Cooperative catalytic reaction induction-based catecholamine substance multicolor visual detection kit and detection method

Abstract

The invention discloses a catecholamine substance multicolor visual detection kit and detection method based on synergistic catalytic reaction induction, and the method comprises the following steps: firstly, enabling catecholamine and AgNPLs to be subjected to a sufficient chemical adsorption reaction, and then adding hydrogen peroxide, so that the Ag NPLs are etched to different degrees and show different colors. According to the reaction of silver ions and urease and the cascade urease catalytic reaction, the color of pH indicator phenol red is changed, so that the colors of Ag NPLs and phenol red are compounded, the color recognition degree after the etching reaction is enhanced, and catecholamine substances are detected according to different colors. The method is simple, convenient, efficient, sensitive and high in naked eye recognition degree, and can be used for detecting diseases such as depression.

Description

Technical Field

[0001] The present invention belongs to the technical fields of nanomaterials science and optical detection, and particularly relates to a multi-color visualization detection kit and detection method for catecholamine substances induced by a synergistic catalytic reaction. Background Art

[0002] Colorimetric visualization sensing has become a common target detection method due to its advantages such as simple reading, low cost, and rapid detection. Among them, noble metal nanomaterials such as gold and silver have good local surface plasmon resonance phenomena in the visible light region, and the extinction coefficient is 1000 times higher than that of organic dyes, with high sensitivity. Especially, the etching-based method can change the particle size and shape to make the solution color change richly, and has been widely used in food safety detection, drug detection, biomarker detection, environmental monitoring and other aspects. The three elements of color include brightness, saturation, and hue. However, due to the large subjective error in the direct observation of similar colors by the naked eye, the detection accuracy is limited in practical applications. And currently, the quantitative method for color enhancement is relatively complex to calculate and not simple and intuitive enough. Therefore, improving color resolution and simply quantifying color signals has great research significance.

[0003] Currently, some colorimetric methods based on the etching of noble metal nanomaterials have been disclosed. However, because they mostly rely on a single signal (such as the color change of the nanomaterials themselves) and lack a signal amplification or composite mechanism, the color resolution is low. The subtle differences in the three elements of color (brightness, saturation, hue) rely on the direct judgment of the naked eye, and the ability to distinguish similar colors is limited, with a large subjective error, resulting in limited detection accuracy. For example, the Chinese patent document with the publication number CN115197996A discloses a colorimetric glucose analysis method based on triangular gold nanosheets and glucose oxidase. It only detects glucose through the color change of triangular gold nanosheets etched by hydrogen peroxide. Although it can also cause changes in the solution color and ultraviolet wavelength and quantitatively detect glucose, single-signal detection often results in insufficient sensitivity or discrimination. Another example is the Chinese patent document with the publication number CN116203235A, which discloses a rapid method for multi-color detection of Staphylococcus aureus based on nanozyme catalysis. It relies on the single color signal of nanozyme catalysis and TMB 2+ etching gold nanorods. The above methods have limitations in sensitivity or multi-target discrimination ability and are difficult to cope with the detection of complex samples or low-concentration targets.

[0004] In addition, some existing technologies rely on the functionalization of specific nanomaterials (such as glucose oxidase immobilized on the surface of gold nanosheets in CN115197996A), or require complex pretreatment steps (such as immunomagnetic nanoprobe capture in CN116203235A), resulting in limited signal stability or universality and making it difficult to be extended to the detection of different types of targets.

[0005] Therefore, there is an urgent need for a new visualization detection method to overcome the lack of intuitiveness and universality in existing technologies. Summary of the Invention

[0006] The object of the present invention is to provide a method for multi-color visualization detection of catechol substances based on co-catalytic reaction induction in view of the deficiencies of existing technologies. Among them, silver nanoplates will be etched by hydrogen peroxide to generate silver ions, and the chemical adsorption between catechol substances and silver nanoplates will reduce the etching degree. Therefore, different concentrations of catechol substances result in different intensities of chemical adsorption, which in turn leads to different etching effects, generating different color changes and different concentrations of silver ions. Urease catalyzes the decomposition of urea to produce alkaline ammonia, increasing the pH. Adding the pH indicator phenol red produces different color changes. And silver ions will inhibit the activity of urease, thus correlating the dopamine concentration with the color of phenol red. Combining the color of etched silver nanoplates with the color of phenol red produces more obvious color changes. The method is simple and rapid, with high visual recognition, and has great prospects in the detection of disease markers.

[0007] In the first aspect, the present invention provides a method for multi-color visualization detection of catecholamine substances based on co-catalytic reaction induction, including the following steps:

[0008] Mix different concentrations of catecholamine samples with silver nanoplates (Ag NPLs) in water and incubate to obtain a first mixture;

[0009] Add hydrogen peroxide to the first mixture for incubation. After incubation, add catalase to it, and mix evenly to obtain a second mixture;

[0010] Add urease to the second mixture for incubation, then sequentially add urea and phenol red solution, mix evenly and react at room temperature;

[0011] After the reaction is complete, extract the H value of the color of the reactant and measure the phenol red absorption spectrum of the reactant using an ultraviolet spectrophotometer, and interpret the detection result according to the H value and absorbance.

[0012] Preferably, the concentration of hydrogen peroxide is 1-5 mM, and the concentration of catalase is 200-800 U / mL.

[0013] Preferably, the volume ratio of the first mixture, hydrogen peroxide, and catalase is 8:(1 - 1.5):(1 - 1.5), more preferably 8:1:1.

[0014] Preferably, the volume ratio of the second mixture, urease, urea, and phenol red solution is 100:(10 - 15):(50 - 55):(20 - 25), more preferably 100:12:50:20.

[0015] Preferably, the concentration of urease is 0.5 - 5 mg / mL, the concentration of urea is 0.1 - 10 M, and the concentration of phenol red is 0.1 - 1 mM.

[0016] Preferably, the catecholamine sample is dopamine.

[0017] In the detection method provided by the present invention, catecholamine first undergoes chemisorption with AgNPLs, and then hydrogen peroxide is added for etching, resulting in a change in its surface plasmon resonance characteristics, thereby inducing a color change; at the same time, the Ag + triggered by the etching generates ammonia through the urease cascade catalytic reaction, combined with the pH color development of phenol red, forming a composite color signal.

[0018] In a second aspect, the present invention provides a multi - color visualization detection kit for catecholamine substances based on a synergistic catalytic reaction, including: silver nanopyramid solution, hydrogen peroxide solution, catalase, urease, urea, phenol red solution, and auxiliary reagent. Among them, the concentration of hydrogen peroxide is 1 - 5 mM, the concentration of catalase is 200 - 800 U / mL, the concentration of urease is 0.5 - 5 mg / mL, the concentration of urea is 0.1 - 10 M, and the concentration of phenol red is 0.1 - 1 mM.

[0019] Preferably, the auxiliary reagent is water.

[0020] Preferably, when using the kit, the above - mentioned method is adopted.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] (1) The present invention uses silver nanopyramids as the etching material. Silver nanomaterials have a low reduction potential, and the three - dimensional structure of the nanopyramids has extreme anisotropy, showing stronger plasmon resonance and more sensitive reactions.

[0023] (2) The present invention utilizes the color change of the urease - catalyzed reaction and the color combination of the etching reaction, making the original color change of the etching more obvious and improving the visual recognition degree of the detection.

[0024] (3) The present invention uses a visualization method to detect catecholamine substances, which is simple, convenient, and has a low cost.

[0025] In summary, through the new multi-signal collaborative enhancement mechanism and signal design, the present invention realizes signal amplification based on the chemical adsorption, etching, and pH cascade reaction of small molecules, and can further perform highly sensitive and highly distinguishable multi-color visual detection on catecholamine substances, effectively solving the problems of low color resolution, complex quantification, single signal, and insufficient discrimination in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a TEM image of silver nanoplisms, and the scale bar is 100 nm.

[0027] Figure 2 It is the UV-visible absorption change of silver nanoplisms after etching for different times.

[0028] Figure 3 It is the UV-visible absorption change of phenol red caused by urease at different concentrations.

[0029] Figure 4 It is the UV-visible absorption change of silver nanoplisms etched by dopamine at different concentrations.

[0030] Figure 5 It is the color difference of silver nanoplisms etched by dopamine at different concentrations.

[0031] Figure 6 It is the UV-visible absorption change of phenol red after the reaction of dopamine at different concentrations with urease.

[0032] Figure 7 It is the color difference of phenol red after the reaction of dopamine at different concentrations with urease. DETAILED DESCRIPTION OF THE INVENTION

[0033] As mentioned above, in view of the deficiencies of the prior art, the inventors of this case have put forward the technical solution of the present invention through long-term research and a large number of practices. The main bases thereof at least include:

[0034] The present invention detects catecholamine substances by using the color change generated by etching silver nanoplisms and the color combination of urease-catalyzed reactions. The present invention performs detection according to the different colors and UV peaks shown in the results, which is simple, convenient, highly efficient and sensitive, improves the naked-eye recognition degree of colors, and can be simply applied to the early diagnosis of diseases.

[0035] In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0036] The specific implementation scheme of the present invention is as follows:

[0037] Step (1): Take 10 μL of catecholamine substances with different concentrations and mix them with 50 μL of silver nanorod solution, and dilute to 120 μL with deionized water. Oscillate and mix evenly, and incubate at room temperature for 5 - 30 minutes to obtain mixture A1; the catecholamine substances include but are not limited to dopamine.

[0038] Step (2): Add 10 μL of 2 mM hydrogen peroxide to mixture A1, mix evenly and incubate at room temperature for 5 - 30 minutes, then add 10 μL of 500 U / mL catalase, and incubate at room temperature for 5 minutes to terminate the etching reaction. Obtain mixture A2.

[0039] Step (3): Add 12 μL of 88 μg / L urease to mixture A2, mix evenly and incubate at room temperature for 5 - 30 minutes to allow the silver ions and urease to react fully. Then add 50 μL of 0.2 M urea and 30 μL of 0.15 mM phenol red in sequence, mix evenly and incubate at room temperature for 3 - 10 minutes to make the phenol red develop color.

[0040] Step (4): Use an ultraviolet spectrophotometer to measure the absorption spectrum of the phenol red solution, and detect the catecholamine substances according to the relative change in absorbance of the absorption peak; visually identify the catecholamine substances according to the color change of the phenol red solution.

[0041] In addition, it should be noted that the following specific embodiments of the present invention do not constitute a limitation on the protection scope of the present invention. Any other corresponding changes and deformations made according to the technical concept of the present invention should be included in the protection scope of the claims of the present invention.

[0042] Example 1: Preparation of silver nanorod solution

[0043] The silver nanorods were directly synthesized by the one-pot method.

[0044] Add 0.08 mL of 0.1 M silver nitrate solution, 1.2 mL of 0.1 M trisodium citrate solution and 0.25 mL of 9.8 M hydrogen peroxide solution to 78.496 mL of water in sequence, stir vigorously at 1200 rpm for 10 minutes, then add 0.8 mL of 0.1 M sodium borohydride solution, stir vigorously at 1200 rpm for 2 minutes, and let stand at room temperature for at least 60 min for standby.

[0045] Figure 1 It is the TEM characterization of the obtained silver nanorods.

[0046] Example 2

[0047] To increase the reaction sensitivity and accuracy, the experimental conditions of the method for the synergistic visual detection of catechol substances based on the etching of AgNPLs and urease-catalyzed reactions were optimized by the single-variable method. Since the etching time is directly related to the degree of etching, and the urease concentration will result in different amounts of NH3 produced, thereby changing the color of phenol red, the etching time and urease concentration are two conditions that must be optimized for the present invention.

[0048] Optimization of the etching time:

[0049] Ag NPLs and catecholamine were added to ultrapure water and incubated for 10 min, and then 2 mM hydrogen peroxide was added for etching for 0, 3, 6, 9, 12, 15, 18 min. The absorption spectrum was measured by an ultraviolet spectrophotometer and the color difference was observed with the naked eye. The optimal etching time was determined to be 15 min.

[0050] Figure 2 It is the ultraviolet change of the etching degree of Ag NPLs at different reaction times. As the time prolongs, the etching degree of AgNPLs is continuously increasing, and the ultraviolet peak is continuously blue-shifted. In order to ensure the complete etching of AgNPLs, a reaction time of 15 min was selected as the subsequent reaction condition.

[0051] Optimization of the urease concentration:

[0052] Ag NPLs and dopamine were added to ultrapure water and incubated for 10 min, then 2 mM hydrogen peroxide was added for etching for 15 min, then 500 U / mL catalase and different concentrations of urease (0, 22, 44, 66, 88, 111, 133 μg / L) were added and incubated for 10 min, and finally urea and phenol red were added for color development. The absorption spectrum was measured by an ultraviolet spectrophotometer and the color difference was observed with the naked eye. The optimal urease concentration was determined to be 88 μg / L.

[0053] Figure 3 It is the ultraviolet change of phenol red at different urease concentrations. As the urease concentration increases, the catalytic reaction of urea is promoted, resulting in an increase in the amount of NH3 produced, and the color of phenol red changes from yellow to red. The absorbance at 525 nm of ultraviolet continuously increases and reaches a plateau at 88 μg / L. The urease concentration of 88 μg / L was selected as the subsequent reaction condition.

[0054] Example 3

[0055] In this example, dopamine was used as the catecholamine substance.

[0056] Mix 10 μL of dopamine at different concentrations with 60 μL of AgNPLs respectively, dilute to 120 μL with ultrapure water, and incubate at room temperature for 10 min. Then add 2 mM hydrogen peroxide, shake and mix evenly, incubate at 37 °C for 15 min to allow hydrogen peroxide to fully etch the material. After 15 min, add 500 U / mL catalase to terminate the reaction, and measure the absorption spectrum with a UV spectrophotometer. The color change can also be distinguished by the naked eye.

[0057] Figure 4 and Figure 5 are the UV and color changes of the etching degree of Ag NPLs by dopamine at different concentrations respectively. Dopamine at different concentrations causes Ag NPLs to be etched to different degrees. The UV peak blue-shifts from 650 nm to 570 nm, and the color changes from blue to light purple.

[0058] Take 100 μL of the above-mentioned reacted solution and add 88 μg / L urease solution, incubate at room temperature for 10 min to allow Ag + and urease to react fully. Then add 50 μL of 0.2 M urease and 20 μL of 0.15 mM urease, react for 3 min, measure the absorption spectrum with a UV spectrophotometer, and the color change can also be distinguished by the naked eye.

[0059] Figure 6 and Figure 7 are the UV and color changes of pH indicator phenol red by dopamine at different concentrations respectively. Dopamine at different concentrations causes different urease activities, thus producing different amounts of NH3. The absorbance at 525 nm of the UV changes, and at the same time, after color combination, the color gradually changes from yellowish-green to purplish-red.

[0060] Application Example 1

[0061] Detect catecholamines in mouse cerebrospinal fluid, PC-12 cells and mouse serum according to the method in Example 3 above:

[0062] Dilute the extracted mouse cerebrospinal fluid directly by 10 times for standby. Add the cultured PC-12 cells to 0.1 M K2CO3 and 1 mM CaH2PO4, place them in an incubator to react for 20 - 50 min to release catecholamines, then centrifuge and take the supernatant for standby. The obtained mouse serum is directly used for standby.

[0063] Mix 10 μL of the actual sample with 60 μL of AgNPLs, dilute to 120 μL with ultrapure water, and incubate at room temperature for 10 min. Then add 2 mM hydrogen peroxide, shake and mix evenly, incubate at 37 °C for 15 min to allow hydrogen peroxide to fully etch the material. After 15 min, add 500 U / mL catalase to terminate the reaction. Take 100 μL of the above-mentioned reacted solution and add 88 μg / L urease solution, incubate at room temperature for 10 min to allow Ag +React fully with urease. Then add 50 μL of 0.2 M urea and 20 μL of 0.15 mM phenol red, and react for 3 min. Measure the absorption spectrum using an ultraviolet spectrophotometer. The color change can also be distinguished by the naked eye, thereby detecting catecholamines.

[0064] The above embodiments are not limitations on the present invention. The present invention is not limited to the above embodiments. As long as it meets the requirements of the present invention, it falls within the protection scope of the present invention.

Claims

1. A multi-color visual detection method for catecholamine substances induced by cooperative catalytic reaction, characterized in that, The detection method includes the following steps: Mix catecholamine samples with different concentrations and silver nanoplates in water and incubate to obtain a first mixture; Add hydrogen peroxide to the first mixture for incubation. After the incubation, add catalase to it, and mix evenly to obtain a second mixture; Add urease to the second mixture for incubation, then add urea and phenol red solution in sequence, mix evenly and react at room temperature; After the reaction is complete, extract the H value of the reactant color and measure the phenol red absorption spectrum of the reactant using an ultraviolet spectrophotometer, and interpret the detection result based on the H value and absorbance.

2. The method for multi-color visualization detection of catecholamine substances induced by cooperative catalytic reaction according to claim 1, wherein The concentration of hydrogen peroxide is 1-5 mM, and the concentration of catalase is 200-800 U / mL.

3. The method for multicolor visualization detection of catecholamine substances induced by cooperative catalytic reaction according to claim 1, characterized in that The volume ratio of the first mixture, hydrogen peroxide and catalase is 8:(1-1.5):(1-1.5).

4. The multi-color visual detection method for catecholamine substances induced by cooperative catalytic reaction according to claim 1, wherein The volume ratio of the second mixture, urease, urea and phenol red solution is 100:(10-15):(50-55):(20-25).

5. The method for multi-color visualization detection of catecholamine substances induced by cooperative catalytic reaction according to claim 1, characterized in that, The concentration of urease is 0.5-5 mg / mL, the concentration of urea is 0.1-10 M, and the concentration of phenol red is 0.1-1 mM.

6. The method for multi-color visualization detection of catecholamine substances induced by cooperative catalytic reaction according to claim 1, wherein The catecholamine sample is dopamine.

7. A kit for multi-color visual detection of catecholamine substances induced by cooperative catalytic reaction, characterized in that, It includes: Silver nanoprism solution, hydrogen peroxide solution, catalase, urease, urea, phenol red solution and auxiliary reagent, wherein the concentration of hydrogen peroxide is 1-5 mM, the concentration of catalase is 200-800 U / mL, the concentration of urease is 0.5-5 mg / mL, the concentration of urea is 0.1-10 M, and the concentration of phenol red is 0.1-1 mM.

8. The kit for multicolor visualization detection of catecholamine substances induced by cooperative catalytic reaction according to claim 7, wherein, The auxiliary reagent is water.

9. The kit for multi-color visual detection of catecholamine substances induced by cooperative catalytic reaction according to claim 7, characterized in that, When the kit is used, the detection method described in any one of claims 1-6 is adopted.

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