G4-ThT Fluorescent Probe for Malachite Green Detection and Its Detection Method

Abstract

The present invention discloses a G4-ThT fluorescent probe for malachite green detection and its detection method. The G4-ThT fluorescent probe is formed by mixing a G4 strand and thioflavin T; the G4 strand, thioflavin T and the malachite green solution to be measured are added together to a buffer solution, shaken and mixed evenly, reacted at room temperature in the dark, the fluorescence spectrum is measured, and the presence and amount of malachite green are judged according to the fluorescence spectrum. The fluorescent probe G4-ThT assembled in the present invention has an obvious response to malachite green. The method only requires one-step reaction, and then a microplate reader is used for high-throughput and rapid detection. The detection time can be controlled within a short time. It has the characteristics of economy, simplicity, rapidity and accuracy, and can accurately quantify the concentration of malachite green as low as 6.28 μg / kg.

Description

Technical Field

[0001] The invention belongs to a rapid detection method of malachite green, and specifically relates to a substance and a method for detecting malachite green by utilizing a G4-ThT fluorescent probe having a strong binding force with malachite green. Background Art

[0002] Malachite Green (MG), also known as Chinese Green and Aniline Green, is a triphenylmethane industrial dye used as a dye for silk, leather and paper. In the 1930s, people discovered that malachite green can be used to kill bacteria, fungi and parasites on the surface of fish, especially for Saprolegniasis. Many countries use malachite green as an insecticide and fungicide in aquaculture to prevent and treat Saprolegniasis, gill mold disease and Ichthyophthirius punctatus in various aquatic animals. Since the 1990s, researchers at home and abroad have successively discovered that after malachite green enters the body of aquatic animals, it will be quickly metabolized into fat-soluble leucomalachite green (LMG). Malachite green and leucomalachite green have a long residual time in aquatic products and the environment. Its chemical functional group "triphenylmethane" has toxic side effects such as carcinogenicity, teratogenicity and mutagenicity (tri-causing). In addition, MG residues in aquaculture water may also cause serious pollution to water resources. However, due to the good prevention and treatment effect of malachite green on fish diseases, low price, and no effective alternatives, some producers and operators are driven by profit and add malachite green to water during the breeding, transportation, and temporary storage of edible aquatic products. The illegal problem is still prominent.

[0003] Therefore, rapid and sensitive detection of malachite green in water is one of the effective means to monitor and ensure the quality and safety of fresh aquatic products and food. With the in-depth research of nanomaterials, nano-assembly systems and artificially assembled and synthesized nanoscale material systems have attracted more and more attention, which means that the research of nanomaterials can already design, assemble and create new systems according to people's wishes, and more purposefully endow the system with the desired characteristics. The technological leap has further opened the market for the application of nanomaterials and formed a large number of high-tech products in a wide range of fields. For example, a Eu(III)-functionalized nano-MOF probe based on fluorescence resonance energy transfer has been designed for the reversible sensing of trace malachite green (Food Chemistry 2021, 354:129584); there is also a dual-emission ratio sensor constructed using two luminescent materials for the quantitative and visual detection of MG. Adding a certain amount of MG to the solution can change the color of the ratio sensor from red to blue, and the quantitative analysis of malachite green can be achieved by measuring the change in fluorescence intensity (F460 nm / 666 nm) (Food Chemistry 2022, 390:133156). An aptamer sensor for the detection of malachite green constructed based on the method of dual-cleavage functional nucleic acid allostery has good detection performance, with good sensitivity and specificity (Journal of Hazardous Materials 2022, 425:127976). However, due to the cumbersome detection steps and poor stability of these methods, it is still difficult to meet the requirements of simple, rapid and sensitive detection of trace malachite green in production and supervision. Summary of the Invention

[0004] In order to solve the problems existing in the background technology, the present invention provides a new method for sensitively detecting malachite green by using the binding of G4-ThT to malachite green and the occurrence of an inner filter effect between ThT and malachite green.

[0005] The present invention obtains a DNA strand rich in G bases, and the formed G-quadruplex (G4) structure has good affinity with malachite green. After mixing the G4 strand with thioflavin T (ThT), a fluorescent probe G4-ThT is assembled, and its fluorescence has an obvious response to malachite green.

[0006] The method of the present invention can achieve sensitive detection of malachite green without the assistance of other materials, and is more economical, simple and time-saving compared with other methods.

[0007] The technical solution of the present invention includes:

[0008] 1. A G4-ThT fluorescent probe for the detection of malachite green:

[0009] It is formed by mixing a G4 strand and thioflavin T (ThT) to form a G4-ThT structure.

[0010] The G4 chain sequence described is SEQ ID NO.1, i.e., 5'-TGAGGGTGGGTAGGGTGGGTAA-3'.

[0011] The G4 chain obtained in the present invention has a strong affinity for malachite green, and this fluorescent probe only needs to be mixed at room temperature for self-assembly without other steps and instruments.

[0012] Application in the detection of malachite green.

[0013] II. Method for detecting malachite green using the G4-ThT fluorescent probe:

[0014] The method steps for detecting malachite green using the G4-ThT fluorescent probe are as follows:

[0015] Take the G4 chain, thioflavin T (ThT) and the malachite green solution with the concentration to be measured and add them together to a buffer solution, shake and mix well, react at room temperature in the dark, measure the fluorescence spectrum with a microplate reader, and judge the concentration of malachite green according to the fluorescence intensity.

[0016] The G4 chain of the present invention has a good affinity for malachite green. After binding to ThT, ThT emits strong fluorescence, while malachite green can be detected by causing quenching of the G4-ThT fluorescence.

[0017] The detection of the malachite green is based on the change in the fluorescence intensity of G4-ThT affected by the affinity of the target to be measured; when there is no malachite green, ThT and the G4 chain produce strong fluorescence. After adding malachite green, the structure of the G4 chain becomes more stable, promoting the fluorescence inner filter effect between malachite green and ThT, and the ThT fluorescence decreases.

[0018] The fluorescence decrease caused by malachite green in the present invention is not caused by destroying the structure of G4-ThT, but the fluorescence inner filter effect between malachite green and ThT. Since malachite green has a good affinity for the G4 chain, malachite green is adsorbed onto G4-ThT, which will be more conducive to the inner filter effect between malachite green and ThT, thus making the fluorescence decrease more obvious. The G4 chain plays a role in promoting and amplifying the detection of malachite green.

[0019] The stoichiometric ratio of the G4 chain to thioflavin T is 1:2 (molar ratio).

[0020] The buffer solution described is a hepes buffer solution containing 10 mM K + (25 mM hepes, 0.5% v / v Triton X-100, pH 5.2).

[0021] The method of the present invention reacts at room temperature in the dark for 15 minutes, that is, the detection time after adding malachite green is 15 minutes.

[0022] The reaction is directly carried out at room temperature of 25 °C. The detection method of the method of the present invention is a one-step mixed detection. The G4 chain, ThT and malachite green are directly mixed together or mixed arbitrarily one after another. The change in the sample addition order has almost no influence on the detection result.

[0023] Measure the fluorescence spectrum between 450 - 600 nm at an excitation wavelength of 425 nm with an enzyme-labeled instrument, and judge the presence and amount of malachite green in the malachite green solution to be detected according to the decrease degree of the fluorescence value at 488 nm in the fluorescence spectrum.

[0024] According to the fluorescence spectrum, the fluorescence quenching rate at 488 nm is calculated by the following formula. The greater the concentration of malachite green, the more the fluorescence quenching rate decreases, and there is a linear relationship within a certain range:

[0025] (F0 - F) / F0

[0026] Wherein, F0 is the fluorescence intensity without adding malachite green, and F is the fluorescence intensity after adding malachite green;

[0027] According to the fluorescence quenching rate at 488 nm, combined with the relationship between the fluorescence quenching rate and the concentration of malachite green drawn by pre-experiment calibration, determine the concentration of malachite green in the malachite green solution to be detected.

[0028] The present invention discovers that there is an approximate linear relationship between the fluorescence quenching rate and the concentration of malachite green, and it is positively correlated. A linear regression curve between the fluorescence quenching rate and the concentration of malachite green can be drawn by pre-experiment calibration as the relationship.

[0029] The pre-drawn linear regression curve is obtained by the following method: React the fluorescent probe with multiple malachite green solutions with different known concentrations respectively, detect the fluorescence intensity of the reaction mixture at 488 nm, calculate the fluorescence quenching rate through the formula, and plot the fluorescence quenching rate of the reaction mixture at 488 nm against the concentration of malachite green to obtain a linear regression curve.

[0030] The present invention utilizes that G-quadruplex can significantly enhance the fluorescence of the water-soluble fluorescent dye thioflavin T (ThT), and the construction of the G-quadruplex-ThT fluorescent probe is low-cost, simple and rapid, and uses the G-quadruplex-ThT fluorescent probe to rapidly detect malachite green.

[0031] The present invention obtains a fluorescent probe composed of G4 chain c-MYC22 with high affinity for malachite green and ThT, which has a good response to malachite green.

[0032] In specific implementation, the detection of malachite green was carried out in a hepes buffer solution (25 mM hepes, 0.5% (v / v) Triton X-100, pH 5.2). The detection effects in Tris-HCl (10 mM Tris-HCl, pH 7.4) and hepes buffer solution were compared, and the result showed that the detection effect in the hepes buffer solution was the best.

[0033] And the confirmation research experiment of the action mechanism was to judge the mechanism of fluorescence quenching of malachite green through ultraviolet absorption spectrum, circular dichroism spectrum and fluorescence lifetime measurement, and compare the spectral changes after adding malachite green to judge that the action mechanism of malachite green was fluorescence inner filter effect.

[0034] The optimization of the experimental conditions included the optimization of buffer solution, the ratio of G4 to ThT, the sample addition order, the reaction time and the reaction temperature.

[0035] The specific detection steps of the malachite green were as follows:

[0036] Solution preparation: Prepare a hepes solution with a pH of 5.2, a final concentration of 25 mM and containing 0.5% (v / v) Triton X-100, and prepare a malachite green solution with a concentration of 133 ppm using acetonitrile as the solvent;

[0037] In the hepes solution, add a certain amount of G4 chain and ThT so that the final concentration of the G4 chain is 500 nM, the final concentration of ThT is 1 μM, and the final concentration of malachite green is 200 μg / kg. Excite with light at 425 nm and measure the change in fluorescence intensity of the mixed solution at 488 nm before and after adding the malachite green solution;

[0038] In the hepes solution, add G4 and ThT, and then add different amounts of malachite green solution so that the concentration of the G4 chain is 500 nM, the concentration of ThT is 1 μM, and the final concentrations of malachite green are 10, 20, 50, 100, 200, 400, 600, 800, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 and 10000 μg / kg respectively. Excite with light at 425 nm, use a microplate reader to measure the fluorescence spectrum after adding different concentrations of malachite green solution, observe the change relationship of the fluorescence intensity of the solution at 488 nm with the concentration of malachite green, and draw a standard curve; the result shows that the G4-ThT fluorescence sensor has a good linear relationship in the ranges of 50 - 1000 μg / kg and 400 - 6000 μg / kg, and the linear correlation coefficients are 0.99593 and 0.99336 respectively, and the detection limit is as low as 6.82 μg / kg;

[0039] The change in the fluorescence spectrum is as follows: When excited by light at 425 nm, after adding malachite green, the fluorescence intensity of the detection reaction solution at 488 nm in the fluorescence spectrum decreases significantly, indicating that malachite green can effectively quench the fluorescence of G4-ThT, and the fluorescence intensity of G4-ThT decreases with the increase in the concentration of malachite green, indicating that malachite green can be quantitatively detected by the change in fluorescence value.

[0040] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0041] The present invention constructs a label-free, fluorescent, homogeneous biosensor for malachite green based on easily available G4 strands and ThT;

[0042] The present invention adopts a one-step method, and all reagents are added in one step and mixed for reaction for 15 minutes, indicating that this method is simple to operate and the detection reaction is rapid;

[0043] The present invention utilizes the specific adsorption between malachite green and the G4 strand, which not only enhances the stability of the G4 strand but also brings malachite green closer to ThT, enhancing the inner filter effect between malachite green and ThT;

[0044] The malachite green biosensor proposed by the present invention can achieve simple, rapid, low-cost and ultrasensitive detection of malachite green, and has certain versatility and industrialization capabilities;

[0045] The malachite green biosensor proposed by the present invention has a good linear relationship in the ranges of 50 - 1000 μg / kg and 400 - 6000 μg / kg, and the lowest detection limit can reach 6.82 μg / kg. Description of the Drawings

[0046] Figure 1 It is a schematic diagram of the G4-ThT fluorescence probe for rapid detection of malachite green;

[0047] Figure 2 It is a characterization diagram of the mechanism of action of the G4-ThT fluorescence probe for detecting malachite green. (A) Circular dichroism spectra before and after adding malachite green; (B) Overlap spectrum of the absorption spectrum of malachite green and the excitation spectrum of ThT; (C) Fluorescence lifetime spectra before and after adding malachite green.

[0048] Figure 3Response diagram of the G4-ThT fluorescence sensor for detecting malachite green. Figure (A) is the fluorescence spectrum of the G4-ThT fluorescence sensor after adding malachite green at different concentrations; Figure (B) is the scatter plot drawn based on the fluorescence changes of malachite green at different concentrations; Figure (C) is the relationship between the fluorescence quenching rate of the fluorescence probe at 488 nm and the concentration of malachite green when the concentration of malachite green gradually increases from 50 μg / kg to 1000 μg / kg; Figure (D) is the relationship between the fluorescence quenching rate of the fluorescence probe at 488 nm and the logarithm of the concentration of malachite green when the concentration of malachite green gradually increases from 400 μg / kg to 6000 μg / kg.

[0049] Figure 4 Result diagram of the response of the fluorescence probe to oxidized leucomalachite green.

[0050] Figure 5 Specificity result diagram of the fluorescence probe of the present invention for malachite green. Detailed implementation manners

[0051] The present invention will be further described below in conjunction with the accompanying drawings and embodiments. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0052] The embodiments of the present invention are specifically as follows:

[0053] Embodiment 1

[0054] Research on confirming the detection principle of the G4-ThT fluorescence probe for malachite green.

[0055] As Figure 1 shown, the present invention utilizes the characteristics that the G4 chain binds to ThT to emit strong fluorescence and the selected G4 chain has good affinity with malachite green, and based on the phenomenon that the fluorescence quenching is caused by the inner filter effect between malachite green and ThT, a method for rapidly detecting malachite green is developed.

[0056] As Figure 2 (A) shows that after adding malachite green, the circular dichroism spectrum of G4-ThT does not change, indicating that the addition of malachite green does not change the secondary structure of G4-ThT, thus excluding the possible reason that the addition of malachite green destroys the structure of the fluorescence probe and causes the fluorescence to decrease. Figure 2 (B) shows that there is a large area of overlap between the absorption spectrum of malachite green and the excitation spectrum of ThT, indicating that there may be an inner filter effect or a fluorescence resonance energy transfer effect between the two. Combining Figure 2 (C) in the fluorescence lifetime spectrum diagram, the fluorescence lifetime does not change after adding malachite green, indicating that the reason for the fluorescence change is not caused by fluorescence resonance energy transfer, and its mechanism of action is the inner filter effect between malachite green and ThT.

[0057] S1: Take 40 μL of G4 and 20 μL of ThT, with their final concentrations being 1 μM and 2 μM respectively, and measure the circular dichroism spectra before and after adding malachite green respectively.

[0058] S2: Measure the absorption spectrum of malachite green and the excitation spectrum of G4 - ThT on an ultraviolet spectrophotometer, and set the wavelength scanning range to 200 - 700 nm.

[0059] S3: Set the concentration of G4 to 500 nM, the concentration of ThT to 1 μM, and the concentration of malachite green to 400 μg / kg, mix them in a buffer solution, set the total volume to 1.5 mL, and then measure the fluorescence lifetime before and after adding malachite green with an integrated fluorescence spectrometer.

[0060] Conduct test analysis:

[0061] A. Response analysis of the optical probe to malachite green at different concentrations.

[0062] Figure 3 As shown in (A), as the final concentration of malachite green increases from 0 to 10000 μg / kg (from low to high, in sequence: 0, 20, 50, 100, 200, 400, 600, 800, 1000, 2000, 4000, 6000, 8000, and 10000 μg / kg), the fluorescence intensity of the fluorescent probe G4 - ThT at 488 nm gradually weakens.

[0063] As Figure 3 As shown in (B), make a scatter plot of the quenching rate ((F0 - F) / F0) of the fluorescence intensity of G4 - ThT at 488 nm versus the concentration of malachite green. Through linear fitting analysis, it can be observed that when the concentration of malachite green is in the ranges of 50 to 1000 μg / kg and 400 - 6000 μg / kg, the fluorescence quenching rate at 488 nm has a linear relationship with the concentration of malachite green. As Figure 3 As shown in (C)(D), the linear regression equations of the concentration of malachite green and the fluorescence quenching rate are y = 5.63378x - 9.48111; the linear regression equation of the logarithm of the concentration of malachite green and the fluorescence quenching rate is y = 60.91582x - 144.07006, and the linear correlation coefficients R 2 are 0.99593 and 0.99336 respectively.

[0064] B. Detection test of the fluorescent probe for leucomalachite green.

[0065] In a buffer solution containing 500 nM G4 and 1 μM ThT, 10 ppm of leucomalachite green was added, and 40 μM of 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) was added. Compared with the combination of directly adding 10 ppm of malachite green, using ultraviolet-visible absorption spectroscopy ( Figure 4 (A)) and its effect on fluorescence intensity ( Figure 4 (B)), it can be seen that the leucomalachite green after oxidation by DDQ was successfully converted into malachite green, and had the same fluorescence quenching effect as malachite green with the same concentration.

[0066] C. Specific detection of malachite green by fluorescence probe.

[0067] Malachite green, leucomalachite green, oxytetracycline, tetracycline, doxycycline, crystal violet and mixtures of these antibiotics and malachite green were respectively added to the G4-ThT fluorescence probe. The concentration of malachite green was 10 μM, and the concentrations of other antibiotics were 10 times that of malachite green except for crystal violet. The responses of the fluorescence probe to different antibiotics and mixtures of other antibiotics and malachite green were explored. As Figure 5 shown, malachite green can effectively quench the fluorescence of G4-ThT, and the responses of other mixed antibiotics containing the same concentration of malachite green were almost the same, while other antibiotics such as leucomalachite green had almost no effect on the fluorescence of G4-ThT. It shows that the fluorescence probe has good specificity for malachite green, and the presence of other antibiotics will not interfere with the detection of malachite green.

[0068] C. Addition recovery experiments in pond water, small yellow croaker and prawn.

[0069] S1: A certain concentration of malachite green was added to pond water, small yellow croaker and prawn respectively, and the three addition concentrations were 50, 500 and 1000 μg / kg.

[0070] S2: For the pond water sample, directly take the added sample in S1 for detection. In a 600 μL centrifuge tube, successively add 40 μL of tap water or pond water added with malachite green, 90 μL of ultrapure water, 20 μL of 10-fold concentrated hepes buffer solution, 20 μL of 10 mM KCl, 10 μL of 10 μM c-MYC22, and 20 μL of 10 μM ThT, shake and mix well, and react at room temperature in the dark for 15 min; for the small yellow croaker and prawn samples, only take the meat of the bought fish and shrimp and mash them into minced meat; weigh 2 g of the sample into a 15 mL centrifuge tube, add a certain concentration of malachite green, then add 6 mL of acetonitrile and shake and mix well. Weigh 2 g of acidic alumina, add it to the centrifuge tube and then centrifuge at a speed of 4000 r / min for 5 min; take the supernatant and filter it through a 0.22 μm organic filter membrane, then take 1 mL of the filtrate and blow it with air at 70 °C until it is completely dried; add 100 μL of acetonitrile to dissolve the malachite green and the sample matrix adhering to the tube wall, shake and mix well, and then take 20 μL for detection.

[0071] S3: Use a pipette to take out 185 μL of the reaction solution in S2 and add it to the sample well of a 96-well microplate suitable for measuring fluorescence spectra. Set the excitation wavelength of the microplate reader to 425 nm, set the emission spectrum acquisition range to 450 - 600 nm, set the step size to 2 nm, and scan the fluorescence emission spectrum.

[0072] S4: According to the steps of S2 - S3 above, the sample solution of malachite green with the same added concentration is measured in parallel 5 times.

[0073] S5: Calculate the recovery rate and relative standard deviation in combination with the results in Example 2.

[0074] Table 1 Results of the addition recovery test

[0075]

[0076] Note: The added concentration is the concentration calculated after adding a known amount of malachite green to the sample; the detected concentration is the concentration detected in the added sample. The addition recovery test is used to verify the reliability of the method.

[0077] It can be seen that in the detection method of the present invention, the DNA strand does not require any modification, and only one-step reaction is needed, and then a microplate reader is used for high-throughput and rapid detection. The detection time can be controlled within 15 min, and it has the characteristics of economy, simplicity, rapidity and accuracy. It can accurately quantify the concentration of malachite green as low as 6.28 μg / kg, and has good application prospects.

[0078] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions.

[0079] Although specific embodiments of the present invention have been given, it should be understood that the present invention can be further improved. In general, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements of the present invention, including those that depart from the scope disclosed in this application and are made using conventional techniques known in the art.

[0080] The gene sequences related to the present invention are as follows:

[0081] SEQ ID No.1;

[0082] Name: G4 strand sequence

[0083] DNA type: other DNA

[0084] Organism source: artificial sequence (Artificial Sequence) / synthetic construct

[0085] TGAGGGTGGGTAGGGTGGGTAA.

Claims

1. A G4-ThT fluorescent probe for malachite green detection, characterized in that: Formed by mixing G4 chain and thioflavin T; the stoichiometric ratio of the G4 chain to thioflavin T is 1:2; The G4 chain sequence is SEQ ID NO.

1.

2. Use of the G4-ThT fluorescent probe according to claim 1, characterized in that: Application in the detection of malachite green.

3. A method for detecting malachite green using the G4-ThT fluorescent probe according to claim 1, characterized in that: The method steps are as follows: Take the G4 chain, thioflavin T and the malachite green solution to be detected and add them together to a buffer solution, shake and mix well, react at room temperature in the dark, measure the fluorescence spectrum, and judge the concentration of malachite green according to the fluorescence intensity; The stoichiometric ratio of the G4 chain to thioflavin T is 1:2; The buffer solution is a hepes buffer solution containing 10 mM K + .

4. The method for detecting malachite green by the G4-ThT fluorescent probe according to claim 3, wherein: The method reacts at room temperature in the dark for 15 min.

5. The method for detecting malachite green by the G4-ThT fluorescent probe according to claim 3, wherein: The reaction is directly carried out at room temperature of 25 °C.

6. The method for detecting malachite green by the G4-ThT fluorescent probe according to claim 3, wherein: Use a microplate reader to measure the fluorescence spectrum between 450~600 nm at an excitation wavelength of 425 nm, and judge the presence and amount of malachite green in the malachite green solution to be detected according to the degree of decrease in the fluorescence value at 488 nm in the fluorescence spectrum.

7. The method for detecting malachite green by the G4-ThT fluorescent probe according to claim 6, wherein: According to the fluorescence spectrum, the fluorescence quenching rate at 488 nm is calculated using the following formula: (F0 - F) / F0 Wherein, F0 is the fluorescence intensity without adding malachite green, and F is the fluorescence intensity after adding malachite green; Determine the concentration of malachite green in the malachite green solution to be detected according to the fluorescence quenching rate at 488 nm in combination with the pre-established relationship between the fluorescence quenching rate and the concentration of malachite green.

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