A fluorescent probe for detecting triphenylmethane compounds, a test paper, and a preparation method and application thereof

Abstract

The present invention relates to the technical field of fluorescent probes, and in particular to a fluorescent probe, test paper, and preparation method and application thereof for detecting malachite green, a triphenylmethane compound. The fluorescent probe comprises a fluorescent ion pair formed by rhodamine B (RB) and perfluorinated tetraphenylborate ion (F5TPB); the triphenylmethane compounds are malachite green (MG), crystal violet (CV), and methyl violet (MV); the detection limit for malachite green is 0.1776 μmol / L (3σ / k, n=11); the present invention also provides a test paper for detecting triphenylmethane compounds, which can simply and conveniently detect malachite green and its residual amount.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and in particular to a fluorescent probe for detecting triphenylmethane compounds, a test paper, and a preparation method and application thereof. Background Art

[0002] In recent years, my country's total fishery output has steadily increased, making it one of the world's leading fishing nations. Aquaculture is a key sector of the fishery. However, the continued abuse of banned fishery drugs, such as malachite green and crystal violet, poses risks to aquatic product quality and impacts consumer health and safety. MG is widely used in aquaculture due to its low price and ease of use. However, due to its high toxicity, high residual, teratogenic, carcinogenic, and mutagenic properties, it can affect the normal growth and development of fish and pose a serious threat to human health. Many countries, including China, have listed MG as a banned drug in aquaculture.

[0003] Among the numerous methods for detecting MG, high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), electrochemical methods, and spectrophotometry are the more commonly used. HPLC offers high separation efficiency and sensitivity. The national standard methods for detecting MG are HPLC and liquid chromatography-mass spectrometry, with detection limits of 2.0 μg / kg and 0.5 μg / kg, respectively. However, the HPLC method requires multiple extraction and centrifugation pretreatment, which is complex, time-consuming, and requires large amounts of solvent. ELISA offers high selectivity, sensitivity, and rapidity. However, traditional monoclonal antibodies can only detect the residual amount of a single drug in a sample, making it difficult to objectively reflect the total residual amount of MG and latent malachite green. This means that while quantitative, it can only detect a single indicator.

[0004] Spectrophotometry allows for qualitative and quantitative analysis of substances by measuring light absorption within a specific range or wavelength. While simple and easy to perform, spectrophotometry suffers from high detection limits and low sensitivity and selectivity. A common strategy involves using solid-phase extraction (SPE) to enrich MG before testing. This yields a high response and effectively improves spectrophotometric sensitivity.

[0005] Among the numerous instrumental analysis techniques, fluorescence analysis is an emerging spectral analysis method with the advantages of ease of use, high sensitivity, wide linear range, high accuracy, and the ability to simultaneously detect multiple channels or track substances. Summary of the Invention

[0006] In order to solve the problem of inconvenient detection of triphenylmethane compounds in the above-mentioned prior art, the present invention provides a fluorescent probe and test paper for detecting triphenylmethane compounds;

[0007] In order to solve the above technical problems, one of the technical solutions provided by the present invention is as follows:

[0008] A fluorescent probe for detecting triphenylmethane compounds, comprising: a fluorescent ion pair formed by rhodamine B (RB) and perfluorinated tetraphenylborate ion (F5TPB);

[0009] The triphenylmethane compounds are malachite green, crystal violet and methyl violet.

[0010] The second technical solution provided by the present invention is as follows:

[0011] A method for preparing a fluorescent probe for detecting triphenylmethane compounds comprises:

[0012] (1) dissolving rhodamine B (RB) in a first solvent;

[0013] (2) dissolving perfluorinated tetraphenylborate in a second solvent;

[0014] (3) prepared by mixing the solution of (1) and (2);

[0015] The triphenylmethane compounds are malachite green, crystal violet and methyl violet;

[0016] Preferably, the concentration of Rhodamine B (RB) is 1-4 mg / ml;

[0017] More preferably, the concentration of Rhodamine B (RB) is 2 mg / ml;

[0018] Preferably, the concentration of the perfluorinated tetraphenylborate is above 0.25 mg / ml;

[0019] More preferably, the concentration of the perfluorinated tetraphenylborate is 1 mg / ml.

[0020] In one embodiment, before mixing the solutions of (1) and (2), polymethyl methacrylate (PMMA) powder is dissolved in a third solvent and mixed with the solutions of (1) and (2);

[0021] Preferably, the first solvent is selected from acetonitrile;

[0022] Preferably, the second solvent is selected from acetonitrile;

[0023] Preferably, the third solvent is selected from acetonitrile.

[0024] In one embodiment, the concentration of the polymethyl methacrylate (PMMA) is 10-30 mg / ml;

[0025] Preferably, the concentration of the polymethyl methacrylate (PMMA) is 20 mg / ml.

[0026] The third technical solution provided by the present invention is as follows:

[0027] A test paper for detecting triphenylmethane compounds, comprising:

[0028] filter paper;

[0029] a fluorescent layer located on the filter paper;

[0030] The fluorescent layer comprises the fluorescent probe as described above, or the fluorescent layer comprises the fluorescent probe prepared by the preparation method as described above.

[0031] In one embodiment, the test paper further comprises: a light stabilizing layer located on the fluorescent layer;

[0032] Preferably, the light stabilizing layer comprises SiO2 nanospheres;

[0033] Preferably, the particle size of the SiO2 nanospheres is 100-300 nm.

[0034] The fourth technical solution provided by the present invention is as follows:

[0035] A method for manufacturing a test paper for detecting triphenylmethane compounds, comprising:

[0036] (1) dissolving rhodamine B (RB) in a first solvent;

[0037] (2) dissolving perfluorinated tetraphenylborate in a second solvent;

[0038] (3) dissolving polymethyl methacrylate (PMMA) powder in a third solvent;

[0039] (4) mixing the solutions of (1), (2) and (3) to prepare a fluorescent probe;

[0040] (5) loading the fluorescent probe onto the filter paper to form a fluorescent layer;

[0041] Preferably, the first solvent is selected from acetonitrile;

[0042] Preferably, the second solvent is selected from acetonitrile;

[0043] Preferably, the third solvent is selected from acetonitrile.

[0044] In one embodiment, it further includes:

[0045] preparing a color fixing agent, and coating the color fixing agent on the surface of the fluorescent layer to form a light stabilizing layer;

[0046] Preferably, the light stabilizing layer comprises SiO2 nanospheres;

[0047] Preferably, the particle size of the SiO2 nanospheres is 100-300 nm;

[0048] Preferably, the color fixing agent includes component A and component B; the component A includes aqueous ammonia and anhydrous ethanol, and the component B includes silicic acid and anhydrous ethanol.

[0049] The fifth technical solution provided by the present invention is as follows:

[0050] Application of the fluorescent probe as described above in the field of detection / removal of triphenylmethane compounds.

[0051] Based on the above, the fluorescent probe provided by the present invention has a detection limit of 0.1776 μmol / L (3σ / k, n=11) for malachite green, and can simply and conveniently detect malachite green (MG), crystal violet (CV) and methyl violet (MV) as well as their residues.

[0052] Other features and beneficial effects of the present invention will be described in the following description, and in part will become apparent from the description, or will be understood by practicing the present invention. The objectives and other beneficial effects of the present invention can be achieved and obtained by the structures particularly pointed out in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0054] Figure 1 A flow chart for preparing test paper for detecting triphenylmethane compounds provided in an embodiment of the present invention;

[0055] Figure 2 The effect of PMMA concentration on the fluorescence performance of the test paper provided in the embodiment of the present invention;

[0056] Among them, the first row is the fluorescent test strips without F5TPB added; the second row is the fluorescent test strips with F5TPB added and the PMMA concentrations from left to right are 0, 5, 10, 20, and 30 mg / ml respectively;

[0057] Figure 3 The effect of the concentration of RB on the fluorescence performance of the test paper provided in the embodiment of the present invention;

[0058] Among them, the first row is the fluorescent test strips without F5TPB added; the second row is the fluorescent test strips with F5TPB added and the RB concentrations from left to right are 0.25, 0.5, 1.0, 2.0, 4.0, and 6.0 mg / ml;

[0059] Figure 4 The effect of the concentration of F5TPB on the fluorescence performance of the test paper provided in the embodiment of the present invention;

[0060] Among them, the concentrations of F5TPB from left to right are 0, 0.25, 0.5, 1.0, and 2.0 mg / ml;

[0061] Figure 5 Fluorescence emission spectra (left) and fluorescence intensity at 590 nm (right) of test papers with different loadings and RB solution (10 μmol / L);

[0062] Figure 6 is the relative fluorescence intensity of different test strips after being placed on different days;

[0063] Figure 7 The infrared spectra of the test paper containing RB+PMMA, the test paper containing RB+PMMA+F5TPB, and the test paper containing RB+PMMA+F5TPB+fixing agent are shown in FIG.

[0064] Figure 8 This is a scanning electron microscope image of the test paper prepared in Example 1;

[0065] Among them, the upper left is a blank test strip with a scale of 1μm; the upper right is a blank test strip with a scale of 10μm; the lower left is a fluorescent test strip with a scale of 1μm; the lower right is a fluorescent test strip with a scale of 10μm;

[0066] Figure 9 The fluorescence response of the fluorescent test paper of Example 1 of the present invention to different aquaculture fish medicines;

[0067] Figure 10 The fluorescence quenching effect of different concentrations of MG on the test paper prepared in the embodiment of the present invention;

[0068] Figure 11 is the relationship between quenching efficiency (F0 / F) and MG concentration;

[0069] Figure 12 The figure shows the change of fluorescence quenching coefficient of the fluorescent test paper prepared in the embodiment of the present invention after adding MG for different time periods;

[0070] Figure 13 This is a fluorescence lifetime spectrum diagram of the test strip prepared in an embodiment of the present invention. DETAILED DESCRIPTION

[0071] In the description of the present invention, it should be noted that all terms used in the present invention (including technical terms and scientific terms) have the same meanings as those generally understood by ordinary technicians in the field to which the present invention belongs, and should not be understood as limiting the present invention; it should be further understood that the terms used in the present invention should be understood to have meanings consistent with the meanings of these terms in the context of this specification and in the relevant fields, and should not be understood in an idealized or overly formal sense, unless explicitly defined as such in the present invention.

[0072] The present invention provides a fluorescent probe for detecting triphenylmethane compounds, comprising: a fluorescent ion pair formed by rhodamine B and perfluorinated tetraphenylborate ion (F5TPB);

[0073] In the present invention, the fluorescent ion pair used comprises a cation of an organic dye and a bulky anion. The anion serves to isolate the fluorescent dye and simultaneously gathers the excitation light, thereby greatly enhancing the fluorescence of the dye.

[0074] The triphenylmethane compounds are malachite green (MG), crystal violet (CV) and methyl violet (MV).

[0075] The present invention provides a method for preparing a fluorescent probe for detecting triphenylmethane compounds, comprising:

[0076] (1) dissolving rhodamine B in a first solvent; preferably, the first solvent is selected from acetonitrile;

[0077] (2) dissolving perfluorinated tetraphenylborate in a second solvent; preferably, the second solvent is selected from acetonitrile; preferably, the perfluorinated tetraphenylborate is selected from at least one of potassium tetrakis(pentafluorophenyl)borate and sodium tetrakis(pentafluorophenyl)borate;

[0078] (3) dissolving PMMA powder in a third solvent; preferably, the third solvent is selected from acetonitrile;

[0079] (4) prepared by mixing a solution of (1), (2) and (3);

[0080] The triphenylmethane compounds are malachite green, crystal violet and methyl violet.

[0081] In this invention, the anionic backbone is composed of tetrakis(pentafluorophenyl)borate (F5TPB) ions and the organic dye used is rhodamine B. Further dispersing both in polymethyl methacrylate (PMMA) powder significantly enhances the fluorescence intensity of the fluorescent ion pair. Furthermore, this method significantly suppresses the ACQ effect and offers the advantages of easy preparation, low cost, and a short production cycle.

[0082] Therefore, the fluorescent ion pairs provided by the present invention can be used in test strips to effectively promote the rapid detection of drug residues in aquatic products.

[0083] The present invention provides a test paper for detecting triphenylmethane compounds, comprising:

[0084] filter paper;

[0085] a fluorescent layer located on the filter paper;

[0086] The fluorescent layer includes the fluorescent probe for detecting triphenylmethane compounds as described above.

[0087] In a preferred embodiment of the present invention, the fluorescent layer further comprises PMMA.

[0088] In a preferred embodiment of the present invention, the test paper further includes: a photostabilizing layer located on the fluorescent layer; preferably, the photostabilizing layer contains SiO2 nanospheres. In this embodiment, the test paper is modified by covering a layer of transparent SiO2 nanospheres using sol-gel technology, thereby improving the photostability of the fluorescent ion pairs; preferably, the particle size of the SiO2 nanospheres is 100-300. If the particle size of the microspheres is too small, the surface coverage effect is relatively insignificant, which can easily cause the loss of rhodamine B. If the particle size of the microspheres is too large, the directional arrangement of SiO2 may produce structural colors in the visible light wavelength range, causing interference.

[0089] refer to Figure 1 RB and F5TPB can form a fluorescent ion pair. Due to the spatial isolation effect of F5TPB, RB can maintain an extremely high fluorescence intensity. In the embodiment of the present invention, ordinary test paper is used as a carrier, the RB-F5TPB composite system is loaded on the filter paper, and PMMA is added as a dispersant to further improve the fluorescence intensity. Finally, sol-gel polymerization is performed to form a light-stabilizing layer on the surface of the fluorescent layer.

[0090] The present invention provides a method for manufacturing a test paper for detecting triphenylmethane compounds, comprising:

[0091] (1) dissolving rhodamine B in a first solvent; preferably, the first solvent is selected from acetonitrile;

[0092] (2) dissolving perfluorinated tetraphenylborate in a second solvent; preferably, the second solvent is selected from acetonitrile; preferably, the perfluorinated tetraphenylborate is selected from at least one of potassium tetrakis(pentafluorophenyl)borate and sodium tetrakis(pentafluorophenyl)borate;

[0093] (3) dissolving PMMA powder in a third solvent; preferably, the third solvent is selected from acetonitrile.

[0094] (4) mixing the solutions of (1), (2) and (3) to prepare a fluorescent probe;

[0095] (5) loading the fluorescent probe onto the filter paper to form a fluorescent layer;

[0096] (6) A color fixing agent is prepared and applied to the surface of the fluorescent layer to form a light stabilizing layer.

[0097] refer to Figure 2 In the present embodiment, the concentration of RB in the mixed solution was fixed at 1 mg / mL, the concentration of F5TPB was fixed at 1 mg / mL, the concentration of PMMA was 0, 5, 10, 20, and 30 mg / ml, and the amount of the fixing agent added was 50 uL. In addition, a group of blank mixed solutions were prepared under the same conditions except that F5TPB was not added. The fluorescence test results were as follows: Figure 2 As shown in the figure, the test strips containing F5TPB emit orange-yellow fluorescence visible to the naked eye. Within a certain range, the higher the PMMA concentration, the greater the fluorescence intensity of the test strips.

[0098] Based on the above, the concentration of PMMA is preferably 10-30 mg / ml, and the concentration can specifically be 10, 15, 20, 25, 30 mg / ml, or any point value between the above two. When the PMMA concentration is 20 mg / ml, the fluorescence intensity is at a high point, and the subsequent further increase in the PMMA concentration does not significantly change the fluorescence intensity. Therefore, more preferably, the PMMA concentration is 20 mg / ml.

[0099] refer to Figure 3 In the present embodiment, the PMMA concentration in the mixed solution was fixed at 20 mg / mL, the F5TPB concentration was fixed at 1 mg / mL, the RB concentrations were 0.25, 0.5, 1.0, 2.0, 4.0, and 6.0 mg / ml, and the amount of the fixing agent added was 50 uL. In addition, a group of blank mixed solutions were prepared under the same conditions except that F5TPB was not added. The fluorescence test results are shown in Figure 2. Figure 3 As shown. Within a certain range, the higher the concentration of RB, the greater the fluorescence intensity of the test strip;

[0100] Based on the above, the concentration of rhodamine B is preferably 1-4 mg / ml, and the concentration can be specifically 1, 2, 3, 4 mg / ml, or any point value between the above two. More preferably, the concentration of rhodamine B is 2 mg / ml, and the fluorescence intensity of the test paper is the largest.

[0101] refer to Figure 4 In the embodiment of the present invention, the PMMA concentration in the mixed solution is fixed at 20 mg / mL, the RB concentration is fixed at 2 mg / mL, the F5TPB concentrations are 0, 0.25, 0.5, 1.0, and 2.0 mg / ml, and the amount of the fixing agent added is 50 uL. The fluorescence test results are as follows Figure 4It can be found that adding a small amount of F5TPB has a significant fluorescence enhancement effect. Within a certain range, the higher the concentration of F5TPB, the greater the fluorescence intensity of the test strip.

[0102] Based on the above, it is preferred that the concentration of the perfluorinated tetraphenylborate is above 0.25 mg / ml, and the concentration can specifically be 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 mg / ml, or any point value between two of the above; when the F5TPB concentration is 1 mg / ml, the fluorescence intensity is at a high point, and the subsequent further increase in the F5TPB concentration does not significantly change the fluorescence intensity. Therefore, it is more preferred that the concentration of the perfluorinated tetraphenylborate is 1 mg / ml.

[0103] In a preferred embodiment, the color fixing agent includes component A and component B; component A includes ammonia water and anhydrous ethanol, and component B includes silicic acid and anhydrous ethanol; wherein, after component A and component B are mixed, the tetraethyl silicate therein will spontaneously hydrolyze and cross-link under the alkaline conditions of ammonia water to form SiO2 nanospheres.

[0104] In a preferred embodiment, the light stabilizing layer comprises SiO2 nanospheres; preferably, the particle size of the SiO2 nanospheres is 100-300 nm.

[0105] Application of the fluorescent probe as described above in the field of detection / removal of triphenylmethane compounds.

[0106] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments; the technical features designed in different implementation modes of the present invention described below can be combined with each other as long as they do not conflict with each other; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0107] Preparation of fixing agent

[0108] Component A: Add 25 ml of pure water, 9 ml of ammonia water and 16 ml of anhydrous ethanol to a 200 ml round-bottom flask and stir in a 30°C water bath at 600 rpm on a magnetic stirrer for 5 minutes.

[0109] Component B: Add 4.5 ml of silicic acid and 45.5 ml of anhydrous ethanol to a 200 ml round-bottom flask and stir in a 30 ° C water bath at 600 rpm with a magnetic stirrer for 5 minutes;

[0110] Use a clean glass rod to quickly drain component B into component A and mix evenly.

[0111] Example 1

[0112] This example is used to prepare fluorescent test paper:

[0113] Cut regular qualitative filter paper into rectangular strips 3 cm long and 1.1 cm wide, roughly equal in width to the diagonal length of a 1 cm cuvette. Disperse RB solution, F5TPB solution, and PMMA in a centrifuge tube to form a mixture containing 20 mg / mL PMMA, 2 mg / mL RB, and 1 mg / mL F5TPB. Add 100 μl of this mixture dropwise onto the filter paper strip. Wait 10 minutes for the filter paper strip to dry, then add 50 μl of color fixative. Allow the solvent on the filter paper to evaporate naturally to obtain fluorescent test paper.

[0114] Example 2

[0115] In this example, the fluorescent test paper prepared in Example 1 was tested for fluorescence performance;

[0116] Test Method: Measure the fluorescence intensity of the test strip in a fluorescence spectrophotometer. Then, place the strip in a UV chamber (wavelength 312nm) to view and photograph the fluorescence. Fluorescence testing conditions are an excitation wavelength of 564nm, an excitation slit of 2.5nm, an emission slit of 5nm, and a photomultiplier tube (PMT) voltage of 430V. Place the test strip vertically and diagonally in a 1cm fluorescence cuvette, with the load facing the light source.

[0117] The test papers with F5TPB (containing PB solution and PMMA), the test papers without F5TPB solution (containing RB solution and PMMA) and the pure RB solution (10 μmol / L) were used as comparisons. The fluorescence intensity was tested under the same conditions. Figure 5 As can be seen, the fluorescence of the test strips without F5TPB increased by more than 10-fold. Under the fluorescence test conditions, the RB solution is almost non-luminescent, with its spectral curve almost coinciding with the coordinate axes. This is also true for the fluorescence spectra of other RB solutions, such as 1 μmol / L and 5 μmol / L. The fluorescence of the test strips without F5TPB is also stronger than that of the RB solution. This is because the PMMA component on the test strips effectively disperses the RB molecules, preventing aggregation and fluorescence quenching.

[0118] The fluorescent test strips prepared in Example 1 were stored in the dark at room temperature. The fluorescence intensity of the test strips at an emission wavelength of 590 nm was measured at intervals of a certain period of time under an excitation wavelength of 564 nm, an excitation slit of 2.5 nm, an emission slit of 5 nm, and a photomultiplier voltage of 430 V. The fluorescence intensity of the freshly prepared test strips was taken as 100%. The relative fluorescence intensity of the test strips after different storage days was calculated. The results are shown in FIG. Figure 6 shown.

[0119] As can be seen, the SiO2 nanospheres covering the test paper act as an air barrier. After a month, the fluorescence intensity of the fluorescent test strips treated with color fixation remained relatively stable, indicating that the color fixation treatment effectively prevented RB from fading due to light. In contrast, the stability of the test strips without the color fixation agent was poor.

[0120] Example 3

[0121] In this embodiment, infrared detection is performed on a test paper containing RB+PMMA, a test paper containing RB+PMMA+F5TPB, and a test paper containing RB+PMMA+F5TPB+fixing agent.

[0122] To three small glass dishes, add 500 μl of the RB+PMMA mixed solution, 500 μl of the RB+PMMA+F5TPB solution, and 700 μl of the RB+PMMA+F5TPB+fixing agent solution (500 μl of RB+PMMA+F5TPB and 200 μl of fixing agent). After the solution dries, gently scrape off the powder adhering to the glass dish with a spatula, grind it with a certain amount of KBr, and press it into a pellet for infrared detection.

[0123] The results are as follows Figure 7 As shown, for RB, the CH stretching vibration is located at 2960 cm -1 The stretching vibration of carboxyl group is located at 1728cm -1 , the N-aryl band is located at 1450 cm -1 , CO vibration is located at 1150cm -1 After adding PMMA solution, F5TPB solution and fixing agent, the functional groups of RB still exist, that is, the high fluorescence activity of RB is still retained.

[0124] Example 4

[0125] In this example, the fluorescent test paper prepared in Example 1 was subjected to SEM scanning electron microscopy;

[0126] This example shows the SEM images of a blank test paper and a test paper loaded with RB+F5TPB+PMMA+fixing agent. Figure 8As shown in the figure, the blank test papers (upper left and right) show no microspheres on their surface. However, the test papers loaded with RB, F5TPB, PMMA, and a color-fixing agent (lower left and right) show numerous microspheres, likely silica nanospheres in the color-fixing agent. Otherwise, the morphologies of the two test papers are essentially identical, indicating that RB, F5TPB, and PMMA are well dispersed throughout the test papers.

[0127] Example 5

[0128] This example tests the fluorescence response of the fluorescent test paper prepared in Example 1 to different aquaculture fish medicines;

[0129] The response results of different fish drugs to fluorescent test paper can be found in Figure 9 As can be seen, malachite green (MG), crystal violet (CV), and methyl violet (MV) have a significant quenching effect on RB-F5TPB. Other fish drugs have no significant quenching effect on the fluorescent test paper. This is because MG, CV, and MV have similar structures and their absorption wavelengths overlap to varying degrees with the absorption peak of the fluorescent ion for RB-F5TPB (590 nm), resulting in varying degrees of fluorescence resonance energy transfer.

[0130] Example 6

[0131] This example is a quantitative test of malachite green (MG) by the fluorescent test paper prepared in Example 1;

[0132] After the fluorescence strips are quenched by MG, the emission peak wavelength of the fluorescent strips moves slightly forward, and the fluorescence intensity decreases as the concentration of MG increases. The corresponding fluorescence intensity of the fluorescent strips at an emission wavelength of 590nm is tested when the final concentration of MG is 0.5, 1, 5, 10, 20, and 50umol / l, and a standard curve is drawn. The results are as follows: Figure 10 shown.

[0133] F0 represents the fluorescence intensity of the test strip at a wavelength of 590 nm when MG is not added, and F represents the fluorescence intensity of the test strip at a wavelength of 590 nm when MG is added. A standard curve was established using the quenching efficiency (F0 / F) and MG concentration. The relationship between F0 / F and MG concentration was roughly linear, as shown in Figure 2. Figure 11 shown.

[0134] At low concentrations, the linear equation is F0 / F=0.4107c+0.4418 (0.5-10 μmol / l, R 2 =0.9598);

[0135] At high concentrations, the linear equation is F0 / F=2.1114c-6.1992 (10-50 μmol / l, R 2=0.9966);

[0136] The detection limit was 0.18 μmol / L (3σ / k, n=11).

[0137] Example 7

[0138] This example is a test of the fluorescence stability time of malachite green (MG) using the fluorescent test paper prepared in Example 1;

[0139] After adding MG solution (10μmol / L), the fluorescence intensity of the test paper was measured at different times. Since it takes at least 5 minutes for the solution to evaporate completely, the first measurement starts from the 5th minute. Figure 12 It can be seen that from the 5th minute to the 20th minute, the fluorescence tends to be stable.

[0140] Furthermore, the fluorescence response mechanism of malachite green (MG) on the test strip;

[0141] Sample 1 is a fluorescent test strip, and sample 2 is a fluorescent test strip to which 50ul of MG solution (50umol / L) is slowly added. After 10 minutes, the solvent is naturally evaporated and the fluorescence lifetime is measured. The results are as follows: Figure 13 shown.

[0142] Perform fluorescence lifetime fitting, the fitting formula is:

[0143]

[0144] The fitting results are as follows:

[0145] The fluorescence lifetime of the fluorescent test strip was 3.7883±0.0109 ns, with a parameter B1 of 2451.1580±8.6780. The fluorescence lifetime of the fluorescent test strip with MG added was 2.4994±0.0140 ns, with a parameter B1 of 957.6770±6.7675. Both fluorescence lifetimes were in the nanosecond range, very short, indicating that RB emitted fluorescence rather than phosphorescence. Adding the quencher MG shortened the fluorescence lifetime, indicating that the fluorescence quenching of the ion pair by MG is dynamic quenching. The change in fluorescence lifetime indicates that fluorescence resonance energy transfer (FRET) occurs between RB and MG, rather than an inner filter effect. Because RB and MG are both molecular, they can be close together, within a distance of less than 10 angstroms. Furthermore, there is a certain degree of overlap between the emission spectrum of RB and the absorption spectrum of MG, satisfying the requirements for FRET.

[0146] In addition, those skilled in the art should understand that, although there are many problems in the prior art, each embodiment or technical solution of the present invention may be improved in only one or several aspects, without having to simultaneously solve all the technical problems listed in the prior art or background art. Those skilled in the art should understand that any content not mentioned in a claim should not be construed as limiting the claim.

[0147] The terms "first," "second," and the like (if any) in the description and claims of the embodiments of the present invention are used to distinguish similar objects and are not necessarily used to describe a particular sequence or order.

[0148] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A test paper for detecting triphenylmethane compounds, characterized in that: include: filter paper; a fluorescent layer located on the filter paper; The fluorescent layer comprises a fluorescent probe, and the fluorescent probe comprises a fluorescent ion pair formed by rhodamine B and perfluorinated tetraphenylborate ion; The triphenylmethane compounds are malachite green, crystal violet and methyl violet.

2. The test paper for detecting triphenylmethane compounds according to claim 1, wherein The preparation method of the fluorescent probe comprises: (1) dissolving rhodamine B in a first solvent; (2) dissolving perfluorinated tetraphenylborate in a second solvent; (3) Prepared by mixing the solutions of (1) and (2).

3. The test paper for detecting triphenylmethane compounds according to claim 2, wherein The concentration of the rhodamine B is 1-4 mg / ml.

4. The test paper for detecting triphenylmethane compounds according to claim 2, wherein The concentration of the rhodamine B is 2 mg / ml.

5. The test paper for detecting triphenylmethane compounds according to claim 2, wherein The concentration of the perfluorinated tetraphenylborate is above 0.25 mg / ml.

6. The test paper for detecting triphenylmethane compounds according to claim 2, wherein The concentration of the perfluorinated tetraphenylborate was 1 mg / ml.

7. The test paper for detecting triphenylmethane compounds according to claim 2, wherein Before mixing the solutions of (1) and (2), polymethyl methacrylate powder is dissolved in a third solvent and mixed with the solutions of (1) and (2).

8. The test paper for detecting triphenylmethane compounds according to claim 7, wherein The first solvent is selected from acetonitrile; the second solvent is selected from acetonitrile; and the third solvent is selected from acetonitrile.

9. The test paper for detecting triphenylmethane compounds according to claim 7, wherein The concentration of the polymethyl methacrylate is 10-30 mg / ml.

10. The test paper for detecting triphenylmethane compounds according to claim 7, wherein The concentration of the polymethyl methacrylate is 20 mg / ml.

11. The test paper for detecting triphenylmethane compounds according to claim 1, wherein The test paper further includes a light-stabilizing layer located on the fluorescent layer.

12. The test paper for detecting triphenylmethane compounds according to claim 11, wherein The light stabilizing layer comprises SiO2 nanospheres.

13. The test paper for detecting triphenylmethane compounds according to claim 12, characterized in that, The particle size of the SiO2 nanoparticles is 100-300nm.

14. A method for manufacturing a test paper for detecting triphenylmethane compounds according to any one of claims 1 to 13, characterized in that: include: (1) dissolving rhodamine B in a first solvent; (2) dissolving perfluorinated tetraphenylborate in a second solvent; (3) dissolving polymethyl methacrylate powder in a third solvent; (4) mixing the solutions of (1), (2) and (3) to prepare a fluorescent probe; (5) The fluorescent probe is loaded onto the filter paper to form a fluorescent layer.

15. The manufacturing method according to claim 14, characterized in that: Also includes: A color fixing agent is prepared and coated on the surface of the fluorescent layer to form a light stabilizing layer.

16. The manufacturing method according to claim 15, characterized in that: The color fixing agent comprises component A and component B; the component A comprises ammonia water and anhydrous ethanol, and the component B comprises tetraethyl silicate and anhydrous ethanol.

17. Use of the test paper according to any one of claims 1 to 13 in the field of detection / removal of triphenylmethane compounds.

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