Tetrahedral DNA-silver cluster probe for detecting Cu < 2 + > by double red fluorescence ratio and preparation method of tetrahedral DNA-silver cluster probe
By preparing a tetrahedral DNA-silver cluster probe with dual red fluorescence ratio detection, the problems of complexity and high cost in the detection of trace/ultra Cu2+ in the prior art are solved, realizing rapid, simple and accurate Cu2+ detection, which is suitable for visualization and ratio fluorescence detection of trace Cu2+ in water.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SICHUAN UNIVERSITY OF SCIENCE AND ENGINEERING
- Filing Date
- 2026-02-06
- Publication Date
- 2026-05-12
AI Technical Summary
Existing technologies struggle to achieve rapid, simple, accurate, and low-cost detection of trace/ultra-level Cu2+, and traditional methods require complex pretreatment and are costly.
The tetrahedral DNA-silver cluster probe, which uses dual red fluorescence ratio detection, is prepared in an aqueous solution system using a DNA template with a specific sequence. The probe is then reacted with Cu2+ at room temperature, and the fluorescence emission spectrum is recorded for detection.
It achieves rapid, simple, accurate and low-cost Cu2+ detection. The probe preparation conditions are mild, the response time is short, and the detection results are consistent with the national standard method. It is suitable for visualization and ratio fluorescence detection of trace Cu2+ in water.
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Figure CN122012084A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of fluorescent nanocluster material preparation technology, and more specifically, this invention relates to the detection of Cu by dual red fluorescence ratio. 2+ Tetrahedral DNA-silver cluster probe and its preparation method. Background Technology
[0002] Divalent copper ions (Cu) 2+ Excessive presence of Cu ions in water bodies not only disrupts the enzyme activity and cell membrane structure of aquatic organisms, inducing imbalances in aquatic ecosystems, but also threatens human health through bioaccumulation in the food chain, causing a series of symptoms such as gastrointestinal irritation and liver damage. As a core indicator for water quality assessment, accurate detection of copper ions provides crucial scientific evidence for tracing water pollution sources, optimizing treatment plans, and ensuring the implementation of relevant water quality regulations. Therefore, conducting research on Cu ion content in aquatic environments is essential. 2+ The detection research has important practical significance and application value.
[0003] Currently used for detecting trace / ultra-scale Cu 2+ Classical methods for detection mainly include atomic emission spectroscopy, atomic absorption spectroscopy, ultraviolet-visible spectrophotometry, and inductively coupled plasma mass spectrometry. These detection methods require complex sample pretreatment, are time-consuming and labor-intensive, have high testing costs, and are difficult to implement for rapid on-site detection.
[0004] Currently reported detection of Cu based on DNA-metal nanoclusters 2+ Probe preparation time and detection of Cu 2+ The time required is relatively long (usually greater than 30 minutes), making it difficult to achieve trace Cu levels. 2+ Rapid on-site detection. Due to its high accuracy, high sensitivity, and good selectivity, ratiometric fluorescence analysis has been widely used in environmental safety monitoring and other fields in recent years. However, most reported ratiometric fluorescence methods require the preparation of two or more fluorescent probes, and the probe preparation methods are time-consuming and cumbersome. 2+ The testing process is time-consuming and costly. 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 dual red fluorescence ratio detection method for Cu. 2+ The tetrahedral DNA-silver cluster probe contains a tetrahedral DNA template that can simultaneously emit strong red fluorescence and weak near-infrared red fluorescence. The tetrahedral DNA template includes four sequences: S1, S2, S3, and S4. The S1 sequence is specifically: 5'-AAAAAAACCCCCCCCCCCCCGCTTCAGACTTCACAGCTGCATCGGATGGCAGCCCATATTACTCCT-3'; The S2 sequence is specifically: 5'-TTCCAGGACGCACAGAGCCATCCGATGCAGCATCTCAATGCAATGGC-3'; The S3 sequence is specifically: 5'-CTGTGCGTCCTGGAAACAATTCGCTCTCACTAAGGAGTAATATGGGC-3'; The S4 sequence is specifically: 5'-GTGAAGTCTGAAGCGAAGTGAGAGCGAATTGAGCCATTGCATTGAGA-3'.
[0007] A dual red fluorescence ratio detection method for Cu 2+ The preparation method of the tetrahedral DNA-silver cluster probe includes the following steps: Add the hybridized TDF-DNA cluster template solution and AgNO3 solution to Tris-HAc buffer, mix well, and react at room temperature in the dark for a period of time; then add freshly prepared NaBH4 solution and incubate overnight at room temperature in the dark to obtain the TDF-DNA template AgNCs probe, i.e., the dual red fluorescence ratio detection Cu. 2+ Tetrahedral DNA-silver cluster probes should be stored under refrigeration.
[0008] Preferably, the final molar ratio of TDF-DNA cluster template, AgNO3 and NaBH4 is 1:48:12.
[0009] Preferably, the reaction time after mixing Tris-HAc buffer, TDF-DNA cluster template solution and AgNO3 solution is 30-90 minutes at room temperature in the dark.
[0010] Preferably, the method for preparing the TDF-DNA cluster template solution includes: mixing DNA sequences S1, S2, S3, and S4 in equal proportions and concentrations, heating them in a constant temperature water bath at 90-100°C for 10-15 minutes, and then cooling them to obtain the TDF-DNA silver cluster template solution.
[0011] Preferably, the concentration of Tris-HAc buffer is 5~30.0 mmol / L, the pH is 6.5~8.0, the concentration of TDF-DNA cluster template solution is 0.5~2.5 μmol / L, the concentration of AgNO3 solution is 0.5~5.0 mmol / L, and the concentration of NaBH4 solution is 0.5~5.0 mmol / L; the volume ratio of Tris-HAc buffer to TDF-DNA cluster template solution is 1~2:1~4.
[0012] A dual red fluorescence ratio detection method for Cu 2+ The tetrahedral DNA-silver cluster probe is used for rapid ratio and visual fluorescence detection of Cu. 2+ .
[0013] Preferably, the specific application steps include: mixing tetrahedral DNA-silver cluster probe solution with Tris-HAc buffer at a volume ratio of 1~2:1~4, and then adding Cu at different concentrations. 2+ Standard solutions or solutions containing Cu 2+ After mixing the sample solution, allow it to stand at room temperature for 1-30 minutes, and record the reaction time using a mobile phone at different Cu concentrations. 2+ Fluorescence images of the system at a certain concentration; then based on Cu 2+ The fluorescent color chart of the standard solution can be used to quickly and semi-quantitatively or quantitatively determine the Cu content in the sample solution by visual inspection or using the ImageJ mobile app. 2+ concentration.
[0014] Preferably, the specific application steps include: mixing tetrahedral DNA-silver cluster probe solution with Tris-HAc buffer at a volume ratio of 1~2:1~4, and then adding Cu at different concentrations. 2+ Standard solutions or solutions containing Cu 2+ After mixing the sample solution, allow it to stand at room temperature for 1-15 minutes. Measure the double emission fluorescence spectrum of the system. The fluorescence intensity ratio of the red double emission peaks is then compared with that of Cu. 2+ Establish a standard curve based on the relationship between concentration and concentration, and accurately quantify Cu in the sample solution according to the standard curve. 2+ The concentration.
[0015] Preferably, the optimal fluorescence excitation wavelengths of the tetrahedral DNA-silver cluster probe are 540 nm and 620 nm, respectively; and the maximum fluorescence emission wavelengths of the tetrahedral DNA-silver cluster probe are 617 nm and 704 nm, respectively. If the sample solution Cu 2+ If the concentration is too high, dilute the sample appropriately with double-distilled water and retest.
[0016] The present invention has at least the following beneficial effects: The tetrahedral DNA-silver cluster probes of this invention are prepared by using tetrahedral DNA with a specific sequence as a template in an aqueous solution under mild conditions. The tetrahedral DNA-silver cluster probes prepared by this invention emit weak near-infrared red fluorescence (704 nm) and strong red fluorescence (617 nm). This invention utilizes the tetrahedral DNA-silver cluster probe solution as the sole signal and target recognition probe, with different concentrations of Cu... 2+ After reacting with the probe solution at room temperature for 8 minutes, the reaction of the probe with Cu was recorded using a fluorescence spectrophotometer. 2+ Fluorescence emission spectra before and after the reaction. Trace Cu 2+ This causes a rapid decrease in the intensity of strong red fluorescence (617 nm), while the intensity of weak red fluorescence (704 nm) decreases slowly. Therefore, according to Cu... 2+ The ratio of fluorescence intensity changes at two different emission wavelengths can enable rapid and accurate quantitative detection of Cu. 2+ Concentration. Therefore, this invention develops a simple, rapid, accurate, efficient, low-consumption, and environmentally friendly method for preparing tetrahedral DNA-silver cluster probes. These probes serve as unique signal and target identification probes for the treatment of trace Cu in water. 2+ Rapid visualization detection and ratio fluorescence detection.
[0017] The tetrahedral DNA-silver cluster probe of the present invention has mild synthesis conditions (room temperature), the synthesis system is an aqueous solution system, no organic solvents are required, and it has low energy consumption, is environmentally friendly and low cost. Using tetrahedral DNA as a template to prepare dual-emission fluorescent probes, the probe preparation process is simple, the conditions are mild, the controllability is good, and the synthesized probes have good reproducibility and stability of fluorescence performance. The tetrahedral DNA-silver cluster probe detects copper ions at room temperature with a short response time of only 8 minutes. Fluorescence signal detection can be performed using a fluorescence spectrophotometer to record the fluorescence emission spectra of the solution at excitation wavelengths of 540 nm and 620 nm, and to record the intensity of strong red and weak red fluorescence emission at emission wavelengths of 617 nm and 704 nm. The Cu in the sample can then be accurately detected using ratiometric fluorescence. 2+ The content of.
[0018] Actual water samples require no complex pretreatment; only a syringe with a filter membrane is needed. (Water sample Cu) 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] The tetrahedral DNA-silver cluster probe prepared in this invention can be visualized and detected at concentrations as low as 10.0 nmol / LCu. 2+ .
[0020] 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
[0021] Figure 1 For the detection of Cu by dual red fluorescence ratio 2+ A schematic diagram of the preparation process of a tetrahedral DNA-silver cluster probe; Figure 2 For the detection of Cu by dual red fluorescence ratio 2+ Visual detection of Cu using tetrahedral DNA-silver cluster probes 2+ A schematic diagram; Figure 3 The dual red fluorescence ratio detection of Cu described in this invention 2+ Two-dimensional fluorescence spectrum contour plot of tetrahedral DNA-silver cluster probe; Figure 4 The dual red fluorescence ratio detection of Cu described in this invention 2+ High-resolution transmission electron microscopy image of a tetrahedral DNA-silver cluster probe; Figure 5 The dual red fluorescence ratio detection of Cu described in this invention 2+ Tetrahedral DNA-silver cluster probe (a) and TDF-DNA-AgNCs probe + Cu 2+ (b) Fluorescence emission curves (excitation wavelengths of 540 nm and 620 nm, respectively); Figure 6 The dual red fluorescence ratio detection of Cu described in this invention 2+ Tetrahedral DNA-silver cluster probes respond to different concentrations of Cu 2+ Fluorescence double emission curve; Figure 7 It is a dual red fluorescence ratio detection of Cu 2+ Tetrahedral DNA-silver cluster probe ratio fluorescence detection of Cu 2+ The linear range; Figure 8 The dual red fluorescence ratio detection of Cu described in this invention 2+ Tetrahedral DNA-silver cluster probes respond to different concentrations of Cu 2+ Standard fluorescent color chart; Figure 9 The dual red fluorescence ratio detection of Cu described in this invention 2+ The tetrahedral DNA-silver cluster probe exhibits fluorescence response to 12 different metal ions. Detailed Implementation
[0022] 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.
[0023] 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. Silver clusters were synthesized by designing different S1 sequences from TDF-DNA as templates and stabilizers. The fluorescence emission of S1 template-AgNCs from different TDF-DNA sequences was investigated, as shown in Table 1. The fluorescence emission of silver clusters formed by templates with different S1 sequences from TDF-DNA varied greatly: S1-A5 template could only produce silver clusters with single fluorescence emission; S1-A7, S1-A9, and S1-A... 11 Both templates can be used to prepare fluorescent dual-emission silver clusters; however, the weak red fluorescence in S1-A9 dual-emission fluorescence is not obvious, and S1-A... 11 The dual-emission fluorescence is not as strong as that of S1-A7, while the dual-emission silver clusters prepared with the S1-A7 template emit both extremely strong red fluorescence and weak red fluorescence.
[0024] Table 1. Fluorescence emission of AgNCs prepared from ssDNA templates with different sequences. 2. Based on the fluorescence emission of different TDF-DNA template-silver cluster sequences, S1-A7 was selected as the optimal template S1. The TDF-DNA sequence of S1 as the silver cluster template is as follows: S1: 5'-AAAAAAACCCCCCCCCCCGCTTCAGACTTCACAGCTGCATCGGATGGCAGCCCATATTACTCCT-3'; S2: 5'-TTCCAGGACGCACAGAGCCATCCGATGCAGCATCTCAATGCAATGGC-3'; S3: 5'-CTGTGCGTCCTGGAAACAATTCGCTCTCACTAAGGAGTAATATGGGC-3'; S4: 5'-GTGAAGTCTGAAGCGAAGTGAGAGCGAATTGAGCCATTGCATTGAGA-3'.
[0025] All DNA strands involved in this invention can be synthesized using conventional techniques in the art.
[0026] Example 2 Dual red fluorescence ratio detection of Cu 2+The preparation method of the tetrahedral DNA-silver cluster probe includes the following steps: Take 50 μL (100.0 μmol / L) of each of S1 to S4 of DNA, mix them on a mixer for 1 minute, then place them in a 95℃ constant temperature water bath for 15 minutes, and then place them in a 4℃ refrigerator for 4 hours to obtain TDA-DNF solution.
[0027] Add 200 μL of TDF-DNA aqueous solution (2.5 μmol / L) and 24.0 μL of AgNO3 solution (1.0 mmol / L) to 200 μL of Tris-HAc buffer (pH 7.2, 20.0 mmol / L). Mix well for 2 minutes and incubate at room temperature in the dark for 60 minutes. Then add 6.0 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 TDF-DNA-AgNCs probe. Store the probe solution at 4°C for later use.
[0028] Based on this embodiment, the effect of different pH values of Tris-HAc buffer on the intensity of probe dual emission fluorescence was investigated, and the specific results are shown in Table 2.
[0029] Table 2. Effect of different pH values of Tris-HAc buffer on the intensity of dual emission fluorescence of the probe. As shown in Table 2, the red fluorescence of the TDF-DNA-AgNCs probe prepared with a buffer solution at pH 7.2 Tris-HAc (20.0 mmol / L) was the strongest.
[0030] Based on this example, the effect of different concentrations of Tris-HAc buffer on the intensity of probe dual emission fluorescence was investigated, and the specific results are shown in Table 3.
[0031] Table 3. Effect of different concentrations of Tris-HAc buffer on the intensity of probe dual emission fluorescence. As shown in Table 3, the red fluorescence of the fluorescent dual-emission TDF-DNA-AgNCs probe prepared with 20.0 mmol / L Tris-HAc buffer was the strongest.
[0032] Example 3 Dual red fluorescence ratio detection of Cu 2+ The preparation method of the tetrahedral DNA-silver cluster probe includes the following steps: Take 50 μL (100.0 μmol / L) of each of S1 to S4 of DNA, mix them on a mixer for 1 minute, then place them in a 95℃ constant temperature water bath for 15 minutes, and then place them in a 4℃ refrigerator for 4 hours to obtain TDA-DNF solution.
[0033] Add 200 μL of TDF-DNA aqueous solution (2.5 μmol / L) and 24.0 μL of AgNO3 solution (1.0 mmol / L) to 200 μL of Tris-HAc buffer (pH 7.2, 20.0 mmol / L). Mix well for 2 minutes and incubate at room temperature in the dark for 60 minutes. Then add 6.0 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 TDF-DNA-AgNCs probe. Store the probe solution at 4°C for later use.
[0034] Based on this example, the effects of TDF-DNA, AgNO3, and NaBH4 on the dual emission fluorescence intensity of the probe under different molar ratios (final molar ratios) were investigated. The specific results are shown in Table 4.
[0035] Table 4. Effects of different molar ratios of TDF-DNA, AgNO3, and NaBH4 on the dual emission fluorescence intensity of the probe. As shown in Table 4, the TDF-DNA-AgNCs probe prepared with a molar ratio of TDF-DNA, AgNO3 and NaBH4 of 1:48:12 exhibits good red fluorescence.
[0036] Example 4 Dual red fluorescence ratio detection of Cu 2+ The preparation method of the tetrahedral DNA-silver cluster probe includes the following steps: Take 50 μL (100.0 μmol / L) of each of S1 to S4 of the DNA, mix them on a mixer for 1 minute, then place them in a 95℃ constant temperature water bath for 15 minutes, and then place them in a 4℃ refrigerator for 4 hours to obtain TDF-DNA solution.
[0037] Add 200 μL of TDF-DNA aqueous solution (2.5 μmol / L) and 24.0 μL of AgNO3 solution (1.0 mmol / L) to 200 μL of Tris-HAc buffer (pH 7.2, 20.0 mmol / L). Mix well for 2 minutes and incubate at room temperature in the dark for 60 minutes. Then add 6.0 μL of freshly prepared NaBH4 solution (1.0 mmol / L), mix thoroughly, and incubate overnight at room temperature in the dark to obtain the fluorescent dual-emission TDF-DNA-AgNCs probe. Store the probe solution at 4°C for later use.
[0038] Based on this example, the effect of different reaction times of TDF-DNA and AgNO3 after mixing at room temperature in the dark on the intensity of probe dual emission fluorescence was investigated. The specific results are shown in Table 5.
[0039] Table 5. Effect of different reaction times of TDF-DNA + AgNO3 mixture under room temperature and dark conditions on the intensity of probe dual emission fluorescence. As shown in Table 5, the two red fluorescence emission peaks of the dual-emission TDF-DNA-AgNCs probe were the largest when TDF-DNA and AgNO3 were mixed and reacted at room temperature in the dark for 60 minutes.
[0040] Example 5 Dual red fluorescence ratio detection of Cu 2+ The preparation method of the tetrahedral DNA-silver cluster probe includes the following steps: Take 50 μL (100.0 μmol / L) of each of S1 to S4 of the DNA, mix them on a mixer for 1 minute, then place them in a 95℃ constant temperature water bath for 15 minutes, and then place them in a 4℃ refrigerator for 4 hours to obtain TDF-DNA solution.
[0041] Add 200 μL of TDF-DNA aqueous solution (2.5 μmol / L) and 24.0 μL of AgNO3 solution (1.0 mmol / L) to 200 μL of Tris-HAc buffer (pH 7.2, 20.0 mmol / L). Mix well for 2 minutes and incubate at room temperature in the dark for 60 minutes. Then add 6.0 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 TDF-DNA-AgNCs probe. Store the probe solution at 4°C for later use.
[0042] The dual-red fluorescence ratio detection Cu prepared in this embodiment 2+ The fluorescence emission spectrum of the tetrahedral DNA-silver cluster probe is as follows: Figure 3 As shown, the optimal fluorescence excitation wavelengths of the probe are 540 nm and 620 nm, and the optimal fluorescence emission wavelengths are 617 nm and 704 nm, respectively.
[0043] The dual-red fluorescence ratio detection Cu prepared in this embodiment 2+ The morphology of the tetrahedral DNA-silver cluster probe is as follows Figure 4 As shown, the tetrahedral DNA template silver nanoclusters are dispersed and have a uniform particle size, with an average particle size of about 2.4 nm and a lattice spacing of 0.193 nm.
[0044] Example 6 Cu was detected using a double red fluorescence ratio. 2+ Tetrahedral DNA-silver cluster probe dilution solution for detecting Cu 2+ The process includes: TDF-DNA-AgNCs fluorescent probe solution (100 μL, prepared in Example 5) was diluted with 100 μL Tris-HAc buffer solution (pH 7.2), and then 20 μL Cu was added. 2+ The standard solution (50.0 μmol / L) was mixed and allowed to stand at room temperature for 8 min. A series of fluorescence emission spectra of the solution at excitation wavelengths of 617 nm and 704 nm were recorded using a fluorescence spectrophotometer.
[0045] Based on the premise of this embodiment, the interaction between the fluorescent probe solution and Cu was investigated. 2+ The effect of different reaction times on the intensity of dual emission fluorescence of the probe is shown in Table 6.
[0046] Table 6 Nanocluster fluorescent probe solution and Cu 2+ Effect of reaction time on probe dual emission fluorescence intensity Table 6 shows that the probe solution obtained by a volume ratio of TDF-DNA-AgNCs probe solution to Tris-HAc buffer solution of 1:1 is compatible with Cu. 2+ After reacting at room temperature for 8 minutes, the fluorescence quenching at the two red emission wavelengths of the probe was at its maximum, indicating the detection of Cu. 2+ It has the highest sensitivity.
[0047] Based on the premise of this embodiment, the effect of different volume ratios of fluorescent probe solution and Tris-HAc buffer solution on the intensity of probe dual emission fluorescence was investigated. The specific results are shown in Table 7.
[0048] Table 7. Effect of different volume ratios of fluorescent probe solution to Tris-HAc buffer solution on the intensity of dual emission fluorescence of the probe. Table 7 shows that the probe solution obtained by a volume ratio of TDF-DNA-AgNCs probe solution to Tris-HAc buffer solution of 1:1 is compatible with Cu. 2+ After reacting at room temperature for 8 minutes, the fluorescence quenching at the two red emission wavelengths of the probe was at its maximum, indicating the detection of Cu. 2+ It has the highest sensitivity.
[0049] The TDF-DNA-AgNCs fluorescent probe solution prepared in this embodiment was mixed with a blank solution (0 μL Cu was added). 2+Compared to the standard solution, the dual-emission TDF-DNA-AgNCs fluorescent probe solution with the addition of 20.0 μL Cu 2+ After applying the standard solution (50.0 μmol / L), the strong red fluorescence was quenched, and the weak red fluorescence decreased. Figure 5 As shown. Accordingly, the dual red emission fluorescent probe of the present invention can be used in Cu 2+ Rapid ratio fluorescence detection and semi-quantitative visualization identification.
[0050] Example 7 Dual red fluorescence ratio detection of Cu 2+ Tetrahedral DNA-silver cluster probe ratio fluorescence detection of Cu 2+ The process of creating a standard curve: Dual red fluorescence ratio detection of Cu 2+ The tetrahedral DNA-silver cluster probe solution (100.0 μL, prepared in Example 5) was diluted with 100.0 μL of Tris-HAc buffer (pH 7.2), and then 20.0 μL of Cu at different concentrations was added. 2+ Standard solutions (0~100.0 μmol / L) were mixed and allowed to stand at room temperature for 8 min. Fluorescence emission spectra of the standard solutions at excitation wavelengths of 540 nm and 620 nm were recorded using a fluorescence spectrophotometer, as follows: Figure 6 As shown in the figure, the strong red fluorescence emission peak is at 617 nm, and the weak red fluorescence emission peak is at 704 nm. From... Figure 6 It can be seen that with Cu 2+ With increasing content, the intensity of the strong red fluorescence at 617 nm decreased significantly, while the intensity of the weak red fluorescence at 704 nm decreased slowly. The ratio of the emission peak intensity of the probe at 704 nm to that at 617 nm was used to correlate the Cu content. 2+ A standard curve was plotted for concentration. The linear range of copper ion concentration detection using dual-emission probe ratiometric fluorescence was 50–1500.0 nmol / L, with a detection limit of 10 nmol / L. Figure 7 As shown.
[0051] Example 8 Dual red fluorescence ratio detection of Cu 2+ Visual detection of Cu using tetrahedral DNA-silver cluster probes 2+ Preparation of a standard fluorescent colorimetric card: TDF-DNA-AgNCs fluorescent probe solution (100.0 μL, prepared in Example 5) was diluted with 100.0 μL of pH 7.2 Tris-HAc (20.0 mmol / L) buffer, and then 20 μL of Cu at different concentrations was added. 2+Standard solutions (0~100.0 μmol / L) were mixed and allowed to stand at room temperature for 8 min. The fluorescence color of the mixed solution was observed under a handheld UV lamp, and a series of fluorescence photographs of the standard solution were taken with a mobile phone to prepare Cu. 2+ Standard fluorescent color chart, such as Figure 8 As shown. With Cu 2+ With increasing concentration, the fluorescence color of the detection system solution gradually changes from bright red to colorless, showing a significant change. The concentration range for copper ion detection using the dual-emission probe fluorescence visualization is 10.0 nmol / L–10.0 μmol / L. Trace Cu can be detected based on the fluorescence color of the detection system solution. 2+ Visual detection of Cu concentrations as low as 10.0 nmol / L 2+ Residues can be quickly identified with the naked eye.
[0052] Example 9 Standard curve method for ratio fluorescence detection of Cu in real water samples 2+ Steps: Dual red fluorescence ratio detection of Cu 2+ The tetrahedral DNA-silver cluster probe solution (100.0 μL, prepared in Example 5) was diluted with 100.0 μL of Tris-HAc buffer solution (pH 7.2), and then 0.1–10.0 μL of actual water sample solution was added. After mixing, the solution was allowed to stand at room temperature for 8 min. The fluorescence emission spectra of the sample solution at excitation wavelengths of 617 nm and 704 nm were recorded using a fluorescence spectrophotometer. The Cu in the water sample can be accurately quantified using the standard curve method. 2+ content.
[0053] Example 10 Standard addition method for ratio fluorescence detection of Cu in real water samples 2+ Steps: Dual red fluorescence ratio detection of Cu 2+ The tetrahedral DNA-silver cluster probe solution (100.0 μL, prepared in Example 5) was diluted with 100.0 μL of Tris-HAc buffer solution (pH 7.2), and then 1.0~50.0 μL of actual water sample solution was added, followed by 20.0 μL of Cu at different concentrations. 2+ Standard solutions (10.0 nmol / L ~ 10.0 μmol / L) were mixed and allowed to stand at room temperature for 10 min. Fluorescence emission spectra of the sample solutions at excitation wavelengths of 617 nm and 704 nm were recorded using a fluorescence spectrophotometer. The Cu content in the sample can be accurately quantified using the standard addition method. 2+ concentration.
[0054] Example 11 Dual red fluorescence ratio detection of Cu 2+ Tetrahedral DNA-silver cluster probe selectively detects Cu 2+ The process: Dual red fluorescence ratio detection of Cu 2+ A tetrahedral DNA-silver cluster probe solution (100.0 μL, prepared in Example 5) was diluted with 100.0 μL of Tris-HAc (20.0 mmol / L) buffer at pH 7.2. Then, 20.0 μL of a standard solution containing 12 metal ions (mercury, magnesium, calcium, cobalt, barium, lead, bismuth, cadmium, chromium, zinc, nickel, and iron) at a concentration of 40.0 μmol / L was added. After mixing, the mixture was allowed to stand at room temperature for 10 min. The fluorescence emission intensity of a series of mixed solutions was recorded at wavelengths of 617 nm and 704 nm using a fluorescence spectrophotometer. Comparison of the fluorescence color and emission intensity of each mixed solution revealed that only copper ions at concentrations ≥10.0 nmol / L caused a significant change in the fluorescence color of the probe solution; and only copper ions caused a rapid decrease in the red fluorescence intensity of the probe. The experimental results indicate that the dual-emission fluorescent probe is effective against Cu... 2+ It exhibits a high fluorescence selectivity response. (Note: Hg coexists in the sample.) 2+ Sodium borohydride needs to be added for pretreatment. Example 12 Dual-emission fluorescent probe for detecting Cu in real water samples 2+ The dual red fluorescence ratio detection method prepared in Example 5 was used to detect Cu. 2+ The tetrahedral DNA-silver cluster probe, according to the preferred method and steps in Example 3, was used to test Cu in local tap water and river water, respectively. 2+ The content was tested, and the results are shown in Table 8.
[0055] The process for testing tap water and river water according to the national standard method (GB / T 5750.6-2023) is as follows: collect and preserve water samples in accordance with the provisions of GB / T5750.2; process the water samples and determine the copper ion content in accordance with the provisions of GB / T 5750.6-2023.
[0056] Table 8 shows the results of using a dual-emission fluorescent probe and the national standard method to detect copper ion content in real water samples (the experimental results are the average of three parallel determinations). As shown in Table 8, the dual-emission fluorescent probe prepared in this invention can be used for visualization and ratiometric fluorescence methods to detect Cu in real water bodies. 2+The content results were close to those obtained using the national standard method (GB / T 5750.6-2023). The Bland-Altman method evaluation results (P>0.05) showed good consistency between the quantitative detection results of the two methods (dual-emission fluorescent probe ratiometric fluorescence method and the national standard method), indicating that the dual-emission fluorescent probe prepared in this invention can be used for trace Cu in real water samples. 2+ Content detection.
[0057] 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.
[0058] 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 dual-red fluorescence ratio detection method for Cu 2+ The tetrahedral DNA-silver cluster probe is characterized by, The tetrahedral DNA-silver cluster probe contains a tetrahedral DNA template that can simultaneously emit strong red fluorescence and weak near-infrared red fluorescence. The tetrahedral DNA template includes four sequences: S1, S2, S3, and S4. The S1 sequence is specifically: 5'-AAAAAAACCCCCCCCCCCCCGCTTCAGACTTCACAGCTGCATCGGATGGCAGCCCATATTACTCCT-3'; The S2 sequence is specifically: 5'-TTCCAGGACGCACAGAGCCATCCGATGCAGCATCTCAATGCAATGGC-3'; The S3 sequence is specifically: 5'-CTGTGCGTCCTGGAAACAATTCGCTCTCACTAAGGAGTAATATGGGC-3'; The S4 sequence is specifically: 5'-GTGAAGTCTGAAGCGAAGTGAGAGCGAATTGAGCCATTGCATTGAGA-3'.
2. A method for detecting Cu using a dual-red fluorescence ratio as described in claim 1 2+ The method for preparing tetrahedral DNA-silver cluster probes is characterized by, Includes the following steps: Add the hybridized TDF-DNA cluster template solution and AgNO3 solution to Tris-HAc buffer, mix well, and react at room temperature in the dark for a period of time. Then, freshly prepared NaBH4 solution was added, and the mixture was incubated overnight at room temperature in the dark to obtain the TDF-DNA template AgNCs probe, which is the dual red fluorescence ratio detection Cu. 2+ Tetrahedral DNA-silver cluster probes should be stored under refrigeration.
3. The dual red fluorescence ratio detection method for Cu as described in claim 2 2+ The method for preparing tetrahedral DNA-silver cluster probes is characterized by, The final molar ratio of TDF-DNA cluster template, AgNO3, and NaBH4 is 1:48:
12.
4. The dual red fluorescence ratio detection method for Cu as described in claim 2 2+ The method for preparing tetrahedral DNA-silver cluster probes is characterized by, After mixing Tris-HAc buffer, TDF-DNA cluster template solution and AgNO3 solution, the reaction time is 30-90 minutes at room temperature in the dark.
5. The dual red fluorescence ratio detection method for Cu as described in claim 2 2+ The method for preparing tetrahedral DNA-silver cluster probes is characterized by, The preparation method of TDF-DNA cluster template solution includes: mixing DNA sequences S1, S2, S3 and S4 of equal proportion and concentration, heating in a constant temperature water bath at 90~100℃ for 10~15 minutes and then cooling to obtain TDF-DNA silver cluster template solution.
6. The dual red fluorescence ratio detection method for Cu as described in claim 3 2+ The method for preparing tetrahedral DNA-silver cluster probes is characterized by, The concentration of Tris-HAc buffer is 5–30.0 mmol / L, pH is 6.5–8.0, the concentration of TDF-DNA cluster template solution is 0.5–2.5 μmol / L, the concentration of AgNO3 solution is 0.5–5.0 mmol / L, and the concentration of NaBH4 solution is 0.5–5.0 mmol / L; the volume ratio of Tris-HAc buffer to TDF-DNA cluster template solution is 1–2:1–4.
7. A method for detecting Cu using a dual red fluorescence ratio as described in claim 1 2+ The application of tetrahedral DNA-silver cluster probes is characterized by, The tetrahedral DNA-silver cluster probe is used for rapid ratio and visual fluorescence detection of Cu. 2+ .
8. The dual red fluorescence ratio detection method for Cu as described in claim 7 2+ The application of tetrahedral DNA-silver cluster probes is characterized by, The specific application steps include: mixing the tetrahedral DNA-silver cluster probe solution with Tris-HAc buffer at a volume ratio of 1~2:1~4, and then adding different concentrations of Cu. 2+ Standard solutions or solutions containing Cu 2+ After mixing the sample solution, allow it to stand at room temperature for 1-30 minutes, and record the reaction time using a mobile phone at different Cu concentrations. 2+ Fluorescence images of the system at a certain concentration; then based on Cu 2+ The fluorescent color chart of the standard solution can be used to quickly and semi-quantitatively or quantitatively determine the Cu content in the sample solution by visual inspection or using the ImageJ mobile app. 2+ concentration.
9. The dual red fluorescence ratio detection method for Cu as described in claim 7 2+ The application of tetrahedral DNA-silver cluster probes is characterized by, The specific application steps include: mixing the tetrahedral DNA-silver cluster probe solution with Tris-HAc buffer at a volume ratio of 1~2:1~4, and then adding different concentrations of Cu. 2+ Standard solutions or solutions containing Cu 2+ After mixing the sample solution, allow it to stand at room temperature for 1-15 minutes. Measure the double emission fluorescence spectrum of the system. The fluorescence intensity ratio of the red double emission peaks is then compared with that of Cu. 2+ Establish a standard curve based on the relationship between concentration and concentration, and accurately quantify Cu in the sample solution according to the standard curve. 2+ The concentration.
10. The dual red fluorescence ratio detection method for Cu as described in claim 8 or 9 2+ The application of tetrahedral DNA-silver cluster probes is characterized by, The optimal fluorescence excitation wavelengths of the tetrahedral DNA-silver cluster probe are 540 nm and 620 nm, respectively; the maximum fluorescence emission wavelengths of the tetrahedral DNA-silver cluster probe are 617 nm and 704 nm, respectively. If the sample solution Cu 2+ If the concentration is too high, dilute the sample appropriately with double-distilled water and retest.