Preparation method and application of a fluorescence sensor for detecting malachite green compounds
Through a fluorescent sensor without nanomaterial modification, the G-quadruplet is used to form a G-quadruplet with thioflavin T and nucleic acid aptamers, the complexity and sensitivity of malachite green compound detection are solved, and efficient and rapid detection of malachite green compound is achieved, with a detection limit of 5.4ng/L.
Patent Information
- Application Number
- CN202410542881.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-01
- Publication Date
- 2025-07-18
- Estimated Expiration
- 2044-05-01
AI Technical Summary
The existing detection methods of malachite green compounds have problems such as complex operation, expensive equipment, low detection limits, insufficient sensitivity and susceptibility to interference. In particular, chromatography and immunoassay methods are not applicable in daily supervision, and the detection limits of fluorescence analysis methods are also high.
A fluorescent sensor without nanomaterial modification was designed, and a G-tetrakisphenyl-thioflavin T conjugate was used to form a G-tetrakisphenyl-thioflavin T conjugate through fluorescence intensity changes were simplified to rapid detection without separation steps.
It realizes simple, fast and sensitive detection of malachite green compound, with a detection limit of up to 5.4ng/L, with high sensitivity and strong anti-interference, and is suitable for water sample detection.
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Figure CN118583825B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of rapid detection of malachite green, and specifically relates to a preparation method and application of a fluorescence sensor for detecting malachite green compounds. Background Art
[0002] At present, the commonly used detection methods for malachite green compounds mainly include thin-layer chromatography, high-performance liquid chromatography, chemical colorimetry, fluorescence analysis, immunological detection, and electrochemical sensor methods. However, the pretreatment process of chromatography is complex, the detection time is long, the operational requirements for personnel are high, and the price of instrument equipment is expensive, with hysteresis, which is not suitable for daily supervision. The immunological detection method has the disadvantages of high cost for preparing monoclonal antibodies and unstable factors. The detection limit of the chemical colorimetry method is between 2 - 10.0 mg / L, the sensitivity is not high enough, and there is a certain degree of interference. The detection limit of the fluorescence analysis method can reach 10 -4 even 10 -5 mg / L, with simple operation, good selectivity, and can achieve intuitive and rapid quantitative analysis. Therefore, it is crucial to develop a fluorescence analysis method for detecting malachite green compounds. Summary of the Invention
[0003] Aiming at the above technical problems, the present invention provides a preparation method and application of a fluorescence sensor for detecting malachite green compounds. The sensor does not require modification with nanomaterials. When malachite green compounds are present, the fluorescence of the system weakens, and the concentration of malachite green compounds in the sample to be tested can be determined according to the change in fluorescence intensity. When used for detecting malachite green compounds, no separation steps are required, and simple, rapid, and highly sensitive detection of malachite green compounds in water samples can be achieved.
[0004] To solve the above technical problems, the present invention adopts the following technical solutions:
[0005] In the first aspect, the present invention provides a fluorescence sensor, comprising: thioflavin T for generating a fluorescence signal; a nucleic acid aptamer for specifically binding to malachite green compounds; wherein, thioflavin T non-specifically binds to the nucleic acid aptamer to form a G-quadruplex-thioflavin T conjugate, which is the fluorescence sensor; the DNA sequence of the nucleic acid aptamer is shown as SEQ ID No.1.
[0006] The DNA sequence of the nucleic acid aptamer designed by the present invention that can specifically recognize malachite green is shown in SEQ ID No. 1, and SEQ ID No. 1 is: 5′-ATT GGC ACT CCA CGC ATA GGG ACG CGA ATA GCG GAC CTATGT GTG GTG AGC CTA TGC GTG CTA CCG TGA A-3′. The above sequence is prepared by a DNA synthesizer, and the obtained nucleic acid aptamer is a G-quadruplex structure.
[0007] The non-specific dye thioflavin T of the present invention can be embedded in the nucleic acid aptamer to obtain a G-quadruplex-thioflavin T conjugate, that is, a fluorescence sensor. This fluorescence sensor does not require the modification of nanomaterials. When it is used for the detection of malachite green compounds, the detection of malachite green compounds can be realized without any separation steps. It has the characteristics of simplicity, rapidity and high sensitivity, and the detection limit can be as low as 5.4 ng / L.
[0008] Preferably, the non-specific binding mode of thioflavin T to the nucleic acid aptamer is intercalative binding.
[0009] In the present invention, thioflavin T can bind to the nucleic acid aptamer by an intercalative manner to form a G-quadruplex-thioflavin T conjugate, thereby significantly increasing the fluorescence intensity of thioflavin T.
[0010] In the second aspect, the present invention also provides a preparation method of the above fluorescence sensor, including the following steps:
[0011] Step 1: Dissolve the nucleic acid aptamer in a buffer solution, thaw and activate it to obtain a nucleic acid aptamer solution; dissolve thioflavin T in a buffer solution to obtain a thioflavin T solution;
[0012] Step 2: Mix the nucleic acid aptamer solution and the thioflavin T solution, and react at 25 °C for 30 min to form a G-quadruplex-thioflavin T conjugate, that is, a fluorescence sensor.
[0013] The fluorescence of thioflavin T in the buffer solution is weak, and it can bind to the nucleic acid aptamer by an intercalative manner to form a G-quadruplex-thioflavin T conjugate, so that the fluorescence intensity of the G-quadruplex-thioflavin T conjugate gradually increases, and a fluorescence sensor for detecting malachite green is obtained. In the present invention, the nucleic acid aptamer is a DNA fragment, which needs to be stored frozen and thawed and activated before use, generally for 25-30 min.
[0014] Preferably, the buffer solution is a Tris-HCl buffer solution with a concentration of 20-30 mM and a pH of 7.0-7.5.
[0015] More preferably, the buffer is a 20 mM Tris-HCl buffer with a pH of 7.0.
[0016] Preferably, the concentration of the aptamer solution is 0.5 - 1.0 μM.
[0017] More preferably, the concentration of the aptamer solution is 1 μM.
[0018] Preferably, the concentration of the thioflavin T solution is 0.5 - 1 mg / L.
[0019] More preferably, the concentration of the thioflavin T solution is 0.5 mg / mL.
[0020] Preferably, the volume ratio of the aptamer solution to the thioflavin T solution is 16:3.
[0021] In a third aspect, the present invention also provides the use of the above fluorescence sensor or the fluorescence sensor obtained by the above preparation method in the detection of malachite green compounds.
[0022] In a fourth aspect, the present invention also provides a method for detecting malachite green compounds using the above fluorescence sensor or the fluorescence sensor obtained by the above preparation method, comprising the following steps:
[0023] Step 1: Take the sample to be tested, add it to the fluorescence sensor, and mix well by shaking at 25 °C for reaction; the volume ratio of the fluorescence sensor to the sample to be tested is 19:20;
[0024] Step 2: Perform fluorescence testing, with an excitation wavelength of 410 nm, and detect the fluorescence intensity of the reaction mixture in Step 1 at an emission wavelength of 490 nm;
[0025] Step 3: Calculate the concentration of malachite green compounds in the sample solution based on the pre-drawn standard curve and the fluorescence intensity of the reaction mixture at the emission wavelength.
[0026] In the fluorescence sensor of the present invention, the aptamer can specifically bind to the malachite green compounds in the sample to be tested to form a stable complex structure, so that the thioflavin T on the aptamer is released and leaves the surface of the aptamer, forming free thioflavin T. By comparing the fluorescence intensity of the sample to be tested at 490 nm with the standard curve, the concentration of malachite green compounds in the sample to be tested is obtained, where the standard curve is obtained by parallel experiments using standard samples of malachite green compounds with known concentrations. This method has the advantages of simplicity, rapidity, and high sensitivity, and can achieve the detection of malachite green compounds without any separation steps, with a detection limit as low as 5.4 ng / L.
[0027] Preferably, in Step 1, the volume ratio of the fluorescence sensor to the sample to be tested is 19:20.
[0028] Preferably, in step 2, the excitation wavelength is 410 nm, the emission wavelength is 490 nm, and the excitation is carried out continuously or discontinuously.
[0029] Preferably, the sample to be tested is a water sample.
[0030] The sample to be detected in the present invention requires a small amount, and can accurately determine the content of malachite green compounds in the sample to be tested. The water sample includes, but is not limited to, cultured fresh water samples, cultured sea water samples, and transportation water samples. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 It is a characterization diagram of the affinity between DNA aptamer and malachite green compound by isothermal titration method;
[0032] Figure 2 It is the fluorescence spectrum of the standard sample of malachite green compound detected by the present invention at different pH values;
[0033] Figure 3 It is an optimization analysis diagram of the reaction time explored by the present invention;
[0034] Figure 4 It is an analysis diagram of the ultraviolet-visible absorption spectrum explored by the present invention;
[0035] Figure 5 It is the fluorescence spectrum diagram of the standard sample of malachite green compound with different concentrations of the present invention;
[0036] Figure 6 It is the standard curve diagram of the standard sample of malachite green compound with different concentrations of the present invention;
[0037] Figure 7 It is an analysis diagram of the anti-interference ability of the fluorescence sensor to malachite green compound explored by the present invention. DETAILED DESCRIPTION OF THE INVENTION
[0038] In order to make the objectives, technical solutions and advantages of the present invention clearer, the following further describes the present invention in detail with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.
[0039] An aptamer is a DNA or RNA fragment that can specifically bind to a target molecule. It has the characteristics of high specificity, high affinity, easy chemical synthesis and modification, and high stability, and is a more potential target recognition element than an antibody. Thioflavin T is a commercially available water-soluble benzothiazole fluorescent dye that exhibits weak fluorescence emission in the free state.
[0040] Therefore, in the present invention, a nucleic acid aptamer capable of specifically binding to malachite green compound is first designed, and then the nucleic acid aptamer is non-specifically bound to thioflavin T to obtain a fluorescence sensor. Finally, the fluorescence sensor is used for detecting malachite green compound in a sample to be measured.
[0041] Example 1
[0042] (1) Design and synthesis of nucleic acid aptamer
[0043] By referring to relevant literature, a DNA fragment capable of specifically recognizing malachite green compound was designed and prepared by a DNA synthesizer. The DNA sequence of the obtained nucleic acid aptamer is: 5′-ATT GGC ACT CCA CGC ATA GGGACG CGA ATA GCG GAC CTA TGT GTG GTG AGC CTA TGC GTG CTA CCG TGA A-3′ (SEQ ID NO.1).
[0044] (2) Preparation of fluorescence sensor
[0045] 1) First, all glassware used in the experiment was cleaned with ultrapure water and dried in an oven for later use.
[0046] 2) Preparation of nucleic acid aptamer solution. The nucleic acid aptamer was thawed and activated at 25 °C for 30 min and dissolved in Tris-HCl buffer with a pH of 7.0 and a concentration of 20 mM to obtain a stock solution with a concentration of 1 μM, thus obtaining a nucleic acid aptamer solution. Thioflavin T was dissolved in Tris-HCl buffer with a pH of 7.0 and 20 mM to obtain a 0.5 mg / mL thioflavin T solution.
[0047] 3) Preparation of fluorescence sensor. 400 μL of the above nucleic acid aptamer stock solution was taken, and 75 μL of thioflavin T solution with a concentration of 0.5 mg / mL was added. The mixture was allowed to react fully at 25 °C for 30 min to form a G-quadruplex-thioflavin T conjugate, which is the fluorescence sensor.
[0048] (3) Affinity between nucleic acid aptamer and malachite green compound
[0049] 1) 200 μM malachite green titrant was loaded into an isothermal titration syringe, and the concentration of the nucleic acid aptamer was maintained at 5 μM;
[0050] 2) The malachite green titrant was injected, 2 μL each time, for a total of 25 injections. Finally, a 0.4 μL cleaning injection was performed.
[0051] 3) The mixing speed of the titrant and the titrand was adjusted to 350 r / min, and the model was appropriately adjusted according to the dilution heat of the titrant.
[0052] Figure 1 It is a characterization diagram of the affinity between a DNA aptamer and malachite green compound by isothermal titration. From Figure 1 it can be seen that the DNA aptamer of the present invention has excellent affinity with malachite green, indicating that the DNA aptamer provided by the present invention can specifically bind to the malachite green compound.
[0053] Example 2
[0054] 1) First, soak all the glassware used in the experiment in the prepared 20 mM Tris-HCl (pH = 7.0) buffer solution for 24 h, wash it clean with ultrapure water, and dry it in an oven for later use.
[0055] 2) Preparation of the aptamer solution. Dissolve the aptamer in a 20 mM Tris-HCl buffer solution with a pH of 7.0 and thaw and activate it at 25 °C for 30 min. Among them, the concentration of the aptamer solution is 1 μM. Take thioflavin T and dissolve it in a 20 mM Tris-HCl buffer solution with a pH of 7.0 to obtain a 0.5 mg / mL thioflavin T solution.
[0056] 3) Preparation of the fluorescence sensor. Take 400 μL of the above-mentioned 1 μM aptamer stock solution, add 75 μL of a 0.5 mg / mL thioflavin T solution, and react fully at 25 °C for 30 min to form a G-quadruplex-thioflavin T conjugate, that is, the fluorescence sensor is obtained.
[0057] Optimization of the detection conditions in Experimental Example 1
[0058] (1) Optimization of the pH value of the Tris-HCl buffer solution
[0059] To obtain the best detection conditions, prepare Tris-HCl buffer solutions with pH values of 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 respectively. Configure 1 μM aptamer solutions and 0.5 mg / mL thioflavin T solutions with the above-mentioned 20 mM Tris-HCl buffer solutions at different pH values. Take 400 μL of the aptamer solution and 75 μL of the 0.5 mg / mL thioflavin T solution and mix them by shaking. The temperature is 25 °C, and react fully for 30 min to obtain the fluorescence sensor. Then add 500 μL of a 200 μg / L malachite green compound standard sample solution, and make up the volume to 1 mL with the Tris-HCl buffer solution. After reacting at 25 °C, perform fluorescence emission spectrum characterization. The results are as Figure 2 shown.
[0060] Figure 2 It is the fluorescence spectrum of the present invention for detecting the malachite green compound standard sample at different pH values. From Figure 2It can be seen that when the pH of the Tris-HCl buffer solution is 7.0 - 7.5, the obtained fluorescence intensity is relatively high, and when the pH of the Tris-HCl buffer solution is 7.0, the fluorescence intensity is the highest. Therefore, the Tris-HCl buffer solution with a pH of 7.0 is selected as the solvent to ensure the optimal reaction conditions for detecting malachite green compounds in the solution.
[0061] (2) Optimization of reaction time
[0062] Take 475 μL of the fluorescence sensor prepared in Example 1, add 500 μL of the malachite green compound standard sample solution, and make up to 1 mL with Tris-HCl buffer solution to obtain a series of system solutions with malachite green compound concentrations of 10, 20, 30, 60, 120, 250, 375, 500, 750, 1000 μg / L. Perform fluorescence emission spectrum tests on the above system solutions every 1 minute within 10 minutes of the reaction duration, and detect the fluorescence intensity at a wavelength of 490 nm. The results are as Figure 3 shown.
[0063] Figure 3 This is the analysis diagram for exploring the optimization of reaction time in the present invention. From Figure 3 it can be seen that it quickly tends to be stable after reacting with the fluorescence sensor. This indicates that the fluorescence sensor prepared in the present invention can detect malachite green within a short time.
[0064] (3) Selection of excitation wavelength
[0065] Characterize the thioflavin T solution, malachite green solution, and the mixed solution of thioflavin T and nucleic acid aptamer before and after adding malachite green by ultraviolet-visible absorption spectroscopy to select the excitation wavelength. The results are as Figure 4 shown.
[0066] Figure 4 This is the ultraviolet-visible absorption spectrum analysis diagram of the present invention. From Figure 4 it can be seen that the fluorescence sensor shows strong ultraviolet absorption at 405 - 410 nm and reaches a peak at 410 nm. This indicates that the optimal excitation wavelength of the fluorescence sensor prepared in the present invention is 410 nm.
[0067] Experimental Example 2 Establishment of standard curve
[0068] Take 75 μL of 0.5 mg / mL thioflavin T solution and 400 μL of 1 μM aptamer solution. After oscillating for 30 min at 25 °C, add 500 μL of malachite green compound standard sample solutions with different concentrations thereto, and make up the volume to 1 mL with 20 mM Tris-HCl buffer solution (pH = 7.0). Malachite green compound standard sample solutions with concentrations of 10, 20, 30, 60, 120, 250, 375, 500, 750, 1000 μg / L are obtained. After oscillating evenly, the reaction is carried out, and fluorescence emission spectrum characterization is carried out to obtain a fluorescence intensity change diagram as Figure 5 shown. Among them, 0 μg / L is used for the blank experiment to obtain the corresponding fluorescence intensity.
[0069] Figure 5 is the fluorescence spectrum diagram of the malachite green compound standard samples with different concentrations of the present invention; from Figure 5 it can be seen that as the concentration of the malachite green compound increases, the fluorescence intensity of the system decreases accordingly, and the two are negatively correlated; and it reaches the peak at 490 nm.
[0070] According to the fluorescence intensity measured at 490 nm, a standard curve of the malachite green compound is drawn. Figure 6 is the standard curve diagram of the malachite green compound standard samples with different concentrations of the present invention; in the figure, the standard curve is drawn with the fluorescence intensity as the ordinate and the concentration of the malachite green compound as the abscissa. The standard curve equation obtained by fitting is Y = -0.32X + 497.1. Where Y is the fluorescence intensity value and X is the concentration of the malachite green compound, and the linear correlation coefficient R 2 = 0.991, meeting the quantitative requirements.
[0071] The linear range of the detection method established by the present invention is 0 - 1000 μg / L, that is, the fluorescence intensity and the concentration of the malachite green compound show a linear correlation within the range of 0 - 1000 μg / L. The detection limit calculated according to the formula LOD = 3*SD / K is 5.4 ng / L. Among them, "SD" represents the standard deviation of the fluorescence intensity of the blank group, and "k" represents the slope of the fitting curve.
[0072] Experimental Example 3 Analysis of the anti-interference of the fluorescence sensor to the detection of malachite green compound
[0073] Prepare the fluorescence sensor according to Example 1. Add 500 μL of malachite green standard solution (100 μg / L), KCl solution (1 mg / mL), NaCl solution (1 mg / mL), CaCl2 solution (1 mg / mL), MgCl2 solution (1 mg / mL), CV solution (1 mg / mL), BG solution (1 mg / mL), LMG solution (1 mg / mL) to the obtained fluorescence sensor respectively. Oscillate evenly, and after the response, carry out fluorescence emission spectrum characterization. The results are asFigure 7 As shown
[0074] Figure 7 This is an analysis diagram for the present invention to explore the anti-interference performance of the fluorescence sensor against malachite green compounds; from Figure 7 it can be seen that the fluorescence intensity of the solutions containing K + , Na + , Ca 2+ , Mg 2+ , CV, BG, and LMG has little influence on the fluorescence intensity of the system, indicating that the fluorescence sensor prepared in the present invention has strong anti-interference performance against malachite green compounds.
[0075] Example 3
[0076] Use the fluorescence sensor prepared in the above Example 1 to detect the concentrations of malachite green compounds in aquaculture fresh water samples, aquaculture sea water samples, and transportation water samples respectively. Take 475 μL of the fluorescence sensor and add 500 μL of aquaculture fresh water samples, aquaculture sea water samples, and transportation water samples respectively. Mix and react by shaking at 25 °C, and then perform fluorescence detection. The excitation wavelength is 410 nm, and record the fluorescence intensity at 490 nm. The detected amounts of malachite green compounds in the three water samples are all 0.0 μg / L. Therefore, a standard addition recovery experiment is carried out, that is, add 3.0, 6.0, and 12.0 μg / L of malachite green compound standard samples to the aquaculture fresh water samples, aquaculture sea water samples, and transportation water samples respectively, and measure the addition recovery rates of malachite green compounds in the aquaculture fresh water samples, aquaculture sea water samples, and transportation water samples. The results are shown in Table 1.
[0077] Table 1 Detection of real samples of malachite green (n = 3)
[0078]
[0079] The results in Table 1 show that the addition recovery rates of malachite green compounds detected in each sample to be measured by using the fluorescence sensor prepared in the present invention are between 94.0% and 102.0%, and the precisions are all within 2.2%. This indicates that the fluorescence sensor of the present invention has good recovery rates and has high accuracy and precision. This method is applicable to the rapid detection of malachite green compounds.
[0080] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.
Claims
1. A fluorescence sensor, characterized in that, The fluorescent sensor includes: Thioflavin T for generating a fluorescent signal; a nucleic acid aptamer for specifically binding to malachite green compounds; wherein, the Thioflavin T non-specifically binds to the nucleic acid aptamer to form a G-quadruplex-Thioflavin T conjugate, which is the fluorescent sensor; the DNA sequence of the nucleic acid aptamer is as shown in SEQ ID No.
1.
2. The fluorescence sensor according to claim 1, wherein The non-specific binding mode of the Thioflavin T and the nucleic acid aptamer is intercalative binding.
3. The preparation method of the fluorescence sensor according to any one of claims 1-2, characterized in that, It includes the following steps: Step 1: Dissolve the nucleic acid aptamer in a buffer solution, thaw and activate it to obtain a nucleic acid aptamer solution; Dissolve Thioflavin T in a buffer solution to obtain a Thioflavin T solution; Step 2: Mix the nucleic acid aptamer solution and the Thioflavin T solution, and react at 25 °C for 30 min to form a G-quadruplex-Thioflavin T conjugate, thus obtaining the fluorescent sensor.
4. The preparation method of the fluorescence sensor according to claim 3, characterized in that, The buffer solution is a 20 - 30 mM Tris-HCl buffer solution with a pH of 7.0 - 7.
5.
5. The application of the fluorescent sensor described in claim 1 or 2 or the fluorescent sensor obtained by the preparation method of the fluorescent sensor described in claim 3 or 4 in the detection of malachite green compounds in water samples.
6. A detection method for malachite green compounds, characterized in that, Using the fluorescent sensor described in claim 1 or 2 or the fluorescent sensor obtained by the preparation method described in claim 3 or 4, it includes the following steps: Step 1: Take the sample to be tested, add it to the fluorescent sensor, and mix well by shaking at 25 °C for reaction; the volume ratio of the fluorescent sensor to the sample to be tested is 19:20; Step 2: Perform fluorescence testing, with an excitation wavelength of 410 nm, and detect the fluorescence intensity of the reaction mixture in step 1 at an emission wavelength of 490 nm; Step 3: Calculate the concentration of malachite green compounds in the sample solution according to the drawn standard curve and the fluorescence intensity of the reaction mixture at 490 nm.
7. The detection method according to claim 6, characterized in that, The reaction temperature in step 1 is 25 °C; the volume ratio of the fluorescent sensor to the sample to be tested is 19:
20.
8. The detection method according to claim 6, wherein In step 2, the excitation wavelength is 410 nm, the emission wavelength is 490 nm, and the excitation is carried out continuously or discontinuously.
9. The detection method according to claim 6, wherein The sample to be tested is a water sample.