is 4℃, and the storage time is 2~6 hours.
[0009] Preferably, the volume ratio of 3-morpholine propanesulfonic acid buffer, copper sulfate solution, and sodium ascorbate solution is 5~10:5~8:1; In step two, rapid ratio fluorescence detection of Hg 2+ The final molar ratio of hpDNA, copper sulfate, and sodium ascorbate in the hairpin-type DNA-copper cluster probe is 1:25 to 200:25.
[0010] Preferably, in step two, the reaction time after mixing the hpDNA template solution and the copper sulfate solution is 0-50 min at a constant temperature of 20-30°C. Add to sodium ascorbate solution, and maintain a constant reaction temperature of 20°C. o C~30o The constant temperature reaction time is 0~50 min.
[0011] Preferably, in step one, the concentration of the 3-morpholinopropanesulfonic acid buffer solution is 5.0~15.0 mmol / L, and the pH is 6.0~8.5.
[0012] A rapid ratio fluorescence detection method for Hg 2+ The application of the hairpin-type DNA-copper cluster probe, wherein the hairpin-type DNA-copper cluster probe is used to detect Hg. 2+ .
[0013] Preferably, the specific application steps are as follows: take the prepared rapid ratio fluorescence detection Hg 2+ Hairpin-type DNA-copper cluster probe solution, followed by the addition of different concentrations of Hg 2+ Standard solutions or solutions containing Hg 2+ The sample solution was diluted with 3-morpholinopropanesulfonic acid buffer (MOPS); after mixing, the mixture was allowed to stand at room temperature for 0-30 min, and the fluorescence emission spectrum of the system was measured. The fluorescence emission peak intensity ratio was then compared with Hg. 2+ A standard curve was established based on the relationship between concentration and concentration, and the Hg in the sample solution was accurately quantified accordingly. 2+ The concentration.
[0014] Preferably, the optimal fluorescence excitation wavelength of the hairpin-type DNA-copper cluster probe is 341 nm; the fluorescence emission wavelengths of the hairpin-type DNA-copper cluster probe are 400 nm and 635 nm, respectively.
[0015] Preferably, if the sample solution Hg 2+ If the concentration is too high, dilute the sample solution appropriately with double-distilled water and retest.
[0016] The rapid ratio fluorescence detection of Hg described in this invention 2+ The hairpin DNA-copper cluster probes (hpDNA-CuNCs) are prepared using hairpin DNA sequences as templates in an aqueous solution under mild conditions. These hairpin DNA-copper cluster probes emit weak blue fluorescence and strong red fluorescence. This invention utilizes the hpDNA-CuNCs solution as the sole signal and target recognition probe, with different concentrations of Hg... 2+ After reacting with the probe solution at room temperature for 5 min, the reaction of the probe with Hg was recorded using a fluorescence spectrophotometer. 2+ Fluorescence emission spectra before and after the reaction. Trace Hg 2+ This causes a rapid decrease in red fluorescence intensity, while blue fluorescence remains unchanged (blue fluorescence serves as an internal reference signal). Therefore, according to Hg... 2+The ratio of fluorescence intensity changes at two different emission wavelengths can enable rapid and accurate quantitative detection of Hg. 2+ Concentration. Therefore, this invention develops a simple, rapid, accurate, efficient, low-consumption, and environmentally friendly method to prepare fluorescent dual-emission hpDNA-CuNCs. This probe serves as both a unique signal and a target identification probe for trace Hg in water. 2+ Rapid ratio fluorescence detection.
[0017] The present invention has at least the following beneficial effects: (1) The rapid ratio fluorescence detection of Hg of the present invention 2+ The hairpin-type DNA-copper cluster probe is synthesized under mild (room temperature) conditions, in an aqueous solution system, without the need for organic solvents, with low energy consumption, and is environmentally friendly and low-cost. (2) Only one hairpin DNA is needed as a template and stabilizer to prepare hairpin DNA-copper cluster probe. The probe preparation process is simple, the conditions are mild, the controllability is good, and the fluorescence performance of the synthesized probe is reproducible and stable. (3) The hairpin-type DNA-copper cluster probe detects mercury ions at room temperature with a short response time of only 5 minutes; (4) Fluorescence signal detection can be performed by using a fluorescence spectrophotometer to simultaneously record the dual fluorescence emission spectrum of the solution at an excitation wavelength of 341 nm, and to record the blue and red fluorescence emission intensities at emission wavelengths of 400 nm and 635 nm. The Hg in the sample can be accurately detected by ratiometric fluorescence method. 2+ The content of.
[0018] (5) Actual water samples do not require complex pretreatment; only a syringe with a filter membrane is needed. (Hg in water samples) 2+ The test results show that the results of this invention are consistent with the national standard method, and the test results are accurate and reliable.
[0019] (6) The hairpin-type DNA-copper cluster probe prepared in this invention can detect concentrations as low as 0.3 nmol / LHg using ratiometric fluorescence detection. 2 + .
[0020] (7) No fructose was used in the process of synthesizing the hairpin-type DNA-copper cluster probe in this invention. By optimizing and adjusting other experimental parameters, the stability and reproducibility of the hairpin-type DNA-copper cluster probe synthesized with less raw material are still good.
[0021] (8) The hairpin-type DNA-copper cluster probe prepared in this invention is effective against Hg. 2+ The detection time is shorter, which can be reduced to less than 10 minutes, far superior to the detection time of 20 minutes or even more than 30 minutes in the prior art. At the same time, the hairpin-type DNA-copper cluster probe of the present invention has a wider detection linear range and a lower detection limit.
[0022] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description
[0023] Figure 1 This is a schematic diagram of the preparation process of the hpDNA-copper nanocluster fluorescent probe. Figure 2 For the ratio detection of Hg using fluorescent dual-emission probes 2+ A schematic diagram; Figure 3 This is a two-dimensional fluorescence spectrum contour plot of the fluorescent dual-emission probe described in this invention; Figure 4 This is a high-resolution transmission electron microscope image of hpDNA-CuNCs described in this invention; Figure 5 The hpDNA-CuNCs probe (a) and hpDNA-CuNCs probe + Hg described in this invention 2+ (b) Fluorescence emission curve (excitation wavelength 341 nm); inset shows hpDNA-CuNCs probe solution (a) and hpDNA-CuNCs probe + Hg. 2+ Fluorescence photograph of solution (b) under ultraviolet light; Figure 6 The fluorescent probe described in this invention responds to different concentrations of Hg 2+ Fluorescence double emission curve; Figure 7 The hpDNA-CuNCs probe ratio fluorescence detection of Hg 2+ The linear range; Figure 8 The fluorescence response of the hpDNA-CuNCs probe described in this invention to 13 different metal ions is shown. Detailed Implementation
[0024] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.
[0025] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof. Example 1 1. Copper clusters were synthesized by designing different single-stranded DNA sequences as templates and stabilizers. The fluorescence emission of CuNCs with different single-stranded DNA templates was investigated. As shown in Table 1, the fluorescence emission of hpDNA template-copper clusters formed by different single-stranded DNA sequences varied greatly: HP-15T and HP-20T templates could only prepare copper clusters with weak blue and red fluorescence; HP-25T and DHP-30T templates could prepare copper clusters with both weak blue and strong red fluorescence emission, with a peak difference of about 235 nm between the blue and red fluorescence.
[0026] Table 1. Fluorescence emission of CuNCs prepared using hpDNA templates with different sequences. 2. Based on the fluorescence emission of copper clusters from different hpDNA template sequences, the copper clusters prepared using the HP-25T template exhibited the strongest red fluorescence. Therefore, HP-25T was selected as the optimal template. The ssDNA sequence of the HP-25T template is as follows: 5'-TTTTTTTTTTTTTTTTTTTTTTTTTCGCGCCGGATATATCGCGCCG-3'.
[0027] All DNA involved in this invention can be synthesized using conventional techniques in the field.
[0028] Example 2 like Figure 1 As shown, a rapid ratio fluorescence detection method for Hg 2+ The preparation method of the hairpin-type DNA-copper cluster probe includes the following steps: Prepare 200.0 μL of single-stranded DNA (5.0 µM) solution using 10.0 mmol / L 3-morpholinopropanesulfonic acid (MOPS) buffer (pH 7.5), and heat to 95°C in a constant temperature water bath. o C and hold for 12 minutes, then cool to room temperature and store in a 4°C refrigerator for 4 hours to form an hpDNA template solution.
[0029] Subsequently, 150.0 µL of freshly prepared 1.0 mM copper sulfate (CuSO4) aqueous solution was added to the obtained hpDNA template solution, and the mixture was incubated at 25 °C for 5 min. Then, 25.0 µL of freshly prepared 1.0 mM sodium ascorbate (SA) solution was added to the above solution, and the reaction was carried out at 25 °C for 5 min to prepare the hpDNA-CuNCs fluorescent probe. The prepared hpDNA-CuNCs fluorescent probe solution was stored in dark brown centrifuge tubes and refrigerated at 4 °C for later use.
[0030] Based on Example 2, the effects of different pH values and concentrations of MOPS buffer on the intensity of dual emission fluorescence of the probe were investigated. The specific results are shown in Table 2.
[0031] Table 2. Effect of different pH values of MOPS buffer on the intensity of dual emission fluorescence of the probe. As shown in Table 2, the red fluorescence of the dual-emission hpDNA-CuNCs probe was strongest when the MOPS buffer was prepared at pH 7.5 (10.0 mmol / L).
[0032] Example 3 Rapid ratio fluorescence detection of Hg 2+ Preparation of hairpin-type DNA-copper cluster probes: 200.0 μL of hpDNA template solution (5.0 μmol / L) and 150.0 μL of CuSO4 solution (1.0 mmol / L) were mixed for 1 minute using a mixer and then incubated at 25 °C for 5 min. Then, 25.0 μL of freshly prepared SA solution (1.0 mmol / L) was added, mixed, and reacted at 25 °C for 5 min to obtain the fluorescently dual-emitting hpDNA-CuNCs probe. This probe solution was stored at 4 °C for later use.
[0033] Based on Example 3, the effects of hpDNA, CuSO4 and SA on the dual emission fluorescence intensity of the probe under different molar ratios (final molar ratio) were investigated. The specific results are shown in Table 3.
[0034] Table 3. Effects of different molar ratios of hpDNA, CuSO4, and SA on the dual emission fluorescence intensity of the probe. As shown in Table 3, the red fluorescence of the fluorescent dual-emission hpDNA-CuNCs probe was strongest when the molar ratio of hpDNA, CuSO4 and SA was 1:150:25.
[0035] Example 4 Rapid ratio fluorescence detection of Hg 2+ Preparation of hairpin-type DNA-copper cluster probes: 200.0 μL of hpDNA template solution (5.0 μmol / L) and 150.0 μL of CuSO4 solution (1.0 mmol / L) were added to MOPS buffer solution at pH 7.5 (10.0 mmol / L). The mixture was mixed for 1 minute on a mixer and then allowed to simmer for 25 minutes. oIncubate at C for 5 min. Then add 25.0 μL of freshly prepared SA solution (1.0 mmol / L), mix well, and react at 25 °C for 5 min to obtain the fluorescently dual-emitting hpDNA-CuNCs probe. Store the probe solution at 4 °C for later use.
[0036] Based on this example, the effect of different reaction times at 25℃ after mixing hpDNA+CuSO4 and SA on the dual emission fluorescence intensity of the probe was investigated. The specific results are shown in Table 4.
[0037] Table 4. Effect of different reaction times at 25℃ after mixing hpDNA, CuSO4, and SA on the intensity of dual emission fluorescence of the probe. As shown in Table 4, the hpDNA-CuNCs probe prepared by mixing hpDNA, CuSO4 and SA and reacting at 25℃ for 5 minutes exhibits the strongest red fluorescence.
[0038] Example 5 Rapid ratio fluorescence detection of Hg 2+ Preparation of hairpin-type DNA-copper cluster probes: 200.0 μL of hpDNA template solution (5.0 μmol / L) and 150.0 μL of CuSO4 solution (1.0 mmol / L) were added to MOPS buffer solution (pH 7.5, 10.0 mmol / L). The mixture was stirred for 1 minute and then incubated at 25°C for 5 minutes. Then, 25.0 μL of freshly prepared SA solution (1.0 mmol / L) was added, stirred, and reacted at 25°C for 5 minutes to obtain the fluorescently dual-emitting hpDNA-CuNCs probe. This probe solution was stored at 4°C for later use. The preparation process of the hpDNA-CuNCs probe is as follows: Figure 1 As shown.
[0039] The fluorescence emission spectrum of the fluorescent dual-emission probe prepared in this embodiment is as follows: Figure 3 As shown, the optimal fluorescence emission wavelengths of the probe are 400 nm and 635 nm, respectively.
[0040] The morphology of the dual-emission fluorescent probe prepared in this embodiment is as follows: Figure 4 As shown, the average particle size of the spherical, monodisperse, and uniformly sized copper nanoclusters is approximately 2.3 nm.
[0041] Example 6 Rapid ratio fluorescence detection of Hg 2+ Hairpin-type DNA-copper cluster probe dilution solution for Hg detection 2+ The process: 1.0 μL of Hg was added to the hpDNA-CuNCs fluorescent probe solution (40.0 μL, prepared in Example 5). 2+ The standard solution (2.0 μmol / L) was diluted to 200.0 μL with MOPS buffer (pH 7.5), mixed well, and allowed to stand at 25°C for 5 min. The fluorescence emission spectra of the solution at an excitation wavelength of 341 nm were recorded using a fluorescence spectrophotometer. The detection principle is as follows. Figure 2 As shown.
[0042] Based on the premise of this embodiment, the interaction between the fluorescent probe solution and Hg was investigated. 2+ The effect of different reaction times on the intensity of dual emission fluorescence of the probe is shown in Table 5.
[0043] Table 5 Fluorescent probe solution and Hg 2+ Effect of reaction time on probe dual emission fluorescence intensity Table 5 shows that the hpDNA-CuNCs probe solution and Hg 2+ After standing at room temperature for 5 minutes, the red fluorescence of the probe showed significant quenching, followed by a stabilization of the red fluorescence. Therefore, the hpDNA-CuNCs probe responds to Hg. 2+ Choosing a time of 5 minutes provides high sensitivity, good stability, and good reproducibility.
[0044] like Figure 5 As shown, this example was compared with a blank solution (0 μL Hg was added). 2+ Compared to the standard solution, the dual-emission hpDNA-CuNCs fluorescent probe solution was modified by adding 1.0 μL of Hg. 2+ After standard solution (2.0 μmol / L), the red fluorescence (635 nm) was quenched, while the blue fluorescence (400 nm) remained unchanged. Therefore, the dual-emission fluorescent probe of this invention can be used for Hg. 2+ Rapid ratio fluorescence and visualization detection.
[0045] Example 7 Hairpin DNA-copper cluster probe ratio fluorescence detection of Hg 2+ The process of creating a standard curve: 1.0 μL of Hg at different concentrations was added to the hpDNA-CuNCs fluorescent probe solution (40.0 μL, prepared in Example 5). 2+ Standard solutions (10.0 nmol / L ~ 1.0 mmol / L) were diluted to 200.0 μL with MOPS buffer (pH=7.5), mixed well, and allowed to stand at room temperature for 5 min. A series of fluorescence emission spectrophotometers at an excitation wavelength of 341 nm were recorded for each standard solution. Figure 6As shown in the figure. The 400nm peak represents the blue fluorescence emission peak, and the 635nm peak represents the red fluorescence emission peak. From... Figure 6 It can be seen that, with Hg 2+ With increasing concentration, the fluorescence intensity at 400 nm remained unchanged, while the red fluorescence intensity at 635 nm decreased rapidly. The ratio of the probe's red fluorescence intensity to its blue fluorescence intensity was compared with Hg. 2+ A standard curve was plotted for concentration. The linear range of mercury ion concentration detection using dual-emission probe ratiometric fluorescence was 1.0–1200.0 nmol / L. Figure 7 As shown.
[0046] Example 8 Standard curve method for ratio fluorescence detection of Hg in real water samples 2+ Steps: Add 0.1–10.0 μL of actual water sample solution to 40.0 μL of hpDNA-CuNCs fluorescent probe solution (prepared in Example 5), then dilute to 200.0 μL with MOPS buffer solution (pH=7.5). After mixing, allow the solution to stand at room temperature for 5 min. Record the fluorescence emission spectrum of the sample solution at an excitation wavelength of 341 nm using a fluorescence spectrophotometer. The Hg in the water sample can be accurately quantified using the standard curve method. 2+ content.
[0047] Example 9 Standard addition method for ratio fluorescence detection of Hg in real water samples 2+ Steps: Add 0–50.0 μL of actual water sample solution to the hpDNA-CuNCs fluorescent probe solution (40.0 μL, prepared in Example 5), and then add 1.0 μL of Hg at different concentrations. 2+ A standard solution (10.0 nmol / L ~ 1.0 mmol / L) was prepared and diluted to 200.0 μL with MOPS buffer (pH=7.5). After mixing, the solution was allowed to stand at room temperature for 5 min. The fluorescence emission spectra of a series of sample solutions at an excitation wavelength of 341 nm were recorded using a fluorescence spectrophotometer. The Hg content in the sample can be accurately quantified by the standard addition method. 2+ concentration.
[0048] Example 10 Selective detection of Hg using dual-emission fluorescent probes 2+ The process: The hpDNA-CuNCs fluorescent probe solution (40.0 μL, prepared in Example 5) was diluted with 1.0 μL of standard solutions of 13 metal ions, including mercury, cadmium, chromium, iron, barium, silver, copper, zinc, nickel, magnesium, calcium, lead, and bismuth, using a pH 7.5 MOPS (10.0 mmol / L) buffer to a final volume of 200.0 μL. After mixing, the solution was allowed to stand at room temperature for 5 min. The red fluorescence emission intensity of the mixed solution at 341 nm was recorded using a fluorescence spectrophotometer. The final concentrations of each metal ion are as follows: Hg 2+ The concentration was 500.0 nm, Ag + The concentration was 2.5.0 μM, Cu 2+ The concentration was 5.0 μM, Cr 3+ 10.0 μM, Cd 2+ Ba 2+ Fe 3+ The concentration was 50.0 μM, Ni 2+ Zn 2+ Mg 2+ Ca 2+ Pb 2+ Bi 2+ The concentration is 100.0 μM. For example... Figure 8 As shown, comparing the fluorescence emission intensities of the various mixed solutions revealed that only mercury ions caused a significant decrease in the red fluorescence intensity of the probe. The experimental results indicate that the dual-emission fluorescent probe is effective against Hg. 2+ It has a high fluorescence selectivity response.
[0049] Example 11 Rapid ratio fluorescence detection of Hg 2+ Hairpin-type DNA-copper cluster probe for detecting Hg in real water samples 2+ : Using the fluorescent dual-emission probe prepared in Example 5, and following the preferred method and steps in Example 6, Hg was detected in local tap water and river water, respectively. 2+ The content was tested, and the results are shown in Table 6.
[0050] The process for testing tap water and river water according to the national standard method (GB / T 37906-2019) is as follows: collect and preserve water samples in accordance with the provisions of GB / T5750.2; process the water samples and determine the mercury ion content in accordance with the provisions of GB / T 37906-2019.
[0051] Table 6 shows the results of using a fluorescent dual-emission probe and the national standard method to detect mercury ion content in real water samples (the experimental results are the average of three parallel determinations). As shown in Table 6, the rapid ratio fluorescence detection method for Hg prepared in this invention...2+ Hairpin-type DNA-copper cluster probe ratiometric fluorescence method for detecting Hg in real water bodies 2+ The content results were close to those obtained using the national standard method (GB / T 37906-2019). The Bland-Altman method evaluation results (P>0.05) showed good consistency between the quantitative detection results of the two methods (fluorescent dual-emission probe ratiometric fluorescence method and the national standard method), indicating that the fluorescent dual-emission probe prepared in this invention can be used for trace Hg in real water samples. 2+ Content detection.
[0052] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.
[0053] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.
Claims
1. A rapid ratio fluorescence detection method for Hg 2+ The hairpin-type DNA-copper cluster probe is characterized by, The hairpin DNA-copper cluster probe contains a hairpin DNA template that can simultaneously emit strong red fluorescence and weak blue fluorescence, and the sequence of the hairpin DNA template is 5'-TTTTTTTTTTTTTTTTTTTTTTTTTCGCGCGCGATATATCGCGCGCG-3'.
2. A rapid ratio fluorescence detection method for Hg as described in claim 1 2+ The method for preparing hairpin-type DNA-copper cluster probes is characterized by, Includes the following steps: Step 1: Prepare a single-stranded DNA aqueous solution using 3-morpholinopropanesulfonic acid buffer. Heat and keep the single-stranded DNA aqueous solution in a constant temperature water bath, then cool it to room temperature and store it in a refrigerator for a certain period of time to form an hpDNA template solution. Step 2: Add copper sulfate solution to the obtained hpDNA template solution and incubate at a constant temperature for a certain time to obtain a mixed solution; then, add sodium ascorbate solution to the mixed solution and react at a constant temperature for a certain time to allow the Cu atoms coordinated on the hpDNA template to react. 2+ Reduced by sodium ascorbate, a rapid ratio fluorescence detection of Hg was obtained. 2+ Hairpin-type DNA-copper cluster probe; the prepared rapid ratio fluorescence detection of Hg 2+ The hairpin-type DNA-copper cluster probes are stored in dark brown centrifuge tubes and kept at 4°C for later use.
3. The rapid ratio fluorescence detection of Hg as described in claim 2 2+ The method for preparing hairpin-type DNA-copper cluster probes is characterized by, In step one, the concentration of the single-stranded DNA aqueous solution is 1.0~10.0µM; The temperature of the single-stranded DNA aqueous solution was heated in a constant temperature water bath to 90℃~100℃ for 10~15 minutes. The temperature for refrigeration and insulation in a refrigerator is 4 degrees Celsius. o C, the storage time is 2~6 hours.
4. The rapid ratio fluorescence detection of Hg as described in claim 2 2+ The method for preparing hairpin-type DNA-copper cluster probes is characterized by, The volume ratio of 3-morpholine propanesulfonic acid buffer, copper sulfate solution, and sodium oxalate solution is 5~10:5~8:1; In step two, rapid ratio fluorescence detection of Hg 2+ The final molar ratio of hpDNA, copper sulfate, and sodium ascorbate in the hairpin-type DNA-copper cluster probe is 1:25 to 200:
25.
5. The rapid ratio fluorescence detection of Hg as described in claim 2 2+ The method for preparing hairpin-type DNA-copper cluster probes is characterized by, In step two, the hpDNA template solution and copper sulfate solution are mixed and then reacted at a constant temperature of 20℃~30℃ for 0~50min. Add to sodium ascorbate solution, and maintain a constant temperature of 20℃~30℃ for 0~50 min.
6. The rapid ratio fluorescence detection of Hg as described in claim 2 2+ The method for preparing hairpin-type DNA-copper cluster probes is characterized by, In step one, the concentration of 3-morpholinopropanesulfonic acid buffer is 5.0~15.0 mmol / L, and the pH is 6.0~8.
5.
7. A rapid ratio fluorescence detection method for Hg as described in claim 1 2+ The application of hairpin-type DNA-copper cluster probes is characterized by, The hairpin-type DNA-copper cluster probe described above is used to detect Hg. 2+ .
8. The rapid ratio fluorescence detection of Hg as described in claim 7 2+ The application of hairpin-type DNA-copper cluster probes is characterized by, The specific application steps are as follows: Take the prepared rapid ratio fluorescence detection Hg 2+ Hairpin-type DNA-copper cluster probe solution, followed by the addition of different concentrations of Hg 2+ Standard solutions or solutions containing Hg 2+ The sample solution was diluted with 3-morpholinopropanesulfonic acid buffer; after mixing, it was allowed to stand at room temperature for 0-30 min, and the fluorescence emission spectrum of the system was measured. The ratio of red to blue fluorescence emission peak intensity was then used to determine the fluorescence emission spectrum. 2+ A standard curve was established based on the relationship between concentration and concentration, and the Hg in the sample solution was accurately quantified accordingly. 2+ The concentration.
9. The rapid ratio fluorescence detection of Hg as described in claim 7 2+ The application of hairpin-type DNA-copper cluster probes is characterized by, The optimal fluorescence excitation wavelength of the hairpin-type DNA-copper cluster probe is 341 nm; the fluorescence emission wavelengths of the hairpin-type DNA-copper cluster probe are 400 nm and 635 nm, respectively.
10. The rapid ratio fluorescence detection of Hg as described in claim 8 or 9 2+ The application of hairpin-type DNA-copper cluster probes is characterized by, If the sample solution has Hg 2+ If the concentration is too high, dilute the sample solution appropriately with double-distilled water and retest.