Blood ammonia detection kit, preparation method and detection method

Abstract

The invention discloses a blood ammonia detection kit, a preparation method and a detection method. The detection kit comprises a reagent R1, a reagent R2 and a calibrator, the reagent R1 is a freeze-dried solid containing a blood ammonia detection enzyme reaction system and an alkaline gas precursor substance; the R2 reagent is a liquid buffer solution for redissolving the R1 reagent and diluting a sample; wherein the alkaline gas precursor substance is configured to react and continuously release alkaline gas when the reagent R1 is redissolved by the reagent R2, so that a gas-phase protective layer is formed in a reaction system to isolate environmental ammonia pollution. The three technical problems that a traditional blood ammonia detection reagent is poor in stability, prone to being polluted by the environment and tedious in operation are solved, and the integrated breakthrough of room temperature stability, interference resistance and easy operation is achieved.

Description

Technical Field

[0001] This invention relates to the field of clinical testing technology, and in particular to a blood ammonia detection kit, its preparation method, and its detection method. Background Technology

[0002] Blood ammonia testing is a key indicator for the clinical diagnosis of severe liver dysfunction such as hepatic encephalopathy, severe hepatitis, and cirrhosis. Accurate and rapid measurement of blood ammonia concentration is of vital importance for emergency resuscitation and intensive care.

[0003] Currently, clinical laboratories commonly use in vitro diagnostic kits based on enzyme cycling methods for quantitative detection of blood ammonia. The principle is as follows: under the catalysis of glutamate dehydrogenase (GLDH), ammonia in plasma reacts with α-ketoglutarate and reduced coenzyme I (NADH) to produce glutamate and oxidized coenzyme I (NAD). + The concentration of ammonia in the sample can be calculated by monitoring the rate at which the absorbance of NADH decreases at a wavelength of 340 nm. Lactate dehydrogenase (LDH) is usually added to eliminate interference from endogenous pyruvate.

[0004] Although the principle of this method is mature, existing commercial reagent kits have several inherent defects that have not been effectively resolved in practical applications, which seriously affect the reliability, convenience and application scope of the detection: 1. Poor reagent stability and stringent storage and transportation conditions: The core active ingredients of the reagents (GLDH, LDH, and especially the unstable NADH) are highly susceptible to inactivation in liquid environments. Therefore, existing kits must be stored and transported under a cold chain at 2-8°C, resulting in a very short shelf life after opening (typically only a few days). This not only increases logistics costs but also leads to rapid performance degradation of the reagents during laboratory use, increased intra-batch and inter-batch variability, and affects the long-term stability and accuracy of test results.

[0005] 2. Susceptible to environmental ammonia contamination, causing severe interference and requiring extremely high operational standards: Ammonia is a widely present pollutant in the environment (such as human exhalation and cleaning agent evaporation). Current reagent kits are open-label reaction systems, meaning the entire process, from reagent opening and sample addition to reaction measurement, involves exposure to air. Ammonia in the environment readily dissolves into the reaction solution, leading to increased background noise and even false positive results. This necessitates that the test be performed in a dedicated ammonia-free laboratory or fume hood, placing extremely high demands on operators and the environment, thus limiting its application in general emergency rooms, bedside settings, or primary healthcare institutions.

[0006] 3. Cumbersome operation, reliant on large-scale automated instruments: Current mainstream solutions mostly involve multiple bottles of liquid reagents, requiring large, fully automated biochemical analyzers for multiple precise sample additions, mixing, and incubation during testing. Samples typically need to be pre-centrifuged to separate plasma. The entire process is complex and cannot meet the needs of rapid bedside testing for critically ill patients, resulting in high testing costs.

[0007] In summary, existing blood ammonia detection technologies are limited by the instability of the reagents themselves, the susceptibility to contamination during the detection process, and the complexity of operation, making it difficult to provide rapid, reliable, and convenient testing services in a wider range of clinical scenarios. Therefore, there is an urgent need to develop a novel blood ammonia detection kit that maintains high detection performance while possessing excellent storage stability, strong anti-interference capabilities, and ease of operation, in order to address the aforementioned long-standing clinical pain points. Summary of the Invention

[0008] To address the aforementioned technical problems, the present invention aims to provide a blood ammonia detection kit, its preparation method, and its detection method. This invention solves three major technical challenges of traditional blood ammonia detection kits: poor stability, susceptibility to environmental contamination, and cumbersome operation, achieving a breakthrough in integrated operation with room temperature stability, interference resistance, and ease of use.

[0009] To achieve the above-mentioned technical objectives and effects, the present invention is implemented through the following technical solution: A blood ammonia test kit includes reagent R1, reagent R2, and calibrators; The R1 reagent is a lyophilized solid containing a blood ammonia detection enzyme reaction system and an alkaline gas precursor substance; The R2 reagent is a liquid buffer solution used to reconstitute the R1 reagent and dilute the sample; The alkaline gas precursor is configured to react and continuously release alkaline gas when reagent R1 is redissolved by reagent R2, thereby forming a gas-phase protective layer in the reaction system to isolate ammonia pollution from the environment.

[0010] Preferably, the alkaline gas precursor is ammonium carbonate or ammonium bicarbonate.

[0011] Furthermore, the blood ammonia detection enzyme reaction system comprises glutamate dehydrogenase, lactate dehydrogenase, α-ketoglutarate or its soluble salt, and reduced coenzyme I.

[0012] Furthermore, the R1 reagent also contains a carbohydrate protective matrix, and the blood ammonia detection enzyme reaction system and the alkaline gas precursor are dispersed and embedded in a stable structure formed by the carbohydrate protective matrix.

[0013] Furthermore, the glycoprotective matrix comprises trehalose and dextran, the stable structure is a nanogel network formed by molecular self-assembly, and the R1 reagent is a porous freeze-dried microsphere or freeze-dried cake formed by freeze-drying the nanogel network.

[0014] Furthermore, the porous freeze-dried microspheres have a particle size distribution between 0.5 mm and 2 mm, and their reconstitution time does not exceed 30 seconds.

[0015] Furthermore, the R2 reagent is a buffer solution with tris(hydroxymethyl)aminomethane as the buffer system and a pH of 8.4 to 8.8, and contains adenosine diphosphate at a concentration of 1-5 mmol / L, disodium ethylenediaminetetraacetate at a concentration of 0.5-2 mmol / L, bovine serum albumin at a concentration of 0.5-1.5 g / L, and a biological preservative at a concentration of 0.5-1.5 g / L.

[0016] Furthermore, the alkaline gas precursor substance has a mass percentage of 0.1% to 8% in the R1 reagent.

[0017] Another aspect of the present invention is a method for preparing a blood ammonia detection kit according to any one of claims 1 to 8, characterized in that it includes the following steps: (1) Preparation of R1 reagent: The mixture containing the blood ammonia detection enzyme reaction system and the alkaline gas precursor is made into a lyophilized solid; (2) Preparation of R2 reagent: Provide a liquid buffer solution; (3) Preparation of calibrator: Provide a liquid containing a known concentration of ammonia; (4) Assembly: The R1 reagent obtained in step (1), the R2 reagent obtained in step (2), and the calibrator obtained in step (3) are combined and packaged.

[0018] The present invention also provides a method for detecting blood ammonia concentration using the blood ammonia detection kit, comprising the following steps: a) Mix the sample to be tested with the R2 reagent to obtain the detection mixture; b) Add the detection mixture to the R1 reagent and mix well to start the reaction. At this time, the alkaline gas precursor substance releases alkaline gas to form a protective layer. c) Monitor the rate of decrease in absorbance of the reaction system at a wavelength of 340 nm; d) Calculate the blood ammonia concentration in the sample based on the absorbance decrease rate and the calibration curve established by the calibrator.

[0019] The beneficial effects of this invention are as follows: (1) Improved reagent stability and convenience: By preparing the core detection components as lyophilized solids containing alkaline gas precursors (R1 reagent), the kit achieves stable storage and transportation of reagents at room temperature and has the characteristic of being ready to use immediately, significantly reducing storage and transportation requirements and simplifying the operation process.

[0020] (2) It achieves endogenous active protection against environmental pollution: the alkaline gas precursor can react and continuously release alkaline gas when the reagent R1 is redissolved, forming a gas phase protective layer. This characteristic enables the detection system to actively isolate ammonia pollution in the environment, thereby obtaining reliable detection results under normal experimental conditions, which greatly improves the anti-interference ability and universality of the method.

[0021] (3) A stable, interference-resistant, and easy-to-operate integrated detection solution has been created: the combination of reagent R1, reagent R2, and calibrators, along with the aforementioned built-in anti-contamination mechanism, constitutes a complete detection system. While ensuring detection sensitivity and accuracy, this system effectively solves the interconnected technical problems of reagent instability, easy contamination, and cumbersome operation in traditional methods, providing a new technical approach for the rapid and reliable detection of blood ammonia. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] This invention provides a highly stable and interference-resistant blood ammonia detection kit, its preparation method, and detection method. Based on the principle of continuous monitoring, the kit integrates alkaline gas precursors and an enzyme reaction system into a nanogel lyophilized network formed by a glycoprotective matrix, achieving long-term room temperature stability and active, endogenous protection against environmental ammonia contamination.

[0024] The kit includes reagent R1 (lyophilized solid), reagent R2 (liquid buffer), and calibrators. Its core component is reagent R1, a lyophilized solid containing a blood ammonia detection enzyme reaction system and an alkaline gas precursor (such as ammonium bicarbonate). When reconstituted with reagent R2 before use, the alkaline gas precursor reacts rapidly, continuously releasing alkaline gas (such as ammonia), forming a dynamic gaseous protective layer above the liquid surface of the reaction system, effectively preventing the intrusion of ammonia from the external environment.

[0025] Composition of a blood ammonia test kit Reagent R1 is a porous lyophilized solid, preferably porous lyophilized microspheres or lyophilized cake. Its lyophilized precursor solution, based on a final volume of 1000 mL, comprises the following components: Blood ammonia detection enzyme reaction system: Sodium α-ketoglutarate dihydrate: 1.0-2.0 g, serving as a key substrate for ammonia binding; Reduced coenzyme I (NADH): 0.3-0.4g, used as a reaction indicator, its oxidation rate is directly proportional to the ammonia concentration; Lactate dehydrogenase (LDH): 30,000-40,000 U, used to eliminate interference from endogenous pyruvate.

[0026] Glutamate dehydrogenase (GLDH): 5,000-8,000 U, the core enzyme that catalyzes the reaction of ammonia with α-ketoglutarate.

[0027] Alkaline gas precursor: Ammonium bicarbonate or ammonium carbonate, 0.5-1.5g. This substance is stable in a dry state. After redissolving in a reagent, it decomposes in an alkaline environment, continuously releasing ammonia gas. It forms a positive micro-pressure gas phase barrier above the reaction liquid, which is the core of achieving active resistance to ammonia pollution in the environment.

[0028] The glycoprotective matrix consists of 15-25g of trehalose and 5-15g of dextran (average molecular weight approximately 40,000). These two components self-assemble in water through intermolecular forces to form a nanogel network, uniformly encapsulating and immobilizing the aforementioned enzymes, coenzymes, and gas precursors. During subsequent freeze-drying, this network protects the active conformation of the biomolecules through a "water substitution" mechanism, which is fundamental to achieving long-term stability at room temperature.

[0029] Preparation and Conversion: The above components were thoroughly dissolved in process water and brought to a final volume of 1000 mL to obtain the R1 lyophilized precursor solution. This solution was then aliquoted according to the predetermined single-test dosage and immediately lyophilized. The lyophilization process removed moisture, ultimately yielding individual R1 lyophilized products containing a fixed amount of active ingredient.

[0030] By encapsulating the active ingredients in a nanogel and then lyophilizing it, reagent R1 achieves a transition from a liquid to a solid state, enabling long-term stable storage (e.g., 18 months) at room temperature (≤25°C), overcoming cold chain limitations. The nanogel network formed by trehalose and dextran protects the activity of enzymes and coenzymes during lyophilization and immobilizes the alkaline gas precursor. When reagent R2 reconstitutes the lyophilized product, the gas precursor reacts rapidly to form a protective layer.

[0031] R2 reagent (liquid buffer dilution): Reagent R2 is used to reconstitute the lyophilized solid R1, dilute the sample, and provide optimal reaction conditions. Its composition is as follows (for 1000 mL): Buffer system: Tris(hydroxymethylaminomethane) 3.0-5.0 g, Tris(hydroxymethylaminomethane) hydrochloride (Tris-HCl) 10.0-13.0 g, pH adjusted to 8.6-8.8. This buffer system is used to provide and maintain a stable alkaline pH environment required for the reaction, with a preferred pH range of 8.4 to 8.8.

[0032] Enzyme activator: Adenosine diphosphate (ADP) 0.4-2.2 g (final concentration approximately 1-5 mmol / L), as a specific activator of GLDH, significantly improves reaction rate and sensitivity.

[0033] Metal ion chelating agent: Disodium ethylenediaminetetraacetate (EDTA-2Na) 0.2-0.75g (final concentration approximately 0.5-2mmol / L), which chelates metal ions in the sample that may inhibit enzyme activity and improves anti-interference properties.

[0034] Stabilizers and biological preservatives: Bovine serum albumin (BSA) 0.5-1.5g, used as a stabilizer to further stabilize enzyme proteins; biological preservatives preferably areothiazolinone preservatives (such as ProClin series), 0.5-1.5g, used to inhibit microbial growth and replace traditional toxic sodium azide.

[0035] After mixing the above components, process water is added to 1000 mL, and then the mixture is dispensed in equal or quantitative amounts into individual containers that match the R1 lyophilized product.

[0036] When reagent R2 is mixed with lyophilized product R1, it instantly provides the optimal pH for the reaction, activates enzyme activity, chelates interfering ions, and provides a stable environment for proteins and protection against spoilage.

[0037] Calibrators and quality control products: Diluent (matrix solution): Contains Tris 10-15 g / L, sodium chloride 5-7 g / L, bovine serum albumin 0.5-1.5 g / L, isothiazolinone preservative 0.5-1.5 g / L, pH 8.0±0.1. This matrix simulates the human serum environment to ensure calibration accuracy.

[0038] Calibrator / Control Samples: Obtained by precisely diluting the ammonium chloride stock solution using the diluent described above. For example, the calibrator concentration is set to approximately 70 μmol / L. This kit may also include control samples, designed with both low and high concentration levels.

[0039] The preparation method of this kit includes the following steps: (1) Preparation of R1 reagent: According to the formula, dissolve each component in sequence (add the enzyme last at low temperature and mix gently), and let stand at 2-8℃ to allow trehalose and dextran to self-assemble into a nanogel encapsulating all active ingredients, to obtain a uniform R1 lyophilized precursor solution. Subsequently, dispense the precursor solution into single-dose unit containers (e.g., vials or microplate wells) and freeze-dry to obtain a lyophilized solid; the lyophilized solid can be a lyophilized cake formed directly by precision dispensing, or a porous lyophilized microsphere obtained by pre-forming microspheres using microdroplet forming technology and then freeze-drying. Store in a sealed container after lyophilization; (2) Preparation of R2 liquid reagent: Dissolve each component according to the formula, adjust the pH, filter to remove bacteria and then dispense; (3) Preparation of calibrators and quality control samples: ammonium chloride solutions of different concentrations are precisely prepared using matrix solution, filtered and dispensed; (4) Assembly: Combine and package the R1 reagent, R2 reagent, calibrator and quality control products.

[0040] Method for detecting blood ammonia using this kit On an automated biochemical analyzer, the continuous monitoring method (rate method) is used for detection, and the steps are as follows: Measurement parameters: main wavelength 340nm, secondary wavelength 405nm (or 700nm); temperature 37℃; reaction direction is decreasing absorbance; measurement time 180 seconds; Sample addition and reaction: Under instrument control, the sample to be tested (or calibrator, quality control sample) and reagents are added to the reaction vessel in a preset volume ratio. When reagent R2 (liquid buffer) and reagent R1 (lyophilized solid) are mixed in the reaction vessel, reagent R1 is rapidly reconstituted, releasing the complete enzyme reaction system. Simultaneously, the alkaline gaseous precursor contained therein (such as ammonium bicarbonate) reacts immediately in the alkaline environment provided by reagent R2, continuously releasing ammonia gas. This forms a dynamic gas-phase protective layer above the reaction liquid, effectively isolating ammonia contamination from the environment and ensuring that the reaction proceeds in a stable microenvironment.

[0041] Monitoring and Calculation: The instrument continuously monitors the decrease in absorbance (A) of the reaction mixture at 340 nm over time (t) at the set wavelength. During the linear reaction period, the rate of decrease in absorbance (ΔA / min) is directly proportional to the concentration of blood ammonia in the sample.

[0042] The instrument automatically calculates the concentration of blood ammonia (AM) in the sample according to the following formula: ; In the formula: ΔA T / min Average absorbance change of the sample under test per minute; ΔA S / min Average absorbance change of calibrator per minute; C S The known labeled concentration (μmol / L) of blood ammonia (AM) in the calibrator.

[0043] Calculation principle: The analyzer first measures ΔA / min for calibrators of different concentrations, establishing a standard curve (usually a straight line passing through the origin) between concentration (Cs) and reaction rate (ΔAs / min). Then, based on the measured ΔAT / min of the sample to be tested, the accurate ammonia concentration can be obtained by calculating using the above formula or by direct interpolation from the standard curve. The entire detection process is completed in approximately 5 minutes.

[0044] Example 1. Preparation of the reagent kit (1) Preparation of R1 lyophilized microspheres Prepare 1000 mL of R1 lyophilized precursor solution with the following composition: 20.0 g trehalose, 10.0 g dextran (average molecular weight 40 kDa), 1.43 g α-ketoglutarate disodium salt dihydrate, 0.354 g reduced coenzyme I (NADH), 1.5 g ammonium bicarbonate, 35,000 units of lactate dehydrogenase (LDH), and 6,500 units of glutamate dehydrogenase (GLDH). Adjust the pH of the solution to approximately 7.0 using dilute hydrochloric acid, and then add water for injection to make up the volume. After dissolving and mixing the above components (the enzymes were added last at low temperature), the solution was allowed to stand at 2-8 °C to allow trehalose and dextran to self-assemble into a gel system encapsulating the active ingredients. This gel system is a homogeneous dispersion with appropriate flowability. Subsequently, the gel system was formed into microbeads with a particle size of approximately 1.0 mm using a microdroplet generator, and then dropped into liquid nitrogen for instantaneous freezing. The resulting porous lyophilized microspheres were then obtained through freeze-drying. All lyophilized microspheres prepared from the 1000 mL lyophilized precursor solution were divided into 1000 portions, each portion was aliquoted into a vial and sealed. The active ingredient in each lyophilized microsphere was derived from 1.0 mL of the R1 precursor solution.

[0045] (2) Preparation of R2 liquid buffer Prepare 1000 mL of R2 reagent, consisting of: 3.64 g Tris, 11.1 g Tris-HCl, 0.6 g ADP, 0.37 g EDTA-2Na, 1.0 g Bovine serum albumin (BSA), and 1.0 g ProClin 950 preservative. Dissolve and bring to volume with water for injection, adjust pH to 8.6 with hydrochloric acid, and filter sterilize through a 0.22 μm filter. Divide the 1000 mL R2 reagent into 1000 aliquots, each 1.0 mL, and dispense into reagent bottles.

[0046] (3) Preparation of calibrators and quality control samples Using Tris-HCl buffer (pH 8.0) containing bovine serum albumin as the matrix, ammonium chloride standard stock solution was diluted to prepare a calibrator with a concentration of 70.0 μmol / L. Simultaneously, two quality control levels were prepared: a low-value (30.0 μmol / L) and a high-value (150.0 μmol / L) calibrator.

[0047] 2. Performance Testing and Results The performance of the reagent kit (experimental group) prepared in this embodiment was evaluated: Accelerated stability test: The kit was placed in a 37°C incubator and removed on days 0, 7, and 14, and the reaction rate (ΔA / min) of the same calibrator was measured. The results showed that the reaction rate decreased by only 2.1% after 14 days, indicating that the R1 lyophilized microspheres have excellent thermal stability and can be stored at room temperature for a long time.

[0048] Environmental ammonia interference test: A trace amount of ammonia water (25%) was placed 30 cm away from the test area in the laboratory to simulate environmental ammonia pollution. Under this environment, a low-value mixed human serum sample (theoretical value approximately 30 μmol / L) was repeatedly tested using the experimental group kit. The intra-assay coefficient of variation (CV) of the test results was 2.9%, and the recovery rate was 98.5%, which was not significantly different from the test results in a clean environment (CV=2.5%).

[0049] Basic analytical performance: The kit has a detection linearity range of 10 to 500 μmol / L (correlation coefficient r>0.999), and both intra- and inter-assay imprecision (CV) are less than 3.5%.

[0050] Comparative example: Reagent kit without basic gaseous precursors Except for the absence of ammonium bicarbonate in the preparation of the R1 precursor solution, the other formulations, preparation processes, R2 reagents, and detection methods are exactly the same as in the examples.

[0051] Comparative test results: Under the same simulated ammonia contamination environment, the intra-assay coefficient of variation (CV) of the kit used in this comparative test was as high as 8.7% for the above low-value serum samples, and the mean value was systematically higher than the theoretical value by about 12%.

[0052] These results demonstrate that the alkaline gas precursor contained in the R1 lyophilized microspheres of the embodiment is an indispensable core component for generating the gas-phase protective layer and achieving active resistance to environmental ammonia pollution. Without this component, the detection accuracy of the kit under conventional laboratory conditions will be significantly affected.

[0053] Application example: Blood ammonia concentration detection This application example details a method for detecting blood ammonia concentration in a clinical plasma sample using the kit prepared in the examples on a Hitachi 7180 fully automated biochemical analyzer.

[0054] 1. Preparation before testing Reagent preparation: Take one R1 lyophilized vial (containing one lyophilized sample derived from 1.0 mL of the R1 lyophilized precursor solution) and one R2 reagent vial (1.0 mL / vial) from the kit. Place the R2 reagent vial in the designated location in the analyzer's reagent compartment. Place the R1 lyophilized vial in the instrument's specific reaction cup position or auxiliary sample position. Calibration and quality control preparation: Bring the calibrator (70.0 μmol / L) and the high and low level quality control samples that come with the kit to room temperature; Sample preparation: Collect venous blood, use EDTA-K2 for anticoagulation, centrifuge at 3000 rpm for 10 minutes to separate the plasma sample to be tested.

[0055] 2. Instrument parameter settings In the operating software of the fully automated biochemical analyzer, create or call up preset test items. The key parameter settings are as follows: Analysis method: Rate method; Main wavelength / secondary wavelength: 340nm / 700nm; Reaction temperature: 37℃; Monitoring time: After adding reagent R2, monitoring will begin 10 seconds later and continue for 180 seconds; Reagent ratio: 5 μL sample, 1000 μL (1.0 mL) of reagent R2, and reagent R1 is a whole lyophilized microsphere (containing a lyophilized product made from 1.0 mL of R1 precursor solution).

[0056] 3. Sample addition and reaction process The instrument automatically executes the following steps according to the set program: a) Sample addition: Add 5 μL of sample (or calibrator / quality control) to the reaction vessel.

[0057] b) Resolution and formation of a protective gas layer: Add 1.0 mL of reagent R2 to the reaction vessel containing the lyophilized microspheres of R1. Reagent R2 (pH 8.6) rapidly and completely resolutions the entire lyophilized microspheres, while simultaneously decomposing the ammonium bicarbonate in it, continuously releasing ammonia gas, and forming an effective protective gas layer above the liquid surface in the sealed space of the reaction vessel, thereby isolating the environment from ammonia pollution; c) Reaction initiation: The ammonia in the sample initiates the enzyme cycle reaction in the homogeneous reaction system after reconstitution; d) Signal acquisition: The instrument continuously monitors the decrease in absorbance caused by NADH oxidation at 340 nm and calculates the reaction rate (ΔA) during the linear period. T / min).

[0058] 4. Calibration, calculation, and quality control Use the matching calibrator (70.0 μmol / L (C) S (The same procedure is followed to calibrate and obtain the reaction rate ΔA). S / min; the instrument automatically calculates the sample concentration according to the formula; The test results of the high and low quality control products in the same batch all fell within the preset acceptable range, indicating that the test was under control.

[0059] This invention presents a novel blood ammonia detection solution by encapsulating an alkaline gaseous precursor and a complete blood ammonia detection enzyme system within a trehalose-dextran nanogel, preparing a room-temperature stable lyophilized solid reagent (R1), and complementing it with a liquid buffer reagent (R2) to provide an optimal reaction environment. This solution overcomes the inherent limitations of traditional reagents: the lyophilization process and protective matrix ensure long-term room-temperature stability, eliminating reliance on a cold chain; the built-in gaseous precursor generates a gaseous protective layer in situ upon reconstitution, providing active, endogenous resistance to environmental ammonia contamination; and the ready-to-use design of R1 / R2 ensures ease of operation. This invention integrates stability, interference resistance, and convenience, enabling rapid and accurate detection of blood ammonia concentration in samples.

[0060] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0061] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A blood ammonia detection kit, characterized in that, This includes reagents R1, R2, and calibrators; The R1 reagent is a lyophilized solid containing a blood ammonia detection enzyme reaction system and an alkaline gas precursor substance; The R2 reagent is a liquid buffer solution used to reconstitute the R1 reagent and dilute the sample; The alkaline gas precursor is configured to react and continuously release alkaline gas when reagent R1 is redissolved by reagent R2, thereby forming a gas-phase protective layer in the reaction system to isolate ammonia pollution from the environment.

2. The blood ammonia detection kit according to claim 1, characterized in that, The alkaline gas precursor is ammonium carbonate or ammonium bicarbonate.

3. The blood ammonia detection kit according to claim 1 or 2, characterized in that, The blood ammonia detection enzyme reaction system contains glutamate dehydrogenase, lactate dehydrogenase, α-ketoglutarate or its soluble salts, and reduced coenzyme I.

4. The blood ammonia detection kit according to claim 3, characterized in that, The R1 reagent also contains a carbohydrate protective matrix, and the blood ammonia detection enzyme reaction system and the alkaline gas precursor are dispersed and embedded in a stable structure formed by the carbohydrate protective matrix.

5. The blood ammonia detection kit according to claim 4, characterized in that, The glycoprotective matrix comprises trehalose and dextran, the stable structure is a nanogel network formed by molecular self-assembly, and the R1 reagent is a porous freeze-dried microsphere or freeze-dried cake formed by freeze-drying the nanogel network.

6. The blood ammonia detection kit according to claim 5, characterized in that, The porous freeze-dried microspheres have a particle size distribution between 0.5 mm and 2 mm, and their reconstitution time does not exceed 30 seconds.

7. The blood ammonia detection kit according to claim 1, characterized in that, The R2 reagent is a buffer solution with tris(hydroxymethyl)aminomethane as the buffer system and a pH of 8.4 to 8.8, containing 1-5 mmol / L adenosine diphosphate, 0.5-2 mmol / L disodium ethylenediaminetetraacetate, 0.5-1.5 g / L bovine serum albumin, and 0.5-1.5 g / L biological preservative.

8. The blood ammonia detection kit according to claim 1, characterized in that, The alkaline gas precursor is present in the R1 reagent at a mass percentage of 0.1% to 8%.

9. A method for preparing a blood ammonia detection kit according to any one of claims 1 to 8, characterized in that, Includes the following steps: (1) Preparation of R1 reagent: The mixture containing the blood ammonia detection enzyme reaction system and the alkaline gas precursor is made into a lyophilized solid; (2) Preparation of R2 reagent: Provide a liquid buffer solution; (3) Preparation of calibrator: Provide a liquid containing a known concentration of ammonia; (4) Assembly: The R1 reagent obtained in step (1), the R2 reagent obtained in step (2), and the calibrator obtained in step (3) are combined and packaged.

10. A method for detecting blood ammonia concentration using the blood ammonia detection kit according to any one of claims 1-8, characterized in that, Includes the following steps: a) Mix the sample to be tested with the R2 reagent to obtain the detection mixture; b) Add the detection mixture to the R1 reagent and mix well to start the reaction. At this time, the alkaline gas precursor substance releases alkaline gas to form a protective layer. c) Monitor the rate of decrease in absorbance of the reaction system at a wavelength of 340 nm; d) Calculate the blood ammonia concentration in the sample based on the absorbance decrease rate and the calibration curve established by the calibrator.