CN112924425B - Method for detecting pH value in red blood cells by utilizing red blood cell autofluorescence - Google Patents
Method for detecting pH value in red blood cells by utilizing red blood cell autofluorescence Download PDFInfo
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- 239000006228 supernatant Substances 0.000 claims description 20
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 16
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 12
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- DANUORFCFTYTSZ-UHFFFAOYSA-N epinigericin Natural products O1C2(C(CC(C)(O2)C2OC(C)(CC2)C2C(CC(O2)C2C(CC(C)C(O)(CO)O2)C)C)C)C(C)C(OC)CC1CC1CCC(C)C(C(C)C(O)=O)O1 DANUORFCFTYTSZ-UHFFFAOYSA-N 0.000 claims description 6
- DANUORFCFTYTSZ-BIBFWWMMSA-N nigericin Chemical compound C([C@@H]1C[C@H]([C@H]([C@]2([C@@H](C[C@](C)(O2)C2O[C@@](C)(CC2)C2[C@H](CC(O2)[C@@H]2[C@H](C[C@@H](C)[C@](O)(CO)O2)C)C)C)O1)C)OC)[C@H]1CC[C@H](C)C([C@@H](C)C(O)=O)O1 DANUORFCFTYTSZ-BIBFWWMMSA-N 0.000 claims description 6
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- 238000004364 calculation method Methods 0.000 claims description 4
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 3
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
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Abstract
The invention discloses a method for detecting the pH value in erythrocytes by utilizing the autofluorescence of erythrocytes, which comprises the steps of preparing calibration liquids with different pH values, collecting fluorescent intensity signals of the calibration liquids, drawing a standard curve of average fluorescent intensity and pH value to obtain a linear equation, and substituting the average fluorescent intensity in a sample of erythrocytes to be detected into the standard curve to obtain the pH value in erythrocytes to be detected. The method provided by the invention is used for detecting the pH value in the red blood cells, and has the advantages of simplicity in operation, short time consumption, stable detection result and the like. According to the invention, by utilizing the autofluorescence characteristic of the red blood cells, a standard curve of the change of the fluorescence intensity of the red blood cells under different pH environments is established without adding or preparing fluorescent dyes, and the corresponding pH values in the red blood cells with different fluorescence intensities are calculated in turn. The method has the advantages that the pH precision value can reach 0.01 pH unit, and the precision is high.
Description
Technical Field
The invention relates to the technical field of analytical chemistry, in particular to a method for detecting the pH value in erythrocytes by utilizing the autofluorescence of erythrocytes.
Background
Body fluid accounts for 60% -70% of human body, wherein 2/3 of the body fluid is located in cells. The pH value (pH), especially the pH value of intracellular fluid, plays an important role in various cell biological behaviors, such as enzyme activity, cell proliferation and apoptosis, information transmission, tumor cell drug resistance and the like, and is an important microenvironment index affecting cell functions.
However, the clinical blood gas analysis technology detects the pH value of serum, which is equivalent to the pH value of extracellular fluid, and the relationship between the acid-base change of intracellular fluid, which is the vast majority of body fluid, and the acid-base balance of intracellular and extracellular fluids is rarely studied. However, in recent years, the importance of intracellular environmental stabilization, especially erythrocytes carrying oxygen and carbon dioxide and delivering them to various tissues and organs of the human body, has been increasingly realized, and changes in the environment (especially acid-base) within erythrocytes have an important influence on the structure and function of intracellular hemoglobin, so that pH detection and monitoring of changes in the environment affected within erythrocytes are particularly important. The existing common intracellular pH detection means mainly adopts ratio type nano probes with sensitive incubation pH, but erythrocytes have an autofluorescence effect, are easily affected when being applied to intracellular pH detection of erythrocytes, and have large detection error.
Disclosure of Invention
The invention aims to provide a method for detecting the pH value in erythrocytes by utilizing the autofluorescence of erythrocytes, which is simple to operate, short in time consumption and stable in measurement result.
The technical scheme of the invention is as follows:
A method for detecting pH in erythrocytes using erythrocyte autofluorescence, comprising the steps of:
s1, taking whole blood, centrifuging, and discarding supernatant to obtain red blood cells;
S2: washing the red blood cells prepared in the step S1 by using PBS (phosphate buffered saline), centrifuging, and discarding the supernatant;
S3: adding PBS into the suspension after S2 supernatant removal, re-suspending, and then split-charging the suspension in a plurality of EP pipes in equal quantity;
S4: centrifuging the liquid in the EP tube, and discarding the supernatant;
S5: adding calibration liquids with different pH values and sample buffer solutions into an EP tube, and incubating to prepare red blood cell calibration liquid samples with different pH values;
s6: collecting fluorescent signals from the S5 sample by using a flow cytometer;
S7: drawing a standard curve of average fluorescence intensity and pH according to the fluorescence signals collected in the step S6 to obtain a linear equation;
s8: and (3) preparing a red blood cell sample to be detected according to the steps S1-S4, and substituting the average fluorescence intensity of the red blood cell sample to be detected, which is collected by a flow cytometer, into an S7 equation for calculation to obtain the pH value in the red blood cell to be detected.
Further, it comprises the steps of:
s1, taking whole blood, centrifuging at 1500rpm for 5min, and discarding supernatant to obtain red blood cells;
s2: washing the red blood cells prepared in the step S1 by using PBS (phosphate buffer solution), centrifuging at 1500rpm for 5min, and discarding the supernatant;
S3: adding PBS into the suspension after S2 supernatant removal, re-suspending, and then split-charging the suspension in a plurality of EP pipes in equal quantity;
S4: centrifuging the liquid in the EP tube for 10min, and discarding the supernatant;
S5: adding calibration solutions with different pH values and sample buffer solutions into an EP tube, and incubating for 15min at 37 ℃ to prepare red blood cell calibration solution samples with different pH values;
s6: collecting fluorescent signals from the S5 sample by using a flow cytometer;
S7: drawing a standard curve of average fluorescence intensity and pH according to the fluorescence signals collected in the step S6 to obtain a linear equation;
s8: and (3) preparing a red blood cell sample to be detected according to the steps S1-S4, and substituting the average fluorescence intensity of the red blood cell sample to be detected, which is collected by a flow cytometer, into an S7 equation for calculation to obtain the pH value in the red blood cell to be detected.
Further, the preparation of the calibration liquid comprises the following steps:
mother liquor: 120mM KCl, 30mM NaCl, 1mM CaCl 2、0.5mM MgSO4、1mM NaH2PO4, 5mM 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 which is 2mM solution dissolved in absolute ethanol and is used at present, and the volume ratio of the nigericin to the mother solution is 1:200.
Further, in step S6, the flow cytometer uses 488nm excitation and BL3 channel (PerCP-Cy5.5) to collect fluorescent signals.
By adopting the technical scheme, the beneficial effects are as follows:
The method provided by the invention is used for detecting the pH value in the red blood cells, and has the advantages of simplicity in operation, short time consumption, stable detection result and the like. The invention utilizes the autofluorescence characteristic of red blood cells: the autofluorescence signal of the red blood cells and the pH value are in a linear positive correlation, fluorescent dyes are not needed to be additionally or alternatively prepared, the red blood cells are treated by adding calibration solutions and sample buffers with different pH values, so that the pH values inside and outside the red blood cells are the same, a standard curve which changes with the fluorescence intensity of the red blood cells under different pH environments is established by collecting the fluorescence signals of red blood cell samples with different pH values, and the pH values corresponding to different fluorescence intensities are calculated in turn. The method has the advantages that the pH precision value can reach 0.01 pH unit, and the precision is high.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a standard graph of red blood cell fluorescence intensity as a function of pH in accordance with the present invention.
FIG. 2 is a distribution of erythrocytes in a sample of interest in an embodiment of the invention;
FIG. 3 is a distribution of erythrocytes in a sample of erythrocytes to be tested according to an embodiment of the invention;
FIG. 4 is a histogram of fluorescence intensity of red blood cells in an embodiment of the invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Examples: a method for detecting pH in erythrocytes using erythrocyte autofluorescence, comprising the steps of:
S1, taking 50 μl of whole blood, centrifuging at 1500rpm for 5min, and discarding supernatant to obtain red blood cells;
s2: washing the red blood cells prepared in the step S1 by using PBS (phosphate buffer solution), centrifuging at 1500rpm for 5min, and discarding the supernatant;
S3: adding PBS into the suspension after S2 supernatant removal, re-suspending, and then split-charging the suspension in a plurality of EP pipes in equal quantity;
S4: centrifuging the liquid in the EP tube at 300 Xg for 10min, and discarding the supernatant;
S5: adding calibration solutions with different pH values and sample buffer solutions into an EP tube, and incubating for 15min at 37 ℃ to prepare red blood cell calibration solution samples with different pH values; the distribution of erythrocytes in the calibration sample was characterized, and the characterization result is shown in fig. 2. Then figure 2 reflects the distribution of red blood cells after equilibration of the pH calibration solution, and cell aggregation may occur during equilibration.
S6: collecting fluorescent signals from the S5 sample by using a flow cytometer;
s7: drawing a standard curve of average fluorescence intensity and pH according to the fluorescence signals collected in the step S6 to obtain a linear equation, wherein the drawn standard curve and a fitting linear equation are shown in figure 1;
S8: according to the steps S1-S4, preparing a red blood cell sample to be detected, characterizing the distribution of red blood cells in the red blood cell sample to be detected, wherein the characterization result is shown in a figure 3, the figure 3 reflects the distribution condition of the red blood cells in a sample buffer solution, the average fluorescence intensity of the red blood cell sample to be detected, which is collected by a flow cytometer, is substituted into an S7 equation to calculate, so that the pH value in the red blood cells to be detected can be obtained, wherein the fluorescence intensity histogram of the red blood cells in the sample is detected by the flow cytometer, the figure 4 shows the fluorescence distribution collected in a wave band where the red blood cells autofluorescence is located, the single peak has complete shape, the fluorescence emitted in a receiving range is uniform, and the value of the abscissa corresponding to the peak is the fluorescence intensity.
The formula of the calibration liquid is as follows:
mother liquor: 120mM KCl, 30mM NaCl, 1mM CaCl 2、0.5mM MgSO4、1mM NaH2PO4, 5mM 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 which is 2mM solution dissolved in absolute ethanol and is used at present, and the volume ratio of the nigericin to the mother solution is 1:200.
In S8, 101 red blood cell samples to be detected (the samples are derived from whole blood samples of kidney-penetrating patients) are prepared, the average fluorescence intensity is collected by a flow cytometer, and the collected average fluorescence intensity is substituted into FIG. 1 to obtain the pH value in red blood cells in each sample, which is shown in Table I.
Table one: detection result of pH in erythrocytes in erythrocyte sample to be detected
The invention 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 lower than the pH value (7.35-7.45) of the whole blood, namely the detected pH value is slightly acidic.
The present invention and its embodiments have been described above with no limitation, and the embodiments of the present invention are shown in the above examples, and the actual structure is not limited thereto. In summary, if one of ordinary skill in the art is informed by this disclosure, a structural manner and an embodiment similar to the technical solution should not be creatively devised without departing from the gist of the present invention.
Claims (4)
1. A method for detecting the pH value in red blood cells by utilizing the autofluorescence of the red blood cells is characterized in that,
Which comprises the following steps:
s1, taking whole blood, centrifuging, and discarding supernatant to obtain red blood cells;
S2: washing the red blood cells prepared in the step S1 by using PBS (phosphate buffered saline), centrifuging, and discarding the supernatant;
S3: adding PBS into the suspension after S2 supernatant removal, re-suspending, and then split-charging the suspension in a plurality of EP pipes in equal quantity;
S4: centrifuging the liquid in the EP tube, and discarding the supernatant;
S5: adding calibration solutions with different pH values and sample buffer solutions into an EP tube, and incubating for 15min at 37 ℃ to prepare red blood cell calibration solution samples with different pH values;
s6: collecting fluorescent signals from the S5 sample by using a flow cytometer;
S7: drawing a standard curve of average fluorescence intensity and pH according to the fluorescence signals collected in the step S6 to obtain a linear equation;
s8: and (3) preparing a red blood cell sample to be detected according to the steps S1-S4, and substituting the average fluorescence intensity of the red blood cell sample to be detected, which is collected by a flow cytometer, into an S7 equation for calculation to obtain the pH value in the red blood cell to be detected.
2. A method for detecting pH in erythrocytes by utilizing autofluorescence of erythrocytes according to claim 1, characterized in that it comprises the following steps:
s1, taking whole blood, centrifuging at 1500rpm for 5min, and discarding supernatant to obtain red blood cells;
s2: washing the red blood cells prepared in the step S1 by using PBS (phosphate buffer solution), centrifuging at 1500rpm for 5min, and discarding the supernatant;
S3: adding PBS into the suspension after S2 supernatant removal, re-suspending, and then split-charging the suspension in a plurality of EP pipes in equal quantity;
S4: centrifuging the liquid in the EP tube for 10min, and discarding the supernatant;
S5: adding calibration solutions with different pH values and sample buffer solutions into an EP tube, and incubating for 15min at 37 ℃ to prepare red blood cell calibration solution samples with different pH values;
s6: collecting fluorescent signals from the S5 sample by using a flow cytometer;
S7: drawing a standard curve of average fluorescence intensity and pH according to the fluorescence signals collected in the step S6 to obtain a linear equation;
s8: and (3) preparing a red blood cell sample to be detected according to the steps S1-S4, and substituting the average fluorescence intensity of the red blood cell sample to be detected, which is collected by a flow cytometer, into an S7 equation for calculation to obtain the pH value in the red blood cell to be detected.
3. The method for detecting the pH value in the red blood cells by utilizing the autofluorescence of the red blood cells according to claim 1 or 2, wherein the preparation of the calibration solution is as follows:
mother liquor: 120mM KCl, 30mM NaCl, 1mM CaCl 2、0.5mM MgSO4、1mM NaH2PO4, 5mM 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 which is 2mM solution dissolved in absolute ethanol and is used at present, and the volume ratio of the nigericin to the mother solution is 1:200.
4. A method for detecting pH in erythrocytes by using the autofluorescence of erythrocytes according to claim 1 or 2, characterized in that the flow cytometer is activated with 488nm and the BL3 channel (PerCP-cy 5.5) collects the fluorescence signal in step S6.
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