CN108956726B - Cytochrome c detection method and kit - Google Patents
Cytochrome c detection method and kit Download PDFInfo
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- CN108956726B CN108956726B CN201810336002.6A CN201810336002A CN108956726B CN 108956726 B CN108956726 B CN 108956726B CN 201810336002 A CN201810336002 A CN 201810336002A CN 108956726 B CN108956726 B CN 108956726B
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Abstract
The invention discloses a cytochrome c detection method and a kit, wherein the inherent peroxidase activity of cytochrome c is combined with a screen printing carbon electrode detection technology to construct an in-situ cytochrome c detection method, and a destructive sample is not needed, so that the method has the advantage of in-situ detection; the method has the advantages of better detection performance, higher sensitivity, detection limit of 0.816nM, simple and quick process and capability of carrying out on-site detection.
Description
Technical Field
The invention belongs to the field of biological detection, and relates to a cytochrome c detection method and a kit.
Background
Cytochrome c, composed of iron, proteins and porphyrins, is an important component of the mitochondrial respiratory chain and a marker indicative of apoptosis. In addition, cytochrome c has many other important functions, such as assisting microorganisms in transferring electrons generated by intracellular oxidation of organic substances to poorly soluble metal (hydr) oxides and solid electrodes.
Traditional cytochrome c detection methods, such as hemoglobin staining and western blotting, have the advantages of visualization and quantification, but their sensitivity is low and the detection process is complex. Spectroscopy, while simple and easy to use, is limited to qualitative monitoring of the redox state of cytochrome c. Electrochemical techniques are applied to the detection of cytochrome c due to their advantages of simple operation, high sensitivity and quantitative analysis. However, electrochemical techniques rely on expensive enzymes or metal nanoparticles for signal amplification. In addition, due to their low electron transfer rate at the electrode/solution interface, complex and cumbersome electrode pretreatment and modification processes are required. In order to overcome these disadvantages, it is necessary to develop new methods for detecting cytochrome c, such as exploring the possible biological activity of cytochrome c and detecting cytochrome c by detecting its substrate molecules. It has been shown that the heme prosthetic group of cytochrome c is structurally similar to peroxidase (both containing Fe (III) coordinated to a porphyrin and one or two axial ligands) and thus possesses peroxidase-like activity. Indeed, the peroxidase-like activity of heme has been applied to the color reaction of gel staining and colorimetric detection. The chromogenic reaction of these detection methods requires the use of a redox-active substrate molecule, named 3,3',5,5' -Tetramethylbenzidine (TMB). The studies show that tetramethylbenzidine has reversible electrochemical activity. In situ electrochemical detection using the catalase activity of cytochrome c has not been reported. Therefore, the development of a novel method for rapidly detecting the cytochrome c of the microorganism in situ is of great significance.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to combine the intrinsic peroxidase activity of cytochrome c with a screen printing carbon electrode detection technology to construct an in-situ cytochrome c detection method. The method has the advantages of high specificity, simple operation and rapid detection.
The purpose of the present invention is to provide a method for detecting cytochrome c.
Another object of the present invention is to provide a cytochrome c detection kit.
The technical scheme adopted by the invention is as follows:
a kit for detecting cytochrome c comprises washing buffer solution, screen-printed carbon electrode, hydrogen peroxide solution, and detection reagent
Buffer solution and tetramethyl benzidine powder.
Furthermore, the washing buffer solution is 0.01M-0.03M, pH 5.4.4-9.4 phosphate buffer solution.
Furthermore, the detection buffer solution is 0.1M-0.3M, pH 3.0.0-7.0 phosphoric acid citric acid buffer solution.
A detection method for detecting cytochrome c, comprising the steps of:
1) dripping the microbial cell to be detected on the surface of a working electrode of the screen printing carbon electrode, and drying to prepare the microbial modified screen printing carbon electrode;
2) dropwise adding the tetramethylbenzidine solution to a screen-printed carbon electrode modified by microorganisms, reacting for 11 minutes, detecting electrochemical reaction by using an instrument, and detecting the generated current intensity;
3) and (4) judging whether the cell to be detected contains cytochrome c according to current intensity analysis.
Further, the microbial cell sap in the step 1) consists of microbial cells and PBS buffer.
Further, the drying temperature in the step 1) is 18-27 ℃.
Further, the drying time in the step 1) is 60-120 min.
Further, the instrument in the step 2) is an electrochemical workstation.
Further, in the step 2), the voltage for detecting the electrochemical reaction is-0.30V to-0.10V.
Further, the tetramethylbenzidine solution in the step 3) contains 0.05-0.2 mg/ml of tetramethylbenzidine, 1-10% v/v of anhydrous ethanol, and 0.3-0.7 mM of H2O230-70% v/v PBS buffer solution, and the balance of water.
Further, the amount of the tetramethylbenzidine solution is 30-70 uL.
Further, the specific method for judging whether the cell to be detected contains cytochrome c according to the current intensity analysis comprises the following steps: the current intensity value at 10 seconds after the start of the detection was observed, and when the current intensity value was-0.23. mu.A or more, it was found that the sample contained cytochrome c.
The invention has the beneficial effects that:
(1) the cytochrome c detection method has the advantage of in-situ detection, and any destructive sample preparation process is not needed;
(2) the cytochrome c has peroxidase activity, can catalyze and oxidize the tetramethylbenzidine in the presence of hydrogen peroxide, and an oxidation product can be directly reduced on the surface of the screen printing carbon electrode, so that the aim of in-situ detection is fulfilled;
(3) the cytochrome c detection method has high sensitivity, the detection limit of the cytochrome c of the model analyte Shewanella S.oneidensis MR-1 is 40.78fmol, the detection process is convenient and rapid, and the cytochrome c detection method can be used for field detection.
Drawings
FIG. 1 is a schematic of in situ detection of cytochrome c;
FIGS. 2(A) and (B) are scanning electron micrographs of a microbially modified screen printed carbon electrode; (C) and (D) is a fluorescence microscopy electron micrograph; (E) is a time-current graph;
FIG. 3(A) is a cytochrome c peroxidase activity assay; (B) is a cyclic voltammogram;
FIG. 4 is an optimization plot of working concentration of hydrogen peroxide;
FIG. 5 is a graph showing the optimization of reaction time of the cytochrome c oxidation of tetramethylbenzidine;
FIG. 6(A) is a regression curve of cytochrome c detection; (B) the standard curve for cytochrome c detection is shown.
Detailed Description
The present invention will be further described with reference to the following specific examples.
Example 1: preparation of microbial modified silk screen carbon electrode
Inoculating the microorganism and the culture solution at a ratio of 1:100 into 500ml conical flask, and shake-culturing for 16h in a constant temperature shaking table at 30 deg.C and 180 rpm. The culture broth was removed, centrifuged at 5000g for 5min, the pellet was collected, washed 3 times with PBS (phosphate buffer, 6 in 0.02M, pH), and the pellet was resuspended in PBS. And (3) dropwise adding 7uL of microbial cell suspension to the surface of a working electrode of the screen printing carbon electrode, and drying at room temperature (18-27 ℃) for 60-120min to enable the microbial cells to be combined to the surface of the working electrode.
In order to verify whether microbial cells modify the surface of the screen-printed carbon electrode, the invention is characterized by a scanning electron microscope, and the result shows that the surface of the screen-printed carbon electrode before modification is relatively flat and has no rod-shaped thallus (see fig. 2 (A)); rod-shaped microbial cells can be seen on the surface of the screen-printed carbon electrode for modifying the microorganisms (see fig. 2(B)), which shows that the droplet coating method of the present invention can modify the microbial cells on the surface of the carbon electrode.
Next, to verify the activity of the microorganisms modified to the surface of the screen-printed carbon electrode, LIVE/DEAD was usedTMBacLightTMThe bacterial viability detection kit stains the carbon electrode. The dyed screen printing carbon electrode modified by the microorganism is observed and analyzed by a fluorescence microscope, and a large number of rod-shaped microorganisms present green fluorescence to indicate that the microbial membrane cells are intactAnd is a living cell (see FIG. 2 (C)). Only a small number of red rods were observed in fig. 2(D), and some of the cells had died or were about to die, and it was further statistically found that 4.35% of the bacterial cells were dead. These results indicate that the microbial modified screen-printed carbon electrode prepared by the drop coating method has higher cell activity.
As shown in FIG. 3(A), Shewanella sp.oneidensis MR-1, a model microorganism, is pink (centrifuge tube a), indicating that it contains a higher cytochrome c.the concentration of 5.0 × 108After cfu/mL of S.oneidensis MR-1 was added to the tetramethylbenzidine solution, the colorless tetramethylbenzidine solution turned blue (centrifuge tube b). Addition of H2SO4After this time, the blue solution was found to turn into a yellow solution (centrifuge tube c). By using cyclic voltammetry, the results are shown in fig. 3 (B). Both the microbially modified screen printed carbon electrode (red dashed line) and the bare screen printed carbon electrode (black dashed line) were current signal free when measured in buffer solution; however, when tested in tetramethylbenzidine solution, the peak reduction current was higher for the microbial modified screen printed carbon electrode (red solid line) than for the bare screen printed carbon electrode (black solid line), demonstrating that microbial cytochrome c contains peroxidase activity.
In order to confirm the feasibility of the detection method, the tetramethylbenzidine solution is dripped on the surfaces of the microorganism modified screen printing carbon electrode and the naked screen printing carbon electrode, the potential is set to be-0.20V, and a chronoamperometric test is carried out. This potential was determined by analyzing a cyclic voltammogram (see FIG. 3(B)), in which a peak potential of the reduction current of 0.056V was observed. A more negative potential (e.g., -0.20V) will provide better detection performance because it can sufficiently reduce oxidized tetramethylbenzidine to produce a stronger response current signal. Results as shown in fig. 2(E), the microbially modified screen printed carbon electrodes (red lines) exhibited significant reduction current, while the bare screen printed carbon electrodes without bacterial modification (grey lines) exhibited only weak background signals. The response current signal was only generated in the presence of microorganisms, indicating that the detection method is feasible.
Example 2: in situ detection of microbial cytochrome c
The schematic diagram of the method for detecting cytochrome c in situ is shown in FIG. 1, and the specific operation steps are as follows:
1) dripping 7uL of microbial cell sap to be detected onto the surface of a working electrode of the screen printing carbon electrode, and drying at room temperature (18-27 ℃) for 60-120min to combine microbial cells onto the surface of the working electrode to obtain the screen printing carbon electrode modified by microorganisms;
2) connecting the prepared screen-printed carbon electrode modified by the microorganisms to a CHI660D type electrochemical workstation (Shanghai Chenghua instruments, Inc., China), selecting a detection mode as a time-current curve mode by operating software in the electrochemical workstation, and setting the voltage to-0.20V;
3) preparing tetramethylbenzidine solution containing 0.2mg/ml tetramethylbenzidine, 10% v/v anhydrous ethanol, 0.7mMH2O270% v/v (PBS) phosphate buffer, the balance being water;
4) dropwise adding the tetramethylbenzidine solution into the screen printing carbon electrode modified by the microorganisms in the step 2) to fully react;
5) when the reaction time reaches 11 minutes, an electrochemical reaction detection is carried out by an electrochemical workstation, the current intensity value of the electrochemical reaction detection is detected at the 10 th second of the time-current curve, and if the current intensity value is more than-0.23 muA, the sample contains cytochrome c.
Example 3: in situ detection of microbial cytochrome c
The schematic diagram of the method for detecting cytochrome c in situ is shown in FIG. 1, and the specific operation steps are as follows:
1) dripping 7uL of microbial cell sap to be detected onto the surface of a working electrode of the screen printing carbon electrode, and drying at room temperature (18-27 ℃) for 60-120min to combine microbial cells onto the surface of the working electrode to obtain the screen printing carbon electrode modified by microorganisms;
2) connecting the prepared screen-printed carbon electrode modified by the microorganisms to a CHI660D type electrochemical workstation (Shanghai Chenghua instruments, Inc., China), selecting a detection mode as a time-current curve mode by operating software in the electrochemical workstation, and setting the voltage to-0.20V;
3) preparing tetramethylbenzidine solution containing 0.05mg/ml tetramethylbenzidine, 1% v/v anhydrous ethanol, 0.3mMH2O230% v/v (PBS) phosphate buffer, balance water;
4) dropwise adding the tetramethylbenzidine solution into the screen printing carbon electrode modified by the microorganisms in the step 2) to fully react;
5) when the reaction time reaches 11 minutes, an electrochemical reaction detection is carried out by an electrochemical workstation, the current intensity value of the electrochemical reaction detection is detected at the 10 th second of the time-current curve, and if the current intensity value is more than-0.23 muA, the sample contains cytochrome c.
Example 4: condition optimization of cytochrome c detection method
In order to obtain the best detection effect, the present embodiment further optimizes the parameters (hydrogen peroxide concentration and reaction time) affecting the detection effect.
The assay of the invention utilizes cytochrome c catalase activity for catalytic oxidation of TMB, whereas cytochrome c peroxidase activity is dependent on hydrogen peroxide concentration, using Shewanella S.oneidensis MR-13.575 × 109cfu/mL was used as a model analyte, and 5 different concentrations of hydrogen peroxide (0.1, 0.3, 0.5, 0.7, 0.9mM) were set to examine the effect of different concentrations of hydrogen peroxide on the detection signal. As a result, as shown in FIG. 4, the response current signal rapidly increased as the hydrogen peroxide concentration increased from 0.1mM to 0.5 mM. The current signal gradually decreased as the hydrogen peroxide concentration increased from 0.5mM to 0.9 mM. The response current signal was maximal at a hydrogen peroxide concentration of 0.5 mM. Therefore, 0.5mM is the optimum hydrogen peroxide concentration.
The detection method depends on the peroxidase activity of cytochrome c, so that the detection performance of the detection method is influenced by the time of TMB oxidation reaction catalyzed by cytochrome c to 3.575 × 109Shewanella S.oneidensis MR-1 cfu/mL is an analyte, the concentration of hydrogen peroxide is 0.5mM, 6 different reaction times (3, 5, 7, 9, 11 and 13min) are set, and response signals under different enzymatic reaction times are detected. The results are shown in FIG. 5, where the current response signal is dependent on the enzymatic reaction timeThe enzyme reaction time was increased from 3min to 11min, and the response current was gradually increased. When the enzyme reaction reached 11min, a maximum current signal was observed. Further increase to 13min of enzyme reaction time, the response current signal slowed, indicating that cytochrome c catalyzed TMB oxidation reached equilibrium. Therefore, 11min is the optimal enzyme reaction time.
Example 5 optimal protocol for in situ detection of microbial cytochrome c
According to the optimization of each parameter, the method for obtaining the optimal cytochrome c in situ detection comprises the following operation steps:
1) dripping 7uL of microbial cell suspension to be detected on the surface of a working electrode of the screen printing carbon electrode, and drying at room temperature (18-27 ℃) for 100min to prepare the screen printing carbon electrode modified by microorganisms;
2) connecting the prepared screen-printed carbon electrode modified by the microorganisms to a CHI660D type electrochemical workstation (Shanghai Chenghua instruments, Inc., China), selecting a detection mode as a time-current curve mode by operating software in the electrochemical workstation, and setting the voltage to-0.20V;
3) preparing tetramethylbenzidine solution containing 0.1mg/ml tetramethylbenzidine, 5% v/v anhydrous ethanol, 0.5mM H2O250% v/v PBS buffer solution, and the balance of water;
4) dropwise adding the tetramethylbenzidine solution into the screen printing carbon electrode modified by the microorganisms in the step 2) to fully react;
5) when the reaction time reaches 11 minutes, the electrochemical detection is started at the electrochemical workstation, the current intensity value of the electrochemical detection at the 10 th second of the time-current curve is detected, and if the current intensity value is more than-0.23 muA, the sample contains the cytochrome c.
The effect of the cytochrome c detection method and the detection kit of the present invention was further evaluated as follows.
Example 6 sensitivity detection
The current intensity values of samples containing model strain S.oneidensis MR-1 of different cell concentrations were examined by the method of the invention. S.oneidenesis MR-1 was quantitatively determined by spectroscopy to have a cytochrome c content of 2.70×10-19mole/cell. The cell concentration of the test sample was converted to the cytochrome c content of 51.70fmol to 6.64pmol (see FIG. 6(A) for the results). The curve is plotted in fig. 6(B) with the logarithm of the cytochrome C content as the abscissa and the current intensity value as the ordinate, and the regression equation is-1.956C +2.757 (R) by analyzing the curve20.990), the detection limit of the method of the invention is 40.78fmol (judged by a signal-to-noise ratio equal to 3), and the linear detection range is 51.7fmol to 6.64 pmol. For comparison with other detection methods, the detection limit of 40.78fmol of the present invention was converted to a cytochrome c concentration of 0.816nM, based on the volume of tetramethylbenzidine solution used in the detection being 50. mu.l.
Example 7 repeatability and stability testing
The invention carries out 5 independent detection experiments, and the detection results show similar current intensity values, the relative standard deviation of which is 4.57 percent, which shows that the method has good repeatability. The screen-printed carbon electrode modified by the microorganisms is stored at a low temperature of 4 ℃ to study the stability of the screen-printed carbon electrode, and when the screen-printed carbon electrode modified by the microorganisms is stored for 7 days, 92% of response current is still kept, which indicates that the screen-printed carbon modified by the method has better stability.
Example 8 commonality detection
The concentration is 5.0 × 108The bacteria of cfu/mL, Pseudomonas aeruginosa, P.aeruginosa and C.guiandongensis were respectively modified on the working electrode of the screen printing carbon electrode, and the cytochrome c contents of P.aeruginosa and C.guiandongensis were respectively 2.295 × 10-20And 3.593 × 10-20The results of this detection are essentially consistent with the reduction (dithionite) -oxidation (air) spectroscopy detection, where cytochrome c of p.aeruginosa and c.guandongensis is 1.841 × 10-20And 2.743 × 10-20mole/cell. These results indicate that the established method can be universally used for detecting cytochrome c of microorganisms.
Compared with an electrochemical immunosensor method, an aptamer biosensor and a fluorescence detection method (the minimum detection limits are respectively 30nM, 20nM and 15nM), the method disclosed by the invention has the advantages of better detection performance, higher sensitivity and detection limit of 0.816nM, is simple and rapid in detection process, and can be used for in-situ detection of cytochrome c contained in microorganisms, animal cells and human cells.
The above embodiments are merely preferred examples to illustrate the present invention, and it should be apparent to those skilled in the art that any obvious variations and modifications can be made without departing from the spirit of the present invention.
Claims (6)
1. A detection method for detecting cytochrome c, comprising the steps of:
1) dripping the microbial cell to be detected on the surface of a working electrode of the screen printing carbon electrode, and drying to prepare the microbial modified screen printing carbon electrode;
2) dropwise adding the tetramethylbenzidine solution to a screen-printed carbon electrode modified by microorganisms, reacting for 11 minutes, detecting electrochemical reaction by using an instrument, and detecting the generated current intensity;
3) and (4) judging whether the cell to be detected contains cytochrome c according to current intensity analysis.
2. The detection method according to claim 1, wherein the microbial cell sap of step 1) is composed of microbial cells and a phosphate buffer.
3. The detection method according to claim 1, wherein the instrument in step 2) is an electrochemical workstation, and the voltage for detecting the electrochemical reaction is-0.30V to-0.10V; in the electrochemical reaction, the washing buffer solution is 0.01M-0.03M, pH 5.4.4-9.4 of phosphate buffer solution; the detection buffer solution is 0.1M-0.3M, pH 3.0.0-7.0 phosphoric acid citric acid buffer solution.
4. The detection method according to claim 1, wherein the tetramethylbenzidine solution in step 2) contains 30 to 70. mu.L of 0.05 to 0.2mg/ml tetramethylbenzidine, 1 to 10% v/v absolute ethanol, 0.3 to 0.7mM H2O230-70% v/vPBS buffer solution, and the balance of water.
5. The detection method according to claim 1, wherein the amount of the tetramethylbenzidine solution is 30 to 70 μ L.
6. The detection method according to claim 1, wherein the specific method for determining whether or not cytochrome c is contained in the test cell based on the current intensity analysis comprises: the current intensity value at 10 seconds after the start of the detection was observed, and when the current intensity value was-0.23. mu.A or more, it was found that the sample contained cytochrome c.
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