Freeze-drying reagent for citric acid determination, biochemical analysis chip and determination method thereof

Abstract

The invention discloses a freeze-drying reagent for citric acid determination. The freeze-drying reagent is prepared from a first freeze-drying reagent ball and a second freeze-drying reagent ball which are obtained by freeze-drying liquid drops of a first citric acid determination reagent and a second citric acid determination reagent, the first citric acid determination reagent comprises citrate lyase, lactic dehydrogenase, malate dehydrogenase, a first buffer solution, an additive, a first cryoprotectant and a first surfactant; the second citric acid determination reagent comprises reduced coenzyme, a second surfactant, a second buffer solution and a second cryoprotectant. The biochemical analysis chip comprises a chip body, the chip body is provided with a colorimetric hole and a diluent groove, the first freeze-drying reagent ball and the second freeze-drying reagent ball are contained in the colorimetric hole, and diluent is contained in the diluent groove. The method for measuring the citric acid comprises the following steps: treating a sample, and adding the treated sample into a heated colorimetric hole to react with a freeze-drying reagent ball; testing the absorbance variation and / or the decline rate of the absorbance value of the solution in the colorimetric hole under the specific wavelength; based on this, a citric acid concentration value is determined.

Description

Technical Field

[0001] The invention relates to the technical field of citric acid determination, and in particular to a freeze-dried reagent, a biochemical analysis chip and a determination method thereof for citric acid determination. Background Art

[0002] Citrate / citric acid plays an important role in the metabolism of organisms and is an important component of pharmaceutical preparations, foods, beverages and various industrial products. Citric acid is used as a preservative, flavor enhancer, acidulant and fermentation substrate in the natural and processed food industry; in the pharmaceutical industry, citric acid is used as a stabilizer for various pharmaceutical preparations, as a counter anion in the preparation of active ingredients, and in the form of an acid in combination with the effervescent carbonates and bicarbonates. Citric acid is an important biomarker for the human body, and its determination is an important topic in biomedicine and clinical medicine. The determination of citric acid is used to diagnose various diseases and pathological conditions associated with abnormal citric acid concentrations, such as for detecting carcinogenesis in prostate tumors, or for assessing the risk of kidney disease and kidney stones. Other medical uses of citric acid include its use as an anticoagulant in coagulation experiments and blood bank storage.

[0003] Clinically, the main methods for detecting citric acid include enzymatic method, spectrophotometry, fluorescence spectroscopy, liquid chromatography, liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry, nuclear magnetic resonance and capillary electrophoresis. Among them, the enzymatic method has become the most commonly used method because of its high selectivity. The reliable and reproducible two-step spectrophotometric method for determining citric acid using citrate lyase was first proposed by Tomisek et al. in 1966. Ten years later, McWinney et al. further improved the method to achieve automation. This method has been successfully used to quantify the concentration of citric acid in blood and urine, and its performance is comparable to that of the gold standard: liquid chromatography. At present, the determination has become the reference method for the determination of citrate and citric acid (see European Standard EN1137: 1994-12).

[0004] All of the above methods require a series of sample pretreatment processes and require large-scale instruments for assistance. They have the disadvantages of complicated detection process, long time consumption, high cost, and poor thermal stability of reagents. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects of the prior art, and to provide a freeze-dried reagent for citric acid determination, a biochemical analysis chip and a determination method thereof in view of the problems of the prior art, such as the cumbersome detection process, long time consumption, high cost, and poor thermal stability of the reagent. The method is simple in sample pretreatment, greatly simplifies the detection procedure, and the whole process only takes 5-15 minutes. It has the advantages of simple detection process, short time consumption, cost saving, good thermal stability of the reagent, etc. The method can be used to quantitatively detect citrate in the sample.

[0006] A first aspect of the present invention is to provide a lyophilized reagent for citric acid determination, which is used for the determination of citric acid in a biological sample or a non-biological sample, wherein the lyophilized reagent is made of first lyophilized reagent spheres and second lyophilized reagent spheres formed by freeze-drying droplets of a first citric acid determination reagent and a second citric acid determination reagent;

[0007] The first citric acid assay reagent comprises the following components: citrate lyase, lactate dehydrogenase, malate dehydrogenase, a first buffer, an additive, a first cryoprotectant, and a first surfactant;

[0008] The second citric acid assay reagent comprises the following components: reduced coenzyme, a second surfactant, a second buffer and a second cryoprotectant.

[0009] Preferably, the citrate lyase includes citrate lyase prepared by integrating the genes of a microorganism into a microorganism through gene recombination technology, and citrate lyase prepared by a microorganism with improved properties through genetic modification;

[0010] The lactate dehydrogenase includes L-lactate dehydrogenase or D-lactate dehydrogenase; the source of the L-lactate dehydrogenase includes animal or microbial sources; the D-lactate dehydrogenase includes D-lactate dehydrogenase of microbial origin, Lactobacillus reinhardtii, Pseudomonas aeruginosa or Staphylococcus;

[0011] The malate dehydrogenase includes malate dehydrogenase of animal, plant or microbial origin;

[0012] The first buffer is an alkaline buffer, which includes a buffer that allows the pH value of the solution to be buffered to a range of 7-9.5, including tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)methylglycine, 4-hydroxyethylpiperazinepropanesulfonic acid, 4-hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethaneethanesulfonic acid, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-morpholinepropanesulfonic acid or 1,4-piperazinediethanesulfonic acid and bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane);

[0013] The additives include divalent metal salts, sugars, sugar alcohols, proteins, chelating agents, amines, and basic amino acids or heterocyclic compounds; wherein the divalent metal salts include sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium acetate, or potassium acetate, and the protein includes bovine serum albumin;

[0014] The first cryoprotectant includes sugars, polyols, amino acids, proteins and peptide excipients, and cyclic oligosaccharide-based polymers, wherein the sugars include one or more of glucose, sucrose, lactose, maltose and trehalose; the polyols include one or more of mannitol, inositol, ethylene glycol and polyethylene glycol; the amino acids include trimethylglycine, glycine, arginine or any salt thereof; the protein and peptide excipients include mucopolysaccharide protein, casein or bovine serum albumin; the cyclic oligosaccharide-based polymer includes any one or more combinations of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, β-cyclodextrin sulfobutyl ether and any derivatives thereof;

[0015] The first surfactant includes a nonionic surfactant; the nonionic surfactant includes one or more combinations of polyethylene glycol monooctylphenyl ether, sorbitan monolaurate, sorbitan monopalmitate, polyoxyethylene lauryl ether, polyoxyethylene cetyl ester, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monolaurate, Tween 20, Tween 40, Tween 80 and Tween 60.

[0016] Preferably, the reduced coenzyme comprises reduced β-nicotinamide adenine dinucleotide NADH or any salt thereof, reduced β-nicotinamide adenine dinucleoside phosphate NADPH or any salt thereof, wherein any salt comprises reduced β-nicotinamide adenine dinucleotide disodium salt, reduced β-nicotinamide adenine dinucleotide dipotassium salt or reduced triphosphate pyridine nucleotide tetrasodium salt;

[0017] The second surfactant comprises a nonionic surfactant, and the nonionic surfactant comprises a combination of one or more of polyethylene glycol monooctylphenyl ether, sorbitan monolaurate, sorbitan monopalmitate, polyoxyethylene lauryl ether, polyoxyethylene cetyl ester, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monolaurate, Tween 20, Tween 40, Tween 80 and Tween 60;

[0018] The second cryoprotectant includes sugars, polyols, amino acids, proteins and peptide excipients, wherein the sugars include glucose, sucrose, lactose, maltose or trehalose; the polyols include mannitol, inositol, ethylene glycol or polyethylene glycol; the amino acids include trimethylglycine, glycine, arginine or any salt thereof; the protein and peptide excipients include mucopolysaccharide protein, casein or bovine serum albumin;

[0019] The second buffer includes a buffer capable of buffering the solution pH to a range of 7-10, including tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)methylglycine, 4-hydroxyethylpiperazinepropanesulfonic acid, 4-hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethaneethanesulfonic acid, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-morpholinepropanesulfonic acid, 1,4-piperazinediethanesulfonic acid or bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane.

[0020] Preferably, the preparation method of the first citric acid assay reagent and the second citric acid assay reagent comprises:

[0021] Prepare an enzyme protection solution: comprising a first buffer solution, an additive, a first cryoprotectant and a first surfactant at a certain concentration;

[0022] Prepare enzyme solution: containing a certain concentration of citrate lyase, lactate dehydrogenase, malate dehydrogenase, a first buffer and additives;

[0023] Prepare substrate solution: including a second buffer solution and reduced coenzyme at a certain concentration;

[0024] preparing a substrate protection solution comprising a second surfactant, a second buffer and a second cryoprotectant at a certain concentration;

[0025] The enzyme protection solution is fully mixed with the first citric acid assay reagent in a certain ratio and order;

[0026] The substrate solution and the substrate protection solution are fully mixed in a certain ratio and order to form the second citric acid determination reagent.

[0027] Preferably, the preparation method of the first freeze-dried reagent ball and the second freeze-dried reagent ball comprises:

[0028] Freezing the first citric acid assay reagent and the second citric acid assay reagent to form a first frozen solid and a second frozen solid respectively; comprising: dropping the liquid droplets of the first citric acid assay reagent and the second citric acid assay reagent into liquid nitrogen using a precise quantitative liquid dispensing system, so that the liquid droplets of the first citric acid assay reagent and the second citric acid assay reagent condense into an ice ball in the liquid nitrogen; adjusting the volume of the ice ball by adjusting and controlling the size of the liquid droplets of the first citric acid assay reagent and the second citric acid assay reagent dropped into the liquid nitrogen;

[0029] The first frozen solid and the second frozen solid are placed in a vacuum freeze dryer, and freeze-dried under vacuum to obtain the first freeze-dried reagent balls and the second freeze-dried reagent balls. After nitrogen re-pressurization, the first freeze-dried reagent balls and the second freeze-dried reagent balls are collected and stored in a dry aluminum bottle; wherein the drying is divided into two stages: primary drying and secondary drying; the primary drying is used to remove frozen water or solvent based on sublimation to form a solidified solution; the secondary drying is used to remove unfrozen "bound" water or solvent from the solidified solution based on desorption.

[0030] The second aspect of the present invention is to provide a biochemical analysis chip, which includes a chip body, the first freeze-dried reagent ball and the second freeze-dried reagent ball as in the first aspect, and a diluent, the chip body is provided with a colorimetric hole and a diluent tank, the first freeze-dried reagent ball and the second freeze-dried reagent ball are accommodated in the colorimetric hole, and the diluent is accommodated in the diluent tank of the chip body.

[0031] Preferably, the biochemical analysis chip further comprises a sample quantitative separation structure, a mixing mechanism, a reaction well and a pore modification component.

[0032] The third aspect of the present invention is to provide a method for determining citric acid, which is implemented based on a lyophilized reagent for determining citric acid, comprising:

[0033] Adding the processed biological sample or non-biological sample to the colorimetric well of the biochemical analysis chip of the second aspect; the sample comprises a biological sample or a non-biological sample; the biological sample comprises a blood sample, a urine sample and / or a semen sample;

[0034] heating the biochemical analysis chip to an appropriately selected constant temperature, wherein the appropriately selected constant temperature is selected from 25° C. to 37° C.;

[0035] The treated biological sample or non-biological sample is fully reacted with the first freeze-dried reagent ball and the second freeze-dried reagent ball in the colorimetric well at the appropriately selected constant temperature; the reaction time for the full reaction is 5-15 minutes;

[0036] Using a spectrophotometer or an automatic analytical instrument commonly used in clinical practice to test the absorbance change and / or the rate of decrease of the absorbance value of the solution in the colorimetric well at a specific wavelength of a light source generated by the spectrophotometer or the automatic analytical instrument commonly used in clinical practice based on spectrophotometry;

[0037] The concentration value of citric acid is determined based on the absorbance change and / or the rate of decrease of the absorbance value.

[0038] Preferably, the processing of the biological sample or the non-biological sample comprises:

[0039] The biological sample or the non-biological sample is separated into a certain amount of plasma, urine or seminal plasma sample or non-biological sample plasma through chip centrifugation and quantitative structure;

[0040] The separated plasma, urine or seminal plasma sample or non-biological sample slurry is filled into the mixing tank and diluted with the diluent.

[0041] Preferably, the specific wavelength of the light source is 320-360 nm.

[0042] The beneficial effects of the freeze-dried reagent for citric acid determination, the biochemical analysis chip and the determination method thereof of the present invention are as follows:

[0043] The present invention aims at the problems of complicated detection steps, complicated operation and long time consumption in the prior art. Based on the traditional citric acid lyase method, a freeze-dried reagent ball, a biochemical analysis chip and a method for measuring citric acid are proposed. By simultaneously freeze-drying the reagent ball 1 and the reagent ball 2 with the biochemical analysis chip, timely detection of citric acid from sample to result is achieved. Through simple operation, the citric acid level in the liquid is quantitatively measured with high precision. At the same time, this product establishes a system for micro-determination of citric acid, and the purpose of accurate determination can be achieved using a small amount of sample. The method saves costs, is easy to operate, has simple equipment, a small amount of sampling, good thermal stability of the reagent, and is easy to be promoted and used clinically.

[0044] (1) The present invention provides a freeze-dried reagent ball and a diluent for determining citric acid, wherein the freeze-dried reagent ball is made by freeze-drying droplets of a citric acid determination reagent. Compared with conventional enzymatic analysis, the present invention improves the reagent composition, adds a specific freeze-dried protective agent composition and a specific buffer system and reactant concentration combination to the freeze-dried reagent, realizes the transformation of the conventional reagent into freeze-dried microspheres, innovates the configuration process and formula of the determination reagent, saves the reagent cost, and improves the thermal stability and repeatability of the reagent.

[0045] (2) The present invention provides a biochemical analysis chip, which comprises a chip body and a freeze-dried reagent ball and a diluent as described in the first aspect, wherein the chip body is provided with a colorimetric hole and a diluent tank, wherein the freeze-dried reagent ball is received in the colorimetric hole, and the diluent is received in the diluent tank of the chip body. The chip is the core part of the citric acid determination, and the pretreatment of biological samples, quantitative separation of biological samples such as plasma, urine, and seminal plasma, quantitative mixing of samples and diluent, and the final citric acid quantitative detection reaction are all completed in the chip.

[0046] (3) The present invention provides a citric acid determination method, which is applied to the biochemical analysis chip as described in the second aspect, and the method includes: the biochemical analysis chip as described in the second aspect is combined with an appropriate detection method to complete the quantitative detection of citric acid. Including: controlling the reaction time, reaction temperature, measurement of absorbance change rate, and calculation of citric acid concentration. This method can realize real-time detection of citric acid, and only requires a small amount of whole blood, urine, or seminal plasma sample to be dripped into the sample hole of the chip, and the citric acid concentration value can be obtained after about 5-15 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the related technologies, the drawings required for use in the specific embodiments or the related technical descriptions will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0048] Figure 1 A flow chart of a determination method according to an embodiment of the present invention;

[0049] Figure 2 This is a schematic diagram of the detection ability of the detection method provided in Example 1 of the present invention for citric acid, wherein the ordinate is the change in absorbance per minute at 340 nm, and the abscissa is the citric acid concentration.

[0050] Figure 3 This is a schematic diagram of the detection ability of the detection method provided in Example 2 of the present invention for citric acid, wherein the ordinate is the change in absorbance at 340 nm per minute, and the abscissa is the citric acid concentration. DETAILED DESCRIPTION

[0051] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0053] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0054] This embodiment provides a lyophilized reagent for citric acid determination, which is used for the determination of citric acid in a biological sample or a non-biological sample. The lyophilized reagent is made of a first lyophilized reagent ball and a second lyophilized reagent ball formed by freeze-drying droplets of a first citric acid determination reagent and a second citric acid determination reagent;

[0055] The first citric acid assay reagent comprises the following components:

[0056] Citrate lyase [EC 4.1.3.6], lactate dehydrogenase, malate dehydrogenase [EC 1.1.1.37], a first buffer, an additive, a first cryoprotectant and a first surfactant; wherein:

[0057] The source of the citrate lyase [EC4.1.3.6] is not limited. For example, microbial sources (e.g., Klebsiella pneumoniae, Enterococcus faecalis, Lactobacillus casei) include citrate lyase prepared by integrating the genes of microorganisms into microorganisms such as Escherichia coli through genetic recombination technology, and citrate lyase prepared by microorganisms with improved characteristics through genetic modification. From the perspective of stability, availability and batch difference, in the present invention, it is preferred to use citrate lyase derived from a microorganism belonging to Klebsiella pneumoniae.

[0058] The lactate dehydrogenase includes L-lactate dehydrogenase [EC1.1.1.27] or D-lactate dehydrogenase [EC1.1.1.28].

[0059] The source of the L-lactate dehydrogenase [EC1.1.1.27] is not limited, and L-lactate dehydrogenases from animals (e.g., pig heart, cow heart, rabbit muscle) or microbial sources (e.g., Bacillus subtilis, Staphylococcus aureus, Streptococcus) can be used.

[0060] The source of the D-lactate dehydrogenase [EC1.1.1.28] is not limited, including but not limited to D-lactate dehydrogenase from microbial sources, such as Lactobacillus reinhardtii, Pseudomonas aeruginosa or Staphylococcus.

[0061] The source of the malate dehydrogenase [EC1.1.1.37] is not limited, including malate dehydrogenase from animals (e.g., pig heart, cow heart) or plants (e.g., citrus, soybean, rice, wheat, corn) or microorganisms (e.g., fission yeast, Streptomyces, Bacillus).

[0062] The first buffer is an alkaline buffer, which includes a buffer capable of buffering the pH value of the solution to a range of 7-9.5, such as tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)methylglycine, 4-hydroxyethylpiperazinepropanesulfonic acid, 4-hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethaneethanesulfonic acid, N,N-di(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-morpholinepropanesulfonic acid, 1,4-piperazinediethanesulfonic acid and bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane.

[0063] The additives include divalent metal salts such as chlorides, sulfates and acetates of sodium, potassium, etc., sugars, sugar alcohols, and proteins, including but not limited to bovine serum albumin, as well as chelating agents, amines, basic amino acids or heterocyclic compounds.

[0064] The first cryoprotectant includes sugars, polyols, amino acids, proteins and peptide excipients, and cyclic oligosaccharide-based polymers, sugars include but are not limited to glucose, sucrose, lactose, maltose and trehalose; polyols include but are not limited to mannitol, inositol, ethylene glycol and polyethylene glycol, etc.; amino acids include trimethylglycine, glycine, arginine or any salt thereof; protein and peptide excipients include mucopolysaccharide protein, casein, bovine serum albumin, etc.; cyclic oligosaccharide-based polymers include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, β-cyclodextrin sulfobutyl ether, or any derivative thereof, and any combination thereof. In addition to being used as stabilizers, mannitol and sucrose are also used as excipients, which help to give the freeze-dried reagent balls a good appearance, making the freeze-dried reagent balls loose and porous, and easy to re-dissolve.

[0065] The first surfactant includes a nonionic surfactant; the nonionic surfactant includes polyethylene glycol monooctylphenyl ether (TritonX-100), sorbitan monolaurate (Span20), sorbitan monopalmitate (Span40), polyoxyethylene lauryl ether (Brij35), polyoxyethylene cetyl ester (Brij58), polyoxyethylene sorbitol monolaurate (Brij58), polyoxyethylene sorbitol monolaurate (Span40), Tween 20, Tween 40, Tween 80 and Tween 60. These surfactants can be used alone or in combination of two or more.

[0066] First citric acid assay reagent application example: In a preferred embodiment of the present invention, the reagent contains 1-1000U / ml, preferably 10-200U / ml of citrate lyase; 0.5-500U / ml, preferably 5-100U / ml of lactate dehydrogenase; 0.5-500U / ml, preferably 5-100U / ml of malate dehydrogenase; 0.0001 to 10wt%, preferably 0.001 to 1wt% of surfactant; 0.1-60wt% of excipient; 0.1-30wt% of additive; 1-100mmol / L buffer to adjust the pH to 7.0-10.0. It is particularly preferred that the buffer concentration is 10-500mmol / L to adjust the pH to the range of 7.5-9.5.

[0067] The second citric acid assay reagent comprises the following components: a reduced coenzyme, a second surfactant, a second buffer and a second cryoprotectant; wherein:

[0068] The reduced coenzyme comprises reduced β-nicotinamide adenine dinucleotide (NADH, prototype coenzyme I), reduced β-nicotinamide adenine dinucleoside phosphate (NADPH, prototype coenzyme II), or any salt thereof, including but not limited to reduced β-nicotinamide adenine dinucleotide disodium salt, reduced β-nicotinamide adenine dinucleotide dipotassium salt and reduced triphosphate pyridine nucleotide tetrasodium salt.

[0069] The second surfactant includes a nonionic surfactant. The nonionic surfactant includes polyethylene glycol monooctylphenyl ether (TritonX-100), sorbitol monolaurate (Span20), sorbitan monopalmitate (Span40), polyoxyethylene lauryl ether (Brij35), polyoxyethylene cetyl ester (Brij58), polyoxyethylene sorbitol monolaurate (Brij58), polyoxyethylene sorbitol monolaurate (Span40), Tween 20, Tween 40, Tween 80 and Tween 60. The second surfactant can be used alone or in combination of two or more.

[0070] The second cryoprotectant includes sugars, polyols, amino acids, proteins and peptide excipients, sugars include but are not limited to glucose, sucrose, lactose, maltose and trehalose; polyols include but are not limited to mannitol, inositol, ethylene glycol and polyethylene glycol, etc.; amino acids include trimethylglycine, glycine, arginine or any salt thereof; protein and peptide excipients include mucopolysaccharide protein, casein, bovine serum albumin, etc. In addition to being used as stabilizers, mannitol and sucrose are also used as excipients to help give the freeze-dried reagent balls a good appearance, making the freeze-dried reagent balls loose and porous and easy to re-dissolve.

[0071] The second buffer includes those capable of buffering to a pH in the range of 7 to 10, including but not limited to tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)methylglycine, 4-hydroxyethylpiperazinepropanesulfonic acid, 4-hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethaneethanesulfonic acid, N,N-di(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-morpholinepropanesulfonic acid, 1,4-piperazinediethanesulfonic acid and bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane.

[0072] Application example of the second citric acid assay reagent: In a preferred embodiment of the present invention, the second citric acid assay reagent contains 0.2 to 500 mmol / L, preferably 1 to 100 mmol / L of reduced coenzyme; 0.0001 to 10 wt%, preferably 0.001 to 1 wt% of surfactant; 0.1-60 wt% of excipient; and a buffer is added to adjust the pH to 7.0 to 10.0 and the concentration to 1 to 1000 mmol / L. It is particularly preferred to adjust the pH to the range of 8.0-9.5 and the concentration to the range of 10-100 mmol / L.

[0073] As a preferred embodiment, the preparation method of the first citric acid assay reagent and the second citric acid assay reagent comprises:

[0074] Prepare an enzyme protection solution: comprising a first buffer solution, an additive, a first cryoprotectant and a first surfactant at a certain concentration;

[0075] Prepare enzyme solution: containing a certain concentration of citrate lyase, lactate dehydrogenase, malate dehydrogenase, a first buffer and additives;

[0076] Prepare substrate solution: including a second buffer solution and reduced coenzyme at a certain concentration;

[0077] preparing a substrate protection solution comprising a second surfactant, a second buffer and a second cryoprotectant at a certain concentration;

[0078] The enzyme protection solution is fully mixed with the first citric acid assay reagent in a certain ratio and order;

[0079] The substrate solution and the substrate protection solution are fully mixed in a certain ratio and order to form the second citric acid determination reagent.

[0080] The present embodiment also provides a biochemical analysis chip, which includes a chip body, a first freeze-dried reagent ball and a second freeze-dried reagent ball, and a diluent. The chip body is provided with a colorimetric hole and a diluent tank. The first freeze-dried reagent ball and the second freeze-dried reagent ball are accommodated in the colorimetric hole, and the diluent is accommodated in the diluent tank of the chip body.

[0081] As a preferred embodiment, the biochemical analysis chip further includes a sample quantitative separation structure, a mixing mechanism, a reaction well and a pore modification component.

[0082] This embodiment also provides a method for determining citric acid, which is implemented based on a freeze-dried reagent for citric acid determination, and is used to perform an assay of an analyte from a sample. The overall detection process of the reagent, chip, and instrument forms a complete solution for rapid quantitative detection of citric acid.

[0083] include:

[0084] Adding the processed sample to the colorimetric well of the biochemical analysis chip; the sample includes a biological sample or a non-biological sample; the biological sample includes a blood sample, a urine sample and / or a semen sample;

[0085] heating the biochemical analysis chip to an appropriately selected constant temperature, wherein the appropriately selected constant temperature is selected from 25° C. to 37° C.;

[0086] The diluted fluid sample fully reacts with the first freeze-dried reagent ball and the second freeze-dried reagent ball in the colorimetric well at the appropriately selected constant temperature; the reaction time for the full reaction is 5-15 minutes;

[0087] A spectrophotometer or an automatic analytical instrument commonly used in clinical practice is used to test the absorbance change and / or the rate of decrease of the absorbance value of the solution in the colorimetric well under a specific wavelength of a light source generated by the spectrophotometer or an automatic analytical instrument commonly used in clinical practice based on spectrophotometry; wherein the specific wavelength of the light source is 320-360 nm, preferably 340 nm;

[0088] The concentration value of citric acid is determined based on the absorbance change and / or the rate of decrease of the absorbance value.

[0089] As a preferred embodiment, the processing of the sample comprises:

[0090] The sample is subjected to chip centrifugation and quantitative structure to separate a certain amount of plasma, urine or seminal plasma sample;

[0091] The separated plasma, urine or seminal plasma sample is filled into the mixing tank and diluted with the diluent.

[0092] Application Examples and Comparative Examples

[0093] The embodiment of the present invention provides a lyophilized reagent for determining human citric acid, wherein the lyophilized reagent is prepared by freeze-drying droplets of a citric acid determination reagent 1 and a citric acid determination reagent 2. Specifically, the citric acid determination reagent 1 includes the following components: citrate lyase, lactate dehydrogenase, malate dehydrogenase, buffer, additive, cryoprotectant, and surfactant; the citric acid determination reagent 2 includes the following components: reduced coenzyme, surfactant, buffer, and cryoprotectant.

[0094] In some embodiments, the preparation method of the citric acid assay reagent is as follows: (1) first prepare an enzyme protection solution: comprising a certain concentration of buffer, additives, cryoprotectants, and surfactants; (2) prepare an enzyme solution: comprising a certain concentration of citrate lyase, lactate dehydrogenase, malate dehydrogenase, buffer, and additives; (3) prepare a substrate solution: comprising a certain concentration of buffer and reduced coenzyme; (4) a substrate protection solution: comprising a certain concentration of surfactant, buffer, and cryoprotectants. (1) and (2) are fully mixed in a certain proportion and order to form a lyophilized reagent 1; (3) and (4) are fully mixed to form a lyophilized reagent 2.

[0095] Freeze drying traditionally consists of two major steps: (1) freezing of the solution, and (2) drying of the frozen solid under vacuum. The drying step is further divided into two stages: primary drying and secondary drying. The primary drying step attempts to remove the frozen water or solvent (sublimation), and the secondary drying step attempts to remove the unfrozen "bound" water or solvent from the solidified solution (desorption).

[0096] In some embodiments, the method for preparing the lyophilized reagent pellets comprises the following steps:

[0097] (1) Freezing of the solution: Drops of the citric acid assay reagent are dropped into liquid nitrogen using a precise quantitative dispensing system, so that the droplets of the citric acid assay reagent 1 and reagent 2 form ice balls; for example, droplets of the citric acid assay reagent can be dropped into liquid nitrogen using a dispensing machine, so that the droplets of the citric acid assay reagent condense into ice balls in the liquid nitrogen. Those skilled in the art can adjust the size of the droplets of the mixed solution dropped into the liquid nitrogen according to actual needs, and adjust the volume of the ice ball by controlling the size of the droplets. Optionally, in some embodiments, the volume of the citric acid assay reagent 1 and reagent 2 is 2-10 μl.

[0098] (2) Drying of frozen solids under vacuum: freeze-drying the ice balls to obtain freeze-dried reagent balls;

[0099] In this embodiment, the ice balls are placed in a vacuum freeze dryer for freeze drying to form freeze-dried reagent balls, and after nitrogen re-pressurization, the freeze-dried reagent balls are collected and stored in a dry aluminum bottle.

[0100] Freeze drying is the process of removing water or other solvents by freeze drying, which stabilizes the reagent by greatly reducing the degradation rate of the components. The removal of the solvent is first accomplished by freezing the solution, so that the freezing process separates one or more solvents from the solute and fixes any unfrozen solvent molecules in the interstitial areas between the frozen solvent crystals. The solvent is then removed by sublimation (primary drying) and then by desorption (secondary drying). After freeze drying, the citrate lyase, lactate dehydrogenase, malate dehydrogenase, buffer, additives, cryoprotectants, surfactants in the citric acid assay reagent 1; the reduced coenzyme, surfactant, buffer, cryoprotectants, etc. in the reagent 2 remain in the ice shelf during freezing, so the freeze-dried reagent ball after freeze drying is loose and porous, and its volume remains basically the same as before freeze drying. Since the freeze-dried reagent ball is always in a frozen state before drying, and the ice crystals are evenly distributed in the various components of the citric acid assay reagent. During the sublimation process, the components will not be concentrated due to dehydration. Therefore, the freeze-dried reagent ball after freeze drying is spongy, loose and porous, and is very easy to dissolve in water and return to its original state. The freeze-dried reagent balls provided by the embodiment of the present invention are suitable for large-scale batch production, and the solid freeze-dried reagent balls are more convenient to store and transport.

[0101] The embodiment of the present invention provides a freeze-dried reagent 1 and a freeze-dried reagent 2 for determining the content of citric acid, and the determination principle is as follows: the citric acid in the sample produces oxaloacetic acid under the action of the citrate lyase in the freeze-dried reagent 1, and on the one hand, oxaloacetic acid oxidizes the reduced coenzyme in the reagent 2 to a coenzyme under the action of the malate dehydrogenase in the reagent 1; at the same time, oxaloacetic acid is converted into pyruvic acid under the action of decarboxylation, and pyruvic acid oxidizes the reduced coenzyme in the reagent 2 to a coenzyme under the action of the lactate dehydrogenase in the reagent 1, and the concentration of the reduced coenzyme is reduced in proportion to the concentration of citric acid, thereby causing the reaction solution to decrease in absorbance, so the absorbance value of the reaction solution at 340nm is inversely proportional to the concentration of citric acid within a certain range. Therefore, the concentration of the sample citric acid can be calculated by the rate of decrease in absorbance.

[0102] In this reaction system, by adding divalent metal ions and surfactants of specific concentrations to reagent 1, the sensitivity of the detection reagent citric acid concentration determination can be improved.

[0103] By adding a nonionic surfactant and a metal ion chelating agent to reagent 1 or reagent 2, the reaction process during the determination of the citric acid concentration in the sample can be stabilized, thereby improving the repeatability of the citric acid concentration determination.

[0104] By adding a suitable buffer solution to reagent 2, the reduced coenzyme in reagent 2 can be stabilized and the thermal stability of reagent 2 can be improved.

[0105] The embodiment of the present invention further provides a biochemical analysis chip, which comprises a chip body and the above-mentioned freeze-dried reagent balls 1 and 2. The chip body is provided with a colorimetric well, and the reagent balls 1 and 2 are received in the colorimetric well.

[0106] Figure 1 A schematic diagram of a citric acid determination method provided by an embodiment of the present invention is shown in FIG. Figure 1 The method shown comprises the following steps:

[0107] The sample is added to the biochemical analysis chip;

[0108] The sample comprises a biological sample or a non-biological sample;

[0109] Biological samples include blood samples, urine samples and semen samples;

[0110] The sample is separated into a certain amount of plasma, urine or seminal plasma through chip centrifugation and quantitative structure;

[0111] Dilute the separated plasma, urine or seminal plasma sample with a diluent;

[0112] Fill the diluted sample into the colorimetric well;

[0113] After the diluted sample fully reacts with the freeze-dried reagent balls 1 and 2 in the colorimetric wells, a spectrophotometer is used to test the absorbance change of the solution in the colorimetric wells.

[0114] In the centrifugal microfluidic biochemical analysis system, due to the self-contained centrifugal property, it is not necessary to centrifuge the sample to detect. In certain embodiments, the biochemical analysis chip includes a sample well, a sample tank, a diluent tank, a sample quantitative tank, a diluent quantitative tank and a mixing tank. When the biochemical analysis chip is used for testing, the sample is added to the sample tank from the sample well by a pipette, and the diluent is added to the diluent tank, and the diluent can be distilled water. The diluent is made to enter the diluent quantitative tank from the diluent tank in a centrifugal manner, so that the sample enters the sample quantitative tank from the sample tank, and the diluent in the diluent quantitative tank and the serum sample in the sample quantitative tank enter the mixing tank, so as to dilute the serum sample by the diluent. The serum sample diluted in the mixing tank is then filled into the colorimetric well by a centrifugal manner, and after the diluted sample is fully reacted with the freeze-dried reagent ball 1 and the reagent ball 2 in the colorimetric well, the absorbance change of the solution in the colorimetric well is tested by a spectrophotometer, and the wavelength of the light source generated by the spectrophotometer is 320-360nm, preferably 340nm.

[0115] To further illustrate the technical solution of the present invention, several embodiments of the preparation method of the citric acid determination reagent of the present invention are provided below.

[0116] Embodiment 1:

[0117] The citric acid assay reagent of this example includes the following components: Assay reagent 1 and reagent 2 having the following compositions are prepared, as shown in Table 1.

[0118] Table 1

[0119]

[0120] In this embodiment, the freeze-dried reagent balls are prepared by the above-mentioned preparation method of freeze-dried reagent balls, and the volume of the citric acid determination reagent 1 droplet is 4 μl, and the volume of the reagent 2 droplet is 2 μl.

[0121] The biochemical analysis chip in this embodiment includes a chip body and freeze-dried reagent balls 1 and 2 provided in this embodiment. The above citric acid determination method is used to detect the absorbance change of the sample per minute, and the wavelength of the light source selected during the test is 320-360nm, preferably 340nm.

[0122] Preparation of standards

[0123] Prepare multiple citric acid standards with different concentration gradients for constructing a standard curve. For example, more than 5 citric acid standards with a concentration range of 20 μmol / L-2000 μmol / L can be prepared. In one example, the freeze-dried citric acid standard is dissolved in double distilled water and then diluted to 2000, 1000, 500, 250, 125, 64, 32, and 16 μmol / L with double distilled water to construct a quantitative standard curve. Citric acid standards can be purchased from the China Food and Drug Inspection Institute.

[0124] The biochemical analysis chip of the present invention is used to test the concentration of citric acid. The test results are as follows: Figure 2 As shown in the figure, the relationship between citric acid concentration and absorbance change rate is as follows. In the range of citric acid concentration from 20 μmol / L to 2000 μmol / L, the absorbance change per minute is linearly related to the citric acid concentration.

[0125] Embodiment 2:

[0126] In order to demonstrate the applicability of different lyoprotectants of the reagents of the present invention, the citric acid level was determined in the same manner as in Example 1 except that the following reagents were used. The differences between this example and Example 1 are shown in Table 2 below.

[0127] Table 2

[0128]

[0129]

[0130] The wavelength of the light source generated by the spectrophotometer used in the test is 340nm, and the results obtained are as follows Figure 3 shown.

[0131] The relationship between citric acid concentration and absorbance change rate is shown in the figure. When the citric acid concentration is in the range of 20μmol / L-2000μmol / L, the absorbance change per minute is linearly related to the citric acid concentration.

[0132] Embodiment 3:

[0133] The reagent stability test of the present invention was carried out by taking the existing seminal plasma citric acid determination kit (enzymatic method) as the control group and the biochemical analysis chip and test method provided in Example 2 of the present invention as the experimental group to test the thermal stability of the reagent at 37 degrees. The existing kit formula (control group) is shown in Table 3.

[0134] Table 3

[0135]

[0136] Control group: After the reagents of the above formula are prepared, they are packaged and placed in a 37°C electric constant temperature biochemical incubator. Samples are taken at regular intervals to test the quality control products on the Mindray fully automatic biochemical analyzer BS350S. The results are shown in the table below (the units of the test results are all μmol / L). The deviation from day 0 is less than 10% and is considered stable.

[0137] Experimental group: The biochemical analysis chips provided in Example 2 were taken from the same batch and placed in a 37°C electric constant temperature biochemical incubator. Samples were taken at regular intervals and the quality control products were tested using the method provided in Example 1. The results are shown in the table below (the units of the test results are all μmol / L). A deviation of less than 10% from day 0 was considered stable.

[0138] The comparison of the test results of the control group and the experimental group is shown in Table 4.

[0139] Table 4

[0140]

[0141]

[0142] From the results in the above table, we can see that the control group was stable from 0 to 7 days, and the relative deviation from 0 day was less than 10%; compared with the control group, the 37-degree thermal stability test results of the experimental group were better, and it was still very stable at 37°C for 60 days, keeping the deviation below 6%, which was far better than the control group.

[0143] Embodiment 4:

[0144] Precision test: The biochemical analysis chip and test method provided in Example 2 of the present invention were used to perform sample detection, and a solution containing 2000 μmol / L citric acid was used as a standard. Samples with known citric acid concentrations of 100 μmol / L and 2000 μmol / L were tested 40 times each.

[0145]

[0146] Where Xi is the concentration value measured for the i-th time, and n is the number of tests.

[0147] According to the above formula, the samples with citric acid concentration of 100 μmol / L and 2000 μmol / L were tested 40 times respectively, and the average value of the measured value was The results are as follows: 105.16 μmol / L and 1978.25 μmol / L, the standard deviation (SD) is 1.65 μmol / L and 22.42 μmol / L, and the coefficient of variation (CV) is 2.15% and 3.27%, respectively. This indicates that the biochemical analysis chip and the test method provided by the present invention have sufficient precision.

[0148] Through the description of the above implementation methods, those skilled in the art can clearly understand that the above embodiments can be implemented by software, or by means of software plus necessary general hardware platforms. Based on such understanding, the technical solutions of the above embodiments can be embodied in the form of software products, which can be stored in a non-volatile storage medium (which can be a CD-ROM, a USB flash drive, a mobile hard disk, etc.), including several instructions for enabling a computer device (which can be a personal computer, a server, or a network device, etc.) to execute the methods described in the various embodiments of the present invention.

[0149] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A freeze-dried reagent for citric acid determination, characterized in that: Used for the determination of citric acid in biological samples or non-biological samples, the lyophilized reagent is made of first lyophilized reagent balls and second lyophilized reagent balls formed by freeze-drying droplets of a first citric acid determination reagent and a second citric acid determination reagent; The first citric acid assay reagent comprises the following components: citrate lyase, lactate dehydrogenase, malate dehydrogenase, a first buffer, an additive, a first cryoprotectant, and a first surfactant; The second citric acid assay reagent comprises the following components: reduced coenzyme, a second surfactant, a second buffer and a second cryoprotectant.

2. The freeze-dried reagent for citric acid determination according to claim 1, characterized in that The citrate lyase includes citrate lyase prepared by integrating the genes of microorganisms into microorganisms through gene recombination technology, and citrate lyase prepared by microorganisms with improved properties through genetic modification; The lactate dehydrogenase includes L-lactate dehydrogenase or D-lactate dehydrogenase; the source of the L-lactate dehydrogenase includes animal or microbial sources; the D-lactate dehydrogenase includes D-lactate dehydrogenase of microbial origin, Lactobacillus reinhardtii, Pseudomonas aeruginosa or Staphylococcus; The malate dehydrogenase includes malate dehydrogenase of animal, plant or microbial origin; The first buffer is an alkaline buffer, which includes a buffer that allows the pH value of the solution to be buffered to a range of 7-9.5, including tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)methylglycine, 4-hydroxyethylpiperazinepropanesulfonic acid, 4-hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethaneethanesulfonic acid, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-morpholinepropanesulfonic acid or 1,4-piperazinediethanesulfonic acid and bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane); The additives include divalent metal salts, sugars, sugar alcohols, proteins, chelating agents, amines, and basic amino acids or heterocyclic compounds; wherein the divalent metal salts include sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium acetate, or potassium acetate, and the protein includes bovine serum albumin; The first cryoprotectant includes sugars, polyols, amino acids, proteins and peptide excipients, and cyclic oligosaccharide-based polymers, wherein the sugars include one or more of glucose, sucrose, lactose, maltose and trehalose; the polyols include one or more of mannitol, inositol, ethylene glycol and polyethylene glycol; the amino acids include trimethylglycine, glycine, arginine or any salt thereof; the protein and peptide excipients include mucopolysaccharide protein, casein or bovine serum albumin; the cyclic oligosaccharide-based polymer includes any one or more combinations of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, β-cyclodextrin sulfobutyl ether and any derivatives thereof; The first surfactant includes a nonionic surfactant; the nonionic surfactant includes one or more combinations of polyethylene glycol monooctylphenyl ether, sorbitan monolaurate, sorbitan monopalmitate, polyoxyethylene lauryl ether, polyoxyethylene cetyl ester, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monolaurate, Tween 20, Tween 40, Tween 80 and Tween 60.

3. The freeze-dried reagent for citric acid determination according to claim 2, characterized in that: The reduced coenzyme comprises reduced β-nicotinamide adenine dinucleotide NADH or any salt thereof, reduced β-nicotinamide adenine dinucleoside phosphate NADPH or any salt thereof, wherein any salt comprises reduced β-nicotinamide adenine dinucleotide disodium salt, reduced β-nicotinamide adenine dinucleotide dipotassium salt or reduced triphosphate pyridine nucleotide tetrasodium salt; The second surfactant comprises a nonionic surfactant, and the nonionic surfactant comprises a combination of one or more of polyethylene glycol monooctylphenyl ether, sorbitan monolaurate, sorbitan monopalmitate, polyoxyethylene lauryl ether, polyoxyethylene cetyl ester, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monolaurate, Tween 20, Tween 40, Tween 80 and Tween 60; The second cryoprotectant includes sugars, polyols, amino acids, proteins and peptide excipients, wherein the sugars include glucose, sucrose, lactose, maltose or trehalose; the polyols include mannitol, inositol, ethylene glycol or polyethylene glycol; the amino acids include trimethylglycine, glycine, arginine or any salt thereof; the protein and peptide excipients include mucopolysaccharide protein, casein or bovine serum albumin; The second buffer includes a buffer capable of buffering the solution pH to a range of 7-10, including tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)methylglycine, 4-hydroxyethylpiperazinepropanesulfonic acid, 4-hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethaneethanesulfonic acid, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-morpholinepropanesulfonic acid, 1,4-piperazinediethanesulfonic acid or bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane.

4. The freeze-dried reagent for citric acid determination according to claim 3, characterized in that: The preparation methods of the first citric acid assay reagent and the second citric acid assay reagent include: Prepare an enzyme protection solution: comprising a first buffer solution, an additive, a first cryoprotectant and a first surfactant at a certain concentration; Prepare enzyme solution: containing a certain concentration of citrate lyase, lactate dehydrogenase, malate dehydrogenase, a first buffer and additives; Prepare substrate solution: including a second buffer solution and reduced coenzyme at a certain concentration; preparing a substrate protection solution comprising a second surfactant, a second buffer and a second cryoprotectant at a certain concentration; The enzyme protection solution is fully mixed with the first citric acid assay reagent in a certain ratio and order; The substrate solution and the substrate protection solution are fully mixed in a certain ratio and order to form the second citric acid determination reagent.

5. The freeze-dried reagent for citric acid determination according to claim 4, characterized in that: The preparation method of the first freeze-dried reagent ball and the second freeze-dried reagent ball comprises: Freezing the first citric acid assay reagent and the second citric acid assay reagent to form a first frozen solid and a second frozen solid respectively; comprising: dropping the liquid droplets of the first citric acid assay reagent and the second citric acid assay reagent into liquid nitrogen using a precise quantitative liquid dispensing system, so that the liquid droplets of the first citric acid assay reagent and the second citric acid assay reagent condense into an ice ball in the liquid nitrogen; adjusting the volume of the ice ball by adjusting and controlling the size of the liquid droplets of the first citric acid assay reagent and the second citric acid assay reagent dropped into the liquid nitrogen; The first frozen solid and the second frozen solid are placed in a vacuum freeze dryer, and freeze-dried under vacuum to obtain the first freeze-dried reagent ball and the second freeze-dried reagent ball. After nitrogen re-pressurization, the first freeze-dried reagent ball and the second freeze-dried reagent ball are collected and stored in a dry aluminum bottle; wherein the drying is divided into two stages: primary drying and secondary drying; the primary drying is used to remove frozen water or solvent based on sublimation to form a solidified solution; the secondary drying is used to remove unfrozen "bound" water or solvent from the solidified solution based on desorption.

6. A biochemical analysis chip, characterized in that: The biochemical analysis chip includes a chip body, the first freeze-dried reagent ball and the second freeze-dried reagent ball as claimed in any one of claims 1 to 5, and a diluent, the chip body is provided with a colorimetric hole and a diluent tank, the first freeze-dried reagent ball and the second freeze-dried reagent ball are accommodated in the colorimetric hole, and the diluent is accommodated in the diluent tank of the chip body.

7. A biochemical analysis chip according to claim 6, characterized in that: The biochemical analysis chip also includes a sample quantitative separation structure, a mixing mechanism, a reaction hole and a hole modification component.

8. A method for determining citric acid, characterized in that: The freeze-dried reagent for citric acid determination according to any one of claims 1 to 5 is implemented, comprising: Adding the processed biological sample or non-biological sample to the colorimetric well of the biochemical analysis chip according to any one of claims 6 to 7; the sample comprises a biological sample or a non-biological sample; the biological sample comprises a blood sample, a urine sample and / or a semen sample; heating the biochemical analysis chip to an appropriately selected constant temperature, wherein the appropriately selected constant temperature is selected from 25° C. to 37° C.; The treated biological sample or non-biological sample is fully reacted with the first freeze-dried reagent ball and the second freeze-dried reagent ball in the colorimetric well at the appropriately selected constant temperature; the reaction time for the full reaction is 5-15 minutes; Using a spectrophotometer or an automatic analytical instrument commonly used in clinical practice to test the absorbance change and / or the rate of decrease of the absorbance value of the solution in the colorimetric well at a specific wavelength of a light source generated by the spectrophotometer or the automatic analytical instrument commonly used in clinical practice based on spectrophotometry; The concentration value of citric acid is determined based on the absorbance change and / or the rate of decrease of the absorbance value.

9. A method for determining citric acid according to claim 8, characterized in that: The processing of the biological sample or the non-biological sample comprises: The biological sample or the non-biological sample is separated into a certain amount of plasma, urine or seminal plasma sample or non-biological sample plasma through chip centrifugation and quantitative structure; The separated plasma, urine or seminal plasma sample or non-biological sample slurry is filled into the mixing tank and diluted with the diluent.

10. A method for determining citric acid according to claim 8, characterized in that: The specific wavelength of the light source is 320-360 nm.

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