A chromogenic solution and kit for detecting the content of free ferrous protoporphyrin in cells by visual colorimetry

Abstract

This invention discloses a chromogenic solution and kit for visual colorimetric detection of free ferrous protoporphyrin in cells. The chromogenic solution consists of reagents A, B, and C: Reagent A is prepared with deionized water and contains NaCl, Proclin 300, and glycerol; Reagent B is prepared with an acetate-sodium acetate buffer at pH 4.0 and contains glycerol, Triton X-100, β-cyclodextrin, disodium EDTA, citric acid, ethanol, and 3,3',5,5'-tetramethylbenzidine; Reagent C is prepared with an acetate-sodium acetate buffer at pH 4.0 and contains 2.0-4.0% hydrogen peroxide and 35.0-50.0% ethanol. The chromogenic solution of this invention has a lower background value and can detect the content of free ferrous protoporphyrin in cells within 10 minutes by visual colorimetry, with a detection limit as low as 10 nmol / L. The chromogenic stability is maintained for 12 hours, making it suitable for rapid assessment of cellular pathological conditions.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry, specifically relating to a colorimetric solution and kit for detecting the content of free ferrous protoporphyrin in cells using a visual colorimetric method. Background Technology

[0002] Ferrous protoporphyrin (FH), also known as heme, is a cofactor for many proteins in epithelial cells. When epithelial cells undergo degeneration, necrosis, apoptosis, or inflammation, their bound FH is released, becoming free FH. Studies have confirmed that FH levels are closely related to the degree of carcinogenesis in tumor cells; higher levels indicate more severe carcinogenesis. For example, detecting free FH based on liquid-based cytology sampling combined with FH immunostaining has important predictive value for cervical cancer screening. Therefore, developing sensitive, accurate, stable, and easy-to-use FH detection methods and kits has significant social and economic value.

[0003] In FH detection methods, 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic solution is widely used due to its high specificity, low cost, ease of use, and visual interpretation of results. The principle is that TMB reacts specifically with hydrogen peroxide in the presence of peroxidase, turning blue, and then turns yellow or yellow-green upon contact with acid. In this reaction, FH can replace the peroxidase as a catalytic agent, enabling quantitative detection. TMB is a safe and mature chromogenic reagent, and chromogenic solutions prepared using TMB as the chromogenic source are important chromogenic substrates in immunodiagnostic kits. Currently, there are hundreds of related ELISA (enzyme-linked immunosorbent assay) kits.

[0004] Unlike ELISA, free free hydroxylamine (FH) exists inside cells. Detecting FH based on a TMB-based colorimetric reaction requires first disrupting the cell membrane to allow the chromogenic agent to rapidly enter the cell and react with the FH. Therefore, the chromogenic solution must contain cell-membrane-disrupting components. Furthermore, TMB itself can be oxidized by air, and the oxidizing agent (hydrogen peroxide) and reducing agent (TMB) in this colorimetric reaction are in the same solution. Therefore, the stability and sensitivity of the solution are the most prominent issues affecting its application.

[0005] Currently, there are numerous patents and literature related to TMB chromogenic solutions, with formulations mainly consisting of single-component and two-component solutions. Firstly, single-component solutions mitigate the auto-reaction of oxidants and reductants by adding TMB stabilizers, eliminating the need for premixing and reducing batch-to-batch operational errors. However, their long-term storage stability and sensitivity are still inferior to two-component solutions. Secondly, while adding synergists to improve TMB chromogenic sensitivity, it also reduces stability; similarly, adding stabilizers to improve TMB stability reduces chromogenic sensitivity. Finally, during long-term storage, TMB itself oxidizes and discolors, inevitably increasing the background value of the detection solution and affecting chromogenic sensitivity. Therefore, balancing stability and sensitivity is the core technical challenge in developing efficient TMB detection solutions. Summary of the Invention

[0006] To address the problems existing in the aforementioned background technology, the purpose of this invention is to provide a more stable chromogenic solution and kit for detecting the content of free ferrous protoporphyrin in cells using a visual colorimetric method that has extremely low background values ​​and does not reduce the sensitivity of the colorimetric reaction. This is achieved through the following technical solution:

[0007] A colorimetric reagent for visually detecting the content of free ferrous protoporphyrin in cells, comprising reagent A, reagent B, and reagent C, wherein:

[0008] Reagent A is a cell collection solution prepared with deionized water, containing 20.0-50.0 g / L glycerol, 9.0 g / L NaCl, and 0.2-0.5 mg / L Proclin 300. 0.9% NaCl serves as an isotonic solution for collecting cells and maintaining their integrity; glycerol stabilizes the solution; and Proclin 300, as a highly effective antibacterial agent, ensures a shelf life of up to 3 years at room temperature.

[0009] Reagent B is an ethanol-water solution of 3,3',5,5'-tetramethylbenzidine, prepared with an acetate-sodium acetate buffer at pH 4.0, containing 20.0-50.0 g / L glycerol, 2.0-5.0 g / L Triton X-100, 3.0-5.0 g / L β-cyclodextrin, 0.3-1.1 g / L disodium ethylenediaminetetraacetate, 0.6-3.8 g / L citric acid, 35.0-50.0% ethanol, and 0.5-1.5 g / L 3,3',5,5'-tetramethylbenzidine; wherein,

[0010] Glycerol-β-cyclodextrin is used in combination to stabilize the aqueous solution and reduce its resistance to photodegradation during storage; disodium EDTA is used to improve the stability of the TMB colorimetric product and extend the reading time. This reagent innovatively uses ethanol and citric acid to coordinate the stability and colorimetric sensitivity of TMB. Detection results are obtained directly by visual colorimetry, eliminating the need for equipment detection, simplifying operation, and possessing greater practical application value and significance.

[0011] The high ethanol content in this invention provides additional H+. + The protonation effect shifts the TMB colorimetric reaction to the right, reducing the detection limit by visual colorimetry from 30 nmol / L to 10 nmol / L, and improving the colorimetric sensitivity by about three times. Secondly, citric acid, ethanol, and the triethyl citrate formed by their mixture have a synergistic antioxidant effect on TMB, and the combination of the two results in the solution's antifungal and antioxidant functions being superimposed, thus allowing the test solution to have a shelf life of more than one year at room temperature. Thirdly, while citric acid itself has a decolorizing effect, it does not have any decolorizing effect on the TMB colorimetric product in this system. This is different from other technologies that use dimethyl sulfoxide and vitamin C, which have a decolorizing effect on the TMB colorimetric product. The test solution has a lower background value and its sensitivity is not affected, thus making the test solution superior in performance.

[0012] Reagent C is an ethanol-water solution of hydrogen peroxide, prepared using an acetate-sodium acetate buffer solution at pH 4.0, containing 2.0-4.0% hydrogen peroxide and 35.0-50.0% ethanol. Ethanol acts as a stabilizer for hydrogen peroxide, avoiding the need for fresh preparation before use.

[0013] Furthermore, the preparation method of reagent A is as follows: add the prescribed amounts of glycerol, NaCl and Proclin 300 to 900 mL of deionized water, dissolve them, and then make up the volume to 1000 mL with deionized water to obtain reagent A.

[0014] Furthermore, reagent B is prepared using the following method:

[0015] 1) Prepare aqueous solutions of sodium acetate and glacial acetic acid with deionized water to a concentration of 0.2 mol / L, and mix them at a volume ratio of sodium acetate solution: acetic acid solution = 4:1 to prepare an acetic acid-sodium acetate buffer solution with a pH of 4.0;

[0016] 2) Add the prescribed amounts of glycerol, Triton X-100, β-cyclodextrin, disodium EDTA dihydrate, and citric acid to 450 mL of the buffer solution prepared in step 1), dissolve and mix well;

[0017] 3) Take the prescribed amount of 3,3',5,5'-tetramethylbenzidine and add it to 350-500 mL of anhydrous ethanol, dissolve and mix well;

[0018] 4) Mix the solutions obtained in steps 2) and 3) completely, and bring the volume up to 1000 mL with an acetate-sodium acetate buffer solution with a pH of 4.0 to obtain reagent B.

[0019] Furthermore, reagent C is prepared using the following method:

[0020] 1) Add 66.7-133.3 mL of commercially available 30% hydrogen peroxide aqueous solution to 300 mL of acetate-sodium acetate buffer solution with a pH of 4.0;

[0021] 2) Add 350-500 mL of anhydrous ethanol to the acetate-sodium acetate buffer prepared in step 1), and adjust the volume to 1000 mL with acetate-sodium acetate buffer at pH 4.0 to obtain reagent C.

[0022] A reagent kit comprising the above-described colorimetric solution.

[0023] The two-component TMB chromogenic solution of this invention can rapidly detect the content of free FH in cells within 10 minutes by visual colorimetry, with a detection limit as low as 10 nmol / L. The chromogenic stability can be maintained for 12 hours without decay, thus enabling a simple assessment of the degree of cell lesions. Attached Figure Description

[0024] Figure 1 The background absorbance values ​​of the detection solution of this invention under different storage times (room temperature, light-proof, and sealed conditions) are shown. Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments in order to better understand the technical solution.

[0026] To demonstrate the economy and practicality of this invention, all chemical reagents used in this application are of analytical grade. The cervical cancer cell line HeLa, lung cancer cell line PC9, mouse colon cancer cell line CT26, primary lung cancer cells from malignant effusions of lung cancer patients, and their primary normal fibroblasts used in subsequent embodiments are all materials previously used in research funding projects in which the inventors participated, and the collection of these materials complies with medical ethics requirements.

[0027] The specific steps of this invention for detecting the content of free ferrous protoporphyrin in cells are as follows:

[0028] Step 1: Prepare reagents A, B, and C respectively according to the specific preparation method of this invention;

[0029] Step 2: Determine the content of free FH in cells, including the following steps:

[0030] a) Add 1.0 mL of reagent A to the cell sample obtained by cell precipitation or liquid-based cell sampling method;

[0031] b) Add 0.5 mL of reagent B and 0.5 mL of reagent C to the solution in a) above;

[0032] c) After 10 minutes, visually determine the content of free FH in the cells based on the color intensity of the color bar: if the solution turns blackish-blue or a black precipitate subsequently forms, the FH content is greater than 1.0 μmol / L; if the solution turns dark blue, the FH content is 0.5-1.0 μmol / L; if the solution turns blue, the FH content is 0.1-0.5 μmol / L; if the solution turns light blue, the FH content is 50.0-100.0 nmol / L; if the solution turns pale greenish-blue, the FH content is 10.0-50.0 nmol / L; if the solution does not change color, the FH content is less than 10.0 nmol / L.

[0033] This invention, through stability testing of the new formulation, shows that under room temperature and light-protected conditions, the signal attenuation of the detection solution after one year of storage is only about 20%, and the background value after attenuation is far below the detection limit without affecting sensitivity. Therefore, the detection solution is superior to other commercially available similar products in balancing TMB stability and colorimetric sensitivity. Figure 1 .

[0034] To verify the effectiveness of the colorimetric solution of the present invention in determining free FH in cells, this application describes the application using heme standards and various tumor cells.

[0035] Example 1: Colorimetric results of different concentrations of heme chloride solutions detected by colorimetric reagent.

[0036] Using heme chloride as a simulated standard, the color development of the colorimetric solution in the presence of different concentrations of heme chloride was determined, demonstrating the sensitivity of the colorimetric solution described in this invention in detecting FH. The steps are as follows:

[0037] Step 1: Prepare reagents A, B, and C according to the first step in the technical solution;

[0038] Step 2, following step a) in the technical solution, prepare heme chloride solutions of different concentrations using 1.0 mL of reagent A respectively;

[0039] Step 3: Mix reagents B and C according to step b) of the technical solution, and add them to the above-mentioned heme chloride solutions of different concentrations respectively;

[0040] Step 4: Following step c) of the technical solution, observe the color development of the solution. The results are shown in Table 1.

[0041] Table 1 shows the visual color development results of the colorimetric solution in the presence of different concentrations of heme chloride.

[0042]

[0043]

[0044] Visual inspection revealed that after 10 minutes of color development with FH detection solution, heme chloride solutions of different concentrations exhibited varying shades of blue, decreasing in concentration: When the heme chloride concentration was greater than 1.0 μmol / L, the solution appeared dark blue, with higher concentrations accompanied by the precipitation of a black precipitate; when the heme chloride concentration was between 0.5 and 1.0 μmol / L, the solution appeared deep blue; when the heme chloride concentration was between 0.1 and 0.5 μmol / L, the solution appeared blue; when the heme chloride concentration was between 50.0 and 100.0 nmol / L, the solution appeared light blue; when the heme chloride concentration was between 10.0 and 50.0 nmol / L, the solution appeared pale greenish-blue; and when the heme chloride concentration was less than 10.0 nmol / L, no color change was visually apparent. This color gradient can be used as a reference concentration for the content of free FH in cells.

[0045] The results also showed that after the colorimetric solution was placed at room temperature in the dark for 12 hours, the detection limit was still 10.0 nmol / L; after 18 hours, the detection limit decreased to 20.0 nmol / L; and after 24 hours, the detection limit decreased to 50.0 nmol / L. This indicates that the colorimetric effect of the solution can remain stable for a long time, avoiding batch-to-batch errors in readings.

[0046] Example 2: Colorimetric results of detecting free FH in tumor cells using a colorimetric reagent.

[0047] To demonstrate the effectiveness of the chromogenic solution of this invention in detecting free FH in cells, the content of free FH in different numbers of tumor cells was first detected using human cervical cancer cell line HeLa, human lung cancer cell line PC9, and mouse colorectal cancer cell line CT26 as diseased cells. The steps are as follows:

[0048] Step 1: Prepare reagents A, B, and C according to the first step in the technical solution;

[0049] Step 2: Culture tumor cells in vitro and collect them quantitatively. The steps are as follows:

[0050] a) Tumor cell lines HeLa, PC9, and CT26 were cultured in vitro using RPMI 1640 medium until the cell density reached 80%.

[0051] b) Digest tumor cells with 0.25% trypsin and resuspend the cells in PBS (pH 7.4) buffer;

[0052] c) After microscopic counting, the tumor cells were prepared into suspensions containing different numbers of tumor cells.

[0053] d) After centrifuging the cells at 300g for 5 minutes, discard the supernatant;

[0054] Step 3, following step a) of the second step in the technical solution, resuspend the above-mentioned different numbers of tumor cells in 1.0 mL of reagent A respectively;

[0055] Step 4: Mix reagents B and C according to step b) of the technical solution, and then add them to the solutions containing different numbers of tumor cells.

[0056] Step 5: Following step c) of the technical solution, observe the color development of the solution. The results are shown in Table 2.

[0057] Table 2 shows the colorimetric results of the chromogenic solution in detecting free FH in tumor cells.

[0058]

[0059] The results showed that free FH was detected in all tumor cell lines, and the content of free FH varied among different tumor cell lines. When the chromogenic solution is used for the detection of free FH in tumor cells, the cell count in the sample must be greater than 1 × 10⁻⁶ cells / year to ensure the accuracy of the results. 4 The index solution can be used as one of the parameters of the colorimetric solution during application.

[0060] To further illustrate the effectiveness of the chromogenic solution in detecting free FH in cells, the inventors, adhering to medical ethics, used primary lung cancer cells collected from malignant effusions of 30 lung cancer patients as diseased cells in previous research, and 5 primary normal fibroblasts as controls, to test the chromogenic effect of the chromogenic solution in actual cell samples, demonstrating its practical application effect. The steps are as follows:

[0061] Step 1: Prepare reagents A, B, and C according to the first step in the technical solution;

[0062] Step 2: Culture primary cells in vitro and collect them quantitatively. The steps are as follows:

[0063] a) Following commonly used procedures, primary lung cancer cells and primary fibroblasts were cultured in vitro in RPMI 1640 medium until the density reached 80%.

[0064] b) Primary cells were digested with 0.25% trypsin and resuspended in PBS (pH 7.4) buffer;

[0065] c) After microscope counting, each sample was prepared to contain 5×104 A suspension of individual cells;

[0066] d) After centrifuging the cell solution, discard the supernatant;

[0067] Step 3, following step a) of the second step in the technical solution, resuspend the above primary cells with 1.0 mL of reagent A;

[0068] Step 4: Mix reagents B and C according to step b) of the technical solution, and then add them to the primary cell solution mentioned above.

[0069] Step 5: Following step 2(c) in the technical solution, observe the color development of the solution. If the solution changes color (i.e., the FH content is greater than 10.0 nmol / L), the cell sample is positive; if the solution does not change color (i.e., the FH content is less than 10.0 nmol / L), the cell sample is negative. The results are shown in Table 3.

[0070] Table 3 shows the colorimetric results of the chromogenic solution in detecting free FH in primary cells.

[0071] sample Positive Negative Primary lung cancer cells 27 3 Primary normal fibroblasts 0 5

[0072] The results showed that the detection rate of the test solution reached 90% in the detection of actual tumor cell samples, with no false positive rate, which demonstrates the effectiveness of the chromogenic solution in detecting actual diseased cells.

Claims

1. A colorimetric solution for detecting the content of free ferrous protoporphyrin in cells using a visual colorimetric method, characterized in that, The colorimetric solution consists of reagent A, reagent B, and reagent C, wherein: Reagent A is a cell collection solution prepared with deionized water, containing 20.0-50.0 g / L glycerol, 9.0 g / L NaCl, and 0.2-0.5 mg / L Proclin 300; Reagent B is an ethanol-water solution of 3,3',5,5'-tetramethylbenzidine, prepared with an acetate-sodium acetate buffer at pH 4.0, containing 20.0-50.0 g / L glycerol, 2.0-5.0 g / L Triton X-100, 3.0-5.0 g / L β-cyclodextrin, 0.3-1.1 g / L disodium ethylenediaminetetraacetate, 0.6-3.8 g / L citric acid, 35.0-50.0% ethanol, and 0.5-1.5 g / L 3,3',5,5'-tetramethylbenzidine. Reagent C is an ethanol-water solution of hydrogen peroxide, prepared with an acetate-sodium acetate buffer solution at pH 4.0, containing 2.0-4.0% hydrogen peroxide and 35.0-50.0% ethanol.

2. The colorimetric solution for detecting the content of free ferrous protoporphyrin in cells by visual colorimetry as described in claim 1, characterized in that... The preparation method of reagent A is as follows: add the prescribed amount of glycerol, NaCl and Proclin 300 to 900 mL of deionized water, dissolve and then make up to 1000 mL with deionized water to obtain reagent A.

3. The colorimetric solution for detecting the content of free ferrous protoporphyrin in cells by visual colorimetry as described in claim 1, characterized in that... Reagent B is prepared using the following method: 1) Prepare aqueous solutions of sodium acetate and glacial acetic acid with deionized water to a concentration of 0.2 mol / L, and mix them at a volume ratio of sodium acetate solution: acetic acid solution = 4:1 to prepare an acetic acid-sodium acetate buffer solution with a pH of 4.0; 2) Add the prescribed amounts of glycerol, Triton X-100, β-cyclodextrin, disodium EDTA dihydrate, and citric acid to 450 mL of the buffer solution prepared in step 1). Dissolve and mix well; 3) Take the prescribed amount of 3,3',5,5'-tetramethylbenzidine and add it to 350-500 mL of anhydrous ethanol, dissolve and mix well; 4) Mix the solutions obtained in steps 2) and 3) completely, and bring the volume up to 1000 mL with an acetate-sodium acetate buffer solution with a pH of 4.0 to obtain reagent B.

4. The colorimetric solution for detecting the content of free ferrous protoporphyrin in cells by visual colorimetry as described in claim 1, characterized in that... Reagent C is prepared using the following method: 1) Add 66.7-133.3 mL of commercially available 30% hydrogen peroxide aqueous solution to 300 mL of acetate-sodium acetate buffer solution with a pH of 4.0; 2) Add 350-500 mL of anhydrous ethanol to the acetate-sodium acetate buffer prepared in step 1), and adjust the volume to 1000 mL with acetate-sodium acetate buffer at pH 4.0 to obtain reagent C.

5. A reagent kit, characterized in that, The kit includes the colorimetric solution according to any one of claims 1-4.

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