Colorimetric detection method for content of toluenesulfonyl on surface of magnetic bead

Abstract

The invention relates to a colorimetric detection method for the content of toluenesulfonyl on the surface of a magnetic bead, and belongs to the technical field of nano material analysis and sensing. According to the colorimetric detection method provided by the invention, arginine reacts with toluenesulfonyl on the surface of a magnetic bead to be detected, then the magnetic bead is separated, and the content of residual arginine is detected by using a ninhydrin colorimetric method, so that the content of toluenesulfonyl on the surface of the magnetic bead to be detected is further determined. By utilizing the superparamagnetism of the toluenesulfonyl magnetic beads, the magnetic beads can be quickly separated from arginine, meanwhile, complicated pretreatment is avoided, the operation is simple, large-scale equipment is not needed in the whole detection process, semi-quantitative analysis on the toluenesulfonyl on the surfaces of the magnetic beads is realized through color change, and the detection accuracy is high. Quantitative detection of toluenesulfonyl on the surface of the magnetic bead is realized through an ultraviolet spectrophotometer, the sensitivity is high, the detection speed is high, and the method has great research significance and practical application value.

Description

Technical Field

[0001] The invention relates to the technical field of nano material analysis and sensing, in particular to a colorimetric detection method for toluenesulfonyl content on the surface of magnetic beads. Background Art

[0002] Tosyl magnetic beads are a kind of magnetic nanomaterial with wide application prospects in the field of biological separation and purification. 3 O 4 The core is a tosyl functional group modified on the surface of the magnetic beads. The principle is based on the characteristics of the active tosyl groups on the surface of the magnetic beads to react with other compounds. These groups can be chemically coupled with biomolecules (such as antibodies, DNA, etc.) containing amino groups and other functional groups, thereby achieving specific recognition and separation of target biomolecules. Tosyl magnetic beads have the advantages of good magnetic responsiveness, biocompatibility, high specific surface area and easy surface functionalization, which make tosyl magnetic beads show good stability and selectivity in biological separation and purification processes. Under the action of specific reagents, the rich tosyl functional groups can bind biological ligands such as proteins to the surface of the magnetic beads in a covalently bonded manner, thereby achieving rapid separation and enrichment of the target from the sample, and are widely used in DNA and RNA extraction, purification and separation in genetic engineering, cell separation, enrichment and detection in the field of biotechnology, and biomarker detection, pathogen separation and identification in medical diagnosis. In addition, tosyl magnetic beads can also be used for enrichment and separation of target substances in the fields of environmental monitoring and food safety. With the continuous development of nanotechnology and biotechnology, the application prospects of tosyl magnetic beads will be broader.

[0003] During the application process, the content of tosyl groups on the surface of magnetic beads greatly affects the performance of magnetic beads. Specifically, a higher tosyl density means that there are more active binding sites on the surface of magnetic beads, which is conducive to the efficient coupling and specific recognition of biomolecules (such as antibodies, proteins, etc.), thereby improving the stability and selectivity of magnetic beads in biological separation, purification and detection applications. On the contrary, if the tosyl density is insufficient, it may cause the binding ability of magnetic beads to decrease, the selectivity to decrease, and then affect the accuracy and reliability of the experiment. Therefore, when preparing and using tosyl magnetic beads, it is necessary to strictly control the density of tosyl groups to ensure that the magnetic beads have excellent performance and meet experimental needs.

[0004] At the same time, during the use of magnetic beads, the toluenesulfonyl groups on their surface will react with amino groups (-NH 2) or thiol (-SH) and other functional groups to undergo coupling reactions, thereby achieving the fixation and separation of biomolecules. During this chemical reaction, the tosyl group will gradually detach from the surface of the magnetic beads, resulting in loss. In addition, environmental factors such as the pH value, temperature, and ionic strength of the solution, as well as the storage and use conditions of the magnetic beads, such as repeated freezing and thawing, may also affect the stability and loss of the tosyl group. After the loss, the performance of the tosyl magnetic beads may be affected. Specifically, with the reduction of tosyl groups, the active binding sites on the surface of the magnetic beads will also be reduced accordingly, which will reduce the binding ability and selectivity of the magnetic beads with biomolecules, thereby affecting the effect of biological separation and purification. At the same time, the loss may also lead to changes in the surface properties of the magnetic beads, such as reduced hydrophilicity, which further affects the application effect of the magnetic beads in biological experiments. Therefore, developing and optimizing the detection method for tosyl groups and accurately detecting the content of tosyl groups can ensure the quality stability and batch-to-batch consistency of the magnetic beads, provide reliable tools and guarantees for biological experiments, and have extremely high value and far-reaching significance for the prevention and control of diseases and early diagnosis in clinical practice.

[0005] At present, a variety of methods for detecting tosyl groups have been developed, mainly including high performance liquid chromatography, gas chromatography, ion chromatography, electrochemical analysis, etc. High performance liquid chromatography performs well in accurately determining tosyl groups in complex samples. However, for tosyl groups on the surface of magnetic beads, the pretreatment steps may be relatively complicated, and effective extraction technology is required to ensure that the tosyl groups on the surface of magnetic beads can be completely released and dissolved in the mobile phase. In addition, the operating cost of high performance liquid chromatography is also high, including solvent consumption, chromatographic column maintenance, etc. Gas chromatography is suitable for the analysis of volatile compounds, but when directly analyzing non-volatile or low-volatile tosyl groups, derivatization treatment is usually required, which increases the complexity and time cost of the operation. For tosyl groups on the surface of magnetic beads, the difficulty of gas chromatography lies in how to effectively extract them from the surface of magnetic beads and convert them into a form suitable for analysis. Ion chromatography has advantages in detecting ionic compounds. To detect tosyl groups on the surface of magnetic beads by ion chromatography, they need to be converted into ionic forms first, which increases the complexity of the analysis. Electrochemical methods such as voltammetry and conductivity methods have the characteristics of rapid response and low cost, but when detecting tosyl groups, they may be affected by electrode selectivity and interfering substances. For tosyl groups on the surface of magnetic beads, electrochemical methods need to solve the problem of how to effectively enrich and detect them on the electrode surface. These analytical detection methods have the advantages of high accuracy and low sample cross-interference, but they also have the disadvantages of high detection technology requirements, long time consumption, and expensive equipment. Therefore, it is of great research significance and practical application value to develop an accurate, fast and efficient detection method for tosyl groups on the surface of magnetic beads. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a colorimetric detection method for the tosyl content on the surface of magnetic beads, by allowing arginine to react with the tosyl groups on the surface of the magnetic beads to be tested, then utilizing the superparamagnetism of the tosyl magnetic beads to separate the magnetic beads, and using the ninhydrin colorimetric method to detect the content of the remaining arginine, thereby further determining the content of the tosyl groups on the surface of the magnetic beads to be tested.

[0007] The first object of the present invention is to provide a colorimetric detection method for the tosyl content on the surface of sulfonyl magnetic beads, wherein arginine reacts with the tosyl groups on the surface of the magnetic beads to be tested, the magnetic beads are separated, and the content of the remaining arginine is detected by the ninhydrin colorimetric method to determine the content of the tosyl groups on the surface of the magnetic beads to be tested.

[0008] Further, the reaction principle is as follows:

[0009]

[0010] Among them, 1 is tosyl magnetic beads, 2 is arginine, 3 is acylated product, 4 is ninhydrin hydrate, and 5 is colored product.

[0011] Furthermore, the method specifically includes the following steps:

[0012] Step S1, mixing the magnetic beads and the arginine solution under alkaline conditions for reaction, and separating the magnetic beads from the remaining arginine by magnetic attraction after the reaction is completed;

[0013] Step S2, adding a buffer solution and a ninhydrin-ascorbic acid mixed solution to the remaining arginine, and heating to form a colored product;

[0014] Step S3, establishing a standard curve according to the absorbance of the UV-visible absorption spectrum at 569 nm;

[0015] Step S4, obtaining the consumed arginine content through the corresponding relationship between the arginine concentration and the absorbance value on the standard curve, and determining the tosyl content on the surface of the magnetic beads to be tested.

[0016] Furthermore, the arginine is D-arginine.

[0017] Furthermore, the magnetic beads are tosyl magnetic beads, silica tosyl magnetic beads or polystyrene tosyl magnetic beads.

[0018] In one embodiment of the present invention, the magnetic beads are silica tosyl magnetic beads.

[0019] Furthermore, the volume ratio of the magnetic beads to arginine is 2-4:100.

[0020] Preferably, the volume ratio of the magnetic beads to arginine is 3:100.

[0021] Furthermore, the volume ratio of ninhydrin to L-ascorbic acid is 5-20:1.

[0022] Preferably, the volume ratio of ninhydrin to L-ascorbic acid is 9:1.

[0023] Furthermore, the buffer comprises potassium dihydrogen phosphate and disodium hydrogen phosphate.

[0024] Furthermore, the molar concentration ratio of potassium dihydrogen phosphate to sodium dihydrogen phosphate in the buffer is 1:6.2-19.5.

[0025] Preferably, the volume ratio of potassium dihydrogen phosphate to disodium hydrogen phosphate is 1:19.

[0026] Furthermore, the pH value of the buffer solution is 8.0-8.5.

[0027] Preferably, the pH value of the buffer solution is 8.04.

[0028] Furthermore, the heating time is 15-20 minutes.

[0029] Preferably, the heating time is 15 minutes.

[0030] In one embodiment of the present invention, the heating is boiling water bath heating.

[0031] Beneficial effects of the present invention:

[0032] The colorimetric detection method for the tosyl content on the surface of magnetic beads of the present invention utilizes the superparamagnetism of tosyl magnetic beads to separate the magnetic beads from arginine in the simplest way, while avoiding complex pretreatment, and is simple to operate. No large-scale equipment is required in the whole detection process, and semi-quantitative analysis of tosyl on the surface of magnetic beads is achieved through color change, and quantitative detection of tosyl on the surface of magnetic beads is achieved through an ultraviolet spectrophotometer. The method has high sensitivity and fast detection speed, and has great research significance and practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0034] Figure 1 The present invention relates to a reaction process, wherein 1 is tosyl magnetic beads, 2 is arginine, 3 is an acylated product, 4 is ninhydrin hydrate, and 5 is a colored product;

[0035] Figure 2 : are photos taken under natural light before and after the reaction of different concentrations of arginine and ninhydrin in the embodiment of the present invention, wherein A is the image before the reaction and B is the image after the reaction;

[0036] Figure 3 is the UV-visible absorption spectrum of ninhydrin in the embodiment of the present invention in response to different concentrations of arginine;

[0037] Figure 4 is a linear fitting relationship diagram between ultraviolet absorbance value and arginine concentration in an embodiment of the present invention;

[0038] Figure 5 The SiO used in the embodiment of the present invention 2 @Fe 3 O 4 Polarized light microscope image of magnetic beads;

[0039] Figure 6 In the embodiment of the present invention, SiO 2 @Fe 3 O 4 After the reaction between magnetic beads and arginine is completed, the images before and after magnetic separation;

[0040] Figure 7 It is the UV-visible absorption spectrum of ninhydrin in response to arginine after reacting with different samples in the comparative example of the present invention. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0042] The reagent configuration method involved in the embodiment is as follows:

[0043] Preparation of potassium dihydrogen phosphate and disodium hydrogen phosphate buffer: weigh 0.4535 g of potassium dihydrogen phosphate on an analytical balance, dilute with a small amount of ultrapure water, transfer to a 50 mL volumetric flask, add ultrapure water and make up to the scale; weigh 1.1938 g of disodium hydrogen phosphate on an analytical balance, dilute with a small amount of ultrapure water, transfer to a 50 mL volumetric flask, add ultrapure water and make up to the scale; take 2.5 mL of potassium dihydrogen phosphate solution and 47.5 mL of sodium dihydrogen phosphate solution and mix evenly to obtain potassium dihydrogen phosphate and disodium hydrogen phosphate buffer.

[0044] Preparation of ninhydrin-ascorbic acid mixed solution: 0.01 g L-ascorbic acid was weighed on an analytical balance and dispersed in 10 mL ultrapure water to obtain an ascorbic acid solution; then 0.1 g hydrated ninhydrin was weighed and diluted with 5 mL ultrapure water, ultrasonicated at 25°C for 15-20 min, 0.5 mL ascorbic acid solution was added, and then 4.5 mL ultrapure water was added to obtain a ninhydrin-ascorbic acid mixed solution.

[0045] Preparation of 1 mg / mL arginine solution: Use an analytical balance to weigh 0.05 g of arginine solid powder and add it to a small amount of ultrapure water for dilution. Transfer it to a 50 mL volumetric flask, add ultrapure water and dilute to the mark to obtain a 1 mg / mL arginine solution.

[0046] Example 1

[0047] Add 0.5 mL of potassium dihydrogen phosphate and disodium hydrogen phosphate buffer, 1 mL of arginine solutions with concentrations of 110 μg / mL, 140 μg / mL, 170 μg / mL, 200 μg / mL, 230 μg / mL, 260 μg / mL, 290 μg / mL, and 320 μg / mL to the centrifuge tube, then add 0.5 mL of ninhydrin-ascorbic acid mixed solution, heat in a boiling water bath for 15 min, and add 3 mL of ultrapure water after cooling.

[0048] After mixing evenly, the fully reacted solution was tested by UV-visible spectrophotometer. The images of the solution before and after the reaction were as follows: Figure 2 As shown in FIG. 1 , the concentration data of arginine and the absorbance data at 569 nm obtained during its detection are plotted to obtain the colorimetric working curve of arginine. The results are shown in FIG. Figure 3 and Figure 4 ,in Figure 3 is the UV-visible absorption spectrum in response to different concentrations of arginine, Figure 4 The figure is a linear relationship between the UV absorbance value and the arginine concentration. The absorbance value at 569nm and the arginine concentration show a good linear relationship, and the linear equation of the working curve is y=0.0069[Arg]-0.7914, R 2 =0.9914, where [Arg] represents the concentration of arginine and y is the absorbance at 569 nm.

[0049] Example 2

[0050] Take 30 μL of 1 mg / mL tosyl magnetic beads (polarizing microscope image as shown in Figure 5 The solution was dispersed into a 200 μg / mL arginine solution and vibrated at 500 rpm using a micro-oscillator for 15-20 min. The magnetic beads were separated from the arginine solution by magnetic attraction. The images before and after magnetic attraction separation are shown in FIG. Figure 6 shown.

[0051] Add 0.5 mL of potassium dihydrogen phosphate and disodium hydrogen phosphate buffer, 1 mL of the remaining arginine solution obtained after separation, and then add 0.5 mL of ninhydrin-ascorbic acid mixed solution to the centrifuge tube, heat in a boiling water bath for 15 min, add 3 mL of ultrapure water after cooling, mix well, and test the fully reacted solution with a UV-visible spectrophotometer. According to the colorimetric working curve established in Example 1, the arginine content reacting with the tosyl group on the surface of the magnetic beads is 26.7 μg.

[0052] The calculation formula of tosyl groups on the surface of magnetic beads is: Wherein M(Ts)=155.17, M(Arg)=174.20, V=30 μL, thus the content of tosyl groups on the surface of the magnetic beads was obtained to be 0.7928 mg.

[0053] Comparative Example

[0054] The amount of tosyl groups on the surface of three different tosyl magnetic beads was detected using the method provided in Example 2. The detection results are shown in Table 1. The UV-visible absorption spectra of ninhydrin in response to arginine after reacting with different magnetic bead samples are as follows: Figure 7 shown.

[0055] Since there is no accurate quantitative method for tosyl groups on the surface of magnetic beads in the prior art, the amount of tosyl groups on the surface of three different tosyl magnetic beads is indirectly characterized by chemiluminescence method. The specific steps are as follows:

[0056] (1) Replace the magnetic bead buffer: dilute the magnetic beads to 1 mg / mL with 0.1 mol PB solution, wash them by magnetic suction three times, and finally adjust the volume to 200 μL;

[0057] (2) Dilute TG to 1000 ng / mL, add 200 μL to the washed magnetic beads, then place in a 37°C incubator and incubate overnight with shaking. After 18 hours, remove the beads and wash three times with 0.1 mol PB;

[0058] (3) Add 600 μL of blocking solution (2.5% BSA, 2% sucrose in 0.1 mol PB solution) and incubate in a 37°C incubator for 1 h. After 1 h, remove and discard the blocking solution;

[0059] (4) Dilute TGAb to 12.5 ng / mL (the concentration depends on the range of the final luminescence instrument and is not unique), add 200 μl to each tube, incubate in a 37°C incubator for 1 h, take out after 1 h and wash with PBT solution three times;

[0060] (5) Dilute HRP-labeled goat anti-human antibody IgG to 1000 ng / mL, add 200 μL to each tube, incubate in a 37°C incubator for 1 h, take out after 1 h and wash with PBT solution 5 times;

[0061] (6) Prepare luminol luminescent solution (1:1), add 100 μL of the prepared luminescent solution to each tube, pipette it several times, then add the ELISA plate to the luminometer to read the final result.

[0062] Table 1 Results of different methods for detecting three different tosyl magnetic beads

[0063]

[0064] From the above results, it can be seen that the tosyl content on the surface of magnetic beads is detected using the method provided by the present invention, and the detection result is consistent with the chemiluminescence method. It can be seen that the method provided by the present invention is accurate and reliable, and can be actually applied to quantitatively detect the tosyl content on the surface of tosyl magnetic beads.

[0065] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A colorimetric detection method for the tosyl content on the surface of magnetic beads, characterized in that: Arginine reacts with the tosyl groups on the surface of the magnetic beads to be tested, the magnetic beads are separated, and the content of the remaining arginine is detected using the ninhydrin colorimetric method to determine the content of the tosyl groups on the surface of the magnetic beads to be tested.

2. The colorimetric detection method according to claim 1, characterized in that The specific steps include: Step S1, mixing the magnetic beads and the arginine solution under alkaline conditions for reaction, and separating the magnetic beads from the remaining arginine by magnetic attraction after the reaction is completed; Step S2, adding a buffer solution and a ninhydrin-ascorbic acid mixed solution to the remaining arginine, heating, and forming a colored product; Step S3, establishing a standard curve according to the absorbance of the UV-visible absorption spectrum at 569 nm; Step S4, obtaining the consumed arginine content through the corresponding relationship between the arginine concentration and the absorbance value on the standard curve, and determining the tosyl content on the surface of the magnetic beads to be tested.

3. The colorimetric detection method according to claim 1, characterized in that: The arginine is D-arginine.

4. The colorimetric detection method according to claim 1, wherein: The magnetic beads are tosyl magnetic beads, silicon oxide tosyl magnetic beads or polystyrene tosyl magnetic beads.

5. The colorimetric detection method according to claim 1, wherein: The volume ratio of the magnetic beads to arginine is 2-4:

100.

6. The colorimetric detection method according to claim 1, characterized in that: The volume ratio of ninhydrin to L-ascorbic acid is 5-20:

1.

7. The colorimetric detection method according to claim 1, characterized in that: The buffer comprises potassium dihydrogen phosphate and disodium hydrogen phosphate.

8. The colorimetric detection method according to claim 7, characterized in that: The molar concentration ratio of potassium dihydrogen phosphate to disodium hydrogen phosphate in the buffer is 1:6.2-19.

5.

9. The colorimetric detection method according to claim 7, characterized in that: The pH value of the buffer solution is 8.0-8.

5.

10. The colorimetric detection method according to claim 1, characterized in that: The heating time is 15-20 minutes.

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