CN113484256A - High-flux concentration determination method for bloody hemoglobin of blood clam - Google Patents
High-flux concentration determination method for bloody hemoglobin of blood clam Download PDFInfo
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- 102000001554 Hemoglobins Human genes 0.000 title claims abstract description 70
- 108010054147 Hemoglobins Proteins 0.000 title claims abstract description 70
- 241001339782 Scapharca broughtonii Species 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 25
- 210000004369 blood Anatomy 0.000 claims abstract description 83
- 239000008280 blood Substances 0.000 claims abstract description 83
- 239000003085 diluting agent Substances 0.000 claims abstract description 46
- 238000002835 absorbance Methods 0.000 claims abstract description 34
- 238000005259 measurement Methods 0.000 claims abstract description 25
- 238000001514 detection method Methods 0.000 claims abstract description 14
- 241000237506 Scapharca Species 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 241000224239 Tegillarca granosa Species 0.000 claims description 8
- 238000010521 absorption reaction Methods 0.000 claims description 8
- 238000012546 transfer Methods 0.000 claims description 7
- 238000010009 beating Methods 0.000 claims description 6
- 238000007664 blowing Methods 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 5
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 4
- 238000004090 dissolution Methods 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 241001465754 Metazoa Species 0.000 abstract description 2
- 238000012417 linear regression Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 31
- 239000012085 test solution Substances 0.000 description 6
- 241000628923 Anadara sativa Species 0.000 description 5
- 235000020639 clam Nutrition 0.000 description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 241000237519 Bivalvia Species 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 150000004032 porphyrins Chemical class 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 235000015170 shellfish Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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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
- G01N1/38—Diluting, dispersing or mixing samples
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Abstract
The invention discloses a method for high-flux measurement of concentration of hemoglobin in blood of blood clam, which forms a stable solution which has a linear relation with the concentration of hemoglobin under a certain absorbance through a specific diluent, and realizes the rapid and high-efficiency measurement of the concentration of the hemoglobin in the blood clam of blood clam by establishing a mathematical linear regression model of quantitative indexes of the concentration of the hemoglobin and the absorbance of the blood clam of blood clam. The error between the concentration of the bloody clam haemoglobin measured by the high-throughput measuring method and the detection result of the existing SLS-Hb kit is small, once the standard curve is established, the absorbance detection can be carried out through the microplate reader, the haemoglobin concentration of the blood of the bloody clam to be detected can be conveniently and rapidly calculated according to the standard curve, each sample does not need to wait for one minute as the existing SLS-Hb kit, and the method has wide application and popularization values. Besides being used for the rapid and high-efficiency measurement of the concentration of the hemoglobin in the blood clam, the method can also be used for the measurement of the concentration of the hemoglobin in other animals.
Description
Technical Field
The invention belongs to the field of biology, and particularly relates to a method for high-flux determination of concentration of hemoglobin in blood of scapharca granosa.
Background
The blood clam is one of four traditional cultured shellfish in China, and has delicious taste and rich nutrition. As a few invertebrates with red blood, the blood clam has hemoglobin similar to human structure, and is rich in porphyrin iron easily absorbed by human bodies, so that people have higher and higher attention on research on blood hemoglobin of the blood clam. The high-flux measuring method for the concentration of the hemoglobin in the blood clams provides technical support for the research on the mechanism of formation of the blood color character of the blood clams and the cultivation of new species of the blood clams with high hemoglobin content.
When the common SLS-Hb kit on the market is used for measuring the concentration of hemoglobin, the influence of reaction time is great, and the measurement must be carried out for one minute, so that the SLS-Hb kit cannot realize high-flux measurement of the concentration of the hemoglobin in the blood clam. However, in the existing literature, no related method for high-flux measurement of the concentration of the hemoglobin in the blood clam is reported at present.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for measuring the concentration of the bloody protein of the blood clam with high flux aiming at the defects of the prior art.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for high-flux measurement of concentration of hemoglobin in blood of blood clam of scapharca granosa comprises the following steps:
(1) preparing diluent and detecting absorption peak of diluent dissolved with blood of blood clam
(1-1) taking 20mL of triton solution with the volume percentage concentration of 1% as a solution A, and adding the solution A into a beaker;
(1-2) taking 180mL of 1.5 mass percent sodium chloride solution as a solution B, adding the solution B into the beaker filled with the solution A in the step (1-1), and uniformly mixing to obtain a solution C, wherein the solution C is a diluent;
(1-3) taking part of diluent, mixing randomly selected fresh blood of scapharca granosa with the taken diluent in a volume ratio of 1:20 to obtain diluent dissolved with blood, carrying out full-wavelength scanning on the diluent dissolved with blood by using a spectrophotometer, and measuring that the diluent dissolved with blood has highest absorption peaks at the wavelengths of 540nm and 580 nm;
(2) establishing a standard curve
Taking N fresh blood samples of Arca granosa with known hemoglobin concentration but different hemoglobin concentrations, wherein N is a positive integer not less than 5, dissolving the blood samples by using diluent respectively, detecting the absorbance of the solution by using a spectrophotometer for the solution obtained after each dissolution, and establishing a hemoglobin concentration-absorbance standard curve according to the hemoglobin concentrations and the corresponding absorbances of the N fresh blood samples of Arca granosa;
(3) measurement of blood hemoglobin concentration of blood clam
(3-1) adding 1mL of diluent into a clean 1.5mL centrifuge tube, adding 50 μ L of blood of the clean 1.blood of the blood of;
and (3-2) adding 200 mu L of detection liquid into an enzyme label plate, measuring the absorbance Abs of the detection liquid at 540nm, and calculating the hemoglobin concentration of blood of the tegillarca granosa to be measured according to the standard curve established in the step (2).
The blood-soluble diluent is prepared by mixing 1% of triton solution and 1.5% of sodium chloride solution, can quickly dissolve blood of air of.
In the diluent prepared by the invention, the sodium chloride with low concentration can promote the water absorption and the burst of blood cell membranes of the blood clam; triton X-100 is a non-ionic surfactant, has the effects of dissolving erythrocytes and free hemoglobin and preventing hemolytic turbidity, can maintain the stability of protein by virtue of low-concentration Triton X-100, and ensures that blood of blood clams after being dissolved in diluent has a stable absorption peak at a 540nm wavelength, so that the accuracy of a standard curve is ensured, and further, the high-flux measurement of the concentration of the hemoglobin of the blood clams is realized by the method.
Preferably, the specific operation of step (2) is as follows:
(2-1) adopting the existing SLS-Hb kit to measure the hemoglobin concentration N of the N fresh blood samples of the scapharca granosa, wherein the unit is g/L;
(2-2) respectively adding 1mL of diluent into N clean 1.5mL centrifuge tubes, adding 50 mu L of fresh blood sample of scapharca granosa into each centrifuge tube, blowing and beating uniformly by using a liquid transfer gun after adding, standing for one minute, and obtaining N different solution samples after blood of scapharca granosa is completely dissolved;
(2-3) respectively measuring the absorbance Abs of the N different solution samples obtained in the step (2-2) at 540nm by using a microplate reader;
(2-4) establishing a hemoglobin concentration-absorbance standard curve according to the hemoglobin concentration N and corresponding absorbance Abs of the N fresh blood samples of the blood of the: abs 0.0039n +0.0439, R2The hemoglobin concentration n is 0.9941, and the measurement range is 10-60 g/L.
Compared with the prior art, the invention has the following advantages: according to the invention, the blood of the blood clams forms a stable solution which has a linear relation with the hemoglobin concentration of the blood clams under a certain absorbance through the specific diluent, and the hemoglobin concentration of the blood clams is rapidly and efficiently measured by establishing a mathematical linear regression model of quantitative indexes of the hemoglobin concentration and the absorbance of the blood clams. The error between the concentration of the bloody clam haemoglobin measured by the high-throughput measuring method and the detection result of the existing SLS-Hb kit is small, once the standard curve is established, the absorbance detection can be carried out through the microplate reader, the haemoglobin concentration of the blood of the bloody clam to be detected can be conveniently and rapidly calculated according to the standard curve, each sample does not need to wait for one minute as the existing SLS-Hb kit, and the method has wide application and popularization values. Besides being used for the rapid and high-efficiency measurement of the concentration of the hemoglobin in the blood clam, the method can also be used for the measurement of the concentration of the hemoglobin in other animals.
Drawings
FIG. 1 is a full wavelength scanning chart of the diluent in which blood from Arca granosa is dissolved in example 1;
fig. 2 is a standard curve of hemoglobin concentration-absorbance established in example 1.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples. The following described embodiments are illustrative only and are not to be construed as limiting the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1: establishing a standard curve
(1) Preparing diluent and detecting absorption peak of diluent dissolved with blood of blood clam
(1-1) taking 20mL of triton solution with the volume percentage concentration of 1% as a solution A, and adding the solution A into a beaker;
(1-2) taking 180mL of 1.5 mass percent sodium chloride solution as a solution B, adding the solution B into the beaker filled with the solution A in the step (1-1), and uniformly mixing to obtain a solution C, wherein the solution C is a diluent;
(1-3) taking part of diluent, mixing randomly selected fresh blood of scapharca granosa with the taken diluent in a volume ratio of 1:20 to obtain diluent dissolved with blood, carrying out full-wavelength scanning on the diluent dissolved with blood by using a spectrophotometer, wherein a full-wavelength scanning diagram is shown in figure 1, and measuring that the diluent dissolved with blood has highest absorption peaks at the wavelengths of 540nm and 580 nm;
(2) taking 7 fresh blood samples of the blood clams with known hemoglobin concentrations but different hemoglobin concentrations, respectively dissolving the blood samples by using diluent, detecting the absorbance of the solution by using a spectrophotometer for the solution obtained after each dissolution, and establishing a hemoglobin concentration-absorbance standard curve according to the hemoglobin concentrations and the corresponding absorbance of the 7 fresh blood samples of the blood clams; the specific operation is as follows:
(2-1) respectively adopting the existing SLS-Hb kit to measure the hemoglobin concentration n of 7 fresh blood samples of the scapharca subcrenata, wherein the unit is g/L;
(2-2) respectively adding 1mL of diluent into 7 clean 1.5mL centrifuge tubes, adding 50 mu L of fresh blood sample of scapharca granosa into each centrifuge tube, blowing and beating uniformly by using a liquid transfer gun after adding, standing for one minute, and obtaining 7 different solution samples after blood of scapharca granosa is completely dissolved;
(2-3) respectively measuring the absorbance Abs of 7 different solution samples at 540nm by using a microplate reader;
(2-4) according to 7 fresh blood clamsThe hemoglobin concentration n and the corresponding absorbance Abs of the sample establish a hemoglobin concentration-absorbance standard curve: abs 0.0039n +0.0439, R2The hemoglobin concentration n is measured in a range of 10-60 g/L (0.9941), and a specific standard curve is shown in FIG. 2. The hemoglobin concentration n and the corresponding absorbance Abs of 7 fresh blood samples of blood of scapharca subcrenata are shown in table 1.
TABLE 1
| Absorbance of the solution | 0.1162 | 0.1338 | 0.1459 | 0.1478 | 0.1162 | 0.2176 | 0.2361 |
| Concentration (g/L) | 20.1 | 22.8 | 25.9 | 26.2 | 31.5 | 43.9 | 50.6 |
Example 2: the accuracy of the standard curve established in example 1 was verified. Adding 1mL of the diluent prepared in the embodiment 1 into 15 clean 1.5mL centrifuge tubes respectively, then randomly taking 15 fresh blood samples of the blood of. For each detection liquid sample, 200 μ L of detection liquid is added into the elisa plate, the absorbance Abs at 540nm is measured, and the hemoglobin concentration of 15 fresh blood samples of blood of Arca subcrenata is calculated according to the standard curve established in example 1. And then, the hemoglobin concentration of the 15 fresh blood samples of the scapharca granosa is respectively determined by adopting the existing SLS-Hb kit. The statistical difference in hemoglobin concentration measurements for both methods was analyzed using SPSS software to give P ═ 0.913> >0.05, indicating that the differences were not very significant for the same samples tested by both methods. The results of the hemoglobin concentration measurements for both methods are shown in Table 2, and the results of the statistical difference analysis are shown in tables 3 and 4, wherein the results of the group statistics are shown in Table 3, and the results of the independent sample tests are shown in Table 4.
TABLE 2
TABLE 3
TABLE 4
Example 3: a repetitive experiment was performed on the standard curve established in example 1. Adding 1mL of the diluent prepared in the embodiment 1 into 20 clean 1.5mL centrifuge tubes respectively, adding 50 mu L of the same fresh blood sample of the scapharca granosa into the 20 centrifuge tubes respectively, adding blood of the scapharca granosa into each tube, blowing and beating the blood sample uniformly by using a liquid transfer gun, and standing for one minute to obtain 20 detection liquid samples. For each sample of the test solution, 200. mu.L of the test solution was added to the microplate, the absorbance Abs at 540nm was measured, and the hemoglobin concentration was calculated according to the standard curve established in example 1, and the average of 20 hemoglobin concentration measurements was 20.5g/L, the standard deviation was 0.9, and the Coefficient of Variation (CV) was 4.5%. The results of 20 hemoglobin concentration measurements are shown in Table 5.
TABLE 5
Example 4: and (3) performing a batch difference experiment on the standard curve established in the embodiment 1, and measuring the hemoglobin concentration of the same blood sample of the scapharca granosa by adopting three batches of continuously prepared diluents in the method in the embodiment 1. Adding 1mL of diluent in different batches into 3 clean 1.5mL centrifuge tubes respectively, adding 50 mu L of the same fresh blood sample of the scapharca granosa into the 3 centrifuge tubes respectively, adding blood of the scapharca granosa into each tube, blowing and beating the blood sample uniformly by using a liquid transfer gun, and standing for one minute to obtain 3 detection liquid samples. For each test solution sample, 200. mu.L of the test solution was added to the microplate, the absorbance Abs at 540nm was measured, and the hemoglobin concentration was calculated according to the standard curve established in example 1, and the average value, standard deviation, and coefficient of variation of the hemoglobin concentration measurement results were calculated 3 times. The above operations were repeated with 5 fresh blood samples of arca subcrenata, and the inter-batch difference variation coefficients and the related measurement results of the 5 fresh blood samples of arca subcrenata are shown in table 6.
TABLE 6
Example 5: in this embodiment, a specific experimental operation is performed, and a total of 14 blood samples of scapharca granosa are measured by the method of the present invention. The specific operation is as follows: randomly selecting 14 fresh and healthy blood clams to be detected for hemoglobin concentration, cleaning the fresh and healthy blood clams by using artificial seawater, and placing 200 mu L of blood taken by each blood clams by using a liquid transfer gun into a clean 1.5mL centrifuge tube. Then, taking 14 clean 1.5mL centrifuge tubes, adding 1mL of the diluent prepared in the embodiment 1, extracting 50 μ L of the diluent from each centrifuge tube filled with 200 μ L of blood, adding the diluent into one centrifuge tube filled with the diluent, adding blood into each tube, blowing and beating the blood uniformly by using a liquid transfer gun, and standing for one minute to obtain 14 detection liquid samples. For each test solution sample, 200. mu.L of the test solution was added to the microplate, and the absorbance Abs at 540nm was measured, and the hemoglobin concentration was calculated according to the standard curve established in example 1, and the measurement results are shown in Table 7.
TABLE 7
| Sample numbering | Absorbance of the solution | Concentration of hemoglobin, g/L |
| 1 | 0.0957 | 13.3 |
| 2 | 0.0885 | 11.4 |
| 3 | 0.1434 | 25.5 |
| 4 | 0.1766 | 34.0 |
| 5 | 0.1205 | 19.6 |
| 6 | 0.0938 | 12.8 |
| 7 | 0.1032 | 15.2 |
| 8 | 0.1162 | 18.5 |
| 9 | 0.1338 | 23.1 |
| 10 | 0.1407 | 24.8 |
| 11 | 0.2176 | 44.5 |
| 12 | 0.2361 | 49.3 |
| 13 | 0.1662 | 31.4 |
| 14 | 0.1459 | 26.2 |
In conclusion, the difference between the concentration of the bloody clams hemoglobin measured by the high-flux measuring method and the detection result of the existing SLS-Hb kit is small, and the repeated measurement result is stable, reliable and high in accuracy.
Claims (2)
1. A method for high-flux measurement of concentration of hemoglobin in blood of blood clam is characterized by comprising the following steps:
(1) preparing diluent and detecting absorption peak of diluent dissolved with blood of blood clam
(1-1) taking 20mL of triton solution with the volume percentage concentration of 1% as a solution A, and adding the solution A into a beaker;
(1-2) taking 180mL of 1.5 mass percent sodium chloride solution as a solution B, adding the solution B into the beaker filled with the solution A in the step (1-1), and uniformly mixing to obtain a solution C, wherein the solution C is a diluent;
(1-3) taking part of diluent, mixing randomly selected fresh blood of scapharca granosa with the taken diluent in a volume ratio of 1:20 to obtain diluent dissolved with blood, carrying out full-wavelength scanning on the diluent dissolved with blood by using a spectrophotometer, and measuring that the diluent dissolved with blood has highest absorption peaks at the wavelengths of 540nm and 580 nm;
(2) establishing a standard curve
Taking N fresh blood samples of Arca granosa with known hemoglobin concentration but different hemoglobin concentrations, wherein N is a positive integer not less than 5, dissolving the blood samples by using diluent respectively, detecting the absorbance of the solution by using a spectrophotometer for the solution obtained after each dissolution, and establishing a hemoglobin concentration-absorbance standard curve according to the hemoglobin concentrations and the corresponding absorbances of the N fresh blood samples of Arca granosa;
(3) measurement of blood hemoglobin concentration of blood clam
(3-1) adding 1mL of diluent into a clean 1.5mL centrifuge tube, adding 50 μ L of blood of the clean 1.blood of the blood of;
and (3-2) adding 200 mu L of detection liquid into an enzyme label plate, measuring the absorbance Abs of the detection liquid at 540nm, and calculating the hemoglobin concentration of blood of the tegillarca granosa to be measured according to the standard curve established in the step (2).
2. The method for high-flux measurement of concentration of hemoglobin in blood of Anadara granosa according to claim 1, wherein the specific operation of the step (2) is as follows:
(2-1) adopting the existing SLS-Hb kit to measure the hemoglobin concentration N of the N fresh blood samples of the scapharca granosa, wherein the unit is g/L;
(2-2) respectively adding 1mL of diluent into N clean 1.5mL centrifuge tubes, adding 50 mu L of fresh blood sample of scapharca granosa into each centrifuge tube, blowing and beating uniformly by using a liquid transfer gun after adding, standing for one minute, and obtaining N different solution samples after blood of scapharca granosa is completely dissolved;
(2-3) respectively measuring the absorbance Abs of the N different solution samples obtained in the step (2-2) at 540nm by using a microplate reader;
(2-4) establishing a hemoglobin concentration-absorbance standard curve according to the hemoglobin concentration N and corresponding absorbance Abs of the N fresh blood samples of the blood of the: abs 0.0039n +0.0439, R2The hemoglobin concentration n is 0.9941, and the measurement range is 10-60 g/L.
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| WO2023240854A1 (en) * | 2022-06-17 | 2023-12-21 | 深圳安侣医学科技有限公司 | Hemoglobin analysis method and system based on microscopically-magnified digital image |
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