Kit for carbon dioxide detection and detection method

Abstract

The invention relates to the technical field of in-vitro detection, in particular to a kit for carbon dioxide detection and a detection method. By adjusting the pH value of a reaction system, reducing the pH value to 6.0-7.0, properly increasing the PEP feeding amount and reducing the PEPC enzyme dosage, the reaction rate can be remarkably inhibited under the condition, the PEP consumption rate is reduced, and the airborne stability can be improved. Furthermore, an activating agent acetyl coenzyme A is added in a reaction system and can be in synergistic effect with cofactors such as Mg < 2 + > and the like at a detection temperature, so that the activity of PEPC enzyme is obviously activated, and the reactivity during sample detection is improved, thereby improving the analysis sensitivity and test repeatability of the reagent and achieving a relatively ideal level; if the activators acetyl coenzyme A and GDP are added at the same time, the reactivity during sample detection can be remarkably improved, the analysis sensitivity and test repeatability of the reagent are remarkably improved, and the airborne stability and the performance of the reagent both reach the optimal level under the condition.

Description

Technical Field

[0001] This invention relates to the field of in vitro detection technology, specifically to a reagent kit and detection method for carbon dioxide detection. Background Technology

[0002] Blood CO2 is a routine clinical biochemical test that provides a good understanding of the body's acid-base balance. Metabolic acidosis, such as pyloric obstruction and hyperadrenocorticism, and respiratory acidosis, such as emphysema and airway obstruction, can all lead to elevated blood CO2 levels. Conversely, metabolic acidosis, such as uremia, shock, severe diarrhea, and dehydration, and chronic respiratory alkalosis, such as prolonged rapid breathing, can lead to decreased blood CO2 levels.

[0003] Currently, the most commonly used method for carbon dioxide detection is the enzymatic method, which involves phosphoenolpyruvate carboxylase (PEPC) in Mg... 2+ Under the presence of these conditions, phosphoenolpyruvate (PEP) and bicarbonate (HCO3) are catalyzed. - The reaction produces oxaloacetate and phosphoric acid. The oxaloacetate produced is then catalyzed by malate dehydrogenase (MDH) to produce malate, while simultaneously oxidizing NADH analogs to NAD. + The consumption of NADH analogues leads to a decrease in absorbance, which is proportional to the concentration of CO2 in the sample.

[0004]

[0005] Because the carbon dioxide assay reagent reacts with bicarbonate (HCO3) in serum (plasma). - The CO2 reacts with the reagent, and during the opening of the test reagent bottle, the CO2 in the air constantly surrounds the reagent, which will consume the effective components in the reagent and cause the reagent measurement value to deviate. The deviation mainly comes from two aspects: (1) the quality control fluctuates greatly between days (very easy to exceed 2SD), and the sample test results deviate greatly; (2) after many days on the air, due to the consumption of the substrate, the reagent substrate is insufficient, and high-value positive samples are detected as false low values. At present, the CO2 reagent kits of major manufacturers on the market are generally less stable. After opening, the stability in the biochemical reagent chamber is generally claimed to be 7-14 days. In order to ensure the accuracy of the results, laboratory physicians can only increase the calibration frequency (calibrate once every three to five days, or even calibrate daily), or even frequently open new reagent kits, which causes obvious inconvenience and waste of resources.

[0006] Therefore, there is an urgent need to develop a CO2 detection kit and method with good airborne stability and more accurate detection results. Summary of the Invention

[0007] This invention aims to at least partially solve one of the technical problems in related technologies. Therefore, one object of this invention is to provide a reagent kit and method for carbon dioxide detection. By adjusting the pH of the reaction system to a value of 6.0-7.0, appropriately increasing the amount of PEP feed and reducing the amount of PEPC enzyme, the reaction rate is significantly inhibited under these conditions, the PEP consumption rate decreases, and the onboard stability is improved. Furthermore, by adding the activator acetyl-CoA to the reaction system, it can react with Mg at the detection temperature. 2+ The synergistic effect of cofactors significantly activates the activity of PEPC enzyme, improves the reactivity when testing samples, thereby improving the sensitivity and repeatability of reagent analysis to a relatively ideal level. If the activators acetyl-CoA and GDP are added at the same time, the reactivity when testing samples can be significantly improved, and the sensitivity and repeatability of reagent analysis can be significantly improved. Under this condition, the airborne stability and reagent performance both reach the optimal level.

[0008] Therefore, a first aspect of the present invention provides a reagent kit for carbon dioxide detection. In some embodiments of the present invention, the reagent kit includes:

[0009] Phosphoenolpyruvate, phosphoenolpyruvate carboxylase, and reduced coenzyme regeneration system.

[0010] The working concentration of the phosphoenolpyruvate is 6-15 mmol / L.

[0011] The working concentration of the phosphoenolpyruvate carboxylase is 50-300 U / L.

[0012] The pH value is 6.0-7.0.

[0013] Existing enzymatic methods for detecting carbon dioxide have two major problems: (1) After the carbon dioxide detection reagent is opened, CO2 in the air dissolves into the reagent and reacts with the substrate PEP, further consuming NADH analogues, leading to substrate consumption. When testing samples, insufficient substrate available for reaction results in deviations in sample detection results; (2) During airborne testing, CO2 in the air dissolves into the reagent. In order to reduce substrate consumption, it is not desirable for the reaction to proceed smoothly at this time. However, during detection, it is desirable for the reaction to proceed smoothly to obtain the sample detection results.

[0014] To address the issue of airborne stability, the inventors adjusted the pH of the reaction system to 6.0-7.0, appropriately increased the working concentration of PEP, and reduced the amount of PEPC enzyme. Under these conditions, the reaction rate was significantly inhibited, the PEP consumption rate decreased, and airborne stability was improved.

[0015] Furthermore, the inventors discovered that if the working concentration of PEP is outside the scope of this invention, such as being too high, a higher reaction rate, analytical sensitivity, and good precision results can be obtained in the initial stage after opening the container. However, the inventors found that the onboard stability is actually worse. Only by simultaneously adjusting the pH and the amount of PEPC enzyme under these conditions can the reaction rate be significantly inhibited, the PEP consumption rate decrease, and the onboard stability be improved.

[0016] In some embodiments of the present invention, the kit further comprises:

[0017] Acetyl-CoA, at least one of GDP.

[0018] The inventors discovered that adding the activator acetyl-CoA to the reaction system allows it to react with Mg at the detection temperature. 2+ The synergistic effect of cofactors significantly activates the activity of PEPC enzyme, improves the reactivity of samples during testing, and enhances the analytical sensitivity during the testing phase, thereby achieving a relatively ideal level. If the activators acetyl-CoA and GDP are added simultaneously, the reactivity of samples during testing can be significantly improved, and the analytical sensitivity during the testing phase can be significantly enhanced. Under these conditions, the onboard stability and reagent performance both reach the optimal level.

[0019] In some embodiments of the present invention, the working concentration of acetyl-CoA is 2-5 mmol / L.

[0020] In some embodiments of the present invention, the working concentration of GDP is 0.5-3 mmol / L.

[0021] In some embodiments of the present invention, the kit further comprises:

[0022] Mg 2+ Malate dehydrogenase.

[0023] In some embodiments of the present invention, the Mg 2+ The working concentration is 4-45 mmol / L.

[0024] In some embodiments of the present invention, the working concentration of the malate dehydrogenase is 1-5 KU / L.

[0025] In some embodiments of the present invention, the reagents associated with the reduced coenzyme regeneration system include reduced coenzyme, glucose, and glucose dehydrogenase.

[0026] In some embodiments of the present invention, the reduced coenzyme includes one or more of 3-acetylpyridine adenine dinucleotide, NADH, NADPH, thioNADH, and thioNADPH.

[0027] In some embodiments of the present invention, the working concentration of the reduced coenzyme is 0.2-0.8 g / L.

[0028] In some embodiments of the present invention, the working concentration of glucose is 3-15 g / L.

[0029] In some embodiments of the present invention, the working concentration of the glucose dehydrogenase is 50-300 U / L.

[0030] In some embodiments of the present invention, the kit further includes a buffer solution.

[0031] In some embodiments of the present invention, the buffer solution comprises at least one selected from PIPES buffer, ACES, BES, BIS-Tris, ADA, Cacodylate, Citrate, HEPES, Imidazole, Maleate, MES, MOPS, Phosphate, and TES.

[0032] In some embodiments of the present invention, the working concentration of the buffer solution is 15-200 mmol / L.

[0033] In some embodiments of the invention, the kit further includes a preservative.

[0034] In some embodiments of the present invention, the preservative includes at least one of sodium azide, PC300, and gentamicin sulfate.

[0035] A second aspect of the present invention provides the use of the kit described in the first aspect in detecting carbon dioxide in a sample to be tested.

[0036] In some embodiments of the present invention, the sample to be tested includes an ex vivo body fluid sample.

[0037] A third aspect of the present invention provides a method for detecting carbon dioxide in a sample. In some embodiments of the present invention, the method includes detecting the sample using the kit described in the first aspect.

[0038] In some embodiments of the present invention, the method includes:

[0039] S1: Mix the reagents in the kit described in the first aspect with the sample to be tested to obtain a mixture;

[0040] S2: The mixture is tested using a biochemical analysis instrument.

[0041] In some embodiments of the present invention, the main detection wavelength of the biochemical analyzer is set to 340-410 nm.

[0042] In some embodiments of the present invention, step S2 further includes using the biochemical analysis instrument to detect the absorbance of the sample at different time points, and obtaining the change in absorbance or the rate of change in absorbance per unit time.

[0043] Based on the change in absorbance or the rate of change in absorbance per unit time, a standard curve is referenced to obtain the concentration of carbon dioxide in the sample to be tested.

[0044] In some embodiments of the present invention, the sample to be tested includes an ex vivo body fluid sample.

[0045] The beneficial effects of this invention are: (1) By lowering the pH of the reagent to 6.0-7.0, and appropriately increasing the amount of PEP and reducing the amount of PEPC enzyme, the reaction rate of the onboard process can be effectively reduced, the consumption of substrate PEP can be reduced, and the onboard 30-day detection results can be kept under control; the inventors have made the following further optimizations: (2) The addition of acetyl-CoA can synergistically enhance the effect of Mg 2+ (2) Cofactors significantly activate the activity of PEPC enzyme at 37℃, thereby improving the reactivity in the test sample; (3) Similar to the effect of acetyl-CoA, the addition of GDP can regulate the metabolic pathway and improve the reactivity in the test sample. The sample contains PEPCK kinase, the oxaloacetate→PEP pathway will be inhibited by the product of GDP, the oxaloacetate→malate pathway will be further amplified, and the reactivity will be improved; while the onboard process does not have PEPCK kinase, the oxaloacetate→PEP pathway reaction will not proceed, but it will not amplify the oxaloacetate→malate pathway.

[0046] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0047] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0048] Figure 1 Line graphs showing the airborne stability-related test results using Examples 1-11 and Comparative Examples 1-3 are displayed.

[0049] Figures 2-A to 2-E The results of linear correlation tests using Examples 1-11 and Comparative Examples 1-3 on airborne day 0 and day 28 are shown. Detailed Implementation

[0050] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0051] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0052] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0053] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0054] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0055] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0056] In this article, PEP is an abbreviation for phosphoenolpyruvate; PEPC is an abbreviation for phosphoenolpyruvate carboxylase; MDH is an abbreviation for malate dehydrogenase; GDP is an abbreviation for guanosine diphosphate; and PEPCK is an abbreviation for phosphoenolpyruvate carboxylkinase.

[0057] In this article, the reduced coenzyme is NADH or an NADH analogue, which includes, but is not limited to, one or more of NADPH, 3-acetylpyridinium adenine dinucleotide (APADH), thio-NADH, and thio-NADPH. Reduced coenzyme, NADH, and NADH analogues can be substituted for each other.

[0058] In this paper, because the amount of sample added is too small (the sample:reagent R1 ratio is generally 1:(10-200)), the amount of sample added will not significantly affect the concentration of each component of the reagent. Therefore, the working concentration and the reagent preparation concentration can be equivalent. The kit and detection method provided by this invention are based on the following exploratory process.

[0059] The principle of CO2 detection is as follows:

[0060] Oxidant generation: Based on the principle of carbon dioxide fixation by higher plants, CO2 is reacted with phosphoenolpyruvate (PEP) to generate oxaloacetic acid, which can be used as an oxidant in the signal generation step.

[0061] Signal generation: Based on the reaction of NADH or NADH analogues with the oxidant oxaloacetate, NAD is generated by malate dehydrogenase. + or NAD + Analogs and malates. Due to NADH or NADH analogs and NAD + or NAD + The absorbance of analogues differs, allowing us to determine the amount of NADH or NADH analogue consumed in the reaction, thereby determining the carbon dioxide content in the sample (such as blood).

[0062] Under this detection principle, the problem is that the content of NADH or NADH analogues and PEP is unstable. (1) The PEP content is unstable because after the test reagent is opened, CO2 in the air dissolves into the reagent, which automatically starts the oxidant generation step, resulting in a decrease in PEP content and the generation of oxaloacetic acid; (2) The reason for the unstable content of NADH or NADH analogues is ① the easy decay characteristics of their structure itself; ② due to the dissolution of CO2, the PEP content decreases and the oxaloacetic acid concentration increases, which promotes the conversion of NADH or NADH analogues into NAD. + or NAD + Analogs exacerbate the decrease in NADH or NADH analogue levels. Consequently, during formal sample testing, due to the reduced substrate, high-value samples have insufficient substrate available for reaction, resulting in false low values. Other samples also show false low values ​​due to decreased reactivity caused by the reduced substrate.

[0063] Existing technologies generally consider the instability of NADH or NADH analogs to be the main cause of inaccurate detection. The solution adopted is to establish an NADH or NADH analog regeneration system. However, the inventors discovered in actual development that existing solutions cannot completely solve the problem of inaccurate CO2 detection. Even after establishing the aforementioned regeneration system, substrate insufficiency due to the consumption of another substrate, PEP, during onboard processing cannot be avoided. Furthermore, it was found that although increasing the content of PEP could achieve higher reaction rates, analytical sensitivity, and good precision results in the initial stage after opening the container, onboard stability was actually worse. After investigating issues such as reagent reaction systems, raw material stability, and enzyme activity stability, the inventors unexpectedly discovered that using an NADH or NADH analog regeneration system and / or increasing the amount of PEP did not completely solve the problem of dissolved CO2 in the air.

[0064] This invention controls the pH of the detection reagent to 6.0-7.0, adjusts the amount of PEP to 6-15 mmol / L, and adjusts the amount of PEPC to 50-300 U / L. Under these conditions, the reaction rate is significantly inhibited, the PEP consumption rate is reduced, and the onboard stability is improved.

[0065] Based on this, according to a specific embodiment of the present invention, the present invention provides a reagent kit for carbon dioxide detection, comprising:

[0066] Phosphoenolpyruvate, phosphoenolpyruvate carboxylase, and reduced coenzyme regeneration system.

[0067] The working concentration of the phosphoenolpyruvate is 6-15 mmol / L.

[0068] The working concentration of the phosphoenolpyruvate carboxylase is 50-300 U / L.

[0069] The pH value is 6.0-7.0.

[0070] It should be noted that the reduced coenzyme regeneration system described in this invention refers to the process in carbon dioxide detection where, through a series of enzymatic reactions, oxidized coenzymes (such as NAD) are regenerated. + APAD + thioNAD + thioNADP + or NADP + ) is reduced to reduced coenzymes (such as NADH, APAD) + thioNAD + thioNADP +This regeneration system, or NADPH-related reagents, ensures a stable supply of reduced coenzymes in the reaction system, thereby guaranteeing the continuous progress of enzymatic reactions related to carbon dioxide detection.

[0071] According to a specific embodiment of the present invention, the kit further comprises:

[0072] Acetyl-CoA, at least one of GDP.

[0073] Unlike the onboard process that inhibits PEPC enzyme activity, the addition of acetyl-CoA to the detection reagent is because, under the detection conditions (incubation temperature reaching 37°C), acetyl-CoA reacts with Mg. 2+ It synergistically activates the activity of PEPC enzyme, and can quickly convert CO2 in the test sample into oxaloacetic acid even when the pH is low and the amount of PEPC enzyme is small. It enters the linear phase (also known as the zero-order reaction phase) within 1 minute of starting the reaction. By detecting the amount of APADH consumed per unit time (usually 2-5 minutes), the sample results can be accurately detected.

[0074] Furthermore, adding GDP to the detection reagent can improve the sensitivity of the reaction. In gluconeogenesis, oxaloacetate can be decarboxylated to PEP by receiving a phosphate group from GTP under the catalysis of PEPCK enzyme. Since PEPCK enzyme is present in the sample, adding GDP to the reagent can inhibit the formation of PEP from oxaloacetate, causing the detection reaction to proceed only to the second step, thus improving the detection sensitivity.

[0075] According to a specific embodiment of the present invention, the working concentration of acetyl-CoA is 2-5 mmol / L.

[0076] According to a specific embodiment of the present invention, the working concentration of GDP is 0.5-3 mmol / L.

[0077] According to a specific embodiment of the present invention, the kit further comprises:

[0078] Mg 2+ Malate dehydrogenase.

[0079] According to a specific embodiment of the present invention, the Mg 2+ The working concentration is 4-45 mmol / L.

[0080] According to a specific embodiment of the present invention, the working concentration of the malate dehydrogenase is 1-5 KU / L.

[0081] According to a specific embodiment of the present invention, the reagents related to the reduced coenzyme regeneration system include reduced coenzyme, glucose, and glucose dehydrogenase.

[0082] According to a specific embodiment of the present invention, there is no particular limitation on the type of reduced coenzyme, including but not limited to one or more of 3-acetylpyridine adenine dinucleotide, NADH, NADPH, thioNADH, and thioNADPH.

[0083] According to a specific embodiment of the present invention, the working concentration of the reduced coenzyme is 0.2-0.8 g / L.

[0084] According to a specific embodiment of the present invention, the working concentration of glucose is 3-15 g / L.

[0085] According to a specific embodiment of the present invention, the working concentration of the glucose dehydrogenase is 50-300 U / L.

[0086] According to a specific embodiment of the present invention, the kit further includes a buffer. In the kit of the present invention, there are no particular limitations on the type of buffer, which may be selected from at least one of PIPES buffer, ACES, BES, BIS-Tris, ADA, Cacodylate, Carbonate, Citrate, HEPES, Imidazole, Maleate, MES, MOPS, phosphate, and TES.

[0087] According to a specific embodiment of the present invention, the working concentration of the buffer solution is 15-200 mmol / L.

[0088] According to a specific embodiment of the present invention, the kit further includes a preservative. It should be noted that any preservative known in the art that can be used in a carbon dioxide detection kit without affecting the properties of the reagents is covered within the scope of protection of the present invention. For example, the preservative may be any one of sodium azide, PC300, and gentamicin sulfate, preferably sodium azide.

[0089] According to a specific embodiment of the present invention, the present invention provides a kit comprising reagent R1, wherein reagent R1 comprises the following components:

[0090] Phosphoenolpyruvate (PEP), phosphoenolpyruvate carboxylase (PEPC), reduced coenzyme, glucose, GDH (glucose dehydrogenase), malate dehydrogenase (MDH), Mg 2+ Magnesium sulfate, preservatives, acetyl-CoA, GDP, buffer solution, pH 6.0-7.0.

[0091] According to a specific embodiment of the present invention, reagent R1 in the kit provided by the present invention comprises the following components:

[0092] 6-15 mmol / L phosphoenolpyruvate (PEP), 50-300 U / L phosphoenolpyruvate carboxylase (PEPC), 0.2-0.8 g / L reduced coenzyme, 3-15 g / L glucose, 50-300 U / L GDH (glucose dehydrogenase), 1-5 KU / L malate dehydrogenase (MDH), 4-45 mmol / L glucose 2+ 0.05-0.9 g / L preservative, 2-5 mmol / L acetyl-CoA, 0.5-3 mmol / L GDP, 15-200 mmol / L buffer solution, wherein the pH of reagent R1 is 6.0-7.0.

[0093] According to a preferred embodiment of the present invention, reagent R1 in the kit provided by the present invention comprises the following components:

[0094] 10 mmol / L PEP, 150 U / L PEPC, 0.5 g / L APADH, 8 g / L glucose, 150 U / L GDH, 2.5 KU / L LMDH, 2 g / L magnesium sulfate, 0.5 g / L preservative, 3 mmol / L acetyl-CoA, 1.5 mmol / L GDP, 50 mmol / L sodium PIPES, pH 6.5.

[0095] According to one specific embodiment of the present invention, the present invention provides the use of the aforementioned kit in detecting carbon dioxide in a test sample. According to one specific embodiment of the present invention, the test sample includes an ex vivo bodily fluid sample, such as blood or urine.

[0096] According to a specific embodiment of the present invention, the present invention provides a method for detecting carbon dioxide in a sample to be tested, the method comprising detecting the sample to be tested using the aforementioned kit.

[0097] According to a specific embodiment of the present invention, the method includes:

[0098] S1: Mix the reagents in the kit described above with the sample to be tested to obtain a mixture;

[0099] S2: The mixture is tested using a biochemical analysis instrument.

[0100] According to a specific embodiment of the present invention, the detection wavelength of the biochemical analyzer is set to 340-410nm.

[0101] According to a specific embodiment of the present invention, step S2 further includes using the biochemical analysis instrument to detect the absorbance of the sample to be tested at different time points, and obtaining the change in absorbance or the rate of change in absorbance per unit time.

[0102] Based on the change in absorbance or the rate of change in absorbance per unit time, a standard curve is referenced to obtain the concentration of carbon dioxide in the sample to be tested. According to a specific embodiment of the present invention, the sample to be tested includes an ex vivo bodily fluid sample, such as blood or urine.

[0103] According to a more specific embodiment of the present invention, the detection method of the carbon dioxide detection kit is as follows: The test is initiated at a main wavelength of 340 nm and a secondary wavelength of 505 nm. The sample and reagent R1 are added and mixed thoroughly. After incubation at 37°C for 1 minute, the first absorbance A1 is measured, and the second absorbance A2 is measured after 3 minutes. The absorbance change rate (ΔA / min) is calculated.

[0104] The present disclosure will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the disclosure. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0105] 1. Solution preparation

[0106] The common components of the comparative example and the embodiment are: 8 g / L glucose, 150 U / L glucose dehydrogenase, 2.5 KU / L malate dehydrogenase, 2 g / L magnesium sulfate, 50 mM sodium PIPES, and 0.9 g / L sodium azide. The remaining components are prepared according to the formula shown in Table 1.

[0107] Table 1

[0108]

[0109] 2. The test methods for the reagent performance of the examples and comparative examples are as follows:

[0110] CO2 detection methods:

[0111] The test was started at a main wavelength of 340 nm and a secondary wavelength of 505 nm. The sample to be tested and reagent R1, which is composed of the reagents in Table 1, were mixed together (the ratio of sample to reagent R1 was 1:10 to 200). After incubation at 37°C for 1 minute, the first absorbance A1 was measured, and the second absorbance A2 was measured after 3 minutes. The absorbance change rate (ΔA / min) was calculated. The concentration of carbon dioxide in the sample to be tested was obtained by referring to the standard curve.

[0112] ①Airborne stability

[0113] a. The reagents of Examples 1-11 and Comparative Examples 1-3 were opened and placed in a fully automated biochemical analyzer (Shenzhen New Industries Biomedical Engineering Co., Ltd., Biossays C8). On days 0, 3, 7, 11, 14, 21, 28 and 35, the quality control changes at a concentration of 30 mmol / L were continuously tracked. When the quality control level deviation did not exceed ±10%, the onboard stability was considered to be good.

[0114] b. Linearity test samples were obtained by diluting a standard of known concentration with pure water, and the results were presented on day 0 and day 28 before recalibration. The linear range should cover [3.00, 50.00] mmol / L, and the linear correlation coefficient should be ≥0.990 within this range; the absolute deviation should be within ±1 mmol / L within [3.00, 10.00] mmol / L; and the relative deviation should be within ±10% within the [10.00, 50.00] mmol / L range, which indicates good airborne stability.

[0115] c. The test results of samples on day 0 and day 28 are presented respectively. For samples with a concentration <10.00 mmol / L, the absolute deviation should be within ±1 mmol / L. For samples with a concentration >10.00 mmol / L, the relative deviation should not exceed ±10% to be considered as good airborne stability.

[0116] ② Sensitivity and Precision

[0117] a. The reagents from Examples 1-11 and Comparative Examples 1-3 were opened and placed in a fully automated biochemical analyzer (Shenzhen New Industries Biomedical Engineering Co., Ltd., Biossays C8). The reactivity of pure water (ΔR1) and CO2 samples (ΔR2) was measured using different reagent schemes. The reactivity and CO2 sample concentration (C) were then used as the basis for the determination of the reactivity. l ) Converted to 25 mmol / L (C t The analytical sensitivity of ) is calculated using the following formula: The criterion is that if the analytical sensitivity is ≥0.1 Abs / min, then the sensitivity is considered good.

[0118] b. Repeatability testing was performed using two-level samples (25 mmol / L, 35 mmol / L) (10 replicates). A coefficient of variation (CV) ≤ 3.0% was considered to indicate good precision.

[0119] 3. Performance test results of the test reagents for Examples 1-11 and Comparative Examples 1-3

[0120] ①Airborne stability

[0121] The airborne stability-related test results of Examples 1-11 and Comparative Examples 1-3 are as follows:

[0122] a. Changes in airborne quality control

[0123] Comparative Examples 1-3 and Examples 1-11 airborne quality control change data, such as Figure 1 As shown in the figure. The results indicate that: in Comparative Example 1, the negative deviation of the quality control test was already greater than 10% on day 7 of airborne operation; Comparative Example 2, which added excessive PEP to Comparative Example 1, did not improve airborne stability, but rather worsened it. On day 3 of airborne operation, the negative deviation of the quality control test was already close to 30%, and in the later stages of airborne operation, the negative deviation even approached 50%, indicating that simply adding excessive PEP could not effectively solve the problem of airborne stability; similarly, Comparative Example 3, by reducing the amount of PEPC enzyme, could not effectively solve the problem of airborne stability. In contrast, Examples 1-11, by lowering the pH to 6.0-7.0, controlling the PEP dosage to 6-15 mmol / L, and the PEPC enzyme dosage to 50-300 U / L, showed that the deviation of the quality control test was smaller than that of the comparative example from day 11 to day 35 of airborne operation, proving that the airborne stability was superior to that of the comparative example. In particular, Example 2, with its higher pH and higher PEPC enzyme dosage, which helps to consume PEP, can solve the problem of in-flight stability even with a PEP dosage of only 6 mmol / L. This demonstrates that any combination of pH 6.0-7.0, PEP dosage of 6-15 mmol / L, and PEPC enzyme dosage of 50-300 U / L can also solve the problem of in-flight stability.

[0124] b. Linearity test results before recalibration

[0125] The airborne linearity test data for Comparative Examples 1-3 and Examples 1-11 are shown in Tables 2-1, 2-2, 3-1, and 3-2. Figures 2-A to 2-E As shown.

[0126] Table 2-1

[0127]

[0128] Table 2-2

[0129]

[0130] Table 3-1

[0131]

[0132] Table 3-2

[0133]

[0134] The results show that on day 0 of airborne operation, the linear correlation coefficients of the comparative examples and the embodiment were all >0.99, which met the requirements. However, by day 28 of airborne operation, the linear correlation coefficients of comparative examples 1-3 were all <0.99, and about 73% of the test values ​​did not meet the requirements for deviation from the theoretical values. In contrast, the linear correlation coefficients of the embodiment were all >0.99, and about 98% of the test values ​​met the requirements for deviation from the theoretical values. This demonstrates that the airborne stability of the embodiment was significantly improved.

[0135] c. Sample test results

[0136] The sample comparison is shown in the table below, where Table 4 shows the test deviation between the airborne measurements after 28 days and the airborne measurements after 0 days:

[0137] Table 4

[0138]

[0139] The results in Table 4 show that, in Comparative Examples 1-3, on day 28 of airborne operation, approximately 89% of the test results were negatively biased by more than 10% compared to the results on day 0. Furthermore, at high test values ​​(>30 mmol / L, such as samples 13, 14, 1, 12, 15, and 11), the negative bias reached 21% to 36%, severely failing to meet the testing requirements. In contrast, in Examples 1-11, on day 28 of airborne operation, 92% of the tests showed a negative bias of less than 10%, and all test biases in Examples 7-11 were close to less than 5%, demonstrating a significant improvement in airborne stability.

[0140] ② Analytical sensitivity and precision

[0141] a. Analytical sensitivity

[0142] Table 5 below shows the reactivity of Examples 1-11 and Comparative Examples 1-3 when detecting calibrators at 25 mmol / L.

[0143] Table 5

[0144]

[0145]

[0146] The results showed that Examples 7-11 were based on Examples 1-6 with the addition of acetyl-CoA or the simultaneous addition of acetyl-CoA and GDP. It can be seen that the analytical sensitivity of adding acetyl-CoA or the simultaneous addition of acetyl-CoA and GDP was greater than 0.1 Abs / min, proving that adding acetyl-CoA and GDP can effectively improve the detection sensitivity.

[0147] b. Precision sample testing

[0148] Ten tests were performed on repeatable samples 1 and 2 using Examples 1-11 and Comparative Examples 1-3 respectively. The statistical test results are shown in Tables 6-7 below.

[0149] Table 6

[0150]

[0151] Table 7

[0152]

[0153] The results showed that Examples 7-11 were based on Examples 1-6 with the addition of acetyl-CoA or the simultaneous addition of acetyl-CoA and GDP. It can be seen that the coefficient of variation (CV) of adding acetyl-CoA or the simultaneous addition of acetyl-CoA and GDP was less than 3.0%, which proves that acetyl-CoA and GDP help improve the precision of the test.

[0154] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Furthermore, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples, without contradiction. Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A reagent kit for carbon dioxide detection, characterized in that, include: Phosphoenolpyruvate, phosphoenolpyruvate carboxylase, and reduced coenzyme regeneration system. The working concentration of the phosphoenolpyruvate is 6-15 mmol / L. The working concentration of the phosphoenolpyruvate carboxylase is 50-300 U / L. The pH value is 6.0-7.

0.

2. The reagent kit according to claim 1, characterized in that, The kit further includes: At least one of acetyl-CoA and GDP; Optionally, the working concentration of the acetyl-CoA is 2-5 mmol / L; Optionally, the working concentration of GDP is 0.5-3 mmol / L.

3. The reagent kit according to claim 1, characterized in that, The kit further includes: Mg 2+ , malate dehydrogenase; Optionally, the Mg 2+ The working concentration is 4-45 mmol / L; Optionally, the working concentration of the malate dehydrogenase is 1-5 KU / L.

4. The reagent kit according to claim 3, characterized in that, The reagents related to the reduced coenzyme regeneration system include reduced coenzyme, glucose, and glucose dehydrogenase. Optionally, the reduced coenzyme includes one or more of 3-acetylpyridine adenine dinucleotide, NADH, NADPH, thioNADH, and thioNADPH. Optionally, the working concentration of the reduced coenzyme is 0.2-0.8 g / L; Optionally, the working concentration of the glucose is 3-15 g / L; Optionally, the working concentration of the glucose dehydrogenase is 50-300 U / L.

5. The kit according to any one of claims 1-4, characterized in that, The kit further includes a buffer solution and a preservative; Optionally, the buffer solution comprises at least one selected from PIPES buffer, ACES, BES, BIS-Tris, ADA, Cacodylate, Citrate, HEPES, Imidazole, Maleate, MES, MOPS, Phosphate, and TES. Optionally, the working concentration of the buffer solution is 15-200 mmol / L; Optionally, the preservative includes at least one of sodium azide, PC300, and gentamicin sulfate.

6. Use of the kit according to any one of claims 1-5 in detecting carbon dioxide in a sample to be tested; Optionally, the sample to be tested includes an ex vivo body fluid sample.

7. A method for detecting carbon dioxide in a sample, characterized in that, This includes using the kit described in any one of claims 1-5 to detect the sample to be tested.

8. The method according to claim 7, characterized in that, The method includes: S1: Mix the reagents in the kit according to any one of claims 1-5 with the sample to be tested to obtain a mixture; S2: The mixture was tested using a biochemical analysis instrument; Optionally, the sample to be tested includes an ex vivo body fluid sample.

9. The method according to claim 8, characterized in that, The detection wavelength of the biochemical analyzer is set to 340-410nm.

10. The method according to claim 8, characterized in that, Step S2 further includes using the biochemical analysis instrument to detect the absorbance of the sample at different time points, and obtaining the change in absorbance or the rate of change in absorbance per unit time. Based on the change in absorbance or the rate of change in absorbance per unit time, a standard curve is referenced to obtain the concentration of carbon dioxide in the sample to be tested.

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