阅读说明：本技术 一种血肌酐检测试剂球及血肌酐检测芯片 (Blood creatinine detection reagent ball and blood creatinine 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：本发明涉及血肌酐测定技术领域,尤其是涉及一种血肌酐检测试剂球及血肌酐检测芯片。本发明实施例提供了血肌酐检测试剂球和包含该血肌酐检测试剂球的血肌酐检测芯片,能够通过抗坏血酸氧化酶消除检测样本中维生素C的干扰,通过去胆红素干扰剂作为氧化剂消除胆红素的干扰；分别通过肌酸酶、肌氨酸氧化酶和过氧化物酶消除检测样本中存在的内源性肌酸、肌氨酸和过氧化物的干扰。本发明提供的血肌酐检测试剂球和检测芯片对血清中原有的干扰物和患者服用的药物类干扰物均具有较强的抗干扰能力,检测结果精度高。(The invention relates to the technical field of blood creatinine determination, in particular to a blood creatinine detection reagent ball and a blood creatinine detection chip. The embodiment of the invention provides a blood creatinine detection reagent ball and a blood creatinine detection chip comprising the same, wherein the interference of vitamin C in a detection sample can be eliminated through ascorbic acid oxidase, and the interference of bilirubin is eliminated through a bilirubin interference agent as an oxidant; the interference with the detection of endogenous creatine, sarcosine and peroxide present in the sample is eliminated by creatinase, sarcosine oxidase and peroxidase, respectively. The blood creatinine detection reagent ball and the detection chip provided by the invention have strong anti-interference capability on original interferents in serum and drug interferents taken by patients, and the detection result has high precision.)

1. The blood creatinine detection reagent ball is characterized by comprising a first reagent ball made of a first reagent, a second reagent ball made of a second reagent and a third reagent ball made of a third reagent; wherein the content of the first and second substances,

the first reagent comprises the following components: 20-100mmol/L of first buffer solution, 5-10g/L of sodium chloride, 10-50g/L of 2, 4, 6-tribromo-3-hydroxybenzoic acid, 10-50g/L of first stabilizer, 100-500KU/L of creatinase, 50-200KU/L of sarcosine oxidase, 50-200KU/L of ascorbic acid oxidase and 100-200g/L of first excipient;

the second reagent comprises the following components: 20-100mmol/L of second buffer solution, 5-10g/L of sodium chloride, 0.1-10g/L of 4-aminoantipyrine, 0.005-1g/L of bilirubin interference removal agent, 10-50g/L of second stabilizer, 50-200KU/L of peroxidase and 100-200g/L of second excipient;

the third reagent comprises the following components: 0.8-1.2kg/L of the second reagent and 200-500KU/L of creatininase.

2. The blood creatinine detection reagent ball according to claim 1, wherein the first, second and third reagent balls are lyophilized reagent balls.

3. The blood creatinine detection reagent ball according to claim 1, wherein the first or second stabilizer includes at least one of sucrose, fructose, glycerol, bovine serum albumin, and fatty alcohol-polyoxyethylene ether.

4. The reagent ball for detecting creatinine according to claim 1, wherein the bilirubin disrupting agent comprises at least one of potassium ferrocyanide, potassium ferricyanide, and bilirubin oxidase.

5. The blood creatinine measurement reagent ball according to claim 1, wherein the first reagent and the second reagent have a pH of 7.5 to 9.0, and the third reagent has a pH of 7.5 to 8.5.

6. The blood creatinine measurement reagent ball according to any one of claims 1 to 5, wherein the first reagent further comprises a first surfactant, and/or the second reagent further comprises a second surfactant.

7. The blood creatinine measurement reagent ball according to claim 6, wherein when the first reagent includes the first surfactant, the first surfactant is a nonionic surfactant;

when the second reagent comprises the second surfactant, the second surfactant is a nonionic surfactant.

8. A blood creatinine detection chip, characterized in that, the blood creatinine detection chip includes a chip body and the blood creatinine detection reagent ball of any one of claims 1-7, wherein, the chip body is provided with a plurality of colorimetric holes, and the blood creatinine detection reagent ball is accommodated in the colorimetric holes.

9. The blood creatinine detection chip according to claim 8, wherein said plurality of colorimetric wells includes a first colorimetric well and a second colorimetric well; wherein the content of the first and second substances,

the first colorimetric hole is used for accommodating a first reagent ball and the second reagent ball, and the second colorimetric hole is used for accommodating the first reagent ball and the third reagent ball.

10. The blood creatinine detection chip according to claim 8, wherein said chip body is configured to:

measuring a first change value of the absorbance of the detection sample by using the first reagent ball and the second reagent ball which are matched;

measuring a second change value of the absorbance of the detection sample by using the first reagent ball and the third reagent ball;

and determining the concentration of the blood creatinine in the detection sample according to the first change value and the second change value.

Technical Field

The invention relates to the technical field of serum creatinine determination, in particular to a blood creatinine detection reagent ball and a blood creatinine detection chip.

Background

The sources of blood creatinine (Cr) include both exogenous creatinine ingested from food and endogenous creatinine produced by muscle metabolism in the body. Creatinine is slowly formed and released into the blood primarily from creatine through irreversible, non-enzymatic dehydration. Creatinine is a small molecule substance that is filtered through the glomerulus and is poorly absorbed in the tubules. Creatinine produced in the body every day is almost completely excreted with urine, and is generally not affected by urine volume. The content of the blood creatinine is closely related to the total amount of the muscle in the body, and is not easy to be influenced by diet.

Clinically, the detection of the content of serum creatinine is one of the main methods commonly used for understanding renal function, and the important index for measuring the renal function is that the increase of serum creatinine means the damage of renal function. At present, the determination method of the blood creatinine content mainly comprises a Jaffe detection method (chemical method), an enzymatic method, a capillary electrophoresis method, a high performance liquid chromatography and the like.

In the process of implementing the invention, the inventor finds that in the prior art, the measurement result of the blood creatinine content is easily interfered by other substances in the blood, and the substances consume hydrogen peroxide in the Trinder reaction due to strong reducibility or similarity with the structure of a chromogen substrate in the Trinder reaction, thereby influencing the measurement result. For example, pseudocreatinine and a portion of the drug that the patient is taking.

Disclosure of Invention

In order to improve the anti-interference capability of the blood creatinine detection reagent, the embodiment of the invention provides the blood creatinine detection reagent ball and the detection chip, which can eliminate the interference of other substances in blood through specific raw material components and improve the accuracy of a creatinine detection result.

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 a blood creatinine detection reagent ball, including a first reagent ball made of a first reagent, a second reagent ball made of a second reagent, and a third reagent ball made of a third reagent; wherein the content of the first and second substances,

the first reagent comprises the following components: 20-100mmol/L of first buffer solution, 5-10g/L of sodium chloride, 10-50g/L of 2, 4, 6-tribromo-3-hydroxybenzoic acid, 10-50g/L of first stabilizer, 100-500KU/L of creatinase, 50-200KU/L of sarcosine oxidase, 50-200KU/L of ascorbic acid oxidase and 100-200g/L of first excipient;

the second reagent comprises the following components: 20-100mmol/L of second buffer solution, 5-10g/L of sodium chloride, 0.1-10g/L of 4-aminoantipyrine, 0.005-1g/L of bilirubin interference removal agent, 10-50g/L of second stabilizer, 50-200KU/L of peroxidase and 100-200g/L of second excipient;

the third reagent comprises the following components: 0.8-1.2kg/L of the second reagent and 200-500KU/L of creatininase.

Optionally, the first, second and third reagent spheres are lyophilized reagent spheres.

Optionally, the first stabilizer or the second stabilizer includes at least one of sucrose, fructose, glycerol, bovine serum albumin, and fatty alcohol-polyoxyethylene ether.

Optionally, the bilirubin-disrupting agent comprises at least one of potassium ferrocyanide, potassium ferricyanide and bilirubin oxidase.

Optionally, the first reagent and the second reagent have a PH of 7.5 to 9.0, and the third reagent has a PH of 7.5 to 8.5.

Optionally, the first reagent further comprises a first surfactant, and/or the second reagent further comprises a second surfactant.

Optionally, when the first reagent comprises the first surfactant, the first surfactant is a nonionic surfactant;

when the second reagent comprises the second surfactant, the second surfactant is a nonionic surfactant.

In a second aspect, an embodiment of the present invention further provides a blood creatinine detection chip, where the blood creatinine detection chip includes a chip body and the blood creatinine detection reagent ball according to the first aspect, where the chip body is provided with a plurality of colorimetric holes, and the blood creatinine detection reagent ball is accommodated in the colorimetric holes.

Optionally, the plurality of colorimetric holes include a first colorimetric hole and a second colorimetric hole, where the first colorimetric hole is configured to receive a first reagent ball and a second reagent ball, and the second colorimetric hole is configured to receive the first reagent ball and a third reagent ball.

Optionally, the chip body is configured to:

measuring a first change value of the absorbance of the detection sample by using the first reagent ball and the second reagent ball which are matched;

measuring a second change value of the absorbance of the detection sample by using the first reagent ball and the third reagent ball;

and determining the concentration of the blood creatinine in the detection sample according to the first change value and the second change value.

The beneficial effects of the embodiment of the invention are as follows: different from the situation of the prior art, the embodiment of the invention provides a blood creatinine detection reagent ball and a blood creatinine detection chip comprising the blood creatinine detection reagent ball, which can eliminate the interference of vitamin C in a detection sample through ascorbic acid oxidase, and eliminate the interference of bilirubin through a bilirubin interference agent as an oxidant; the interference with the detection of endogenous creatine, sarcosine and peroxide present in the sample is eliminated by creatinase, sarcosine oxidase and peroxidase, respectively. The blood creatinine detection reagent ball and the detection chip provided by the invention have strong anti-interference capability on original interferents in serum and drug interferents taken by patients, and the detection result has high precision.

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 a blood creatinine assay chip according to an embodiment of the present invention;

fig. 2 is a linear range analysis diagram of a blood creatinine detection chip according to an 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 a logical order is shown in the flowcharts, in some cases, the steps shown or described may be performed in an order different than in 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.

In the existing determination method, the principle of Jaffe detection method is that creatinine reacts with picric acid under alkaline condition to generate red reaction products, and the red reaction products are complexes of 1:1 and 1:2 generated by the creatinine and the picric acid. However, the serum contains pseudocreatinine (homologues or derivatives of creatinine), such as guanidinoacetic lactam, 5-methyl guanidinoacetic lactam, acetoacetic acid, pyruvic acid, bilirubin and hydantoin, and the pseudocreatinine can react with picric acid, so that the measurement result of the creatinine content is inaccurate.

The capillary electrophoresis method has the advantages of high separation efficiency, high analysis speed, small use amount of products, high automation degree and the like. However, a small amount of protein sometimes remains on the inner wall of the capillary, resulting in poor reproducibility of the detection result.

The high performance liquid chromatography has high test accuracy, and because the sample is subjected to deproteinization treatment, and because of the high separation performance of the liquid chromatography column, the interference in creatinine determination is greatly reduced. The disadvantage of this method is the high analysis cost; the liquid chromatograph is expensive in price and daily maintenance cost, and has a long analysis time.

The enzymology method has high specificity and accurate result, can eliminate the interference of endogenous creatinine, and is suitable for various automatic analyzers. The creatinine detection kit widely used in clinic generally adopts an enzyme coupling method based on Trinder reaction (coupling end point colorimetry).

Meanwhile, the medicine taken by the patient can affect the detection result, so that the detection result is misinterpreted and even misdiagnosed, and unnecessary additional examination is added. Therefore, it is important to solve the problem of drug interference in detection. Clinically common drugs, such as: calcium dobesilate (used for treating microvascular diseases and the like), etamsylate (a hemostatic drug), methyldopa (an antihypertensive drug), levodopa (used for treating parkinsonism), dopamine (a nerve conduction substance) and the like, because of strong reducibility or similarity to a chromogen substrate structure in a Trinder reaction, hydrogen peroxide is consumed in the Trinder reaction, and the measured value of an object to be measured is low. Moreover, these drugs are structurally stable and are not easily destroyed or degraded, and therefore, they interfere with clinical chemistry tests based on the Trinder reaction, and affect the results of the tests.

Based on this, the embodiment of the invention provides a blood creatinine detection reagent ball and a detection chip, wherein a first reagent ball is respectively matched with a second reagent ball and a third reagent ball for use, peroxidase and 4-aminoantipyrine are added into the second reagent ball, and peroxidase, 4-aminoantipyrine and creatinine are added into the third reagent ball, so that the interference of various substances on the blood creatinine detection result is effectively eliminated. In addition, the reagent ball can be placed in a microfluidic detection chip for detection of a POCT analyzer, the operation is simple, cross contamination is avoided, and the determination result is quicker and more accurate. To facilitate the reader's understanding of the invention, reference will now be made to specific examples.

The embodiment of the invention provides a blood creatinine detection reagent ball, which comprises a first reagent ball, a second reagent ball and a third reagent ball, wherein the first reagent ball, the second reagent ball and the third reagent ball are respectively prepared by freeze-drying a first reagent, a second reagent and a third reagent.

The first reagent comprises the following components: 20-100mmol/L of first buffer solution, 5-10g/L of sodium chloride, 10-50g/L of 2, 4, 6-tribromo-3-hydroxybenzoic acid (TBHBA), 0.1-10g/L of first surfactant, 10-50g/L of first stabilizer, 100-and 500-KU/L of creatinase, 50-200-KU/L of sarcosine oxidase, 50-and 200-KU/L of ascorbic acid oxidase and 100-and 200-gram/L of first excipient. The pH of the first reagent is 7.5-9.0.

In some embodiments, the first reagent is formulated as follows: adding appropriate amount of distilled water into a container (such as a beaker), and weighing the first buffer solution and adding into the container; after the first buffer solution is completely dissolved, sequentially adding sodium chloride, TBHBA, a first surfactant, a first stabilizer, creatinase, sarcosine oxidase and ascorbic acid oxidase; and adjusting the pH value of a container in the solution, finally adding a first excipient, and then fixing the volume to obtain a first reagent.

The second reagent comprises the following components: 20-100mmol/L of second buffer solution, 5-10g/L of sodium chloride, 0.1-10g/L of 4-aminoantipyrine, 0.005-1g/L of decholethrin interfering agent, 0.1-10g/L of second surfactant, 10-50g/L of second stabilizing agent, 50-200KU/L of peroxidase and 100-200g/L of second excipient. The pH of the second reagent is 7.5-9.0.

In some embodiments, the second reagent is formulated as follows: adding a proper amount of distilled water into a container, weighing a second buffer solution component, adding the second buffer solution component into the container, adding sodium chloride, 4-aminoantipyrine, a bilirubin interference removing agent, a second surfactant, a second stabilizer and peroxidase in sequence after the second buffer solution is completely dissolved, finally adding a second excipient, and performing constant volume to obtain a second reagent.

The third reagent comprises the following components: 0.8-1.2kg/L of a second reagent and 200-500KU/L of creatininase. The pH of the third reagent is 7.5-8.5. And adding creatininase into the second reagent to obtain a third reagent. In order to improve the accuracy of the detection result, the third reagent may specifically include 1kg/L of the second reagent.

In the embodiment of the invention, the second reagent ball is added with peroxidase and 4-aminoantipyrine, and the third reagent ball is added with peroxidase, 4-aminoantipyrine and creatinine. The first reagent ball and the second reagent ball are matched for use, the change value of the absorbance of the detected sample measured by the first reagent ball is a first change value, and the first change value is used as the background of endogenous creatine; the first reagent ball and the third reagent ball are matched for use, and the change value of the absorbance of the detection sample measured by the first reagent ball and the third reagent ball is a second change value; and subtracting the background from the second change value to obtain an absorbance change value of the creatinine reaction in the detection sample, thereby partially eliminating the interference of various external factors on the Trinder reaction detection in the blood creatinine detection process.

The determination principle of the blood creatinine detection reagent ball provided by the embodiment of the invention is as follows: hydrogen peroxide (H) produced by the action of an enzyme under alkaline conditions of creatinine₂O₂) In the presence of 4-aminoantipyrine (4-AAP) and Peroxidase (POD), a red quinonimine compound is generated, so that the reaction solution has an absorption peak at a certain wavelength, and the absorbance value of the reaction solution at the wavelength is in direct proportion to the creatinine concentration within a certain range. Therefore, the concentration of creatinine in the blood sample can be calculated from the rate of increase in absorbance.

In the embodiment of the invention, the first reagent, the second reagent and the third reagent are all alkaline. Since the isoelectric point of most proteins in serum is in the acidic range, proteins readily form precipitates in acidic buffers to interfere with creatinine determination. Under alkaline conditions, interference caused by protein precipitation can be avoided.

The buffer solution is a mixed solution composed of weak acid and salt thereof or weak base and salt thereof, and can offset or reduce the influence of external strong acid or strong base on the pH value of the solution to a certain extent, so that the pH value of the solution is kept relatively stable. The buffer solution in the embodiment of the invention comprises a first buffer solution and a second buffer solution, and the first buffer solution and the second buffer solution can be the same or different, and the first buffer solution or the second buffer solution is one of phosphate buffer solution, 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid (HEPES) buffer solution, trihydroxymethyl aminomethanesulfonic acid (TAPS) buffer solution, 3-morpholine propanesulfonic acid (MOPS) buffer solution, N-dihydroxyethylglycine (BICINE) buffer solution or glycine buffer solution.

The first surfactant and the second surfactant in the embodiments of the present invention are nonionic surfactants. The first surfactant and the second surfactant may be the same or different in kind from each other, and are used for improving the stability of the oxidase. Nonionic surfactants have a relatively low critical micelle concentration, and lower concentrations are generally used to achieve enzyme protection. The nonionic surfactant is not ionized in an aqueous solution, and the hydrophilic group thereof is mainly an oxygen-containing group such as an ether group and a hydroxyl group. Compared with ionic surfactants, nonionic surfactants are not in an ionic state in a solution, so that the nonionic surfactants have high stability and are not easily affected by strong electrolytes and pH values. The nonionic surfactant can be one of Tween series active agent (such as Tween-20 and Tween-80), span series active agent (such as span 20 or span 40), or TRITON series (such as Triton X-100 or Triton X-114).

The first stabilizer or the second stabilizer in the practice of the present invention comprises at least one of sucrose, fructose, glycerol, bovine serum albumin, fatty alcohol and polyoxyethylene ether.

Bilirubin as reducing agent and oxidant H in Tinder reaction system₂O₂The reaction is carried out, so that the final product is reduced, and the interference of bilirubin can be eliminated by adding a bilirubin interference removing agent into the reagent. In some embodiments, the bilirubin disrupting agent may specifically be at least one of potassium ferrocyanide, potassium ferricyanide, or bilirubin oxidase.

The excipient can endow the freeze-dried reagent ball with good appearance, so that the freeze-dried reagent ball 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: phosphates, 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 first excipient and the second excipient in the present embodiment may be the same or different. In order to provide the first, second and third reagent beads with a good appearance, the first or second excipient may be at least one of inositol, trehalose, water-soluble starch and mannitol.

In some embodiments, the method for preparing a reagent ball (second or third reagent ball) comprises the steps of:

s11, dropping the droplets of the first reagent (the second reagent or the third reagent) in liquid nitrogen to form the droplets of the first reagent (the second reagent or the third reagent) into ice balls;

for example, droplets of the first reagent (the second reagent or the third reagent) may be dropped in liquid nitrogen by a dispenser, and the droplets may be condensed into ice balls in the liquid nitrogen. The size of the liquid drop 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. Alternatively, in some embodiments, the ice ball has a volume of 2.5-3.5. mu.l, for example, 2.5. mu.l, 3. mu.l or 3.5. mu.l.

S12, freeze-drying the ice ball to obtain a freeze-dried reagent ball;

in this embodiment, the ice ball is placed in a vacuum freeze dryer to be freeze-dried to form a freeze-dried reagent ball, and the freeze-dried reagent ball is collected and stored in a dry aluminum bottle after being repressed by nitrogen gas.

The freeze drying is a drying method which is characterized in that a first reagent, a second reagent and a third reagent containing water are respectively cooled and frozen into solid ice balls in advance, and the ice balls are dehydrated at low temperature under the condition of low temperature and reduced pressure by utilizing the sublimation performance of water so as to achieve the aim of drying. After freeze-drying, each of the raw materials in the first, second, or third reagents remains in the frozen ice shelf. Therefore, the freeze-dried reagent ball after freeze-drying is loose and porous, and the volume of the freeze-dried reagent ball is basically unchanged from that of the ice ball before freeze-drying. Because the freeze-dried reagent ball is always in a frozen state before being dried, the ice crystals are uniformly distributed in each chemical component of the blood creatinine detection reagent ball. In the sublimation process, the components are not concentrated due to dehydration. Therefore, the freeze-dried reagent ball after freeze drying is spongy, loose and porous, and is easy to dissolve in water and restore to the original shape.

The embodiment of the invention also provides a blood creatinine detection chip, which comprises a chip body and a blood creatinine detection reagent ball. A plurality of colorimetric holes are formed in the chip body, and the blood creatinine detection reagent ball is contained in the colorimetric holes. In some embodiments, the plurality of colorimetric apertures comprises a first colorimetric aperture for receiving the first and second reagent balls and a second colorimetric aperture for receiving the first and third reagent balls. And subtracting the background from the second change value of the absorbance measured by the first reagent ball and the third reagent ball to obtain the absorbance change value of the creatinine reaction in the detection sample. The test sample in the embodiments of the present invention includes serum, plasma, or whole blood.

In some embodiments, the blood creatinine detection chip further includes a sample well, a diluent well, a sample tank, a diluent tank, a sample quantification tank, a diluent quantification tank, and a mixing tank. When the blood creatinine detection chip is used for testing, the sample can be detected without centrifugal treatment, so that serum, plasma and whole blood can be used for detection. Firstly, adding a detection sample into a sample groove from a sample hole through a liquid shifter, then tearing a sealing film of the dilution groove, enabling a pre-stored dilution liquid to enter a dilution liquid quantifying groove from the dilution groove in a centrifugal mode, enabling the detection sample to enter the sample quantifying groove from the sample groove, enabling the dilution liquid in the dilution liquid quantifying groove and the detection sample in the sample quantifying groove to enter a mixing groove, and diluting the detection sample through the dilution liquid. And filling the diluted detection 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 detection sample fully reacts with the freeze-dried reagent ball in the colorimetric hole. Specifically, in the above examples, the diluent may be distilled water or physiological saline.

The method for detecting the blood creatinine provided by the embodiment of the invention uses the blood creatinine detection reagent ball provided by the embodiment to detect the blood creatinine in a detection sample, and comprises the following steps:

s11, measuring a first change value of the absorbance of the detection sample through the first reagent ball and the second reagent ball;

s12, measuring a second change value of the absorbance of the detection sample through the first reagent ball and the third reagent ball;

and S13, determining the concentration of creatinine in the detection sample according to the first change value and the second change value.

Diluting the detection sample by using a diluent, and filling the diluted detection sample into the first colorimetric hole and the second colorimetric hole; and after the diluted detection sample fully reacts with the first reagent ball and the second reagent ball in the first colorimetric hole, testing a first change value of absorbance of the solution in the first colorimetric hole by using a spectrophotometer. And after the diluted detection sample fully reacts with the first reagent ball and the third reagent ball in the second colorimetric hole, testing a second variation value of absorbance of the solution in the second colorimetric hole by using a spectrophotometer. And subtracting the first change value from the second change value to obtain an absorbance change value of the creatinine reaction in the detection sample, and calculating the concentration of the creatinine in the detection sample according to the absorbance change value.

To further illustrate the technical solution of the present invention, several examples of the blood creatinine assay reagent ball of the present invention are provided below.

Example 1:

the raw materials of the blood creatinine detection reagent ball of the embodiment comprise the following components:

the components of the first reagent are as follows: 20mmol/L HEPES buffer solution, 10g/L sodium chloride, 40 g/L2, 4, 6-tribromo-3-hydroxybenzoic acid, 10g/L tween-20, 30g/L fructose, 500KU/L creatinase, 60KU/L creatininase, 100KU/L ascorbate oxidase and 150g/L trehalose;

the components of the second reagent are as follows: 100mmol/L HEPES buffer solution, 5g/L sodium chloride, 0.2 g/L4-aminoantipyrine, 1g/L potassium ferrocyanide, 0.5g/L Tween-20, 50g/L bovine serum albumin, 50KU/L peroxidase and 200g/L trehalose;

the third reagent: 1kg/L of said second agent and 200KU/L of creatininase.

In this embodiment, the freeze-dried reagent ball is prepared by the above method for preparing a reagent ball for detecting serum creatinine, and the volumes of the first, second and third reagent balls are about 3.5 ul.

The blood creatinine detection chip in the embodiment comprises a chip body and the freeze-drying reagent ball provided by the embodiment. And testing the absorbance value of the detection sample by adopting the blood creatinine detection method.

Example 2:

the present example differs from example 1 as follows:

the components of the first reagent are as follows: 100mmol/L phosphate buffer solution, 6g/L sodium chloride, 30 g/L2, 4, 6-tribromo-3-hydroxybenzoic acid, 0.1g/L Triton X-100, 40g/L glycerol, 100KU/L creatinase, 200KU/L creatininase, 50KU/L ascorbate oxidase and 200g/L water-soluble starch;

the components of the second reagent are as follows: 20mmol/L of 3-morpholine propanesulfonic acid buffer solution, 10g/L of sodium chloride, 10g/L of 4-aminoantipyrine, 0.005g/L of bilirubin interference removal agent, 10g/L of Triton X-100, 10g/L of glycerol, 200KU/L of peroxidase and 100g/L of water-soluble starch;

a third reagent: 1kg/L of said second agent and 500KU/L of creatininase.

The volume of the first reagent ball, the second reagent ball and the third reagent ball is about 2.5 ul.

Example 3

The present example differs from example 1 as follows:

the components of the first reagent are as follows: 80mmol/L of BICINE buffer solution, 5g/L of sodium chloride, 10g/L of 2, 4, 6-tribromo-3-hydroxybenzoic acid, 5g/L of span 40, 10g/L of polyoxyethylene ether, 300KU/L of creatinase, 100KU/L of sarcosine oxidase, 200KU/L of ascorbic acid oxidase and 100g/L of inositol;

the components of the second reagent are as follows: 600mmol/L of TAPS buffer solution, 10g/L of sodium chloride, 5g/L of 4-aminoantipyrine, 0.5g/L of bilirubin oxidase, 0.1g/L of Tween-80, 30g/L of fatty alcohol, 100KU/L of peroxidase and 150g/L of mannitol;

a third reagent: 1kg/L of said second agent and 350KU/L of creatininase.

The volume of the first reagent ball, the second reagent ball and the third reagent ball is about 3 ul.

The performance of the blood creatinine assay chip obtained in example 1 of the present invention will be described below with reference to the table. It should be noted that, in the embodiments of the present invention, a portable biochemical analyzer is used to measure the change value of the absorbance of the measurement chip injected with the detection sample (i.e., the serum sample) at 37 ℃; and the concentration of the blood creatinine in the detection sample can be calculated and obtained by using a calibration product provided by British Landau company for calibration.

(1) And testing precision: the blood creatinine detection chip provided by the embodiment 1 of the invention is adopted to test the blood creatinine concentration in a detection sample with the known blood creatinine concentration of 127umol/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, X_iThe 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 detection sample for 20 times129umol/L, standard deviation SD 2.331, coefficient of variation CV 1.81%. 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 blood creatinine detection chip provided by the embodiment 1 of the invention is adopted to test a detection sample with known blood creatinine concentration of 404umol/L for three times to obtain the measured blood creatinine concentration value, and the average value of the blood creatinine concentration values obtained by 3 times of measurement is calculated to be 397umol/L, and the relative deviation is-1.82%.

(3) Clinical relevance analysis:

the blood creatinine content of a plurality of detection samples with different blood creatinine concentrations is simultaneously determined by adopting the blood creatinine detection chip provided by the embodiment 1 of the invention and an Abaxis reagent disk. The corresponding measured blood creatinine concentration value (unit umol/L) is shown in Table I, wherein the X column is the blood creatinine concentration measured by the Abaxis reagent disk in the serum sample, and the Y column is the blood creatinine concentration measured by the blood creatinine detection chip provided in embodiment 1 of the present invention. For example, for the test sample No. 1, the concentration of creatinine in blood tested using the Abaxis reagent disk was 168umol/L, while the concentration of creatinine in the blood serum sample No. 1 tested using the blood creatinine assay chip provided in example 1 of the present invention was 155 umol/L.

According to the data in the table one, and obtaining fig. 1, as shown in fig. 1, the correlation equation between the two sets of test results of the blood creatinine detection chip provided by the embodiment of the present invention and the Abaxis reagent disk is as follows:

y＝1.0541x-13.267

the closer the correlation coefficient R is to 1, the stronger the correlation between the two sets of data 0.9947. Therefore, the correlation between the test results of the blood creatinine detection chip provided by the embodiment of the invention and the test results of the Abaxis reagent disk is strong.

Table one: clinical correlation analysis table of the blood creatinine detecting reagent kit.

(4) Linear range test

The test method is as follows: the high concentration test sample and the low concentration test sample were mixed at different ratios into 9 diluted concentration test samples as shown in Table II using a high concentration (activity) sample near the upper limit of the linear range ([50, 2000] umol/L) and a 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:

the blood creatinine detection chip provided by the embodiment 1 of the invention is adopted to respectively test the blood creatinine concentration of 9 detection samples, each detection sample is tested for 3 times, and the average value (y) of the concentration values of the blood creatinine in the 9 detection samples is respectively calculated_i). Concentration (x) after dilution with each sample_i) As an independent variable, perMean value of measured concentration values (y) of individual samples_i) 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, x_iTo dilute the concentration, y_iThe average value of the measurement results is shown.

As shown in fig. 2, the obtained linear regression equation is y-0.9709 x +4.402, and the correlation coefficient R-0.9996.

Generally, when the kit detects a detection sample with the blood creatinine concentration within the [50, 2000] umol/L interval, the linear correlation coefficient R is more than or equal to 0.990, and the requirement is met. Therefore, the blood creatinine detection reagent ball 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 blood creatinine detection reagent ball provided by the 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 the plurality of test chips provided in example 1 were stored in a dark environment at 37 ℃ for 0, 2, 3, 4, 6, and 8 days, the blood creatinine concentrations in two sets of calibrators (sample 1 and sample 2) provided by the british langway company were measured several times, respectively, to analyze the average value and the relative deviation (in umol/L) of the results of the several measurements, thereby analyzing the test accuracy of the test chips, and the test results 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 concentration of the blood creatinine 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 blood creatinine 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	421	417	423	420	404	4.06％
2 days	417	408	411	412	404	1.98％
							3 days	418	416	413	416	404	2.89％
4 days	414	416	411	414	404	2.39％
							6 days	408	406	405	406	404	0.58％
8 days	399	397	400	399	404	-1.32％

Table four: thermal stability analysis table.

Sample 2	1	2	3	Mean value of	Target value	Relative deviation of
							Day 0	129	131	133	131	127	3.20％
2 days	126	130	129	128	127	0.92％
							3 days	121	127	124	124	127	-2.36％
4 days	123	125	122	123	127	-2.89％
							6 days	130	121	124	125	127	-1.57％
8 days	123	126	120	123	127	-3.15％

(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 blood creatinine concentrations in two sets of calibrators (sample 3 and sample 4) provided by the british lambdado company were measured several times to analyze the average value and the relative deviation (in umol/L) of the results of the several measurements, so as to analyze 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	392	401	398	397	404	-1.69％
3 month	411	408	396	405	404	0.25％
							6 month	391	399	399	396	404	-1.90％
9 month	400	394	403	399	404	-1.24％
							12 month	401	391	403	398	404	-1.40％
15 month	363	399	395	386	404	-4.54％

Table six: long term stability analysis table.

Sample 4	1	2	3	Mean value of	Target value	Relative deviation of
							0 month	117	126	130	124	127	-2.10％
3 month	116	113	128	119	127	-6.30％
							6 month	119	123	120	121	127	-4.99％
9 month	120	128	116	121	127	-4.46％
							12 month	124	113	112	116	127	-8.40％
15 month	117	117	126	120	127	-5.51％

Table five and table six are the detection results of the detection chip for detecting the concentration of the blood creatinine 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 value in each table is the actual concentration of the blood creatinine 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 of 2-8 ℃ for a long time.

(7) Anti-interference capability test

By adopting the blood creatinine detection chip provided by the embodiment of the invention, detection samples containing different interferents and having known actual concentrations are respectively detected, and the actual concentration of the blood creatinine in each detection sample is 404 umol/L. The type of interferent is different in each test sample.

And the seventh table is an analysis table of the anti-interference capability of the detection chip provided by the embodiment of the invention on the original substances in the serum. As can be seen from table seven, when the sample to be tested contains ascorbic acid, bilirubin, hemoglobin or triglyceride, the difference between the concentration of creatinine in blood and the actual concentration of creatinine in the sample to be tested, which is determined by the chip for testing blood creatinine provided in the embodiment of the present invention, is no more than ± 10.0%.

Table eight is an analysis table of the anti-interference capability of the detection chip provided in the embodiment of the present invention for the patient to take the medicine. It can be seen from table eight that, when the detection sample contains calcium dobesilate, etamsylate, methyldopa, levodopa, and dopamine, the deviation between the detected concentration of the blood creatinine and the actual concentration of the blood creatinine in the detection sample, which is detected by the blood creatinine detection chip provided by the embodiment of the present invention, is not more than ± 10.0%.

According to the experimental results in the seventh and eighth tables, the blood creatinine detection reagent ball and the detection chip provided by the embodiment of the invention have strong anti-interference capability on the original interferents in serum and the drug interferents taken by patients.

TABLE VII: the interference ability of original substances in antiserum is analyzed.

Table eight: anti-drug interference ability analysis table.

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.