CN113092781B - Preparation method of albumin determination reagent ball, reagent ball and microfluidic chip - Google Patents
Preparation method of albumin determination reagent ball, reagent ball and microfluidic chip Download PDFInfo
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- CN113092781B CN113092781B CN202110347879.7A CN202110347879A CN113092781B CN 113092781 B CN113092781 B CN 113092781B CN 202110347879 A CN202110347879 A CN 202110347879A CN 113092781 B CN113092781 B CN 113092781B
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Abstract
The embodiment of the invention relates to the technical field of medical detection, in particular to a preparation method of a reagent ball for measuring albumin, the reagent ball and a microfluidic chip, wherein the method comprises the steps of mixing a certain dose of buffer solution, a non-ionic detergent, an anionic dye, an excipient and water to form a mixed solution, and adjusting the pH value of the mixed solution to a preset value so as to provide an acidic environment required by detection; and then dropping the mixed solution into liquid nitrogen in a liquid drop form to form an ice ball, and freeze-drying the ice ball to prepare the spherical albumin determination reagent ball. Wherein the mixed solution comprises the following components: 40-200mmol/L of buffer solution, 10-50g/L of nonionic detergent, 1-10g/L of anionic dye and 10-100g/L of excipient. The excipient in the dosage range can ensure the shape and the re-melting solubility of the reagent ball and is beneficial to the complete freeze-drying of the reagent ball. Therefore, the reagent ball has better shape and meltdown solubility, can be completely freeze-dried, and has higher stability and precision.
Description
Technical Field
The embodiment of the invention relates to the technical field of medical detection, in particular to a preparation method of a reagent ball for determining albumin, the reagent ball and a microfluidic chip.
Background
In the human body, albumin is synthesized by hepatic parenchymal cells and plays an important role in maintaining blood colloid osmotic pressure, transport of in vivo metabolic substances, nutrition and the like. Generally, albumin increase is mainly caused by blood concentration, such as dehydration, extensive burns, high fever, acute hemorrhage, chronic adrenal cortex dysfunction, and the like. The albumin reduction is commonly seen in cirrhosis combined with ascites and other serious liver function damages, such as malnutrition, digestive and absorptive dysfunction, liver dysfunction, pernicious anemia, diabetes, hyperthyroidism, severe tuberculosis, nephrotic syndrome, chronic enteritis, congestive heart failure and the like. From this, it is known that albumin measurement is a main test item for visceral diseases.
At present, methods for measuring albumin mainly comprise an ammonium sulfate salting-out method, a dye binding method and an immunoassay method, wherein the ammonium sulfate salting-out method is complicated in operation, poor in specificity and repeatability, high in cost of the immunoassay method, and the dye binding method is difficult to realize instant diagnosis due to the fact that a large-scale biochemical analyzer is required. Although the liquid albumin reagent is prepared into albumin freeze-dried reagent balls to realize instant diagnosis, the conventional albumin freeze-dried reagent balls have poor shapes and are difficult to completely freeze-dry, so that the stability and the accuracy of the conventional albumin freeze-dried reagent balls are low.
Disclosure of Invention
The technical problem mainly solved by the embodiment of the invention is to provide a preparation method of an albumin determination reagent ball, the reagent ball and a microfluidic chip.
In order to solve the above technical problems, in a first aspect, an embodiment of the present invention provides a method for preparing a reagent ball for measuring albumin, including:
mixing a buffer solution, a nonionic detergent, an anionic dye, an excipient and water to form a mixed solution, and adjusting the pH value of the mixed solution to a preset value;
dropping the liquid drops of the mixed liquid into liquid nitrogen to enable the liquid drops to form ice balls;
freeze-drying the ice ball to prepare the albumin determination reagent ball;
wherein the mixed solution comprises the following components: 40-200mmol/L of buffer solution, 10-50g/L of nonionic detergent, 1-10g/L of anionic dye and 10-100g/L of excipient.
In some embodiments, the mixing the buffer, the non-ionic detergent, the anionic dye, the excipient, and the water to form a mixed solution, and adjusting the PH of the mixed solution to a preset value includes:
adding the buffer to a first predetermined amount of water;
after the buffer solution is completely dissolved in the water, sequentially adding the nonionic detergent and the anionic dye to obtain a first solution, and adjusting the pH value of the first solution to the preset value, wherein the preset value is 4.0-4.5;
adding the excipient into the first solution, and adding a second preset amount of water into the first solution to obtain a third preset amount of the mixed solution.
In some embodiments, the excipient comprises at least one of mannitol, inositol, sucrose, trehalose, PEG3350, PEG8000, dextran 1 ten thousand, dextran 4 ten thousand.
In some embodiments, the buffer comprises at least one of a citric acid-trisodium citrate buffer, a succinic acid-sodium succinate buffer, an acetic acid-sodium acetate buffer, a lactic acid-sodium lactate buffer.
In some embodiments, the nonionic detergent comprises at least one of polyoxyethylene lauryl ether, polyoxyethylene octyl phenyl ether, tween 20, trideceth.
In some embodiments, the anionic dye comprises bromocresol green or bromocresol purple.
In some embodiments, the ice ball has a volume of 2.5ul to 3.5ul.
In order to solve the above technical problems, in a second aspect, the present invention provides a reagent ball prepared by the method for preparing a reagent ball for measuring albumin according to the first aspect.
In order to solve the above technical problem, in a third aspect, the present invention provides a microfluidic chip, including a chip body and the reagent ball as described in the second aspect, wherein the reagent ball is disposed inside the chip body.
In order to solve the above technical problem, in a fourth aspect, an embodiment of the present invention provides a biochemical analyzer, including an analyzer body, a reaction tank, and the microfluidic chip according to the third aspect, wherein the reaction tank is disposed in the analyzer body, and the microfluidic chip is mounted in the reaction tank.
The embodiment of the invention has the following beneficial effects: different from the situation of the prior art, in the preparation method of the albumin determination reagent ball provided by the embodiment of the invention, a certain amount of buffer solution, a nonionic detergent, an anionic dye, an excipient and water are mixed to form a mixed solution, and the pH value of the mixed solution is adjusted to a preset value so as to provide an acidic environment required by detection; and then dropping the mixed solution in liquid nitrogen in the form of liquid drops to form ice balls, and freeze-drying the ice balls to prepare the spherical albumin determination reagent balls. Wherein the mixed solution comprises the following components: 40-200mmol/L of buffer solution, 10-50g/L of nonionic detergent, 1-10g/L of anionic dye and 10-100g/L of excipient. The anionic dye can be quantitatively and specifically combined with albumin under certain acidic environment and the environment with the nonionic detergent, so that the content of the albumin can be evaluated, the structure of the albumin can not be damaged by the nonionic detergent, the shape and the re-melting solubility of the reagent spheres can be ensured by the excipient in the dosage range, and the complete freeze-drying of the reagent spheres is facilitated. Therefore, the albumin determination reagent ball prepared by the method has better shape and meltdown solubility, and can be completely lyophilized, thereby having higher stability and precision.
Drawings
One or more embodiments are illustrated by way of example in the accompanying drawings, which correspond to the figures in which like reference numerals refer to similar elements and which are not to scale unless otherwise specified.
FIG. 1 is a schematic flow chart of a method for preparing reagent balls for measuring albumin according to an embodiment of the present invention;
FIG. 2 is a schematic view of a sub-flowchart of step S10 shown in FIG. 1;
FIG. 3 is a schematic line of fit of correlation equations in clinical correlation analysis;
FIG. 4 is a schematic diagram of a fit line of correlation equations in a linear range test.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.
It should be noted that, if not conflicted, the various features of the embodiments of the invention may be combined with each other within the scope of protection of the present application. Additionally, while functional block divisions are performed in apparatus schematics, with logical sequences shown in flowcharts, in some cases, steps shown or described may be performed in sequences other than block divisions in apparatus or flowcharts. Further, the terms "first," "second," "third," and the like, as used herein, do not limit the data and the execution order, but merely distinguish the same items or similar items having substantially the same functions and actions.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The embodiment of the present invention provides a method for preparing a reagent ball for measuring albumin, which can prepare a spherical reagent for measuring albumin, and with reference to fig. 1, the method includes the following steps:
s10: mixing a buffer solution, a nonionic detergent, an anionic dye, an excipient and water to form a mixed solution, and adjusting the pH value of the mixed solution to a preset value.
In this embodiment, a buffer solution, a nonionic detergent, an anionic dye, an excipient, and water (each component) are measured, and then the components are sufficiently mixed to obtain a mixed solution. The pH value of the mixed solution can be tested by methods such as a pH test paper, and the pH value of the mixed solution is adjusted to a preset value by adding citric acid. Wherein the preset value is within the pH value range required by albumin determination, so that the mixed solution meets the acidic environment required by albumin determination.
In order to fully mix the mixed solution and accurately contain the components, in some embodiments, the step S10 specifically includes the following steps:
s11: adding the buffer to a first predetermined amount of water;
s12: after the buffer solution is completely dissolved in the water, sequentially adding the nonionic detergent and the anionic dye to obtain a first solution, and adjusting the pH value of the first solution to the preset value, wherein the preset value is 4.0-4.5;
s13: adding the excipient into the first solution, and adding a second preset amount of water into the first solution to obtain a third preset amount of the mixed solution.
Specifically, 800ml of water is added into a 1L beaker, namely the first preset amount is 800ml, buffer solution components are weighed and added into the beaker containing 800ml of water, and after the buffer solution is completely dissolved in the water, a nonionic detergent and an anionic dye are sequentially added to obtain a first solution. Thereby, the water, the buffer, the nonionic detergent, and the anionic dye can be completely mixed.
Then, adjusting the pH value of the first solution to 4.0-4.5 by adding citric acid, adding an excipient to fully mix the excipient with the first solution, and finally adding a second preset amount of water to fix the volume of the first solution in the beaker to 1L to obtain a 1L (third preset amount) mixed solution.
The amount of water is determined once based on the uncertain amount of citric acid added when the pH value is adjusted, which easily causes the subsequent total amount (namely, the third preset amount) to exceed the standard, so that the content of each component is inaccurate. In the embodiment, the water with the preset amount is added twice, so that the accurate mixed liquid of all the components is favorably prepared, after the buffer solution is completely dissolved, the nonionic detergent and the anionic dye are sequentially added, the pH value is adjusted, and the excipient is added after the pH value is adjusted, so that all the components can be uniformly mixed, and the detection precision is favorably improved.
In addition, the mixed solution comprises the following components: 40-200mmol/L of buffer solution, 10-50g/L of nonionic detergent, 1-10g/L of anionic dye and 10-100g/L of excipient.
The anionic dye can be quantitatively and specifically combined with albumin under certain acidic environment and in the presence of a nonionic detergent to form a complex, the color of the complex is in direct proportion to the content of the albumin, so that the content of the albumin can be reflected, and the structure of the albumin cannot be damaged by the nonionic detergent.
Wherein the excipient does not react with other components, has stable properties, does not produce side effects, and does not influence the precision of the reagent. In addition, the excipient in the dosage range can endow the reagent ball with good appearance, so that the reagent ball is loose and easy to redissolve, the shape and the redissolution solubility of the reagent ball can be ensured, and the good shape is beneficial to completely freeze-drying the reagent ball.
In some embodiments, the excipient comprises at least one of mannitol, inositol, sucrose, trehalose, PEG3350, PEG8000, dextran 1 ten thousand, dextran 4 ten thousand. It is understood that the excipient can give the reagent ball a good appearance, and the reagent ball is loosened, so that the reagent ball has good solubility and sufficiently reacts with the sample after contacting the test sample. For example, excipients include inositol and mannitol, or trehalose and PEG3350, or sucrose and PEG8000.
In some implementations, the buffer includes at least one of a citric acid-trisodium citrate buffer, a succinic acid-sodium succinate buffer, an acetic acid-sodium acetate buffer, a lactic acid-sodium lactate buffer. It will be appreciated that the buffer solution works well to buffer the acid.
In some embodiments, the nonionic detergent comprises at least one of polyoxyethylene lauryl ether, polyoxyethylene octyl phenyl ether, tween 20, trideceth. It is understood that the nonionic detergent provides an environment for the anionic dye to bind to albumin.
In some embodiments, the anionic dye comprises bromocresol green or bromocresol purple. The anionic dye is negatively charged and can be quantitatively and specifically combined with albumin with positive charge in serum. For example, bromocresol green binds quantitatively and specifically to albumin at pH4.2 without interference from other globulins in plasma. The combined compound is in grass green, has a special absorption peak at 630nm, can be distinguished from free dye, and the absorption peak is generally not influenced by other compounds (such as bilirubin, heme and the like) possibly existing in blood plasma.
S20: and dropping the liquid drops of the mixed liquid into liquid nitrogen to enable the liquid drops to form ice balls.
After the mixed liquid is prepared, the liquid drops of the mixed liquid can be dropped in liquid nitrogen through a dispenser, so that the liquid drops are condensed into ice balls in the liquid nitrogen. The size of the liquid drop of the mixed liquid dropped into the liquid nitrogen can be adjusted by those skilled in the art according to actual needs, and the volume of the ice ball can be adjusted by controlling the size of the liquid drop. Optionally, in some embodiments, the ice ball has a volume of 2.5ul to 3.5ul.
S30: and freeze-drying the ice ball to obtain the albumin determination reagent ball.
After obtaining the ice balls, placing the ice balls in a vacuum freeze dryer for freeze drying to obtain albumin determination reagent balls, and collecting and storing the albumin determination reagent balls in a dry aluminum bottle after nitrogen repression.
Wherein, freeze drying means that the ice ball is cooled and frozen into solid in advance, and the sublimation performance of water is utilized under the condition of low temperature and reduced pressure to dehydrate the ice ball at low temperature so as to achieve the drying purpose. After freeze drying, all components (buffer solution, nonionic detergent, anionic dye and excipient) except water in the ice ball are left in an ice rack during freezing, so that the ice ball after freeze drying is loose and porous and has unchanged volume, and the ice ball is always in a frozen state before drying, and meanwhile, ice crystals are uniformly distributed in a substance, so that the concentration phenomenon caused by dehydration in the sublimation process can be avoided. The reagent ball obtained after freeze drying is spongy, loose and porous, the volume of the reagent ball is basically unchanged with that of the ice ball before drying, and the reagent ball is easy to dissolve in water and restore to the original shape.
In the embodiment of the invention, by arranging the excipient with higher content, the shape and the re-melting solubility of the reagent ball can be ensured, and the complete freeze-drying of the reagent ball is facilitated. Therefore, the albumin determination reagent ball prepared by the method has better shape and meltdown solubility, and can be completely lyophilized, thereby having higher stability and precision.
The detection principle of the reagent ball prepared by the preparation method of the albumin determination reagent ball provided by the embodiment of the invention is as follows: the detection sample is contacted with the albumin determination reagent ball, so that the albumin determination reagent ball is dissolved and fully reacts with the detection sample, the pH range of the reagent ball is 4.0-4.5, and a nonionic detergent exists, so that albumin in the detection sample is positively charged and can be quantitatively and specifically combined with negatively charged anionic dye to form a turquoise compound, and the combined compound has a special absorption peak at a wavelength of 630 nm. Within a certain content range, the color of the complex is in direct proportion to the content of albumin, so that the concentration of albumin in the sample can be calculated.
The embodiment of the invention also provides a reagent ball, which is prepared by the preparation method of the albumin determination reagent ball in the above embodiment, and has the same structure and function as the reagent ball prepared by the preparation method of the albumin determination reagent ball in the above embodiment, and the details are not repeated here.
The embodiment of the invention also provides a microfluidic chip which comprises a chip body and the reagent ball provided by any one of the embodiments, wherein the reagent ball is arranged in the chip body. In some embodiments, the chip body is formed by adhering an injection-molded plastic substrate and an optical film through an adhesive layer, and the chip body comprises a sample tank, a dilution liquid tank, a quantification tank, a mixing tank, a waste liquid tank, a liquid flow channel, a colorimetric hole and the like. The chip body also comprises a plurality of colorimetric holes for storing the reagent balls, and the detection sample enters the colorimetric holes and then undergoes chemical reaction with the reagent balls.
In this embodiment, the reagent ball has the same structure and function as the reagent ball in the above-mentioned embodiment, so that, after the test sample enters the colorimetric hole, the reagent ball can be completely dissolved, and the test result is more accurate.
The embodiment of the invention also provides a biochemical analyzer, which comprises an analyzer body, a reaction tank and the microfluidic chip, wherein the reaction tank is arranged in the analyzer body, and the microfluidic chip can be arranged in the reaction tank, so that a detection sample in the microfluidic chip can be analyzed and detected. The structure and the function of the microfluidic chip are completely the same as those of the microfluidic chips in the embodiments, and are not described in detail herein.
To further illustrate the technical solution of the present invention, several examples of the preparation method of the albumin assay reagent ball of the present invention are provided below.
Example 1：
In this example, the buffer comprises trisodium citrate buffer, the nonionic detergent comprises polyoxyethylene lauryl ether, the anionic dye comprises bromocresol green, and the excipient comprises inositol and mannitol. The preparation method of the albumin determination reagent ball comprises the following steps: mixing a certain amount of trisodium citrate buffer solution, polyoxyethylene lauryl ether, bromocresol chloroinositol, mannitol and water to form a mixed solution, adjusting the pH value of the mixed solution to 4.2, dripping liquid drops of the mixed solution into liquid nitrogen to form an ice ball with the volume of about 3.0ul, and freezing and drying the ice ball to obtain the albumin determination reagent ball. The albumin determination reagent ball is worthy of a microfluidic chip.
Specifically, in this embodiment, the mixed solution includes the following components: 40-200mmol/L trisodium citrate buffer solution, 10-50g/L polyoxyethylene lauryl ether, 1-10g/L bromocresol green, 0.1-10g/L inositol and 10-100g/L mannitol.
Example 2：
In this example, the buffer comprises sodium lactate buffer, the non-ionic detergent comprises tween 20, the anionic dye comprises bromocresol green, and the excipient comprises trehalose and PEG3350. The preparation method of the albumin determination reagent ball comprises the following steps: mixing a certain amount of sodium lactate buffer solution, tween 20, bromocresol green, trehalose, PEG3350 and water to form a mixed solution, adjusting the pH value of the mixed solution to 4.0, dripping liquid drops of the mixed solution into liquid nitrogen to form an ice ball with the volume of about 2.8ul, and freeze-drying the ice ball to obtain the albumin determination reagent ball. The albumin determination reagent ball is worthy of a microfluidic chip.
Specifically, in this embodiment, the mixed solution includes the following components: sodium lactate buffer 40-200mmol/L, tween 20:10-50g/L, bromocresol green 1-10g/L, trehalose 1-10g/L, PEG3350:10-100g/L.
Example 3：
In this example, the buffer comprises sodium succinate buffer, the non-ionic detergent comprises trideceth, the anionic dye comprises bromocresol green, and the excipient comprises sucrose and PEG8000. The preparation method of the albumin determination reagent ball comprises the following steps: mixing a certain amount of sodium succinate buffer solution, tridecyl polyoxyethylene ether, bromocresol green, sucrose and PEG8000 with water to form a mixed solution, adjusting the pH value of the mixed solution to 4.0, dripping the mixed solution into liquid nitrogen to form an ice ball with the volume of about 2.8ul, and freeze-drying the ice ball to obtain the albumin determination reagent ball. And the albumin determination reagent ball is adopted to prepare the microfluidic chip.
Specifically, in this embodiment, the mixed solution includes the following components: 40-200mmol/L sodium succinate buffer solution, 10-50g/L trideceth-yl ether, 1-10g/L bromocresol green, 1-10g/L sucrose, PEG800:10-100g/L.
The performance of the reagent ball prepared in example 1 of the present invention will be described below with reference to specific test and comparative experiments.
In an environment with air humidity of 8%, the reagent ball in example 1 is loaded into a chip body of a microfluidic chip, a detection sample is injected into the microfluidic chip, and then, a portable automatic biochemical analyzer vp10 of shenzhen jinrui bio ltd is used for detection, so as to detect a change value of absorbance at a wavelength of 600nm at 37 ℃. And the concentration of the albumin in the detected sample can be calculated by using a calibrator provided by British Landau company for calibration.
1) And (3) testing precision: the microfluidic chip provided by the embodiment 1 of the invention is adopted to detect the detection sample 1# with the known albumin concentration, the albumin concentration of the detection sample 1# is 32g/L, and 20 detected concentration values are obtained after 20 times of detection.
The mean, standard deviation and coefficient of variation of these 20 concentration values were calculated to give a mean of 31.32g/L, standard deviation SD =0.46 and coefficient of variation CV =1.48%.
It can be known that the measured concentration (31.32 g/L) is very close to the actual concentration (32 g/L), the accuracy is high, and the standard deviation and the coefficient of variation are small, which indicates that the stability of the protein determination reagent ball is good.
2) And (3) testing accuracy: by adopting the microfluidic chip provided by the embodiment 1 of the invention, a detection sample 2# with the known albumin concentration of 41.5g/L is tested, the detection is repeated three times to obtain a concentration value, and the average value of the concentration values measured by 3 is calculated to be 41.82g/L, and the relative deviation is 0.77%.
It can be seen that the measured concentration of 41.82g/L is close to the actual concentration of 41.5g/L, with high accuracy and small relative deviation.
3) Clinical relevance analysis
Serum sample sets A1 and A2 with different albumin concentrations are prepared, i.e. A1 includes several serum samples with albumin concentrations, A2 includes several serum samples with albumin concentrations, like A1, and a microfluidic chip in example 1 is used to detect the albumin concentration of each serum sample in sample set A1, and a fully automatic biochemical analyzer hitachi 7180 is used to detect the albumin concentration of each serum sample in sample set A2. As shown in Table 1, for the sample 1 with the same concentration, the concentration of albumin measured by Hitachi 7180 was 24.75g/L, and the concentration of albumin measured by the microfluidic chip provided in example 1 of the present invention was 25.74g/L.
TABLE 1 test results of clinical relevance analysis
Taking the detection concentration value corresponding to Hitachi 7180 as the value of the X axis and the detection concentration value corresponding to the microfluidic chip as the value of the Y axis in Table 1, the correlation equation between the two groups of detection results is obtained as follows:
Y＝0.9702x+1.3617；
the fit line of the correlation equation is shown in fig. 3, where the correlation coefficient R =0.9954, the closer the correlation coefficient is to 1, the stronger the correlation between the two sets of data. Therefore, the micro-fluidic chip provided by the embodiment of the invention has strong correlation with the test result of Hitachi 7180.
4) Linear range test
The test method is as follows: serum samples at 6 dilution concentrations were mixed as shown in table 2 using high concentration (active) samples near the upper end of the linear range and low concentration (active) samples near the lower end of the linear range.
TABLE 2
| Sample numbering | 1 | 2 | 3 | 4 | 5 | 6 |
| High concentration (active) samples | 0 portion of | 1 part of | 2 portions of | 3 portions of | 4 portions of | 5 portions of |
| Low concentration (active) samples | 5 portions of | 4 portions of | 3 portions of | 2 portions of | 1 part of | 0 portion of |
The microfluidic chip provided by the embodiment 1 of the invention is adopted to respectively test the albumin concentration of 6 serum samples, each serum sample is tested for 3 times, and the average value (y) of the concentration values of albumin measured in the 6 serum samples is respectively calculated i ). Concentration (x) after dilution with each sample i ) As independent variable, the mean value of the measured concentration values (y) of each sample is taken i ) Solving a linear regression equation for the dependent variable as follows:
Y＝0.9491x+1.565；
the fit line of the correlation equation is shown in fig. 4, wherein the closer the correlation coefficient R =0.9491 is to 1, the closer the result measured by using the microfluidic chip in the embodiment of the present invention is to the actual result after dilution, that is, the closer the measured value is to the actual value, the higher the accuracy of the microfluidic chip provided in the embodiment of the present invention is, and on the other hand, the accurate measurement can be performed on samples with different concentrations, and the better the linear range is.
5) Thermal stability test
The microfluidic chip of example 1 of the present invention was packaged in an environment with an air humidity of 8%, and stored in a dark environment at 37 ℃ for 0, 2, 3, 4, 6, and 8 days. The accuracy of the microfluidic chip in the embodiment of the invention is tested by taking Landau calibration products and quality control products as detection samples, and the relative deviation is within +/-10.0%. Specifically, two groups of quality control products (sample 1# and sample 2 #) are provided by Landau corporation, the sample 1# and the sample 2# are respectively detected by adopting the microfluidic chip which meets the storage requirement, and the detection is carried out three times for the same type of microfluidic chip.
Table 3 shows the detection results of sample 1# detected by the microfluidic chip after storing each day, and table 4 shows the detection results of sample 2# detected by the microfluidic chip after storing each day. Wherein the average value is the average value of the concentrations detected in three times, and the target value is the actual concentration of albumin in the sample.
As can be seen from tables 3 and 4, the microfluidic chip provided in the embodiment of the present invention has an absolute value of a relative deviation within 10.0% after being stored in an environment for 2, 3, 4, 6, and 8 days, and thus has good thermal stability, and can ensure accuracy of a detection result after being stored in an environment for a plurality of days.
TABLE 3 test results of sample No. 1
| Sample No. 1# | 1 | 2 | 3 | Mean value of | Target value (g/L) | Relative deviation of |
| Day 0 | 45.45 | 44.71 | 45.87 | 45.34 | 45.3 | 0.10％ |
| 2 days | 45.70 | 45.85 | 45.85 | 45.80 | 45.3 | 1.10％ |
| 3 days | 45.09 | 45.60 | 45.68 | 45.46 | 45.3 | 0.35％ |
| 4 days | 45.69 | 44.86 | 44.53 | 45.03 | 45.3 | -0.60％ |
| 6 days | 44.17 | 44.54 | 44.05 | 44.25 | 45.3 | -2.31％ |
| 8 days | 45.40 | 44.21 | 45.50 | 45.04 | 45.3 | -0.58％ |
TABLE 4 test results of sample No. 2
| Sample No. 2# | 1 | 2 | 3 | Mean value of | Target value (g/L) | Relative deviation of |
| Day 0 | 30.86 | 29.27 | 29.37 | 29.83 | 30.5 | -2.19％ |
| 2 days | 30.14 | 29.94 | 30.10 | 30.06 | 30.5 | -1.44％ |
| 3 days | 30.86 | 29.31 | 30.38 | 30.18 | 30.5 | -1.04％ |
| 4 days | 29.80 | 29.80 | 30.77 | 30.12 | 30.5 | -1.23％ |
| 6 days | 29.28 | 29.61 | 30.66 | 29.85 | 30.5 | -2.13％ |
| 8 days | 30.41 | 30.61 | 29.84 | 30.29 | 30.5 | -0.70％ |
6) Long term stability test
The microfluidic chip in example 1 of the present invention was packaged in an environment with an air humidity of 8%, and stored in a dark environment at 2-8 ℃ for 0, 3, 6, 9, 12, and 15 months. When the Landau calibrator and the quality control product are used as detection samples, the accuracy of the microfluidic chip is tested, and the relative deviation is within +/-10.0%. Specifically, two groups of quality control products (sample 3# and sample 4 #) are provided by Landau corporation, the sample 3# and the sample 4# are detected by the microfluidic chip after being stored for 0, 3, 6, 9, 12 and 15 months in a dark environment at the temperature of 2-8 ℃, and the detection is carried out for three times on the same type of microfluidic chip.
Table 5 shows the results of the microfluidic assay on sample # 3 after 0, 3, 6, 9, 12, and 15 months of storage in a light-shielding environment at 2-8 ℃, and table 6 shows the results of the microfluidic assay on sample # 4 after 0, 3, 6, 9, 12, and 15 months of storage in a light-shielding environment at 2-8 ℃. Wherein the average is the average of the three detected concentrations and the target value is the actual concentration of albumin in the sample.
As can be seen from tables 5 and 6, the microfluidic chip provided in the embodiments of the present invention has an absolute value of the relative deviation within 10.0% after being stored in the environment for 0, 3, 6, 9, 12, and 15 months, so that the microfluidic chip has good long-term stability, and can ensure the accuracy of the detection result after being stored in the environment for a long time.
TABLE 5 examination result of sample No. 3
| Sample No. 3# | 1 | 2 | 3 | Mean value of | Target value (g/L) | Relative deviation of |
| 0 month | 39.20 | 43.00 | 42.30 | 41.50 | 41.5 | 0.00％ |
| Month 3 | 41.90 | 41.30 | 41.40 | 41.53 | 41.5 | 0.08％ |
| 6 month | 38.60 | 41.70 | 41.30 | 40.53 | 41.5 | -2.33％ |
| 9 month | 41.30 | 39.80 | 40.00 | 40.37 | 41.5 | -2.73％ |
| 12 month | 39.70 | 40.90 | 39.50 | 40.03 | 41.5 | -3.53％ |
| 15 month | 43.70 | 42.50 | 42.80 | 43.00 | 41.5 | 3.61％ |
TABLE 6 test results of sample No. 4
In summary, in the preparation method of the reagent ball for measuring albumin provided by the embodiment of the invention, a certain amount of buffer solution, a nonionic detergent, an anionic dye, an excipient and water are mixed to form a mixed solution, and the PH value of the mixed solution is adjusted to a preset value to provide an acidic environment required by detection; and then dropping the mixed solution into liquid nitrogen in a liquid drop form to form an ice ball, and freeze-drying the ice ball to prepare the spherical albumin determination reagent ball. Wherein the mixed solution comprises the following components: 40-200mmol/L of buffer solution, 10-50g/L of nonionic detergent, 1-10g/L of anionic dye and 10-100g/L of excipient. The anionic dye can be quantitatively and specifically combined with albumin under certain acidic environment and the environment with the nonionic detergent, so that the content of the albumin can be evaluated, the structure of the albumin can not be damaged by the nonionic detergent, the shape and the re-melting solubility of the reagent spheres can be ensured by the excipient in the dosage range, and the complete freeze-drying of the reagent spheres is facilitated. Therefore, the albumin determination reagent ball prepared by the method has better shape and meltdown solubility, and can be completely lyophilized, thereby having higher stability and precision.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; within the idea of the invention, also technical features in the above embodiments or in different embodiments may be combined, steps may be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.
Claims (6)
1. A method for preparing a reagent ball for measuring albumin is characterized by comprising the following steps:
mixing trisodium citrate buffer solution, polyoxyethylene lauryl ether, bromocresol green, inositol, mannitol and water to form mixed solution, and adjusting the pH value of the mixed solution to a preset value, wherein the preset value is in the range of 4.0-4.5;
dropping the liquid drops of the mixed liquid into liquid nitrogen to enable the liquid drops to form ice balls;
freeze-drying the ice ball to prepare the albumin determination reagent ball;
wherein the mixed solution comprises the following components: 40-200mmol/L of trisodium citrate buffer solution, 10-50g/L of polyoxyethylene lauryl ether, 1-10g/L of bromocresol green, 0.1-10g/L of inositol and 10-100g/L of mannitol.
2. The method for preparing reagent balls for albumin assay according to claim 1, wherein the mixing trisodium citrate buffer, polyoxyethylene lauryl ether, bromocresol green, inositol, mannitol, and water to form a mixed solution, and adjusting the PH of the mixed solution to a predetermined value comprises:
adding the trisodium citrate buffer to a first predetermined amount of water;
after the trisodium citrate buffer solution is completely dissolved in the water, sequentially adding the polyoxyethylene lauryl ether and the bromocresol green to obtain a first solution, and adjusting the pH value of the first solution to the preset value;
and adding the inositol and the mannitol into the first solution, and adding a second preset amount of water into the first solution to obtain a third preset amount of the mixed solution.
3. The method for preparing reagent beads for measuring albumin as set forth in claim 1 or 2, wherein the iceball has a volume of 2.5ul to 3.5ul.
4. A reagent ball prepared by the method for preparing a reagent ball for measuring albumin according to any one of claims 1 to 3.
5. A microfluidic chip comprising a chip body and the reagent ball of claim 4, wherein the reagent ball is disposed inside the chip body.
6. A biochemical analyzer comprising an analyzer body, a reaction chamber and the microfluidic chip of claim 5, wherein the reaction chamber is opened in the analyzer body, and the microfluidic chip is mounted in the reaction chamber.
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