Method for detecting content of manganese ions in cells

Abstract

The invention discloses a method for detecting the content of manganese ions in cells, and relates to the technical field of biological and medical analysis. The method comprises the following steps: performing cell lysis, preparing a standard solution, establishing a standard curve and a regression equation of manganese ion concentration and absorbance difference, and calculating the manganese ion content by measuring the absorbance difference between a sample to be detected and a blank sample and combining with protein concentration. The method is simple and convenient to operate, high in sensitivity and suitable for quantitative detection of trace manganese ions in cells, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical detection technology, specifically relating to a method for detecting intracellular manganese ion content. Background Technology

[0002] Manganese ions (Mn) 2 Mn is an essential trace metal element in living organisms, widely distributed in subcellular structures such as the cytoplasm, mitochondria, and nucleus, playing a crucial role in various life activities. Under physiological conditions, Mn... 2 It participates in the formation of the active sites of many metabolism-related enzymes, such as mitochondrial superoxide dismutase (Mn-SOD), pyruvate carboxylase, and glutamate dehydrogenase, regulating basic physiological processes such as redox balance, energy metabolism, amino acid metabolism, and glucose and lipid metabolism. 2 It is also a key factor in neurotransmitter synthesis, axonal growth, and synaptic plasticity regulation, maintaining normal nervous system function and cognitive abilities.

[0003] Manganese ion metabolism disorders have been proven to be closely related to a variety of diseases. 2 Manganese deficiency can lead to decreased immune function, skeletal development disorders, and impaired reproductive capacity; while excess is even more toxic, especially prone to accumulating in the brain, inducing neurotoxicity, manifesting as motor disorders similar to Parkinson's disease, and even causing manganese toxic encephalopathy. Furthermore, a growing body of research has found a potential link between abnormal manganese metabolism and neurodegenerative or neurodevelopmental disorders such as Alzheimer's disease, autism spectrum disorders, and depression. Therefore, accurately understanding the levels of Mn at the cellular level is crucial. 2 The dynamic changes are of great significance for the study of disease mechanisms, drug screening, and environmental metal toxicity assessment.

[0004] Currently, Mn 2 Detection primarily relies on techniques such as inductively coupled plasma mass spectrometry (ICP-MS), atomic absorption spectrometry (AAS), and electrochemical analysis. While these methods offer high sensitivity and accuracy, they generally suffer from drawbacks including expensive equipment, complex operation, long detection cycles, significant sample destructiveness, and severe matrix interference, limiting their application, especially in biological samples such as live cells, subcellular organelles, and tissue sections. Furthermore, these traditional methods often fail to detect intracellular Mn. 2 Visual positioning is also insufficient to meet the research needs of in vivo imaging, dynamic monitoring, and high-throughput screening.

[0005] Therefore, it is necessary to develop a method that is suitable for biological systems, highly sensitive, selective, easy to operate, and capable of achieving Mn at the cellular or subcellular level. 2Real-time monitoring and detection methods have become a key challenge and research hotspot in analytical chemistry, life sciences, and pharmaceutical research. An ideal detection method should possess the following characteristics: firstly, it should accurately reflect intracellular free Mn without requiring complex sample processing. 2 It exhibits two key characteristics: firstly, it addresses concentration changes; secondly, it possesses high spatiotemporal resolution, enabling it to be used for monitoring Mn. 2 It enables dynamic transport and homeostasis regulation between organelles; thirdly, it possesses good biocompatibility and low toxicity, making it suitable for long-term live-cell imaging and in vivo applications; and fourthly, it exhibits high selectivity, avoiding interference from other metal ions such as Ca. 2 Fe 2 Zn 2 Interference, etc.

[0006] Based on these needs, an increasing number of studies are focusing on novel detection methods such as fluorescent probes, biosensors, and molecular imprinting techniques to achieve the detection of intracellular Mn. 2 This method offers rapid, specific, and highly sensitive detection. It not only expands the technical means for imaging and quantifying metal ions within cells but also provides a powerful tool for elucidating the mechanisms by which manganese plays a role in disease development and is expected to advance the diagnosis and treatment of metal ion-related diseases. Summary of the Invention

[0007] The present invention discloses a method for detecting intracellular manganese ion content, characterized in that the detection method comprises the following steps:

[0008] (1) Cell lysis: For adherent cells, after discarding the culture medium, wash three times with PBS buffer, add RIPA lysis buffer to the cell culture dish, and repeatedly pipette to completely lyse the cells. After centrifugation at 12000 rpm for 20 min at 4°C, the supernatant is obtained as the test sample. The protein concentration of the test sample is detected using a BCA kit. The entire operation must be performed on ice; the number of cells to be tested should not be less than 1.2-2 × 10⁻⁶. 6 indivual.

[0009] (2) Preparation of standard solutions: Add potassium pyrophosphate-sodium acetate buffer and manganese standard solution of known concentration, mix thoroughly, then add potassium periodate solution and mix thoroughly to obtain the standard solution; the manganese standard solution is manganese chloride solution, which is prepared by diluting 1M manganese chloride with ultrapure water to obtain manganese standard solutions of different concentrations, namely 10μM, 15μM, 25μM, 50μM, 100μM, 200μM and 400μM; the final concentrations of potassium pyrophosphate and sodium acetate in the potassium pyrophosphate-sodium acetate buffer are 0.6M and 0.1M, respectively, and the pH is adjusted to about 7 with acetic acid; the concentration of potassium periodate solution is 0.1M; in the preparation of the standard solution, blank sample or test sample, the volume ratio of each reagent is: the volume of potassium pyrophosphate-sodium acetate solution is 20μL and the volume of potassium periodate solution is 10μL.

[0010] (3) Establish a standard curve: Plot the difference in absorbance between standard solutions of different concentrations and blank samples as the ordinate and the concentration of manganese chloride in the standard solution as the abscissa to obtain a standard curve of target concentration and absorbance change; establish a regression equation for the relationship between target manganese ion concentration and absorbance value based on the standard curve; the specific method is to place the standard solution and blank control solution in a 96-well microplate and measure the absorbance value at 525nm using a microplate reader.

[0011] (4) This method is suitable for detecting intracellular manganese ions with a detection limit of 10 μM.

[0012] The detection method described in this invention has the following technical advantages: It is simple to operate and requires no complex instruments. This invention abandons the traditional detection method that relies on large-scale equipment such as ICP-MS, and achieves rapid detection of manganese ions in cells through a novel detection system (such as fluorescent probes / colorimetric methods), lowering the experimental threshold and making it suitable for use under ordinary laboratory conditions; it can detect Mn in living cells. 2+ Dynamic monitoring and visualization: The detection method employed can reflect the spatial distribution and concentration changes of manganese ions in real time at the cellular level, possessing excellent spatiotemporal resolution, making it particularly suitable for studying the dynamic behavior of manganese ions in physiological or pathological states such as nerve cells and microglia; High selectivity and low interference: This method targets Mn 2 It has high selectivity and can effectively distinguish other divalent metal ions (such as Fe). 2 Zn 2 Cu 2 Ca 2This invention avoids the common problem of metal ion cross-interference in traditional methods, thereby improving the accuracy and reliability of detection; it has excellent biocompatibility and is suitable for long-term imaging: the detection system has no obvious toxicity to cells and can be widely used for long-term detection in live cell culture systems, suitable for high-throughput screening, drug efficacy evaluation, metal homeostasis research and other application scenarios; it has strong prospects for promotion and application: the method provided by this invention can not only be used for the study of manganese metabolism and signaling pathways in basic scientific research, but also applied to environmental toxicology, neurodegenerative disease research, heavy metal pollution assessment and other fields, providing technical support for the early diagnosis and intervention of diseases related to metal ion metabolism disorders. Attached Figure Description

[0013] Figure 1 This is the correspondence between manganese chloride and absorbance difference (ΔA / A0) in Example 1.

[0014] Figure 2 This is the correspondence between manganese chloride and absorbance difference (ΔA / A0) in Example 2.

[0015] Figure 3 This is the correspondence between manganese chloride and absorbance difference (ΔA / A0) in Example 3.

[0016] Figure 4 This is the correspondence between manganese chloride and absorbance difference (ΔA / A0) in Example 4. Detailed Implementation

[0017] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0018] Example 1

[0019] Plot a standard curve showing the change in target manganese ion concentration versus absorbance value. The standard curve is as follows: Figure 1As shown, this includes the following steps: preparing a test solution with a known target manganese ion concentration to obtain solution A. Specifically, multiple centrifuge tubes are taken, and 200 μl of manganese chloride solution of different concentrations is added to each tube. Then, 20 μl of potassium pyrophosphate-sodium acetate buffer (final concentrations of 0.6 M and 0.1 M, respectively) is added, and after thorough mixing, 10 μl of... 0.1M potassium periodate solution, thoroughly mixed, becomes the test group, solution A. Take one centrifuge tube and replace the known concentration of manganese chloride solution with ultrapure water, following the same method as preparing solution A to obtain the blank control group, solution B. Take 200 μl of solution A from step ① and solution B from step ② respectively, and place them in a 96-well microplate. Measure the absorption peak at 525 nm using a microplate reader. Process the absorbance values ​​of the blank control (A1) and the sample (A2) using software, and calculate the difference ΔA = A2 - A1 to obtain the absorbance difference. Based on the absorbance values, plot the absorbance difference between solutions A and B of different concentrations as the ordinate and the manganese ion concentration as the abscissa to obtain a standard curve of target concentration versus absorbance change. Based on the standard curve of target concentration versus absorbance change, establish a regression equation for the relationship between target concentration and absorbance change: y = 0.0009x + 0.0038R. 2 =0.9978. Experimental data show that the reaction was most stable when the final concentrations of the potassium pyrophosphate-sodium acetate buffer were 0.6M and 0.1M, respectively.

[0020] To prepare the test solution, solution C is obtained by replacing the target standard solution of known concentration with the actual sample solution. The test solution, C, can be prepared following step ①. The absorbance of solution C at 525 nm is measured. The absorbance difference between solution C and solution B is calculated. Substituting this absorbance difference into the linear regression equation yields the concentration of manganese ions in solution C.

[0021] Example 2

[0022] Plot a standard curve showing the change in target manganese ion concentration versus absorbance value. The standard curve is as follows: Figure 2As shown, this includes the following steps: preparing a test solution with a known target manganese ion concentration to obtain solution A. Specifically, multiple centrifuge tubes are taken, and 200 μl of manganese chloride solution of different concentrations is added to each tube. Then, 20 μl of potassium pyrophosphate-sodium acetate buffer (final concentrations of 0.1 M and 0.1 M respectively) is added, and after thorough mixing, 10 μl of [the solution] is added. 0.1M potassium periodate solution, thoroughly mixed, becomes the test group, solution A. Take one centrifuge tube and replace the known concentration of manganese chloride solution with ultrapure water, following the same method as preparing solution A to obtain the blank control group, solution B. Take 200 μl of solution A from step ① and solution B from step ② respectively, and place them in a 96-well microplate. Measure the absorption peak at 525 nm using a microplate reader. Process the absorbance values ​​of the blank control (A1) and the sample (A2) using software, and calculate the difference ΔA = A2 - A1 to obtain the absorbance difference. Based on the absorbance values, plot the absorbance difference between solutions A and B of different concentrations as the ordinate and the manganese ion concentration as the abscissa to obtain a standard curve of target concentration versus absorbance change. Based on the standard curve of target concentration versus absorbance change, establish a regression equation for the relationship between target concentration and absorbance change: y = 0.0008x + 0.0118R. 2 =0.9895. Experimental data show that the reproducibility of this reaction is poor when the final concentrations of the potassium pyrophosphate-sodium acetate buffer are 0.1M and 0.1M, respectively.

[0023] To prepare the test solution, solution C is obtained by replacing the target standard solution of known concentration with the actual sample solution. The test solution, C, can be prepared following step ①. The absorbance of solution C at 525 nm is measured. The absorbance difference between solution C and solution B is calculated. Substituting this absorbance difference into the linear regression equation yields the concentration of manganese ions in solution C.

[0024] Example 3

[0025] Plot a standard curve showing the change in target manganese ion concentration versus absorbance value. The standard curve is as follows: Figure 3As shown, this includes the following steps: preparing a test solution with a known target manganese ion concentration to obtain solution A. Specifically, multiple centrifuge tubes are taken, and 200 μl of manganese chloride solution of different concentrations is added to each tube. Then, 20 μl of potassium pyrophosphate-sodium acetate buffer (final concentrations of 0.1 M and 0.6 M, respectively) is added, and after thorough mixing, 10 μl of... 0.1M potassium periodate solution, thoroughly mixed, becomes the test group, solution A. Take one centrifuge tube and replace the known concentration of manganese chloride solution with ultrapure water, following the same method as preparing solution A to obtain the blank control group, solution B. Take 200 μl of solution A from step ① and solution B from step ② respectively, and place them in a 96-well microplate. Measure the absorption peak at 525 nm using a microplate reader. Process the absorbance values ​​of the blank control (A1) and the sample (A2) using software, and calculate the difference ΔA = A2 - A1 to obtain the absorbance difference. Based on the absorbance values, plot the absorbance difference between solutions A and B of different concentrations as the ordinate and the manganese ion concentration as the abscissa to obtain a standard curve of target concentration versus absorbance change. Based on the standard curve of target concentration versus absorbance change, establish a regression equation for the relationship between target concentration and absorbance change: y = 0.0007x + 0.0119R. 2 =0.9695. Based on experimental data, the standard curve R of this reaction was [value missing] when the final concentrations of the potassium pyrophosphate-sodium acetate buffer were 0.1M and 0.6M, respectively. 2 <0.99.

[0026] To prepare the test solution, solution C is obtained by replacing the target standard solution of known concentration with the actual sample solution. The test solution, C, can be prepared following step ①. The absorbance of solution C at 525 nm is measured. The absorbance difference between solution C and solution B is calculated. Substituting this absorbance difference into the linear regression equation yields the concentration of manganese ions in solution C.

[0027] Example 4

[0028] Plot a standard curve showing the change in target manganese ion concentration versus absorbance value. The standard curve is as follows: Figure 4As shown, this includes the following steps: preparing a test solution with a known target manganese ion concentration to obtain solution A. Specifically, multiple centrifuge tubes are taken, and 200 μl of manganese chloride solution of different concentrations is added to each tube. Then, 20 μl of phosphate buffer (prepared using sodium dihydrogen phosphate dihydrate and disodium hydrogen phosphate dodecahydrate, with a final concentration of 0.1 M) is added, and after thorough mixing, 10 μl of... 0.1M potassium periodate solution, thoroughly mixed, becomes the test group, solution A. Take one centrifuge tube and replace the known concentration of manganese chloride solution with ultrapure water, following the same method as preparing solution A to obtain the blank control group, solution B. Take 200 μl of solution A from step ① and solution B from step ② respectively, and place them in a 96-well microplate. Measure the absorption peak at 525 nm using a microplate reader. Process the absorbance values ​​of the blank control (A1) and the sample (A2) using software, and calculate the difference ΔA = A2 - A1 to obtain the absorbance difference. Based on the absorbance values, plot the absorbance difference between solutions A and B of different concentrations as the ordinate and the manganese ion concentration as the abscissa to obtain a standard curve of target concentration versus absorbance change. Based on the standard curve of target concentration versus absorbance change, establish a regression equation for the relationship between target concentration and absorbance change: y = 0.0004x + 0.0034R. 2 =0.9979

[0029] To prepare the test solution, solution C is obtained by replacing the target standard solution of known concentration with the actual sample solution. The test solution, C, can be prepared following step ①. The absorbance of solution C at 525 nm is measured. The absorbance difference between solution C and solution B is calculated. Substituting this absorbance difference into the linear regression equation yields the concentration of manganese ions in solution C.

[0030] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles of the present invention, and all such changes and modifications fall within the protection scope of the present invention.

Claims

1. A method for detecting intracellular manganese ion content, characterized in that: The process includes the following steps: (1) Cell lysis: For adherent cells, after discarding the culture medium, wash three times with PBS buffer, add RIPA lysis buffer to the cell culture dish, repeatedly pipette to completely lyse the cells, centrifuge at 4°C for 12000 rpm for 20 min to obtain the supernatant, which is the sample to be tested. Use the BCA kit to detect the protein concentration of the sample to be tested; the entire operation must be performed on ice; (2) Preparation of standard solution: Add potassium pyrophosphate-sodium acetate buffer and manganese standard solution of known concentration, mix thoroughly, then add potassium periodate solution and mix thoroughly to obtain the standard solution; (3) Establishment of standard curve: Plot the difference in absorbance between standard solutions of different concentrations and blank samples as the ordinate and the concentration of manganese chloride in the standard solution as the abscissa to obtain the standard curve of target concentration versus absorbance change; Establish a regression equation for the relationship between the target manganese ion concentration and absorbance value based on the standard curve; (4) Detection of the test sample: Prepare the standard solution of the test sample according to the method in step (2), and then measure its absorbance value at a wavelength of 525nm. Calculate the absorbance difference between the test sample and the blank sample. Substitute the absorbance difference into the linear regression equation and then divide it by the protein concentration (gprot / L) of the test sample before adding it to the detection system to obtain the concentration of manganese ions in the test sample.

2. The method for detecting manganese ion content in cells according to claim 1, characterized in that: The number of cells tested should be no less than 1.2-2 × 10⁻⁶. 6 indivual.

3. The method for detecting manganese ion content in cells according to claim 1, characterized in that: The manganese standard solution mentioned in step (2) is a manganese chloride solution; preparation method: dilute 1M manganese chloride with ultrapure water to prepare manganese standard solutions of different concentrations, namely 10μM, 15μM, 25μM, 50μM, 100μM, 200μM and 400μM.

4. The method for detecting manganese ion content in cells according to claim 1, characterized in that: In step (2), the final concentrations of potassium pyrophosphate and sodium acetate in the potassium pyrophosphate-sodium acetate buffer solution are 0.6M and 0.1M, respectively, and the pH is adjusted to about 7 with acetic acid; the concentration of potassium periodate solution is 0.1M.

5. The method for detecting manganese ion content in cells according to claim 1, characterized in that: In the preparation of the standard solution, blank sample, or test sample, the volume ratio of each reagent is as follows: the volume of potassium pyrophosphate-sodium acetate solution is 20 μL, and the volume of potassium periodate solution is 10 μL.

6. The method for detecting manganese ion content in cells according to claim 1, characterized in that: The blank sample was prepared by replacing the standard solution of known concentration with ultrapure water according to the method in step (2).

7. The method for detecting manganese ion content in cells according to claim 1, characterized in that: In step (3), the absorbance value is obtained by placing the standard solution and blank control solution in a 96-well microplate and measuring the absorbance value at 525 nm using a microplate reader.

8. The method for detecting manganese ion content in cells according to claim 1, characterized in that: The detection limit for intracellular manganese ions is 10 μM.

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