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CN112924425A - Method for detecting pH value in erythrocyte by utilizing erythrocyte autofluorescence - Google Patents

Method for detecting pH value in erythrocyte by utilizing erythrocyte autofluorescence Download PDF

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CN112924425A
CN112924425A CN202110117483.3A CN202110117483A CN112924425A CN 112924425 A CN112924425 A CN 112924425A CN 202110117483 A CN202110117483 A CN 202110117483A CN 112924425 A CN112924425 A CN 112924425A
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CN112924425B (en
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丁小强
宋娜娜
张健
陆雨菲
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Zhongshan Hospital Fudan University
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Abstract

The invention discloses a method for detecting pH value in red blood cells by utilizing red blood cell autofluorescence, which comprises the steps of preparing calibration solutions with different pH values, collecting fluorescence intensity signals of the calibration solutions, drawing a standard curve of average fluorescence intensity and pH value to obtain a linear equation, and substituting the average fluorescence intensity in a red blood cell sample to be detected into the standard curve to obtain the pH value in the red blood cells to be detected. The method for detecting the pH value in the erythrocyte has the advantages of simple operation, short time consumption, stable detection result and the like. The invention utilizes the autofluorescence characteristic of the red blood cells, does not need to add or prepare fluorescent dye, establishes a standard curve of the change of the fluorescence intensity of the red blood cells under different pH environments, and calculates the corresponding pH value in the red blood cells with different fluorescence intensities in turn. The pH precision value of the method can reach 0.01pH unit, and the precision is high.

Description

Method for detecting pH value in erythrocyte by utilizing erythrocyte autofluorescence
Technical Field
The invention relates to the technical field of analytical chemistry, in particular to a method for detecting a pH value in red blood cells by utilizing the autofluorescence of the red blood cells.
Background
Body fluid comprises 60% -70% of human body, wherein 2/3 is located in cell. The pH value (pH), especially the pH value of intracellular fluid, plays an important role in regulating and controlling various cell biological behaviors, such as enzyme activity, cell proliferation and apoptosis, information transmission, drug resistance of tumor cells and the like, and is an important microenvironment index influencing cell functions.
However, the current clinical blood gas analysis technology measures the ph value of serum, which is equivalent to the ph value of extracellular fluid, and studies on the acid-base change of intracellular fluid, which accounts for most of body fluids, and the relationship between the acid-base balance of intracellular fluid and extracellular fluid are few. However, in recent years, the importance of intracellular environment stabilization, especially the erythrocytes whose main physiological functions are to carry oxygen and carbon dioxide and deliver them to various tissues and organs of the human body, has been increasingly recognized, and the change of the environment (especially acid-base) in erythrocytes has an important influence on the structure and function of intracellular hemoglobin, and thus the detection and monitoring of pH values affecting environmental changes in erythrocytes are particularly important. The existing means for detecting the pH value in the common cells mainly adopts a ratio type nano probe sensitive to incubation pH value, but the red blood cells have an autofluorescence effect, are easily influenced when being applied to the detection of the pH value in the red blood cells, and have large detection errors.
Disclosure of Invention
The invention aims to provide a method for detecting the pH value in red blood cells by using the autofluorescence of the red blood cells, which has the advantages of simple operation, short time consumption and stable measurement result.
The technical scheme of the invention is as follows:
a method for detecting pH value in red blood cells by utilizing the autofluorescence of the red blood cells comprises the following steps:
s1, taking whole blood, centrifuging, and removing supernatant to obtain erythrocytes;
s2: washing the red blood cells prepared in the step S1 with PBS, centrifuging, and removing the supernatant;
s3: adding PBS into the suspension liquid obtained by discarding the supernatant of S2, then re-suspending, and subpackaging the suspension liquid into a plurality of EP tubes in equal amount;
s4: centrifuging the liquid in the EP tube, and then discarding the supernatant;
s5: adding calibration solution and sample buffer solution with different pH values into an EP tube, and incubating to prepare erythrocyte calibration solution samples with different pH values;
s6: collecting a fluorescence signal of the S5 sample by using a flow cytometer;
s7: drawing a standard curve of the average fluorescence intensity and the pH according to the fluorescence signal collected in S6 to obtain a linear equation;
s8: preparing a red blood cell sample to be detected according to the steps of S1-S4, and substituting the average fluorescence intensity collected by the red blood cell sample to be detected by adopting a flow cytometer into an S7 equation to calculate the pH value in the red blood cell to be detected.
Further, it comprises the following steps:
s1, taking whole blood, centrifuging at 1500rpm for 5min, and removing supernatant to obtain erythrocytes;
s2: washing the red blood cells prepared in the step S1 with PBS, centrifuging at 1500rpm for 5min, and removing the supernatant;
s3: adding PBS into the suspension liquid obtained by discarding the supernatant of S2, then re-suspending, and subpackaging the suspension liquid into a plurality of EP tubes in equal amount;
s4: centrifuging the liquid in the EP tube for 10min, and then removing the supernatant;
s5: adding calibration solution and sample buffer solution with different pH values into an EP tube, and incubating for 15min at 37 ℃ to prepare erythrocyte calibration solution samples with different pH values;
s6: collecting a fluorescence signal of the S5 sample by using a flow cytometer;
s7: drawing a standard curve of the average fluorescence intensity and the pH according to the fluorescence signal collected in S6 to obtain a linear equation;
s8: preparing a red blood cell sample to be detected according to the steps of S1-S4, and substituting the average fluorescence intensity collected by the red blood cell sample to be detected by adopting a flow cytometer into an S7 equation to calculate the pH value in the red blood cell to be detected.
Further, the preparation of the calibration solution is as follows:
mother liquor: 120mM KCl, 30mM NaCl, 1mM CaCl2、0.5mM MgSO4、1mM NaH2PO45mM glucose, 20mM HEPES, HCl or NaOH; wherein HCl or NaOH is used to adjust the gradient pH (6.9-7.6)
The regulating solution is nigericin, and the using volume ratio of the nigericin to the mother solution is 1:200, wherein the nigericin is 2mM solution dissolved in absolute ethyl alcohol and is used as the ready-to-use solution.
Further, in step S6, the flow cytometer employs 488nm excitation, and BL3 channel (PerCP-Cy5.5) collects fluorescence signals.
By adopting the technical scheme, the beneficial effects are as follows:
the method for detecting the pH value in the erythrocyte has the advantages of simple operation, short time consumption, stable detection result and the like. The invention utilizes the autofluorescence property of red blood cells: the autofluorescence signal of the red blood cells is in a linear positive correlation with the pH value, no fluorescent dye is required to be added or prepared, the red blood cells are treated by adding calibration liquid and sample buffer solution with different pH values, so that the pH values inside and outside the red blood cells are the same, a standard curve of the fluorescence intensity change of the red blood cells under different pH environments is established by collecting the fluorescence signals of the red blood cell samples with different pH values, and the pH values corresponding to different fluorescence intensities are calculated in turn. The pH precision value of the method can reach 0.01pH unit, and the precision is high.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a standard graph of the fluorescence intensity of erythrocytes according to the invention as a function of pH.
FIG. 2 is a graph showing the distribution of red blood cells in a calibration sample according to an embodiment of the present invention;
FIG. 3 is a diagram illustrating the distribution of red blood cells in a red blood cell sample to be tested according to an embodiment of the present invention;
FIG. 4 is a histogram of fluorescence intensity of red blood cells according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example (b): a method for detecting pH value in red blood cells by utilizing the autofluorescence of the red blood cells comprises the following steps:
s1, taking 50 mu l of whole blood, centrifuging for 5min at 1500rpm, and removing supernatant to obtain erythrocytes;
s2: washing the red blood cells prepared in the step S1 with PBS, centrifuging at 1500rpm for 5min, and removing the supernatant;
s3: adding PBS into the suspension liquid obtained by discarding the supernatant of S2, then re-suspending, and subpackaging the suspension liquid into a plurality of EP tubes in equal amount;
s4: centrifuging the liquid in the EP tube at 300 Xg for 10min, and removing the supernatant;
s5: adding calibration solution and sample buffer solution with different pH values into an EP tube, and incubating for 15min at 37 ℃ to prepare erythrocyte calibration solution samples with different pH values; the distribution of the red blood cells in the calibration sample was characterized and the results are shown in FIG. 2. Fig. 2 reflects the distribution of the red blood cells after the pH calibration solution is equilibrated, and the cell aggregation may occur during the equilibration process.
S6: collecting a fluorescence signal of the S5 sample by using a flow cytometer;
s7: drawing a standard curve of the average fluorescence intensity and the pH according to the fluorescence signal collected in S6 to obtain a linear equation, wherein the drawn standard curve and the fitted linear equation are shown in figure 1;
s8: preparing a to-be-detected red blood cell sample according to the steps of S1-S4, characterizing the red blood cell distribution in the to-be-detected red blood cell sample, wherein the characterization result is shown in figure 3, the figure 3 reflects the distribution condition of red blood cells in a sample buffer solution, and the pH value in the to-be-detected red blood cell can be obtained by substituting the average fluorescence intensity collected by the to-be-detected red blood cell sample by a flow cytometer into an S7 equation for calculation, wherein the histogram of the fluorescence intensity of the red blood cells in the sample when the flow cytometer is used for detection is shown in figure 4, and the figure 4 shows the fluorescence distribution collected in the wave band where the red blood cell autofluorescence is located, the unimodal peak shape is complete, which indicates that the emitted fluorescence is uniform in the receiving.
The formula of the calibration solution is as follows:
mother liquor: 120mM KCl, 30mM NaCl, 1mM CaCl2、0.5mM MgSO4、1mM NaH2PO45mM glucose, 20mM HEPES, HCl or NaOH; wherein HCl or NaOH is used to adjust the gradient pH (6.9-7.6)
The regulating solution is nigericin, and the using volume ratio of the nigericin to the mother solution is 1:200, wherein the nigericin is 2mM solution dissolved in absolute ethyl alcohol and is used as the ready-to-use solution.
In S8, 101 red blood cell samples to be tested (samples derived from whole blood samples of renal biopsy patients) were prepared, and the average fluorescence intensities collected by the flow cytometer were used, and the collected average fluorescence intensities were substituted into fig. 1, to obtain the pH values in the red blood cells of the respective samples, which is shown in table one.
Table one: detection result of pH in erythrocytes in erythrocyte sample to be detected
Figure BDA0002921270100000041
The method can accurately detect the pH value in the red blood cells to reach 0.01pH unit, has the advantages of simple operation, short time consumption, stable detection result and the like, and the detected pH value in the red blood cells is slightly lower than the pH value of whole blood (7.35-7.45), namely slightly acidic.
The present invention and the embodiments thereof have been described above, but the description is not limited thereto, and the embodiments shown in the above embodiments are only one of the embodiments of the present invention, and the actual configuration is not limited thereto. In summary, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (4)

1.一种利用红细胞自发荧光检测红细胞内pH值的方法，其特征在于，1. a method utilizing erythrocyte autofluorescence to detect pH value in erythrocytes, is characterized in that, 其包括以下步骤：It includes the following steps: S1:取全血，进行离心，弃上清，得到红细胞；S1: take whole blood, carry out centrifugation, discard supernatant to obtain red blood cells; S2：将步骤S1制备的红细胞用PBS液进行洗涤后，进行离心后，弃上清；S2: after washing the red blood cells prepared in step S1 with PBS solution, after centrifugation, discard the supernatant; S3：将S2弃上清后的悬浮液加入PBS后进行重悬，然后将等量分装在若干个EP管内；S3: The suspension after discarding the supernatant from S2 was added to PBS and resuspended, and then the same amount was dispensed into several EP tubes; S4：将EP管内的液体离心后，弃上清；S4: After centrifuging the liquid in the EP tube, discard the supernatant; S5：向EP管内加入不同pH值的标定液和样品缓冲液，进行孵育，配制出不同pH值的红细胞标定液样品；S5: adding calibration solutions and sample buffers with different pH values into the EP tube, incubating, and preparing red blood cell calibration solution samples with different pH values; S6：将S5样品采用流式细胞仪收集荧光信号；S6: use the S5 sample to collect the fluorescence signal by flow cytometer; S7：根据S6收集的荧光信号，绘制出平均荧光强度与pH的标准曲线，得到线性方程；S7: According to the fluorescence signal collected in S6, draw the standard curve of the average fluorescence intensity and pH, and obtain a linear equation; S8：按照S1-S4步骤制备将待测红细胞样品，将待测红细胞样品采用流式细胞仪收集的平均荧光强度代入S7方程内计算即可得到待测红细胞内pH值。S8: Prepare the red blood cell sample to be tested according to steps S1-S4, and substitute the average fluorescence intensity of the red blood cell sample to be tested collected by the flow cytometer into the S7 equation to calculate the pH value in the red blood cell to be tested. 2.根据权利要求1所述的一种利用红细胞自发荧光检测红细胞内pH值的方法，其特征在于，其包括以下步骤：2. a kind of method utilizing erythrocyte autofluorescence to detect pH value in erythrocytes according to claim 1, is characterized in that, it comprises the following steps: S1:取全血，1500rpm离心5min后，弃上清后，得到红细胞；S1: take whole blood, centrifuge at 1500 rpm for 5 min, discard the supernatant, and obtain red blood cells; S2：将步骤S1制备的红细胞用PBS液进行洗涤后，1500rpm离心5min后，弃上清；S2: after washing the red blood cells prepared in step S1 with PBS, centrifuge at 1500 rpm for 5 min, and discard the supernatant; S3：将S2弃上清后的悬浮液加入PBS后进行重悬，然后将等量分装在若干个EP管内；S3: The suspension after discarding the supernatant from S2 was added to PBS and resuspended, and then the same amount was dispensed into several EP tubes; S4：将EP管内的液体离心10min后，弃上清；S4: After centrifuging the liquid in the EP tube for 10 min, discard the supernatant; S5：向EP管内加入不同pH值的标定液和样品缓冲液，在37℃条件下孵育15min，配制出不同pH值的红细胞标定液样品；；S5: add calibration solutions and sample buffers with different pH values into the EP tube, and incubate at 37°C for 15 minutes to prepare red blood cell calibration solution samples with different pH values; S6：将S5样品采用流式细胞仪收集荧光信号；S6: use the S5 sample to collect the fluorescence signal by flow cytometer; S7：根据S6收集的荧光信号，绘制出平均荧光强度与pH的标准曲线，得到线性方程；S7: According to the fluorescence signal collected in S6, draw the standard curve of the average fluorescence intensity and pH, and obtain a linear equation; S8：按照S1-S4步骤制备将待测红细胞样品，将待测红细胞样品采用流式细胞仪收集的平均荧光强度代入S7方程内计算即可得到待测红细胞内pH值。S8: Prepare the red blood cell sample to be tested according to steps S1-S4, and substitute the average fluorescence intensity of the red blood cell sample to be tested collected by the flow cytometer into the S7 equation to calculate the pH value in the red blood cell to be tested. 3.根据权利要求1或2所述的一种利用红细胞自发荧光检测红细胞内pH值的方法，其特征在于，所述标定液的配制为：3. a kind of method utilizing erythrocyte autofluorescence to detect pH value in erythrocytes according to claim 1 and 2, is characterized in that, the preparation of described calibration solution is: 母液：120mM KCl、30mM NaCl、1mM CaCl2、0.5mM MgSO4、1mM NaH2PO4、5mM葡萄糖、20mMHEPES、HCl或NaOH；其中HCl或NaOH用来调节梯度pH(6.9-7.6)；Stock solution: 120 mM KCl, 30 mM NaCl, 1 mM CaCl 2 , 0.5 mM MgSO 4 , 1 mM NaH 2 PO 4 , 5 mM glucose, 20 mM HEPES, HCl or NaOH; wherein HCl or NaOH is used to adjust the gradient pH (6.9-7.6); 调节液为尼日利亚菌素，其与母液的使用体积配比为1:200，其中尼日利亚菌素为溶于无水乙醇的2mM溶液，其为现配现用。The conditioning solution is nigericin, and its use volume ratio with the mother liquor is 1:200, wherein nigericin is a 2mM solution dissolved in absolute ethanol, which is prepared and used now. 4.根据权利要求1或2所述的一种利用红细胞自发荧光检测红细胞内pH值的方法，其特征在于，步骤S6中流式细胞仪采用488nm激发，BL3通道(PerCP-Cy5.5)收集荧光信号。4. a kind of method utilizing erythrocyte autofluorescence to detect pH value in erythrocytes according to claim 1 and 2, is characterized in that, in step S6, flow cytometer adopts 488nm to excite, and BL3 channel (PerCP-Cy5.5) collects fluorescence Signal.
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