Inorganic phosphorus detection reagent and detection chip
阅读说明：本技术 一种无机磷检测试剂及检测芯片 (Inorganic phosphorus detection reagent and detection chip ) 是由 周慧欣 汪晨宇 牟健 陈明 于 2021-04-19 设计创作，主要内容包括：本发明涉及无机磷检测技术领域,特别是涉及一种无机磷检测试剂及检测芯片。本发明实施例提供的无机磷检测试剂和检测芯片包括麦芽糖、氧化型辅酶、β-磷酸葡萄糖变位酶、麦芽糖磷酸化酶和葡萄糖-6-磷酸脱氢酶；检测样本中的无机磷与麦芽糖反应生成1-磷酸葡萄糖。1-磷酸葡萄糖在磷酸葡萄糖变位酶和/或其辅酶的作用下形成6-磷酸葡萄糖。6-磷酸葡萄糖又在葡萄糖-6-磷酸脱氢酶的作用下被氧化为6-磷酸葡萄糖酸,同时氧化型辅酶被还原为还原型辅酶,利用反应体系在340nm处吸光度的变化可计算出检测样本中无机磷的含量。测试结果表明,本发明实施例提供的无机磷检测试剂和检测芯片对抗坏血酸、胆红素、血红蛋白、甘油三酯、尿酸和肌酐均具有较强的抗干扰能力。(The invention relates to the technical field of inorganic phosphorus detection, in particular to an inorganic phosphorus detection reagent and a detection chip. The inorganic phosphorus detection reagent and the detection chip provided by the embodiment of the invention comprise maltose, oxidized coenzyme, beta-phosphoglucomutase, maltose phosphorylase and glucose-6-phosphate dehydrogenase; and (3) reacting inorganic phosphorus in the detection sample with maltose to generate the glucose-1-phosphate. Glucose-1-phosphate is converted to glucose-6-phosphate by phosphoglucomutase and/or its coenzyme. The 6-phosphoglucose is oxidized into 6-phosphogluconate under the action of glucose-6-phosphate dehydrogenase, meanwhile, the oxidized coenzyme is reduced into reduced coenzyme, and the content of inorganic phosphorus in the detection sample can be calculated by utilizing the change of the absorbance of the reaction system at 340 nm. Test results show that the inorganic phosphorus detection reagent and the detection chip provided by the embodiment of the invention have strong anti-interference capability on ascorbic acid, bilirubin, hemoglobin, triglyceride, uric acid and creatinine.)
1. An inorganic phosphorus detection reagent, characterized in that, the inorganic phosphorus detection reagent comprises a first freeze-dried reagent ball prepared by a first reagent and a second freeze-dried reagent ball prepared by a second reagent;
the first reagent comprises the following components:
the second reagent comprises the following components:
2. the inorganic phosphorus detection reagent of claim 1, wherein the first reagent further comprises a first stabilizer, and/or the second reagent further comprises a second stabilizer.
3. The inorganic phosphorus detection reagent of claim 2, wherein when the first reagent comprises the first stabilizer, the first stabilizer is present in an amount of 1 to 10 g/L;
when the second reagent comprises the second stabilizer, the content of the second stabilizer is 1-10 g/L.
4. The inorganic phosphorus detection reagent of claim 2, wherein the first stabilizer or the second stabilizer comprises at least one of glycerol, bovine serum albumin, propylene glycol-monomethyl ether, 4-methylphenylboronic acid, magnesium chloride, lithium chloride, ammonium sulfate, ethylenediaminetetraacetic acid, glutamate, or reduced glutathione.
5. The inorganic phosphorus detection reagent of any one of claims 1 to 4, wherein the pH of the first reagent and/or the second reagent is 6.5 to 7.5.
6. The inorganic phosphorus detection reagent of any one of claims 1 to 4, wherein the oxidized coenzyme is oxidized nicotinamide adenine dinucleotide or oxidized nicotinamide adenine dinucleotide phosphate.
7. The inorganic phosphorus detection reagent of any one of claims 1-4, wherein the first excipient or the second excipient comprises at least one of mannitol, trehalose, dextran forty thousand and dextran ten thousand.
8. The inorganic phosphorus detection reagent of claim 4, wherein the first buffer and the second buffer comprise piperazine-1, 4-diethylsulfonate buffer, the first stabilizer comprises the ethylenediaminetetraacetic acid and the glycerol, the second stabilizer comprises the ethylenediaminetetraacetic acid, the first excipient comprises trehalose, and the second excipient comprises dextran one thousand;
the first reagent comprises the following components:
20-100mmol/L piperazine-1, 4-diethylsulfonic acid buffer solution, 1-10g/L ethylene diamine tetraacetic acid, 1-10g/L glycerol, 20-50g/L maltose, 20-50g/L oxidized coenzyme I and 100-200g/L trehalose;
the second reagent comprises the following components:
20-100mmol/L piperazine-1, 4-diethylsulfonic acid buffer solution, 1-10g/L ethylene diamine tetraacetic acid, 10-20KU/L beta-phosphoglucomutase, 10-20KU/L maltose phosphorylase, 20-40KU/L glucose-6-phosphate dehydrogenase, 100-150g/L trehalose and 20-50g/L dextran ten thousand.
9. The inorganic phosphorus detection reagent of claim 4, wherein the first buffer solution and the second buffer solution are bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane buffer solution, the first stabilizer and the second stabilizer are bovine serum albumin, the first excipient is mannitol, and the second excipient is composed of dextran tetrapotaxane and mannitol, wherein the content of the dextran tetrapotaxane and the content of the mannitol are both 100-200 g/L.
10. An inorganic phosphorus detection chip, wherein the inorganic phosphorus detection chip comprises a chip body and the inorganic phosphorus detection reagent according to any one of claims 1 to 9 contained in the chip body.
Technical Field
The invention relates to the technical field of inorganic phosphorus detection, in particular to an inorganic phosphorus detection reagent and a detection chip.
Background
Phosphorus has important physiological functions in human body. Most of phosphorus elements in human body are deposited in bones in the form of calcium phosphate, and are important components constituting bones and teeth. Only a small fraction of phosphorus is present in body fluids. The phosphorus element in the blood exists in two forms of inorganic phosphorus and organic phosphorus, wherein the inorganic phosphorus mainly exists in the form of phosphate, and a small part of the phosphorus element and other substances form important organic compounds. The main physiological functions of phosphorus are the participation in carbohydrate, lipid and amino acid metabolism, which constitute energy-transport substances.
Inorganic phosphorus is usually measured in serum. At present, the total phosphorus content of a human body cannot be directly measured, and is mostly reflected by detecting the concentration of phosphate ions in blood plasma or blood serum. Since phosphate ions are stable in the blood, measuring the concentration of phosphate indirectly represents or infers the total amount of inorganic phosphorus.
In the process of implementing the invention, the inventor finds that in the prior art, the inorganic phosphorus detection reagent suitable for enzymatic detection has poor anti-interference capability and is easily interfered by bilirubin, uric acid and other reducing substances in a detection sample.
Disclosure of Invention
In order to overcome the problem of poor interference resistance of an inorganic phosphorus detection reagent, embodiments of the present invention provide an inorganic phosphorus detection reagent and a detection chip, which can detect inorganic phosphorus by maltose, oxidized coenzyme, β -phosphoglucomutase, maltose phosphorylase, and glucose-6-phosphate dehydrogenase, and have strong interference resistance to ascorbic acid, bilirubin, hemoglobin, triglyceride, uric acid, and creatinine.
In order to solve the above technical problem, an embodiment of the present invention provides the following technical solutions:
in a first aspect, embodiments of the present invention provide an inorganic phosphorus detection reagent, where the inorganic phosphorus detection reagent includes a first lyophilized reagent bead prepared from a first reagent and a second lyophilized reagent bead prepared from a second reagent;
the first reagent comprises the following components:
the second reagent comprises the following components:
optionally, the first reagent further comprises a first stabilizer, and/or the second reagent further comprises a second stabilizer.
Optionally, when the first reagent comprises the first stabilizer, the content of the first stabilizer is 1-10 g/L;
when the second reagent comprises the second stabilizer, the content of the second stabilizer is 1-10 g/L.
Optionally, the first stabilizer or the second stabilizer includes at least one of glycerol, bovine serum albumin, propylene glycol-monomethyl ether, 4-methylphenylboronic acid, magnesium chloride, lithium chloride, ammonium sulfate, ethylenediaminetetraacetic acid, glutamate, or reduced glutathione.
Optionally, the oxidized coenzyme is oxidized nicotinamide adenine dinucleotide or oxidized nicotinamide adenine dinucleotide phosphate.
Optionally, the first excipient or the second excipient comprises at least one of mannitol, trehalose, dextran forty thousand and dextran ten thousand.
Optionally, the first buffer and the second buffer comprise piperazine-1, 4-diethylsulfoacid buffer, the first stabilizer comprises the ethylenediaminetetraacetic acid and the glycerol, the second stabilizer comprises the ethylenediaminetetraacetic acid, the first excipient comprises trehalose, and the second excipient comprises dextran one thousand;
the first reagent comprises the following components:
20-100mmol/L piperazine-1, 4-diethylsulfonic acid buffer solution, 1-10g/L ethylene diamine tetraacetic acid, 1-10g/L glycerol, 20-50g/L maltose, 20-50g/L oxidized coenzyme I and 100-200g/L trehalose;
the second reagent comprises the following components:
20-100mmol/L piperazine-1, 4-diethylsulfonic acid buffer solution, 1-10g/L ethylene diamine tetraacetic acid, 10-20KU/L beta-phosphoglucomutase, 10-20KU/L maltose phosphorylase, 20-40KU/L glucose-6-phosphate dehydrogenase, 100-150g/L trehalose and 20-50g/L dextran ten thousand.
Optionally, the first buffer solution and the second buffer solution are bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane buffer solutions, the first stabilizer and the second stabilizer are bovine serum albumin, the first excipient is mannitol, and the second excipient is composed of dextran tetrapotan and mannitol, wherein the contents of the dextran tetrapotan and the mannitol are both 100-200 g/L.
In a second aspect, embodiments of the present invention provide an inorganic phosphorus detection chip, which includes a chip body and the inorganic phosphorus detection reagent according to the first aspect, which is accommodated in the chip body.
The beneficial effects of the embodiment of the invention are as follows: in contrast to the prior art, the embodiment of the present invention provides an inorganic phosphorus detection reagent and a detection chip, including a first lyophilized reagent ball and a second lyophilized reagent ball, wherein the first lyophilized reagent ball includes maltose and an oxidized coenzyme, and the second reagent ball includes beta-phosphoglucomutase, maltose phosphorylase, and glucose-6-phosphate dehydrogenase; and (3) reacting inorganic phosphorus in the detection sample with maltose to generate the glucose-1-phosphate. Glucose-1-phosphate is converted to glucose-6-phosphate by phosphoglucomutase and/or its coenzyme. The 6-phosphoglucose is oxidized into 6-phosphogluconate under the action of glucose-6-phosphate dehydrogenase, meanwhile, the oxidized coenzyme is reduced into reduced coenzyme, and the content of inorganic phosphorus in the detection sample can be calculated by utilizing the change of the absorbance of the reaction system at 340 nm. Test results show that the inorganic phosphorus detection reagent and the detection chip provided by the embodiment of the invention have strong anti-interference capability on ascorbic acid, bilirubin, hemoglobin, triglyceride, uric acid and creatinine.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required to be used in the embodiments of the present invention will be briefly described below. It is obvious that the drawings described below are only some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive effort.
FIG. 1 is a graph of clinical correlation analysis of an inorganic phosphorus detection chip according to one embodiment of the present invention;
FIG. 2 is a graph of a linear range analysis of an inorganic phosphorus detection chip according to one embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention 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 invention and are not intended to limit the invention.
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 invention. Additionally, while functional block divisions are performed in the device diagrams, with logical sequences shown in the flowcharts, in some cases, the steps shown or described may be performed in a different order than the block divisions in the device diagrams, or the flowcharts.
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.
The methods suitable for measuring inorganic phosphorus include phosphomolybdic acid reduction method/non-reduction method, atomic absorption spectrophotometry, enzyme method, dye binding method flow injection analysis method, isotope dilution mass spectrometry and the like.
The phosphomolybdic acid reduction method is characterized in that trichloroacetic acid is used for precipitating protein in a serum sample, an ammonium molybdate reagent is added into a protein-free serum filtrate, and the ammonium molybdate is combined with inorganic phosphorus in a detection sample to generate phosphomolybdic acid; and reducing phosphomolybdic acid into a blue compound by using ferrous sulfate as a reducing agent, and carrying out colorimetric determination. When the protein in the serum sample is not removed, a nonionic surfactant is added to the reagent to avoid turbidity. The phosphomolybdic acid non-reduction method does not need to use a reducing agent, and the complex formed by the action of inorganic phosphorus and ammonium molybdate is directly measured at the wavelength of 340nm or 325 nm. The phosphomolybdic acid reduction method needs to remove protein in a detection sample or add a surfactant, and has complex operation steps and large error. Although the phosphomolybdic acid non-reduction method has stable color development and simple and convenient operation, the phosphomolybdic acid non-reduction method is easily influenced by interference factors such as lipemia, jaundice and hemolysis, and the detection result is inaccurate.
The enzyme method detection kit mainly comprises enzyme raw materials, and the enzyme raw materials are easy to inactivate, so that the problems that the kit is easy to lose efficacy and the like exist; and the detection result is easily interfered by other substances in the detection sample. For example, in an inorganic phosphorus detection kit in which Purine Nucleoside Phosphorylase (PNP) and Xanthine Oxidase (XOD) are coupled and Peroxidase (POD) is used as an indicator, the detection result is easily interfered by reducing substances such as bilirubin and uric acid. And at present, serum phosphorus is mainly detected by various large-scale full-automatic biochemical analyzers, and the defects of high equipment price, complex operation, high requirement on supporting facilities, long detection waiting time and the like exist.
Based on this, the embodiment of the invention provides an inorganic phosphorus determination reagent ball, which is a freeze-dried reagent ball prepared from an inorganic phosphorus detection reagent and capable of being placed in a microfluidic chip, does not need to depend on a large-scale full-automatic biochemical analyzer, can realize rapid diagnosis around a patient, and has the advantages of short test time, simple and convenient operation and the like. Meanwhile, the influence of ascorbic acid, bilirubin, hemoglobin, triglyceride, uric acid and creatinine on the inorganic phosphorus detection result can be effectively solved. To facilitate the reader's understanding of the invention, reference will now be made to specific examples.
The embodiment of the invention provides an inorganic phosphorus detection reagent, which comprises a first reagent ball and a second reagent ball, wherein the first reagent ball is prepared from a first reagent, and the second reagent ball is prepared from a second reagent.
The first reagent comprises the following components:
the second reagent comprises the following components:
the buffer solution is a mixed solution composed of weak acid and salt thereof, weak base and salt thereof, and can maintain the pH value of the reaction system to be relatively stable. The buffer solution in the embodiment of the invention comprises a first buffer solution and a second buffer solution, wherein the first buffer solution or the second buffer solution comprises one of Bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris) buffer solution, piperazine-1, 4-diethylsulfonic acid (PIPES) buffer solution, N- (2-acetamido) -2-aminoethanesulfonic Acid (ACES) buffer solution, 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid (MOPSO) buffer solution, 2- (diethanolamino) ethanesulfonic acid (BES) buffer solution and 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer solution. PIPES buffer, MOPSO buffer and HEPES buffer are zwitterionic (Good's) buffers. Good's buffers have Good PH stability and high polarity, are inert to various chemical reagents and enzymes, do not participate in and do not interfere with biochemical reaction processes. The main component of the HEPES buffer solution is 4-hydroxyethyl piperazine ethanethiosulfonic acid, the pH buffer range is 6.8-8.2, the buffer capacity is good in the pH range of 7.2-7.4, and the constant pH range can be controlled for a long time. In this example, the buffering capacity is better when the concentration of the first buffer solution and/or the second buffer solution reaches 20 to 100 mmol/L.
The excipient can endow the inorganic phosphorus detection reagent with good appearance, so that the inorganic phosphorus detection reagent is loose and porous and is easy to redissolve. The excipient mainly comprises polyhydric alcohols, saccharides, amino acids, inorganic salts, proteins and peptides. The polyalcohol excipient comprises glycerol, sorbitol, mannitol, inositol, adonitol, ethylene glycol, polyethylene glycol, etc. The saccharide excipient comprises monosaccharide excipient, disaccharide excipient and polysaccharide excipient; wherein the monosaccharide excipient comprises glucose; disaccharide excipients include sucrose, lactose, maltose, trehalose, and the like; the polysaccharide excipient comprises water soluble starch, maltodextrin, and dextran, wherein the dextran can be one or more of dextran 1 ten thousand, dextran 2 ten thousand, dextran 4 ten thousand or dextran 7 ten thousand. Amino acid excipients include: sodium glutamate, proline, lysine, alanine, and the like; inorganic salt excipients include: calcium carbonate, manganese sulfate, sodium cholate, sodium acetate and the like; protein and peptide excipients include mucopolysaccharide protein, casein or bovine serum albumin, and the like. The excipients in embodiments of the invention include a first excipient and a second excipient, and the first excipient and the second excipient are any suitable excipients.
Further, in some embodiments, to impart better appearance and reconstitution properties to the first reagent sphere, the first excipient or the second excipient in embodiments of the invention is at least one of mannitol, trehalose, dextran forty thousand or dextran ten thousand.
The oxidized coenzyme in the embodiments of the invention may be oxidized Nicotinamide Adenine Dinucleotide (NAD)+) Or oxidized form of Nicotinamide Adenine Dinucleotide Phosphate (NADP)+)。
The detection principle of the inorganic phosphorus determination reagent ball provided by the embodiment of the invention is as follows: under the action of maltose phosphorylase, inorganic phosphorus in the sample is detected to react with maltose to generate 1-phosphoglucose. Glucose-1-phosphate forms glucose-6-phosphate by the action of Phosphoglucomutase (PGM) and/or its coenzymes. The glucose-6-phosphate is oxidized to gluconic acid-6-phosphate by the action of glucose-6-phosphate dehydrogenase, and the oxidized coenzyme (NAD (P))+) Reduced coenzyme (NAD (P) H) to cause the rise of the absorbance at 340nm, and the change of the absorbance is related to the content of inorganic phosphorus in the detection sample, thereby calculating the content of the inorganic phosphorus in the sample to be detected. The reaction equation is as follows:
in some embodiments, the inorganic phosphorus detection reagent further comprises a stabilizer for improving the stability of the enzyme, e.g., for improving the stability of an oxidized coenzyme, a beta-phosphoglucomutase, a maltose phosphorylase, and/or a glucose-6-phosphate dehydrogenase. The stabilizer in the embodiment of the present invention includes a first stabilizer in the first reagent and a second stabilizer in the second reagent, and the first stabilizer and the second stabilizer include at least one of glycerol, bovine serum albumin, propylene glycol-monomethyl ether, 4-methylphenylboronic acid, magnesium chloride, lithium chloride, ammonium sulfate, ethylenediaminetetraacetic acid, glutamate, or reduced glutathione.
Specifically, in some embodiments, the activity of each enzyme in the inorganic phosphorus detector reagent is best when the pH of the first reagent and/or the second reagent is optimally 6.5 to 7.5. Smaller (acidic) or larger (alkaline) pH values denature and inactivate enzyme proteins. Changes in pH affect the degree of dissociation of the essential groups of the enzyme active center and also affect the degree of dissociation of the reaction substrate and coenzyme, and thus affect the binding and catalysis of the enzyme molecule to the substrate molecule. Only when the pH value is in the range of 6.5-7.5, the dissociation state of the enzyme, the substrate and the coenzyme is optimum for combining with each other and generating catalysis, thereby leading the reaction rate of the enzyme in the reaction (i) -c to reach the maximum value.
Specifically, in some embodiments, in order to improve the testing accuracy, stability and anti-interference capability of the inorganic phosphorus detection reagent, the first reagent comprises 20-100mmol/L PIPES buffer solution, 1-10g/L EDTA, 1-10g/L glycerol, 20-50g/L maltose, 20-50g/L oxidized coenzyme I (NAD +) and 100-200g/L trehalose; the second reagent comprises 20-100mmol/L PIPES buffer solution, 1-10g/L EDTA, 10-20KU/L beta-phosphoglucomutase, 10-20KU/L maltose phosphorylase, 20-40KU/L glucose-6-phosphate dehydrogenase, 100-150g/L trehalose and 20-50g/L dextran. In other embodiments, the first reagent specifically comprises 20-100mmol/L Bis-Tris buffer, 1-10g/L bovine serum albumin, 20-50g/L maltose, 20-50g/L oxidized coenzyme II and 100-200g/L mannitol; the second reagent specifically comprises 20-100mmol/L Bis-Tris buffer solution, 1-10g/L bovine serum albumin, 10-20KU/L beta-phosphoglucomutase, 10-20KU/L maltose phosphorylase, 20-40KU/L glucose-6-phosphate dehydrogenase, 50-100g/L dextran forty thousand and 50-100g/L mannitol. In the embodiment, the inorganic phosphorus detection reagent has high test precision, good correlation with an Abaxis reagent disk, and strong anti-interference capability on ascorbic acid, bilirubin, hemoglobin, triglyceride, uric acid and creatinine; and after the inorganic phosphorus detection reagent is stored for 8 days at 37 ℃ in a dark place and is stored for 15 months at 2-8 ℃ in a dark place, the absolute value of the relative deviation of the detection result is still less than 10 percent, thereby meeting the use requirement.
The embodiment of the invention also provides an inorganic phosphorus detection chip for detecting the content of inorganic phosphorus in serum, which comprises a chip body and the inorganic phosphorus detection reagent. The chip body is provided with a colorimetric hole, and the inorganic phosphorus detection reagent is contained in the colorimetric hole. The detection method of the inorganic phosphorus comprises the following steps: diluting a serum sample by using a diluent, filling the diluted serum sample into a colorimetric hole, and testing the absorbance value of a solution in the colorimetric hole by using a spectrophotometer after the diluted serum sample fully reacts with an inorganic phosphorus detection reagent in the colorimetric hole.
In some embodiments, the detection chip further comprises a sample well, a diluent well, a sample quantification well, a diluent quantification well, and a mixing well. When the detection chip is used for testing, the detection can be carried out without carrying out centrifugal treatment on a sample, so that detection samples such as serum, plasma, whole blood and the like can be used for detection. Specifically can will add the sample groove through the pipettor with serum sample from the sample hole, tear the sealing film in diluent groove again, adopt centrifugal mode to make the diluent that prestores from diluent groove entering diluent ration groove, make serum sample from the sample groove entering sample ration groove to and make the diluent in the diluent ration groove and the serum sample in the sample ration groove get into the mixing tank, dilute serum sample through the diluent. And filling the diluted serum sample in the mixing tank into the colorimetric hole in a centrifugal mode, and testing the absorbance of the solution in the colorimetric hole by using a spectrophotometer after the diluted serum sample fully reacts with the inorganic phosphorus detection reagent in the colorimetric hole. Specifically, the diluent may be distilled water.
The embodiment of the invention provides an inorganic phosphorus detection reagent, which inherits the advantages of good enzyme method selectivity, high sensitivity and capability of realizing automatic analysis. The liquid reagent is prepared into the freeze-dried reagent ball which can be placed in the microfluidic detection chip, a large-scale full-automatic biochemical analyzer is not needed, the rapid diagnosis at the patient side can be realized, and the method has the advantages of short test time, simplicity and convenience in operation and the like.
The embodiment of the invention also provides a preparation method of the inorganic phosphorus detection reagent, which comprises the following steps:
s11, obtaining a first reagent and a second reagent;
in some embodiments, the first reagent is specifically configured as follows: adding a proper amount of distilled water into a container, weighing buffer solution components, sequentially adding maltose and oxidized coenzyme after the buffer solution is completely dissolved, adjusting the pH to 6.5-7.5, finally adding an excipient, and performing constant volume to obtain a first reagent. In some embodiments, the process of configuring the second reagent is as follows: adding a proper amount of distilled water into a container, weighing buffer solution components, sequentially adding beta-phosphoglucomutase, maltose phosphorylase and glucose-6-phosphate dehydrogenase after the buffer solution is completely dissolved, adjusting the pH to 6.5-7.5, finally adding an excipient, and performing constant volume to obtain a second reagent. In this example, the components of the first reagent and the second reagent are mixed in the same ratio as in the above-described example of the inorganic phosphorus detection reagent.
S12, respectively dropping the droplets of the first reagent and the second reagent into liquid nitrogen, so that the droplets of the first reagent and the second reagent are respectively coagulated into a first ice ball and a second ice ball;
for example, droplets of the first reagent (or the second reagent) may be dropped in liquid nitrogen by a dispenser, causing the droplets to coalesce into a first ice ball (or a second ice ball) in the liquid nitrogen. The size of the droplet of the first reagent (or the second reagent) dropped into the liquid nitrogen can be adjusted by those skilled in the art according to actual needs, and the volume of the droplet (or the second ice ball) can be adjusted by controlling the size of the droplet. Alternatively, in some embodiments, to ensure reconstitution of the inorganic phosphorus detection reagent, the first ice ball and the second ice ball have a volume of 2.5-3.5. mu.l, e.g., 2.5. mu.l, 3. mu.l, or 3.5. mu.l.
And S13, respectively carrying out freeze drying on the first ice ball and the second ice ball to obtain a first freeze-dried reagent ball and a second freeze-dried reagent ball.
In this embodiment, the first ice ball and the second ice ball are respectively placed in a vacuum freeze dryer to be freeze-dried to form the inorganic phosphorus detection reagent, and the inorganic phosphorus detection reagent is collected and stored in a dry aluminum bottle after nitrogen gas is repressed.
Freeze drying is a drying method in which a reagent raw material containing water is cooled and frozen into a solid in advance, and the sublimation performance of water is utilized under the condition of low temperature and reduced pressure to dehydrate the reagent raw material at low temperature so as to achieve the purpose of drying. After freeze-drying, the raw material components of the first reagent and the second reagent remain in the frozen ice shelf, so that the freeze-dried inorganic phosphorus detection reagent is loose and porous, and the volume of the freeze-dried inorganic phosphorus detection reagent is basically unchanged from that before freeze-drying. Because the inorganic phosphorus detection reagent is always in a frozen state before being dried, and the ice crystals are uniformly distributed in each component of the raw materials of the reagent. In the sublimation process, the components are not concentrated due to dehydration. Therefore, the freeze-dried inorganic phosphorus detection reagent is spongy, loose and porous, and is easy to dissolve in water and restore to the original shape.
To further illustrate the technical solution of the present invention, several examples of the inorganic phosphorus detection reagent and the detection chip of the present invention are provided below.
Example 1:
the inorganic phosphorus detection reagent in the inorganic phosphorus detection chip provided by the embodiment comprises the following raw material components:
the components of the first reagent are as follows:
PIPES buffer solution 20 mmol/L;
1g/L of ethylene diamine tetraacetic acid;
10g/L of glycerol;
maltose 50 g/L;
oxidized coenzyme I20L;
trehalose is 100 g/L;
the first reagent has a pH of 6.7.
The components of the second reagent are as follows:
PIPES buffer solution 100 mmol/L;
10g/L of ethylene diamine tetraacetic acid;
beta-phosphoglucomutase 10 KU/L;
maltose phosphorylase 10 KU/L;
glucose-6-phosphate dehydrogenase 20 KU/L;
150g/L of trehalose; (ii) a
Dextran ten thousand 50 g/L;
the pH of the second reagent was 6.7.
Example 2:
the present example differs from example 1 as follows:
the components of the first reagent are as follows:
100mmol/L of Bis-Tris buffer solution;
10g/L of bovine serum albumin;
maltose 25 g/L;
oxidized coenzyme II 50 g/L; (ii) a
120g/L of mannitol;
the pH of the first reagent was 7.0.
The components of the second reagent are as follows:
100mmol/L of Bis-Tris buffer solution;
bovine serum albumin 1 g/L;
beta-phosphoglucomutase 20 KU/L;
maltose phosphorylase 20 KU/L;
glucose-6-phosphate dehydrogenase 40 KU/L;
forty thousand glucan 50 g/L;
80g/L of mannitol;
the pH of the second reagent was 7.0.
The performance of the inorganic phosphorus detecting chip obtained in example 1 of the present invention will be described below with reference to the table. The change in absorbance of the measurement chip into which the serum sample was injected at 37 ℃ was measured by a vp10 portable biochemical analyzer. The concentration of inorganic phosphorus in the serum samples was calculated using calibration standards provided by the british langway company.
(1) And testing precision: the detection chip provided by the embodiment 1 of the invention is used for testing the inorganic phosphorus concentration in a serum sample with the known inorganic phosphorus concentration of 1.45mmol/L for 20 times, and the average value of the concentration values is calculated by the following formulaStandard Deviation (SD) and Coefficient of Variation (CV):
wherein, XiThe concentration value was measured for the i-th time, and n is the number of times of measurement.
Obtaining the average value of the measured concentration values obtained by testing the same serum sample for 20 times The standard deviation SD is 0.034 and the coefficient of variation CV is 2.44%. Generally, a larger standard deviation represents a larger difference between most of the test results and their average values; if the standard deviation is small, it means that the test results are closer to the average.
(2) And testing the accuracy: the detection chip provided by the embodiment 1 of the invention is adopted to test a serum sample with known inorganic phosphorus concentration of 2.27mmol/L for three times, the measured inorganic phosphorus concentration value is obtained, the average value of the inorganic phosphorus concentration values measured for 3 times is calculated to be 2.22mmol/L, and the relative deviation is-2.06%.
(3) And clinical relevance analysis:
the detection chip provided by the embodiment 1 of the invention and the Abaxis reagent disk are adopted to simultaneously determine the inorganic phosphorus content of a plurality of serum samples with different inorganic phosphorus concentrations. The corresponding measured inorganic phosphorus concentration value (unit mmol/L) is shown in Table I, wherein the X column number is the inorganic phosphorus concentration in the serum sample measured by the Abaxis reagent disk, and the Y column number is the inorganic phosphorus concentration in the serum sample measured by the detection chip provided by the embodiment 1 of the invention. For example, for the serum sample No. 1, the concentration of inorganic phosphorus tested by using the Abaxis reagent disk is 3.84mmol/L, while the concentration of inorganic phosphorus in the serum sample No. 1 tested by using the inorganic phosphorus detection chip provided in the embodiment 1 of the present invention is 3.99 mmol/L.
The correlation equation between the two groups of test results of the detection chip and the Abaxis reagent disk provided by the embodiment of the invention is as follows:
y＝0.9956x+0.0193
the closer the correlation coefficient R is to 1, the stronger the correlation between the two sets of data 0.9955. Therefore, the detection chip provided by the embodiment of the invention has strong correlation with the test result of the Abaxis reagent disk.
Table one: clinical correlation analysis table of inorganic phosphorus detection kit.
(4) Linear range test
The test method is as follows: the high concentration test sample and the low concentration test sample were mixed in different ratios into 6 diluted concentration test samples as shown in Table II using the high concentration (activity) sample near the upper limit of the linear range ([0.2, 7] mmol/L) and the low concentration (activity) sample near the lower limit of the linear range. Since low concentration (active) samples are difficult to collect, they can be replaced with physiological saline.
Table two:
sample number
1
2
3
4
5
6
High concentration (active) serum sample
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 detection chip provided by the embodiment 1 of the invention is adopted to respectively test the inorganic phosphorus concentration of 6 serum samples, each serum sample is tested for 3 times, and the average value (y) of the concentration values measured by the inorganic phosphorus in the 6 serum samples is respectively calculatedi). Concentration (x) after dilution with each samplei) As independent variable, mean value of measured concentration values (y) of each samplei) Linear regression equations were solved for the dependent variables. Calculating a correlation coefficient R of the linear regression according to a formula (4); equation (4) is as follows:
wherein n is the number of samples to be measured, xiTo dilute the concentration, yiThe average value of the measurement results is shown.
As shown in fig. 2, the linear regression equation obtained is y equal to 0.9985x +0.0343, and the correlation coefficient R is 0.9995.
Generally, when the kit detects a detection sample with inorganic phosphorus concentration in the range of [0.2, 7] mmol/L, the linear correlation coefficient R is more than or equal to 0.990, and the requirement is met. Therefore, the inorganic phosphorus detection reagent provided by the embodiment of the invention has the characteristic of wide linear range.
(5) Thermal stability test
In an environment with 8% air humidity, the inorganic phosphorus detection reagent provided in embodiment 1 of the present invention is loaded into a chip body to form a plurality of detection chips, and then loaded into an aluminum foil bag for sealing.
After storing the plurality of detection chips provided in example 1 in a dark environment at 37 ℃ for 0, 2, 3, 4, 6 and 8 days, the inorganic phosphorus concentrations in two sets of calibrators (sample 1 and sample 2) provided by the british lambdas company were measured several times to analyze the average value and the relative deviation (in mmol/L) of the results of the several measurements, thereby analyzing the detection accuracy of the detection chips, and the results of the measurements are shown in table three and table four. In order to ensure the accuracy of the detection result, the absolute value of the relative deviation should be within ± 10.0%.
The third table and the fourth table are respectively the detection results of the detection chip for detecting the inorganic phosphorus concentration in the sample 1 and the sample 2 for 3 times after storing different time, and the average value and the relative deviation of the calculated detection results, wherein the target value in each table is the actual concentration of the inorganic phosphorus in the sample 1 and the sample 2 correspondingly. In order to ensure the accuracy of the detection result, the absolute value of the relative deviation should be within 10.0%. As can be seen from table three and table four, the absolute value of the relative deviation of the detection results after the detection chip provided by the embodiment of the present invention is stored in an environment at 37 ℃ for 2, 3, 4, 6, or 8 days is still within ± 10.0%, and therefore, the detection chip provided by the embodiment of the present invention has good thermal stability.
Table three: thermal stability analysis table.
Sample 1
1
2
3
Mean value of
Target value
Relative deviation of
Day 0
2.19
2.22
2.27
2.23
2.21
0.75％
2 days
2.24
2.27
2.14
2.22
2.21
0.30％
3 days
2.17
2.20
2.19
2.19
2.21
-1.06％
4 days
2.16
2.11
2.14
2.14
2.21
-3.32％
6 days
2.19
2.07
2.13
2.13
2.21
-3.62％
8 days
2.05
2.21
2.10
2.12
2.21
-4.07％
Table four: thermal stability analysis table.
Sample 2
1
2
3
Mean value of
Target value
Relative deviation of
Day 0
1.45
1.39
1.49
1.44
1.43
0.93％
2 days
1.38
1.41
1.47
1.42
1.43
-0.70％
3 days
1.43
1.41
1.40
1.41
1.43
-1.17％
4 days
1.35
1.39
1.37
1.37
1.43
-4.20％
6 days
1.42
1.34
1.39
1.38
1.43
-3.26％
8 days
1.32
1.38
1.38
1.36
1.43
-4.90％
(6) Long term stability test
After storing the plurality of detection chips provided in example 1 in a dark environment at 2-8 ℃ for 0, 3, 6, 9, 12 and 15 months, the inorganic phosphorus concentrations in two sets of calibrators (sample 3 and sample 4) provided by the british lambdas company were measured several times to analyze the average value and the relative deviation (in mmol/L) of the results of the several measurements, thereby analyzing the detection accuracy of the detection chips, and the results of the measurements are shown in table five and table six.
Table five: long term stability analysis table.
Sample 3
1
2
3
Mean value of
Target value
Relative deviation of
0 month
2.29
2.24
2.16
2.23
2.21
0.84％
3 month
2.16
2.27
2.20
2.21
2.21
-0.03％
6 month
2.13
2.25
2.21
2.20
2.21
-0.66％
9 month
2.14
2.16
2.17
2.16
2.21
-2.41％
12 month
2.12
2.17
2.19
2.16
2.21
-2.32％
15 month
2.07
2.13
2.14
2.11
2.21
-4.43％
Table six: long term stability analysis table.
Sample 4
1
2
3
Mean value of
Target value
Relative deviation of
0 month
1.39
1.45
1.42
1.42
1.43
-0.70％
3 month
1.43
1.37
1.34
1.38
1.43
-3.54％
6 month
1.38
1.40
1.33
1.37
1.43
-4.20％
9 month
1.35
1.32
1.36
1.34
1.43
-6.06％
12 month
1.40
1.28
1.34
1.34
1.43
-6.39％
15 month
1.40
1.27
1.42
1.3
1.43
-4.66％
Table five and table six are the detection results of the detection chip for detecting the inorganic phosphorus concentrations in the sample 3 and the sample 4 for 3 times after storing different times, and the average value and the relative deviation of the calculated detection results, respectively, wherein the target values in the respective tables are the actual concentrations of the inorganic phosphorus in the sample 3 and the sample 4, respectively. It can be seen from tables five and six that, after the detection chip provided by the embodiment of the invention is stored in an environment of 2-8 ℃ for 3, 6, 9, 12 and 15 months, the absolute value of the relative deviation of the detection result is still within ± 10.0%, so that the detection chip provided by the embodiment of the invention has good long-term stability, and the accuracy of the detection result can be ensured after the detection chip is stored in the environment for a long time.
(7) Anti-interference capability test
Taking fresh serum samples of the same patient in the same batch, and dividing the serum samples into a control group and an experimental group, wherein the control group has no artificially added interferents, and the experimental group is added with 6 interferents shown in the seventh table. The inorganic phosphorus detection chip provided by the embodiment of the invention is adopted to respectively detect the detection samples of the experimental group and the control group. And (4) repeatedly detecting each detection sample for three times, and averaging the detection results. The measured concentration of the inorganic phosphorus in the control group is 2.27mmol/L, and the measurement results in the experimental group are shown in the seventh table.
TABLE VII: and (5) an anti-interference capability analysis meter.
In table seven, relative deviation (%) - (measured value of experimental group-measured value of control group)/measured value of control group × 100%. As can be seen from Table seven, the concentration of inorganic phosphorus measured in the experimental group did not deviate more than. + -. 10.0% from that measured in the control group. The inorganic phosphorus detection reagent and the detection chip provided by the embodiment of the invention have strong anti-interference capability on ascorbic acid, bilirubin, hemoglobin, triglyceride, uric acid and creatinine.
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 the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.