Application of acriflavine as catalyst in TMB oxidation chromogenic reaction

Abstract

The invention provides an application of acriflavine as a catalyst in TMB (Tetramethylbenzidine) oxidation chromogenic reaction. According to the method, acridine yellow is used as a catalyst to catalyze dissolved oxygen to oxidize a substrate 3, 3 ', 5, 5'-tetramethyl benzidine (TMB) into oxidation-state TMB (OxTMB), and the color of a solution system is changed from faint yellow of acridine yellow to characteristic blue of OxTMB. A series of experiments prove that acriflavine is different from a traditional enzyme-like catalyst which catalyzes hydrogen peroxide to be decomposed into hydroxyl free radicals, but directly catalyzes dissolved oxygen in a solution to oxidize TMB to generate OxTMB, and the acriflavine is found and applied as a TMB oxidation chromogenic reaction catalyst for the first time. The method does not need expensive reagents and additional oxidants, has the advantages of being environmentally friendly, easy to operate and low in cost, and is very beneficial to being popularized to other fields. Besides, trace colorimetric analysis of acriflavine can be realized by utilizing the property and the color characteristic of OxTMB, the detection linear range is 0.5-5 nM, and the detection limit is 0.036 nM.

Description

Technical Field

[0001] The invention relates to application of acridinium yellow as a catalyst in TMB oxidation color development reaction, belonging to the technical field of colorimetric detection. Background Art

[0002] Acriflavine, as a small molecule fluorescent dye, generally exists in the form of acriflavine hydrochloride, is easily soluble in water, and is commonly used in the field of fluorescent probes and bioimaging. In addition, in clinical application research, acriflavine and its derivatives can be used as antibacterial, anti-inflammatory and antiviral drugs. The latest research has found that acriflavine has certain anti-cancer activity and can be used as a potential anti-cancer drug. As a biological stain and potential drug, the quantitative detection of acriflavine in cells and biological samples is of great significance for studying its biological activity and metabolic pathways. However, few studies have focused on the detection of acriflavine.

[0003] Small molecule catalysts have the characteristics of small molecular weight, simple structure, flexible design, high selectivity, etc., and are widely used in organic synthesis, asymmetric catalysis, materials science and green chemistry. The advantages of small molecule catalysts are easy industrial application, mild reaction conditions, controllable cost, high catalytic efficiency and easy to control properties, which can greatly improve the efficiency of chemical reactions and provide a new path for achieving green sustainable chemistry. At present, there have been research reports on acridinium yellow derivatives as photoinduced electron transfer photocatalysts in the field of organic synthesis, indicating that acridinium yellow and its derivatives have the potential to be used as catalysts. 3,3',5,5'-Tetramethylbenzidine (TMB) is a new substrate for peroxidase and has been widely used in enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA). Currently, many studies on peroxidase catalysis are based on TMB as a substrate. 2 O 2 In the presence of H and peroxidase, TMB can be 2 O 2 Oxidized to oxidized TMB (OxTMB), OxTMB exhibits a specific blue color under acidic conditions and has characteristic absorption peaks at 370 nm and 652 nm. In addition to peroxidase, some precious metal nanoparticles, ferroferric oxide nanoparticles and some carbon nanomaterials can use peroxidase-like properties to achieve the above catalytic effect. However, there are few reports on the study of small molecules as peroxidases. In current research, it is generally believed that only H 2 O 2 TMB will be oxidized only when H is present. However, the present invention can be used in the presence of acridinium yellow as a catalyst and without the need for H 2 O 2 With the participation of , TMB is oxidized to OxTMB using dissolved oxygen.

[0004] In the present invention, we use acridine yellow as a catalyst for TMB oxidation color reaction and find that it can catalyze dissolved oxygen to oxidize the substrate TMB into oxidized TMB (OxTMB). After the oxidation reaction, the color of the solution changes from the light yellow of acridine yellow to the characteristic blue of OxTMB. The experiment confirmed that acridine yellow is different from traditional enzyme-like catalysts in catalyzing H 2 O 2 Instead of decomposing into hydroxyl radicals, it catalyzes the oxidation of TMB to produce OxTMB by dissolved oxygen in the solution without the introduction of additional oxidants. This is the first application and discovery of acridinium yellow as a catalyst for TMB oxidation colorimetric reaction. Using this method, we also achieved trace colorimetric detection of acridinium yellow. Summary of the invention

[0005] The invention aims to provide application of acridinium yellow as a catalyst in TMB oxidation color development reaction.

[0006] The invention provides the use of acridinium yellow as a catalyst in TMB oxidation color development reaction. An acridinium yellow solution is added to an acidic solution of TMB, and the solution is placed in an environment for catalytic reaction. The color of the solution system changes from light yellow to blue; and ultraviolet absorption peaks appear at 370 nm and 652 nm in the solution.

[0007] Add acridinium yellow solution to the acidic solution of TMB and place it in the environment normally. The oxygen molecules in the environment dissolve in the solution to form dissolved oxygen. Acridinium yellow acts as a catalyst to catalyze the dissolved oxygen to oxidize the substrate TMB into OxTMB. The color of the solution system changes from the light yellow of acridinium yellow to the characteristic blue displayed by OxTMB.

[0008] The acidic solution of TMB is an acidic solution after adjusting the pH using citric acid-sodium citrate buffer. The pH of the acidic solution of TMB is adjusted to 3-4. The catalytic reaction time is 10-30 min. 5 nM acriflavine is added to the 0.2 mM-2 mM TMB acidic solution. The molar concentration ratio of the TMB acidic solution to the acriflavine solution is (4×10 4 ~4×10 5 ):1.

[0009] 1. Investigation of the feasibility of acridinium yellow as a small molecule catalyst for TMB oxidation color development reaction Weigh 0.12 g TMB into a 10 mL volumetric flask and dilute to volume with anhydrous ethanol to obtain a 50 mM TMB stock solution; weigh 4.69 mg acridinium yellow and dissolve it in 10 mL ultrapure water to obtain a 1 mM acridinium yellow stock solution; weigh 33.2 mg fluorescein, 47.9 mg rhodamine B and 47.9 mg rhodamine 6G respectively into 10 mL ultrapure water to obtain stock solutions of the corresponding organic dyes with a concentration of 10 mM.

[0010] Figure 1 The feasibility analysis of the oxidation color reaction of TMB catalyzed by acridinium yellow is shown in Figure 1. A is the UV absorption spectra of different solutions. The three curves from bottom to top are the acridinium yellow control solution; the solution after acridinium yellow and TMB mixture; the solution after acridinium yellow, H 2 O 2 Spectra of mixed solutions of A and TMB. All solutions were placed at room temperature without light for 30 min before measurement. B is the color photos of different solutions, where 1 is acridine yellow solution; 2 is acridine yellow and TMB solution; 3 is acridine yellow, H 2 O 2 All solutions were placed at room temperature without protection from light for 30 min before taking pictures.

[0011] TMB solution itself is colorless, very stable in the environment, and has no UV absorption. When 5 nM acridine yellow solution is added to 1 mM TMB acid solution (pH=4), the solution quickly turns into the characteristic blue of OxTMB. As time goes by, the color gradually deepens and gradually stabilizes after about 30 minutes. When the solution is measured by UV-visible spectroscopy, it is found that there are characteristic absorption peaks of OxTMB at 370nm and 652nm ( Figure 1 ). This indicates that the addition of acriflavine will promote the oxidation of TMB to OxTMB, but acriflavine itself has no oxidizing property. Therefore, we speculate that acriflavine plays a catalytic role in the oxidation reaction of TMB. Figure 1 It can be found from the absorption peaks of acridinium yellow itself at 451nm in the three groups of solutions in A that the intensity of the ultraviolet absorption peaks of acridinium yellow remains consistent before and after the production of OxTMB, indicating that the concentration of acridinium yellow does not change before and after the reaction, proving that acridinium yellow does not participate in the reaction as a reactant, but only participates in the oxidation reaction of TMB as a catalyst, which is consistent with the characteristics that the chemical properties and quality of the catalyst do not change before and after the reaction.

[0012] In addition, we found that H 2 O 2 It is not a necessary condition for the oxidation of TMB. As a peroxidase substrate, TMB is generally believed to be oxidized only by H 2 O 2 TMB will be oxidized only when H is present. However, the present invention has found that under the condition of acridinium yellow as a catalyst, no H is required. 2 O 2 The participation of can oxidize TMB in the solution into OxTMB. Figure 1 It can be seen from the color picture and UV absorption spectrum of the solution that when H 2 O 2(concentration of 100 mM) the changes in solution color and UV absorption spectrum were very weak, indicating that H 2 O 2 The addition of has little effect on the TMB oxidation system. The solution system in the present invention is a citric acid-sodium citrate aqueous solution, and the main oxidant in the aqueous solution is the dissolved oxygen formed after the oxygen molecules are dissolved in the aqueous solution. Therefore, under the catalysis of acridinium yellow, the oxidant that causes the oxidation color development reaction of TMB is dissolved oxygen, which will be further verified by subsequent experiments.

[0013] 2. Parameter optimization of catalytic color reaction Figure 2 The conditions for catalytic color development reaction are optimized. A is the optimization of the concentration of the reaction substrate TMB; B is the optimization of the reaction pH conditions; C is the optimization of the reaction time.

[0014] TMB is a colorless solution, but when oxidized to OxTMB, the solution color is characteristic blue under acidic conditions, and there are obvious UV absorption peaks at 652 nm and 370 nm. We optimized the optimal TMB concentration required for the system, such as Figure 2 As shown in A, when the concentration of acriflavine was fixed at 5 nM and the concentration of TMB was 1 mM, the ultraviolet absorption at 652 nm hardly changed, and the concentration of TMB used in subsequent experiments was 1 mM.

[0015] TMB is oxidized to OxTMB under the catalysis of acridinium yellow. OxTMB exhibits a characteristic blue color under acidic conditions and has characteristic UV absorption peaks at 370 nm and 652 nm. Therefore, we optimized the optimal pH required for acridinium yellow to catalyze TMB oxidation. The pH was optimized using citric acid-sodium citrate buffer, and the results were as follows: Figure 2 B, such as Figure 2 As shown in Figure B, at pH = 4, the UV absorption at 370 nm and 652 nm was maximum, so the subsequent experiments were carried out in citric acid-sodium citrate buffer at pH = 4.

[0016] We also optimized the time required for acridinium yellow to catalyze the oxidation of TMB. Figure 2 As shown in Figure C, with the increase of time, the ultraviolet absorption at 370 nm and 652 nm continued to increase and reached equilibrium around 30 min. Therefore, 30 min was selected as the optimal reaction time.

[0017] 3. Study on the mechanism of catalytic color reaction Figure 3 To investigate the mechanism of acridinium yellow catalyzing the oxidation of TMB by dissolved oxygen. Figure 3 A is the solution into which N 2 The changes in solution color and UV absorption after the removal of dissolved oxygen are Figure 3 A verified that the production of OxTMB was due to the catalytic effect of acriflavine and the oxidation effect of dissolved oxygen; Figure 3 B shows the changes in solution color and UV absorption after ascorbic acid is added to the solution, indicating that TMB cannot be oxidized after dissolved oxygen, the only oxidant in the solution, is removed.

[0018] The principle of the present invention is that acridinium yellow, as a catalyst, catalyzes dissolved oxygen to convert the substrate TMB into OxTMB. After the reaction occurs, the color of the solution changes from the light yellow of acridinium yellow to the characteristic blue displayed by OxTMB, which has been described in detail in the feasibility analysis. Oxygen is the main oxidant in the environment, and the main oxidant in the aqueous solution is the dissolved oxygen formed after the oxygen molecules are dissolved in the aqueous solution. Therefore, we speculate that the oxidant that causes the oxidation of TMB should be the dissolved oxygen in the reaction system. The following specific experiments verify our conjecture.

[0019] We conducted two groups of experiments. In one group, TMB and acriflavine solutions were added and placed in the environment normally. In the other group, N was immediately introduced into the solution after TMB and acriflavine were added. 2 To remove dissolved oxygen, after 30 min, observe the color changes of the two groups of solutions and measure the absorbance respectively. Figure 3 In A, we can see that the color of the normally placed TMB and acridinium yellow solution is blue, and there are obvious absorption peaks at 370 nm and 652 nm, indicating that TMB has been oxidized to OxTMB. 2 The color of the solution of TMB and acridinium yellow hardly changed, and there were no UV absorption peaks at 652 nm and 370 nm, indicating that TMB could not be oxidized after the solution was deoxygenated, which further indicated that the oxidative color development reaction of TMB was actually caused by dissolved oxygen.

[0020] To further verify this mechanism, we added ascorbic acid (AA) to the solution to inhibit the oxidation reaction of TMB. One group of experiments added TMB and acriflavine as a control, and the other group added TMB, acriflavine and AA. AA, as a strong reducing agent, can remove the oxidant in the reaction system and prevent the oxidation of TMB. After the two groups of solutions reacted for 30 minutes, the color changes of the solutions were observed and the absorbance of the solutions was measured respectively. The results are as follows: Figure 3 As shown in Figure B, compared with the control solution, the color of the solution with AA added did not change, and there were no UV absorption peaks at 652 nm and 370 nm, indicating that TMB could not be oxidized to OxTMB after the addition of ascorbic acid. This proves that after the only oxidant dissolved oxygen in the system is removed, OxTMB cannot be generated, indicating that the oxidative color development reaction of TMB is actually caused by dissolved oxygen catalyzed by acridine yellow.

[0021] In summary, acriflavine is different from traditional enzyme-like catalysts in catalyzing hydrogen peroxide (H 2 O 2 ) decomposes into hydroxyl radicals, but directly catalyzes the dissolved oxygen in the solution to oxidize TMB to produce OxTMB. We compared the same concentration of acriflavine with the addition of H 2 O 2 The UV absorption spectrum of TMB solution at 652 nm showed that the addition of H 2 O 2 After adding OxTMB into the system, the characteristic ultraviolet absorption at 652 nm did not change significantly, thus confirming that acridinium yellow catalyzed the dissolved oxygen in the solution to achieve the oxidation of TMB.

[0022] 4. Study on the catalytic properties of acridinium yellow Figure 4 The molecular formulas of acriflavine and some common fluorescent dyes (A) and their UV absorption spectra after incubation with TMB solution (B). Figure 4 It can be seen that compared with other fluorescent molecules, acridinium yellow has good catalytic properties, which is related to the symmetry of its molecular structure.

[0023] In the feasibility analysis, we confirmed that acridinium yellow played a role as a catalyst in the oxidation color reaction of TMB. Figure 4 A), Acriflavine has an amino group with two lone pairs of electrons and a symmetrical structure. Therefore, we speculated that its catalytic activity may be related to this structure. Therefore, we selected several organic molecular dyes commonly used in laboratories, namely Rhodamine B, Rhodamine 6G, and Fluorescein, to test the reaction specificity. Figure 4 As shown in Figure B, when the concentration of rhodamine B and rhodamine 6G was 10 times that of acridinium yellow and coexisted with TMB solution, the color of TMB solution did not change after 30 minutes of reaction, and there was no characteristic ultraviolet absorption peak of OxTMB at 370 nm and 652 nm, indicating that both had no catalytic properties. Observation of the molecular structures of the two revealed that neither of them had a symmetrical chemical structure. When the concentration of fluorescein was 10 times that of acridinium yellow and coexisted with TMB solution, there were absorption peaks at 370 nm and 652 nm after 30 minutes, but its intensity was significantly lower than that of acridinium yellow and TMB solution. From the molecular structure of fluorescein, it can be seen that the keto form at the symmetrical position of the hydroxyl group of fluorescein can be converted into an enol form under acidic conditions, thus presenting a symmetrical molecular structure, thereby showing a certain catalytic activity similar to acridinium yellow, but the catalytic effect is significantly lower than that of acridinium yellow. In summary, the good catalytic properties of acridinium yellow are related to the symmetry of its molecular structure.

[0024] 5. Detection and research of acriflavine Figure 5The following is the relationship between the change of acridinium yellow concentration and the change of solution color (A) and the corresponding ultraviolet absorption spectrum (B). Figure 5 A shows that the color of the solution gradually deepens with the increase of the concentration of acridinium yellow; from left to right, the concentrations of acridinium yellow in TMB solution are 0, 0.5 nM, 1.0 nM, 1.5 nM, 2.0 nM, 2.5 nM, 3.0 nM, 3.5 nM, 4.0 nM, and 5 nM. Figure 5 B can be seen that with the increase of acriflavine concentration, the ultraviolet absorption at 370 nm and 652 nm gradually increases. The concentrations of acriflavine from bottom to top of the curve are 0.5 nM, 1.0 nM, 1.5 nM, 2.0 nM, 2.5 nM, 3.0 nM, 3.5 nM, 4.0nM, and 5 nM, respectively.

[0025] Figure 6 The figure is a linear relationship between the concentration of acridinium yellow and the absorbance at 652 nm. It can be seen from the figure that when the concentration of acridinium yellow is 0.5-5nM, it has a good linear relationship with the absorbance value at 652 nm.

[0026] The detection of acridinium yellow is of great significance for studying its biological activity and catalytic performance. Through the above experiments, we proved that acridinium yellow can catalyze the oxidation of TMB to OxTMB by dissolved oxygen, and as the concentration of acridinium yellow increases, the content of OxTMB also gradually increases. This phenomenon provides an effective method for the quantitative detection of acridinium yellow. Therefore, we added different concentrations of acridinium yellow to a 1 mM TMB solution and observed the change in the color of the solution, such as Figure 5 A, it was found that as the concentration of acridinium yellow solution increased, the color of the solution gradually deepened. Subsequently, the ultraviolet absorption spectrum of the solution at 652nm and 370nm was measured, as shown in Figure 5 As shown in Figure B, with the increase of acriflavine concentration, the UV absorption peaks at 652nm and 370nm gradually increased. Figure 6 As shown in the figure, the linear relationship between the absorbance at 652 nm and the acridinium yellow solution was investigated. It was found that when the concentration of the acridinium yellow solution was 0.5-5 nM, it had a good linear relationship with the absorbance at 652 nm. The linear equation was y=0.1153x+0.01253, R 2 =0.9981. Using the formula LOD=3σ / S (σ=0.0014, S=0.1153), it was calculated that the minimum detection limit of this method for acriflavine was 0.036 nM.

[0027] Advantages of the present invention and the beneficial effects produced: The present invention overcomes the shortcomings of the prior art and expands the application field of small molecule catalysts. Acridinium yellow is used as a catalyst for the TMB oxidation color reaction, and it is found that it can catalyze dissolved oxygen to rapidly oxidize TMB to OxTMB, causing the solution color to turn blue. In addition, we confirmed that H 2 O 2 The presence of is not a necessary condition for the oxidation of TMB to OxTMB. TMB can be oxidized by using dissolved oxygen in the solution. This method does not require expensive reagents and additional oxidants. It is green, environmentally friendly, simple to operate, and low-cost, which is very conducive to promotion to other fields. We used this method to achieve rapid colorimetric detection of acridinium yellow with a detection limit as low as nanomolar level, which provides a new strategy and method for simple and rapid detection of organic small molecule dyes. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The present invention provides a feasibility analysis of the acridinium yellow catalyzing the oxidation of TMB to OxTMB.

[0029] Figure 2 The conditions for the color development reaction of TMB catalyzed by acridine yellow are optimized.

[0030] Figure 3 The invention is an investigation on the mechanism of acridinium yellow catalyzing dissolved oxygen to oxidize TMB to OxTMB.

[0031] Figure 4 The molecular formulas of acriflavine of the present invention and some common fluorescent dyes and their ultraviolet absorption spectra after being incubated with TMB solution for 30 minutes are shown.

[0032] Figure 5 The present invention shows the changes in solution color and ultraviolet absorption spectrum with increasing concentration of acridinium yellow.

[0033] Figure 6 The linear relationship diagram of the acriflavine concentration of the present invention and the absorbance at 652 nm. DETAILED DESCRIPTION

[0034] The following is a further description of the application of acridinium yellow as a catalyst for the TMB oxidation color development reaction through specific examples.

[0035] 1. Processing of actual water samples In order to investigate the practical application capability of this method, the present invention uses the standard addition method to test the content of acriflavine in two actual water samples, namely Yellow River water and tap water. First, the different water samples were filtered to remove the precipitate, then filtered with a 0.22 μM water membrane to remove impurities, and finally the pH of the required sample was adjusted to 4 with citric acid and sodium citrate. Different concentrations of acriflavine solution were added to different samples for subsequent measurement.

[0036] 2. UV spectrophotometry The ultraviolet absorption spectra of the actual samples were measured. 1 mM TMB solution was added to the actual water samples containing different concentrations of acridinium yellow. All water samples were reacted at room temperature for 30 min under the condition of pH = 4. The ultraviolet absorption spectra were then measured using a UV-visible spectrophotometer, and the ultraviolet absorption value at 652 nm was recorded.

[0037] 3. Detection of Acriflavine in actual water samples Substitute the ultraviolet absorption value at 652 nm into the linear equation of the present invention y=0.1153x+0.001253, R 2 =0.9981, calculate the concentration of acridinium yellow actually measured. The measurement results of the Yellow River water are as follows: when the acridinium yellow spiked concentration is 1 nM, the concentration measured by the detection method proposed by the present invention is 0.97 nM, and the spiked recovery rate is 97%. When the acridinium yellow spiked concentration is 3 nM, the concentration measured by the detection method proposed by the present invention is 3.1 nM, and the spiked recovery rate is 103%. When the acridinium yellow spiked concentration in tap water is 1 nM, the concentration measured by the detection method proposed by the present invention is 0.93 nM, and the spiked recovery rate is 93%. When the acridinium yellow spiked concentration in tap water is 3 nM, the concentration measured by the detection method proposed by the present invention is 2.92 nM, and the spiked recovery rate is 97%. These data illustrate that the acridinium yellow detection method proposed by the present invention has good accuracy and practical application capabilities.

Claims

1. Application of acridinium yellow as a catalyst in TMB oxidation color development reaction.

2. The use of acridinium yellow as a catalyst in TMB oxidation color development reaction as claimed in claim 1, characterized in that: Acridine yellow solution is added to the acidic solution of TMB and placed in an environment for catalytic reaction. The color of the solution system changes from light yellow to blue.

3. The use of acridinium yellow as a catalyst in TMB oxidation color development reaction as claimed in claim 1, characterized in that: Acridine yellow solution was added to the acidic solution of TMB and placed in an environment for catalytic reaction. The solution showed ultraviolet absorption peaks at 370 nm and 652 nm.

4. The use of acridine yellow as a catalyst in TMB oxidation color development reaction as claimed in claim 2 or 3, characterized in that: Add acridinium yellow solution to the acidic solution of TMB and place it in the environment normally. The oxygen molecules in the environment dissolve in the solution to form dissolved oxygen. Acridinium yellow acts as a catalyst to catalyze the dissolved oxygen to oxidize the substrate TMB into OxTMB. The color of the solution system changes from the light yellow of acridinium yellow to the characteristic blue displayed by OxTMB.

5. The use of acridinium yellow as a catalyst in TMB oxidation color development reaction as claimed in claim 2 or 3, characterized in that: The acidic solution of TMB is an acidic solution whose pH is adjusted using a citric acid-sodium citrate buffer. The pH of the acidic solution of TMB is adjusted to 3-4.

6. The use of acridinium yellow as a catalyst in TMB oxidation color development reaction as claimed in claim 2 or 3, characterized in that: The catalytic reaction time is 10~30min.

7. The use of acridinium yellow as a catalyst in TMB oxidation color development reaction as claimed in claim 2 or 3, characterized in that: The molar concentration ratio of TMB acid solution to acridinium yellow solution is (4×10 4 ~4×10 5 ):

1.

8. The use of acridinium yellow as a catalyst in TMB oxidation color development reaction as claimed in claim 1, characterized in that: When the concentration of acridinium yellow was in the range of 0.5-5 nM, there was a good linear relationship between the concentration of acridinium yellow and the ultraviolet absorbance value of TMB solution at 652 nm. The linear equation was y=0.1153x+0.01253, R 2 =0.9981, where y is the UV absorbance value of TMB solution at 652 nm, and x is the concentration of acridinium yellow, in nM; Acridine yellow solution was added to the acidic solution of TMB and the ultraviolet absorbance value at 652 nm was measured. The concentration of acridine yellow solution was calculated by substituting the linear relationship to achieve trace detection of acridine yellow.

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