CN112505000A - Novel method for quantifying CB (platelet-rich protein) in biological sample based on polyacrylamide gel electrophoresis - Google Patents
Novel method for quantifying CB (platelet-rich protein) in biological sample based on polyacrylamide gel electrophoresis Download PDFInfo
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- CN112505000A CN112505000A CN202011306382.2A CN202011306382A CN112505000A CN 112505000 A CN112505000 A CN 112505000A CN 202011306382 A CN202011306382 A CN 202011306382A CN 112505000 A CN112505000 A CN 112505000A
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Abstract
The invention discloses a method for quantifying carbon black in a biological sample based on polyacrylamide gel electrophoresis. The method comprises the following steps: 1) preparing BSA solutions containing carbon black with different concentrations, respectively carrying out polyacrylamide Gel electrophoresis, carrying out image scanning on an electrophoresis strip corresponding to each carbon black-BSA compound by using a UMAX Magic scanner, measuring the optical density (IOD) of the electrophoresis strip by using Gel-pro image software, and establishing a standard curve of the IOD and the carbon black concentration; 2) pretreating a biological sample to be detected, determining the treated biological sample by adopting the method in the step 1), obtaining the IOD of the corresponding electrophoresis strip, and substituting the IOD into the standard curve in the step 1) to obtain the carbon black concentration of the biological sample to be detected. The method requires few samples and is low in cost, but has high reliability and accuracy.
Description
Technical Field
The invention belongs to the technical field of biological nano analysis, and particularly relates to a novel method for quantifying CB (platelet-rich protein) in a biological sample based on polyacrylamide gel electrophoresis.
Background
Carbon Black (CB) has a wide range of industrial applications and has been used in recent years as a basic model for environmental health research of air Particulate Matter (PM). The characterization of the exposure of carbon black to organisms is the first and most important step in the analysis of the impact of carbon black on human health. However, research efforts have been largely limited due to the lack of effective methods capable of quantifying CB in biological samples.
Currently, several methods are available for quantifying nanoparticles in biological samples, including inductively coupled plasma mass spectrometry (ICP-MS), microscopy, raman spectroscopy, thermo-optical transmittance (TOT), and isotopic/fluorescent labeling. However, there are also some drawbacks that limit their application. For example, in a study of CB kinetics in swiss mice, CB is isotopically labeled with Be for oral administration, and then the concentration of the isotope is measured. But may cause the tag to detach over time. For ICP-MS analysis, it is difficult to distinguish the source of carbon from a biological sample because the nanomaterial must be digested to a soluble form. The same problem exists with TOT, and the results of the test differ due to the lack of a constant temperature heating procedure. Raman spectroscopy is only used to relatively quantify nanomaterials within a single cell. Furthermore, they all require expensive instrumentation and complex pre-processing. Thus, there remains a need for a simple, reliable, label-free quantitative method for measuring CB in biological systems.
Disclosure of Invention
The invention aims to provide a method for quantifying CB in a biological sample based on polyacrylamide gel electrophoresis. The method is economical and convenient, and can be carried out in most biological laboratories. The negative charge on the surface of the CB or CB-protein complex encourages CB to migrate in the electric field and aggregate to form a band at the gel interface (as shown in fig. 1. a). In the quantitative analysis, the load CB amount is correlated with the strip gray scale. The method requires few samples and is low in cost, but has high reliability and accuracy.
The invention provides a method for quantifying CB in a biological sample based on polyacrylamide gel electrophoresis, which comprises the following steps:
1) preparing BSA solutions containing Carbon Black (CB) with different concentrations, respectively carrying out polyacrylamide Gel electrophoresis, carrying out image scanning on an electrophoresis strip corresponding to each carbon black-BSA compound by using a UMAX Magic scanner, measuring the optical density (IOD) of the electrophoresis strip by using Gel-pro image software (Media Cybernetics, USA), and establishing a standard curve of the IOD and the quality of the Carbon Black (CB) in a sample;
2) pretreating a biological sample to be detected, determining the treated biological sample by adopting the method in the step 1), obtaining the optical density (IOD) of the corresponding electrophoresis strip, and substituting the IOD into the standard curve in the step 1) to obtain the Carbon Black (CB) concentration of the biological sample to be detected.
In step 1) of the above method, the concentration of BSA in the BSA solution is 2 mg/mL.
The concentration of Carbon Black (CB) in the BSA solution containing different concentrations of Carbon Black (CB) ranges from 0 to 0.125 mg/mL.
The standard curve in the step 1) can be 226.98x-0.8019, R20.9992, where y is the optical density (IOD) of the corresponding electrophoretic band and x is the mass of carbon black in the loaded sample (directly converted to the mass of carbon black in the loaded sample since the loading volume is fixed).
In step 1), the polyacrylamide gel electrophoresis may be sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or Native-PAGE electrophoresis (Native-PAGE); the gel concentration of the stacking gel for polyacrylamide gel electrophoresis is 5%, and the electrophoresis conditions are as follows: the voltage is 120V and the time is 2 h.
Each sample of the polyacrylamide gel electrophoresis contained 5.0. mu.L BSA, 5.0. mu.L CB standard, 10.0. mu.L 2 Xbuffer (buffer consisting of 4% SDS and 10% 2-mercaptoethanol.).
The resolution of the image scan in step 1) of the above method is 200 dpi.
In step 2) of the above method, the biological sample may be a cell, a tissue or an organ.
The biological sample is cells from any species (such as Raw264.7, A549 cells or MSCs), and the pretreatment method of the cells comprises the steps of cracking the cells and then carrying out polyacrylamide gel electrophoresis analysis on a cracking solution.
The biological sample is a tissue or an organ, and the tissue or the organ is homogenized and then subjected to polyacrylamide gel electrophoresis analysis.
In step 2) of the above method, the protein content of the treated biological sample needs to be determined before loading, and the protein concentration (the concentration can be adjusted by using a loading buffer) is adjusted to 1/2 which is the protein content in the BSA solution in step 1).
In step 2) of the above method, each sample subjected to polyacrylamide gel electrophoresis comprises 10.0. mu.L of the biological sample and 10.0. mu.L of 2 Xbuffer (the buffer is composed of 4% SDS and 10% 2-mercaptoethanol). ).
In the present invention, the inventors developed an SDS-PAGE-based method for quantifying CB in a biological sample with satisfactory reliability and accuracy. The negative surface charge of the carbon black particles causes the carbon black particles to migrate to the anode in an electric field, and the pore diameter of the gel is only 2-5nm, which is much smaller than the size (17 +/-3 nm) of the CB particles, so that the CB particles cannot enter a separation gel layer after reaching the stacked gel layer, and are gathered at the gel interface to form an obvious strip. Raman spectroscopy confirmed that all the CB in the sample was within the band and not dispersed into the other gel layers (fig. 1. a). Can be used for rapid and reliable quantification in a trace amount of sample (1-30. mu.L). The driving force for molecular migration in the gel electric field is the charge. For neutral charged nanoparticles, the SDS coating charges the particles with a negative charge to drive the migration of the nanoparticles. Since zeta potential analysis found negative charges in both the CB in the BSA-coated and in the cell lysates (see table 1), we analyzed the samples using native-PAGE without SDS and mercaptoethanol. CB bands were also formed in native-PAGE gels. After Coomassie blue staining, BSA bands appeared in native-PAGE gels, similar to SDS-PAGE (FIG. 2), indicating that this method can be further simplified in the case of negative charge on CNMs. Taken together, the negative charge carried by the nanoparticles is sufficient to drive the migration of the nanoparticles in the electric field. Since this method relies on band intensity for quantification, it is ideally suited for quantifying gray nanoparticles in biological samples (e.g., tissue/organ homogenates).
TABLE 1 Zeta potentials (ZP, mV) of nanomaterials in BSA solution (2mg/mL) and cell lysates.
Data represent the results of three independent experiments and are presented as mean ± s.d
Compared with the prior art, the invention has the following advantages:
1) the sample preparation is simple, only cells need to be lysed in situ, and the experimental deviation is reduced;
2) the calibration curve can be directly prepared by BSA solution, and can be widely applied to various cells;
3) gel scanning, digitization and quantification of bands only require a UMAX Magic scanner, a routine device for molecular biology experiments;
4) the required samples are few, the cost is low, and the reliability and the accuracy are high.
Drawings
FIG. 1 is a qualitative and quantitative analysis of CB after gel electrophoresis of a sample. (A) And performing Raman scanning on the gel to obtain the distribution of the CB. The low gel image is a coomassie blue stained gel after electrophoresis, showing the presence of CB and BSA bands. (B) A series of concentrations of CB standards in BSA and cell lysis matrices were imaged and quantified by SDS-PAGE. Each sample contained 5.0. mu.L BSA or cell lysate (2mg/mL), 5.0. mu.L CB standard, 10.0. mu.L 2X buffer (consisting of 4% SDS and 10% 2-mercaptoethanol), loaded onto 5% stacking gel, and electrophoresed at 120V for 2 h. The scan image showed that CB-containing material was collected at the interface of the gel and the wells as thin dark bands. Columns 1-7 are standard samples (mix 0-0.125mg/mL CB). Calibration curves are shown below, showing a linear relationship between IOD and CB amounts of the strip. Data are representative of three independent experiments and are presented as mean ± s.d.
FIG. 2 is a comparison of Native-PAGE and SDS-PAGE gel electrophoresis. (A) native-PAGE was scanned at 120V for 2 hours using a UMAX Magic scan. (B) Images of whole channel gels after staining with 0.5% coomassie blue show CB and BSA bands; (a) a gel of SDS-PAGE; (b) native-PAGE gels.
FIG. 3 is a graph of the survival of three types of cells (A549, Raw264.7 and MSCs) after 24 hours of exposure to different concentrations of CB (0-40. mu.g/mL). Cell viability was determined by the WST-1 method. The control group was normal cultured cells. Data are representative of three independent experiments and are expressed as mean ± s.d.
FIG. 4 is a linear plot of cell number versus total protein content for three types of cells (A549, Raw264.7, and MSCs).
Fig. 5 is a graph of CB levels and uptake rates measured in MSC, raw264.7 and a549 cells after 24 hours of exposure to different concentrations of CB. The cell lysate samples were subjected to electrophoresis, digitization and quantification of the calibration curve. (A) A CB band is formed in the gel after electrophoresis, column 1 and a control group; columns 2-6, cell samples were incubated in exposure media containing 1, 2, 4, 10, and 20 μ g/mL CB, respectively. (B) The rate of absorption of CB by three types of cells exposed to different doses of CB. Error bars represent the standard deviation of three independent measurements.
Figure 6 is an image of three cells after CB exposure. MSC cells, raw264.7 and a549 cells were exposed to 20 μ g/mL CB for 24 hours and imaged using laser confocal microscopy. Typical images are shown in the figure, with a scale bar of 20 μm.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified.
Examples 1,
The specific implementation process of the invention is illustrated by three cells, namely epithelial cells (A549), Mesenchymal Stem Cells (MSCs) and macrophages (Raw264.7):
(1) the calibration curve was prepared directly using the BSA solution (see FIG. 1. B);
(2) raw264.7 and A549 cells were derived from American Type Cell Culture (ATCC), and MSCs were isolated from mice (mouse femur was harvested, bone ends were excised, bone marrow cavity was washed with DMEM, and cells were cultured at 75cm2In the culture flask of (DMEM + 10% FBS medium), after 24 hours, nonadherent cells were removed. Then changing the culture solution every 2-3 days until the culture bottle is full of cells. ). Raw264.7 cells were cultured in complete medium (c-RPMI) supplemented with 10% (v/v) heat-inactivated fetal bovine serum and antibiotics (100U/mL penicillin and 100. mu.g/mL streptomycin; A549 cells were cultured in DMEM medium containing the same supplementsCells were set at 37 ℃ and 95% air-5% CO2The incubator of (2) for cultivation.
(3) The WST-1 method is adopted to determine the cell survival rate to determine the non-cytotoxic concentration of CB on Raw264.7, A549 and MSCs cells. The cells were sequentially washed at 2X10 per well4Cell, 1X104Cells and 1X104The density of cells was seeded and attached to 96-well plates. After 24 hours of exposure to various concentrations of CB (0-40. mu.g/mL), the PBS was washed 3 times and incubated in fresh medium containing 10% (v/v) WST-1(Roche, Germany) (see step (2)). After 1 hour, the absorbance of each well was recorded at 450nm using a Thermo Varioskan microplate reader (Winooski, VT, USA).
As shown in fig. 3, at all CB concentrations tested, cell viability of MSCs and a549 cells was unaffected, and cell viability of RAW264.7 cells was gradually increased, but not statistically significant compared to controls, until the concentration was increased to 40 μ g/mL. Therefore, we performed experiments using non-cytotoxic exposure concentrations (0-20. mu.g/mL).
(4) Raw264.7, A549 and MSCs cells were individually plated in 6-well plates (Corning, NY, USA) at 4X10 per well5Cell, 2X105Cells and 8X105Cells were cultured overnight. The following day, exposure was performed with media containing different concentrations (0, 1, 2, 4, 10, and 20 μ g/mL) of CB instead of media.
(5) Total cellular protein was measured, cell counts were performed, and the relationship between cell number and total protein content was established, see FIG. 4.
(6) Preparation of SDS-PAGE gels.
(7) The cells are lysed. The exposed cells were washed 3 times with PBS, lysed and sonicated for 2 min.
(8) The resulting lysates were immediately subjected to SDS-PAGE analysis (or could be stored at-20 ℃ for later use). Before loading, the volume of each lysate was adjusted to ensure the same number of cells based on the total protein amount versus the number of cells. The lysate was mixed with loading buffer (consisting of 4% SDS and 10% 2-mercaptoethanol) in a volume ratio of 1: 1 after boiling for 3min, the samples were loaded in 20.0. mu.L each (standard curve was prepared, each sample contained 5.0. mu.L BSA or cell lysate (2mg/mL), 5.0. mu.L CB standard, 10.0. mu.L 2 Xbuffer; test samples were assayed consisting of 10. mu.L test sample and 10. mu.L 2 Xbuffer). Electrophoresis was performed at a constant voltage of 120V for 2 hours, and the wells were sealed with the same stacking gel for 30 minutes after electrophoresis.
(9) Image scans were performed at a resolution of 200dpi using a UMAX Magic scanner. The optical density measurements (IOD) of the strips were analyzed using Gel-pro imaging software (Media Cybernetics, USA). All the band intensities of the samples were subtracted from the intensity of the blank gel band, which did not contain any sample but was buffered.
(10) Calculating the CB uptake of the three cells according to the standard curve prepared in the step (1).
As shown in FIG. 4.A, after gel electrophoresis, CB formed a distinct black band in the cell lysate. The intensity of the band increased with increasing exposure concentration, indicating that the method can be well applied to the quantitative determination of CB in biological systems, regardless of the species (mouse or human) from which the cells are derived. MSCs cells and raw264.7 cells accumulated CB in a concentration-dependent manner, with minimal CB accumulation in a549 cells (fig. 4. B). When the exposure concentration reaches 20 mug/mL (the total exposure is 40 mug), the number of the MSCs, Raw264.7 and A549 cells with the same protein loading is 1.4 multiplied by 10 respectively5、6.5×105And 8X104. The uptake rates of MSCs, raw264.7 and a549 cells were 3702.7, 1706.4 and 0.3 fg/day, respectively, after normalization to cell number.
To further verify that BSA was actually detected as representative of the cell matrix/sample, the same series of CB standards were mixed with a549 cell lysate and analyzed in the same manner. As shown in fig. 1.B, the results obtained in cell lysates are highly consistent with those obtained in BSA, and the standard curve is almost the same (especially when the content of CB is low), indicating that the BSA established method is suitable for real cell samples.
To assess the accuracy of the method of the invention, a known amount of CB was added to the cell lysate and then analyzed by the methods described above. The data in table 2 shows the actual amount of CB in relation to the measured values and the corresponding recovery. The results show that the calculated values are substantially consistent with the actual values. Through calculation, the recovery rate is 84% -128%, the average recovery rate is 104.7%, and the accuracy of the method is verified.
TABLE 2 relationship between carbon black addition and measurement values, data expressed as mean. + -. s.d
Furthermore, we provide additional evidence using confocal laser scanning microscopy to demonstrate that differences in CB numbers are linked to different cells. As shown in fig. 5, after exposure and PBS washing, the images showed an increasing trend of intracellular CB presentation, ordered as MSC > raw264.7> a549, consistent with the results obtained with our established method (fig. 5).
Claims (10)
1.A method for quantifying carbon black in a biological sample based on polyacrylamide gel electrophoresis comprises the following steps:
1) preparing BSA solutions containing carbon black with different concentrations, respectively carrying out polyacrylamide Gel electrophoresis, carrying out image scanning on an electrophoresis strip corresponding to each carbon black-BSA compound by using a UMAXmagic scanner, measuring the optical density of the electrophoresis strip by using Gel-pro image software, and establishing a standard curve of the IOD and the mass of the carbon black in a sample;
2) pretreating a biological sample to be detected, determining the treated biological sample by adopting the method in the step 1), obtaining the IOD of the corresponding electrophoresis strip, and substituting the IOD into the standard curve in the step 1) to obtain the carbon black concentration of the biological sample to be detected.
2. The method of claim 1, wherein: in the step 1), the concentration of BSA in the BSA solution is 2 mg/mL;
the concentration of carbon black in the BSA solution containing different concentrations of carbon black ranges from 0 to 0.125 mg/mL.
3. The method according to claim 1 or 2, characterized in that: in the step 1), the polyacrylamide gel electrophoresis is sodium dodecyl sulfate-polyacrylamide gel electrophoresis or non-denaturing polyacrylamide gel electrophoresis; the gel concentration of the polyacrylamide gel electrophoresis is 5%, and the electrophoresis conditions are as follows: the voltage is 120V and the time is 2 h.
4. The method according to any one of claims 1-3, wherein: each sample of the polyacrylamide gel electrophoresis contained 5.0. mu.L BSA, 5.0. mu.L CB standard, 10.0. mu.L 2 Xbuffer;
the buffer solution is a buffer solution containing SDS with the mass concentration of 4% and 2-mercaptoethanol with the mass concentration of 10%.
5. The method according to any one of claims 1-4, wherein: the resolution of the image scanning in the step 1) is 200 dpi.
6. The method according to any one of claims 1-5, wherein: in the step 2), the biological sample is a cell, a tissue or an organ.
7. The method of claim 6, wherein: the biological sample is cells from any species, and the pretreatment method of the cells comprises the steps of cracking the cells and then carrying out polyacrylamide gel electrophoresis analysis on a lysate.
8. The method of claim 6, wherein: the biological sample is a tissue or an organ, and the tissue or the organ is homogenized and then subjected to polyacrylamide gel electrophoresis analysis.
9. The method according to claim 7 or 8, characterized in that: in the step 2), the protein content of the processed biological sample needs to be measured before loading, and the protein concentration of the processed biological sample is adjusted to 1/2 which is equal to the protein content of the BSA solution in the step 1).
10. The method according to claims 1-9, characterized in that: in step 2), each sample of polyacrylamide gel electrophoresis comprises 10.0. mu.L of biological sample and 10.0. mu.L of 2 Xbuffer.
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| JP2005240114A (en) * | 2004-02-26 | 2005-09-08 | Choichi Furuya | Method for producing carbon black high-dispersed body, production apparatus therefor and composition for preparing gas diffusion electrode |
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Application publication date: 20210316 |