CN1940550A - Method for determining cell active oxygen and reduced glutathione simultaneouslly - Google Patents

Abstract

The invention relates to the detection of intracellular active oxygen and reduced glutathione, specifically a method for simultaneously measuring cellular active oxygen and reduced glutathione, using a microfluidic chip to detect cellular active oxygen and reduced glutathione Simultaneous determination of prototype glutathione, the concentration of the buffer used in microfluidic chip electrophoresis is 20-50mM, the pH of the buffer is 8.0-10.1, the separation voltage is 200-450V/cm, and the detection point is separated from the injection channel and the separation channel The intersection is 1.0-3.0cm. The amount of sample used in the detection method of the present invention is very small (3-4uL), and the simultaneous determination of intracellular active oxygen and reduced glutathione is realized on the microfluidic chip. The analysis time is only 27 seconds, and the detection sensitivity is relatively high. High (amol-zmol).

Description

A detection method for simultaneous determination of cellular reactive oxygen species and reduced glutathione

technical field

The invention relates to the detection of intracellular active oxygen and reduced glutathione, in particular to a detection method for simultaneously measuring cellular active oxygen and reduced glutathione.

Background technique

Reactive oxygen species (ROS) and reduced glutathione (GSH) are two important signaling molecules in the process of intracellular signal transduction, which are closely related to the intracellular redox state. Active oxygen mainly refers to a class of oxygen molecules with high biological activity produced by exogenous oxidants or intracellular aerobic metabolism, such as O ^2- , NO, H ₂ O ₂ , OH, etc. When the body is in a state of stress, cell hypoxia, damage or apoptosis, intracellular reactive oxygen species are excessively produced. Reduced glutathione is a strong reducing agent with the most content in cells. Its main function is to remove excess reactive oxygen species in the body, prevent protein sulfhydryl oxidation, and maintain intracellular oxidation-reduction balance. The determination of cellular reactive oxygen species and reduced glutathione is an important indicator for studying the relationship between oxygen free radicals and stress response, and human diseases.

The current detection methods for reactive oxygen species and reduced glutathione mainly include high performance liquid chromatography (HPLC), fluorescence method, enzymatic method and flow cytometry. Although these methods have their own strengths, generally speaking, the amount of sample required is large, the sensitivity is not high, and the simultaneous detection of two substances cannot be performed. Microfluidic chip (also known as lab-on-a-chip) is a brand-new analysis technology developed in recent years. It can basically integrate different operating units in the process of biochemical reactions on a chip with a size of only a few square centimeters. It has the characteristics of fast analysis speed, small sample consumption, and easy integration. It is known as one of the most important cutting-edge technologies in the 21st century. In principle, it is suitable for the separation and analysis of nucleic acids, proteins and small molecules, and may be used for the analysis and determination of intracellular complex components such as reactive oxygen species and reduced glutathione. The use of microfluidic chips to simultaneously measure cellular reactive oxygen species and reduced glutathione has not been reported at home and abroad.

Contents of the invention

The purpose of the present invention is to provide a detection method with less sample consumption, high detection sensitivity, and simultaneous determination of cellular reactive oxygen species and reduced glutathione. Determination, the analysis time is only 27 seconds, the sample amount is very small (3-4uL), and the detection sensitivity is high (amol-zmol).

To achieve the above object, the technical solution adopted in the present invention is:

A detection method for simultaneous determination of cellular reactive oxygen species and reduced glutathione. A microfluidic chip is used to simultaneously measure cellular reactive oxygen species and reduced glutathione. The buffer concentration used for microfluidic chip electrophoresis is 20- 50mM, the pH value of the buffer solution is 8.0-10.1, the separation voltage is 200-450V/cm, and the detection point is 1.0-3.0cm away from the intersection of the injection channel and the separation channel.

The specific operations include microfluidic chip design, intracellular reactive oxygen species and glutathione fluorescent labeling and extraction methods, microfluidic chip electrophoresis separation conditions and microfluidic chip laser-induced fluorescence detection methods.

In the present invention, the microfluidic chip is used as the technical platform, the microfluidic chip-laser-induced fluorescence is used as the detection means, the selected laser excitation wavelength is 473nm, and the detection wavelength is 520nm; the probes DHR-123 and NDA are used to mark the intracellular activity respectively Oxygen and reduced glutathione, adopt organic solvent (perchloric acid and acetonitrile mixed solution) to extract intracellular active oxygen and reduced form glutathione; The weight concentration of described perchloric acid and acetonitrile mixed solution is 3.3% perchloric acid and acetonitrile are mixed in a volume ratio of 1:1, wherein the volume ratio of perchloric acid and acetonitrile is 1:1

The chip material of the microfluidic chip can be glass, quartz or other chip materials; the chip design preferably adopts a double T-shaped structure, and the sample injection adopts a clamping method to ensure the accuracy of the sample volume; the buffer can be phosphate buffer , borax buffer, etc., preferably borax buffer;

The advantages of the present invention are:

1. The amount of sample used in the detection method of the present invention is very small (3-4uL), and the simultaneous determination of two substances (intracellular active oxygen and reduced glutathione) has been realized on the microfluidic chip, and the analysis time is only 27 seconds, and the detection sensitivity is high (amol-zmol), and the optimized microfluidic chip electrophoresis conditions are applicable to a wide range (mainly including buffer concentration, pH value, electrophoresis sample injection and separation voltage and other conditions).

2. The present invention provides a general-purpose chip detection method for cellular reactive oxygen species and reduced glutathione, which is suitable for monitoring changes in the redox state of cells when oxidative stress is caused in the body, and can be applied to different types of cells such as tumors The detection of cells, red blood cells, neurons and embryos, etc., the experimental operation is convenient and easy, and it is operable.

Description of drawings

Fig. 1 is a schematic structural diagram of the microfluidic chip adopted in the present invention;

Fig. 2 is a design diagram of the laser-induced fluorescence optical path of the microfluidic chip of the present invention;

Figure 3 is a microfluidic chip electrophoresis image of reactive oxygen species and reduced glutathione in NB4 cells;

Fig. 4 is a microfluidic chip electrophoresis image of reactive oxygen species and reduced glutathione in K562 cells, where the coordinates represent relative fluorescence intensity (mV) and migration time (s), respectively.

Detailed ways

Example

1. Microfluidic chip design:

The design of the microfluidic chip used in the implementation of the present invention is shown in Figure 1 below. The glass chip used is a double T-shaped structure. The four round holes in the figure represent different solution pools, of which 2 is the buffer injection pool and 4 is the buffer. 1 is the sample pool, 3 is the sample waste pool, and 5 is the detection point. The separation channel of the chip is 8.5cm long (0.5cm from the buffer pool to the cross, and 8.0cm from the cross to the separation waste pool), and the sampling channel is 1.0cm long (from the sample pool to the cross and From the intersection to the sample waste pool is 0.5cm), the cross-section of the channel is approximately semi-elliptical, with an upper width of 50 μm, and the distance between the detection point and the intersection is 1.0cm.

2. Microfluidic chip laser-induced fluorescence detector:

The laser excitation wavelength selected for the microfluidic chip-laser-induced fluorescence detector is 473nm, the detection wavelength is 520nm, and the single-point confocal detection method is adopted. The design diagram of the laser-induced fluorescence optical path of the microfluidic chip is shown in Figure 2, where: 6 is the objective lens , 7 is a half mirror, 8 is a beam splitter, 9 is a first bandpass filter, 10 is a convex lens, 11 is a pinhole, 12 is a photomultiplier tube, 13 is a chip, and 14 is a second bandpass filter sheet, 15 is a laser, and 16 is an inductive coupling device.

3. Cell sample preparation:

Taking the human acute promyelocytic line NB4 cells as an example, the cells were cultured in RPMI1640 medium containing 10% neonatal bovine serum in a 5% CO2 incubator at 37 degrees. Collect the cells, centrifuge at 1000rpm for 5 minutes, discard the supernatant, resuspend the cell pellet with PBS solution (pH 7.4) and centrifuge, and count the cells before use.

4. Intracellular reactive oxygen species and reduced glutathione fluorescent labeling and extraction methods:

DHR-123 and NDA were used to fluorescently label intracellular reactive oxygen species and reduced glutathione, respectively. For reactive oxygen species, the labeling concentration of DHR-123 was 20-30uM. Since NDA reacts quickly with reduced glutathione, NDA was added to the running buffer to reach a final concentration of 0.1-0.5mM in the experiment to dynamically label glutathione. The cell density used in the experiment was about 1×10 ⁵ /mL. First, 20uM DHR-123 dye was added to the cell culture medium to act for 20-30 minutes, and then the cell suspension was washed three times with PBS (800 rpm, 6 minutes). Discard the supernatant, keep the precipitate, then add an equal volume of perchloric acid/acetonitrile mixture (3.3%) to the precipitate, and centrifuge at high speed (12,000 rpm) for 5 minutes. Discard the supernatant, repeat the above steps twice, and keep the cell pellet for detection.

5. Microfluidic sheet electrophoresis separation conditions:

Before each run, the chip channel needs to be washed with water, 0.1M sodium hydroxide and buffer respectively. When loading the sample, try to keep the liquid levels of the four buffer pools at the same level, and then insert the electrode. As shown in Figure 1, during the sampling process, ground the sample waste liquid pool 3, apply +200V voltage to the sample pool 1, apply 150V and 600V voltage to the buffer pool 2 and buffer waste pool 4, respectively, and the sampling time is 12s. During the separation process, the buffer waste liquid pool 4 is grounded, the sample pool 1 and the sample waste liquid pool 3 are respectively applied with 2kV voltage, and the buffer pool 2 is applied with +3kV voltage. Firstly, the microfluidic chip electrophoresis separation conditions such as buffer, pH, separation voltage and other conditions were investigated. The optimized electrophoresis conditions are: borax buffer 20-50mM, buffer pH range 8.0-10.1, separation voltage 200-450V/cm.

6. Application examples:

Figure 3 and Figure 4 are microfluidic chip electrophoresis images of reactive oxygen species and reduced glutathione in NB4 cells and K562 cells, respectively. Peaks 1 and 2 in the electrophoresis graph represent cellular reactive oxygen species and reduced glutathione, respectively . The whole separation time is only 27s, the actual sample consumption is only 3-4uL, and the detection sensitivity is high. Table 1 shows the main performance indicators of the microfluidic chip for detecting active oxygen and glutathione. It can be seen from the table that its detection sensitivity is high, and the detection of active oxygen and glutathione can reach pmol and amol levels.

Table 1. Main performance indicators of microfluidic chip detection of reactive oxygen species and reduced glutathione (Table 1.Linearity, reproducibility, and detection limits of ROS and GSH) Measured object Relative standard deviation (t/s) RSD Detection concentration range linear regression equation Regression coefficient (r) Detection limit (S/N=3) ROS 0.4/3.5 2.6× ^10-12-1.3 × ^10-10M Y＝-0.01188+0.27348X 0.9956 1.9pM (0.2zmol) GSH 2.2/3.7 5×10 ^-7 -5×10 ^-5 M Y=-1.67212+0.01469X 0.9967 0.5μM (5.4amol)

Claims (6)

1. detection method of measuring cytoactive oxygen and reduced glutathione simultaneously, it is characterized in that: adopt micro-fluidic chip pair cell active oxygen and reduced glutathione to measure simultaneously, the buffer concentration that micro-fluid control chip electrophoretic adopts is 20-50mM, the pH of buffer value is 8.0-10.1, separation voltage is 200-450V/cm, and check point is 1.0-3.0cm apart from sample intake passage and split tunnel infall.

2. according to the described detection method of measuring cytoactive oxygen and reduced glutathione simultaneously of claim 1, it is characterized in that: be technology platform with the micro-fluidic chip, with micro-fluidic chip-laser-induced fluorescence (LIF) is detection means, and selected laser excitation wavelength is 473nm, detects wavelength 520nm.

3. according to claim 1 or the 2 described detection methods of measuring cytoactive oxygen and reduced glutathione simultaneously, it is characterized in that: described micro-fluidic chip is double-T shaped structure.

4. according to the described detection method of measuring cytoactive oxygen and reduced glutathione simultaneously of claim 1, it is characterized in that: described damping fluid is a borate buffer solution.

5. according to the described detection method of measuring cytoactive oxygen and reduced glutathione simultaneously of claim 1, it is characterized in that: adopt probe DHR-123 and NDA to come active oxygen and reduced glutathione in the labeled cell respectively, adopt perchloric acid and the fine mixed liquor of second to extract intracellular reactive oxygen and reduced glutathione.

6. according to the described detection method of measuring cytoactive oxygen and reduced glutathione simultaneously of claim 5, it is characterized in that: the weight concentration of described perchloric acid and the fine mixed liquor of second is that 3.3% perchloric acid mixed with acetonitrile in 1: 1 by volume, and wherein the fine volume ratio of perchloric acid and second is 1: 1.

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