Biochemical detection reagent and preparation method and biochemical index detection method and kit

Abstract

The present invention discloses a biochemical detection reagent, which has a molecule with the structure shown in formula (1): #imgabs0# After reacting with singlet oxygen, the color of the biochemical detection reagent changes and afterglow light is emitted. Among them, R1 and R2 are each one of hydrogen, halogen, carboxyl group, sulfonic acid group, keto group, aldehyde group, nitro group, carbonyldiimidazolyl group, sulfonyl group, sulfate group, cyano group and phenyl group, and R2 can also be an amino group or an amino-substituted group. This biochemical detection reagent can solve the problem that the color development in microfluidics for biochemical detection is not obvious, and has high sensitivity even when the concentration of the index to be detected is low. The present invention also discloses a preparation method of the biochemical detection reagent, a method for detecting the concentration of biochemical indexes using the biochemical detection reagent, and a biochemical detection kit.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to a biochemical detection reagent and a preparation method thereof, and a method and a kit for detecting the concentration of biochemical indicators using the biochemical detection reagent. Background Art

[0002] Biochemical testing is a technology that diagnoses diseases by detecting chemicals or biomolecules in organisms based on the principle of biochemical reactions. This technology involves quantitative and qualitative analysis of markers such as proteins, nucleic acids, and small molecule metabolites in biological samples such as blood, urine, and tissues in order to detect, monitor, and evaluate the treatment of diseases at an early stage. Biochemical testing technology is an indispensable part of modern medicine and is of great significance for improving the accuracy of disease diagnosis, achieving early diagnosis and treatment, and improving the survival rate and quality of life of patients. In addition, biochemical testing also plays a key role in the development of new drugs, epidemic monitoring, and environmental pollution assessment. In the field of biochemical testing, microfluidics makes it possible to complete complex biochemical reactions in a very small volume, greatly reducing the consumption of reagents and samples, speeding up the detection speed, and improving the detection efficiency. Although microfluidics has shown great potential in biochemical testing, it still faces some challenges in practical applications. One of the main problems is that the color change produced by the color development reaction is not obvious due to the minimization of the sample volume in the microfluidic device, especially when low-concentration samples are detected, this color change may be difficult to detect accurately. This not only affects the sensitivity and accuracy of the detection, but also limits the application scope of microfluidics technology in the detection of low-concentration biomarkers.

[0003] Therefore, the development of new biochemical detection reagents suitable for microfluidic platforms has become an urgent problem to be solved in the field of biochemical detection. The solution to this problem can not only promote the further development of microfluidic technology in the field of biochemical detection, but also broaden its application prospects in clinical diagnosis, disease monitoring and other fields. Summary of the invention

[0004] In order to solve the above technical problems, the embodiment of the present invention discloses a biochemical detection reagent, which has a molecule with a structure as shown in formula (1):

[0005]

[0006] After the molecule reacts with singlet oxygen, the color of the biochemical detection reagent changes and an afterglow is emitted.

[0007] wherein R1 is one of hydrogen, halogen, carboxyl, sulfonic acid, keto, aldehyde, nitro, carbonyldiimidazole, sulfonyl, sulfate, cyano and phenyl,

[0008] R2 is one of hydrogen, halogen, carboxyl, sulfonic acid, keto, aldehyde, nitro, carbonyldiimidazole, sulfonyl, sulfate, cyano, phenyl, amino and amino substituents.

[0009] By adopting the above technical solution, the molecule, under the action of singlet oxygen present in the detection environment, can cause the color of the biochemical detection reagent to change while the afterglow phenomenon occurs, and the color change is relatively obvious, which can solve the problem of unclear color development of biochemical detection in microfluidics. Therefore, even if the concentration of the indicator to be detected is low, the biochemical detection reagent has a high sensitivity.

[0010] Optionally, the biochemical detection reagent has a molecule having a structure as shown in formula (2):

[0011]

[0012] Wherein, R3 and R4 are each independently selected from an alkyl group, an alkane derivative group, an aryl group, a heterocyclic group, a halogen, a carbonyl group, a sulfonic acid group, a carboxylic acid derivative group, an amino group, an ether group, a thiol group, an alkynyl group, an alkenyl group, a ketone group and an ester group.

[0013] Optionally, the alkane derivative group is one of a fatty alcohol group, a fatty acid group, an alkyl sulfonic acid group, an alkyl ether group, an alkylamine group and an alkyl sodium sulfate group, R1 is a halogen or hydrogen, and R3 and R4 are each independently selected from an alkyl group or an alkane derivative group having 1 to 5 carbon atoms.

[0014] Optionally, R1 is halogen or hydrogen, and R2 is one of amino, amino substituent or halogen.

[0015] Optionally, the structure of the molecule is as shown in formula (3):

[0016]

[0017] Optionally, the biochemical detection reagent also includes a horseradish peroxidase solution, which is used to detect the concentration of biochemical indicators. The biochemical indicators generate hydrogen peroxide after the enzymatic reaction, and the hydrogen peroxide decomposes to generate singlet oxygen under the action of the horseradish peroxidase solution. The concentration of the biochemical indicator is detected after the biochemical detection reagent reacts with the singlet oxygen. The concentration range of the horseradish peroxidase solution is 2-10KU / L.

[0018] Optionally, the biochemical detection reagent is used to detect the concentration of at least one biochemical indicator among glucose, total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, creatinine and uric acid.

[0019] Optionally, the biochemical detection reagent can detect at least one of 0.1-20mmol / L glucose, 0-12.9mmol / L total cholesterol, 0.05-11.29mmol / L triglycerides, 0.01-3.9mmol / L high-density lipoprotein cholesterol, 0-26.160mmol / L low-density lipoprotein cholesterol, 0-30mmol / L creatinine and 0-1000μmol / L uric acid.

[0020] According to another specific embodiment of the present invention, the embodiment of the present invention discloses a method for preparing a biochemical detection reagent, comprising the following steps:

[0021] A compound having a molecular structure as shown in formula (a) and a compound having a molecular structure as shown in formula (c) are mixed in a solvent and refluxed to obtain a compound having a molecular structure as shown in formula (1);

[0022]

[0023] The solvent is one or more of benzene, toluene, xylene, ethylbenzene, isopropylbenzene, styrene, chlorobenzene, nitrobenzene, dichlorobenzene and trichlorobenzene, and the reflux time is 5-10 hours.

[0024] By using this method, biochemical detection reagents can be prepared conveniently and efficiently.

[0025] Optionally, the following steps are included:

[0026] A compound having a structure as shown in formula (b) and a compound having a structure as shown in formula (c) are mixed in

[0027] The mixture is mixed in a solvent and refluxed to obtain a compound having a molecular structure as shown in formula (2):

[0028]

[0029]

[0030] Optionally, the following steps are included:

[0031] A molecular compound having a structure as shown in formula (d) and a molecular compound having a structure as shown in formula (c) are mixed in a solvent and refluxed to obtain a molecular compound having a structure as shown in formula (3):

[0032]

[0033] The solvent is toluene and the reflux time is 5 h.

[0034] According to another specific embodiment of the present invention, the embodiment of the present invention discloses a method for detecting the concentration of a biochemical indicator using the biochemical detection reagent of any of the above embodiments, comprising the following steps:

[0035] Preparation of samples to be tested: preparing samples of biochemical indicators to be tested, wherein the biochemical indicators to be tested are at least one of glucose, total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, creatinine and uric acid;

[0036] Preparation of biochemical detection reagents: Prepare biochemical detection reagents, which include molecules having a structure as shown in formula (1) and horseradish peroxidase solution:

[0037]

[0038] Measurement and calculation: In the microfluidic device, a microfluidic pump is used to transport the biochemical indicator samples to be tested and the biochemical detection reagents to the mixing chamber of the microfluidic chip. The absorbance is measured by an optical detection device. The concentration of the biochemical indicator is converted according to the absorbance using the Lambert-Beer law to obtain the concentration of the biochemical indicator to be tested.

[0039] By adopting the above technical solution, the target biochemical index can be sensitively detected using biochemical detection reagents. Regardless of whether it is a low concentration or a high concentration environment, this method can accurately measure the concentration of the target index.

[0040] Optionally, the step of preparing the sample to be tested further includes adding an enzyme to allow the biochemical indicator to be tested to react and generate hydrogen peroxide.

[0041] Optionally, in the measuring and calculating steps, the absorbance is measured within a wavelength range of 450-600 nm.

[0042] An embodiment of the present invention also discloses a biochemical detection kit, which includes the above-mentioned biochemical detection reagents, and the biochemical detection kit includes at least one of a glucose reagent detection kit, a total cholesterol reagent detection kit, a triglyceride reagent detection kit, a high-density lipoprotein cholesterol reagent detection kit, a low-density lipoprotein cholesterol reagent detection kit, a creatinine reagent detection kit and a uric acid reagent detection kit.

[0043] By adopting the above technical solution, the biochemical indexes can be conveniently detected using the biochemical detection kit, and the test results are accurate and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Photographs showing solutions after the detection reagent in Example 1 of the present invention reacts with glucose solutions of different concentrations;

[0045] Figure 2A schematic diagram showing the color blocks of the solutions after the detection reagents react with glucose solutions of different concentrations in Example 1 of the present invention and Comparative Example 1;

[0046] Figure 3 A schematic diagram showing the color blocks of the solutions after the detection reagents react with total cholesterol solutions of different concentrations in Example 2 of the present invention and Comparative Example 2;

[0047] Figure 4 A schematic diagram showing the linear relationship between the measured concentration and the theoretical concentration of high-density lipoprotein cholesterol in Example 4 of the present invention;

[0048] Figure 5 A schematic diagram of a standard curve between absorbance at 550 nm and creatinine concentration in Example 6 of the present invention is shown;

[0049] Figure 6 A schematic diagram of a standard curve between absorbance at 555 nm and uric acid concentration in Example 7 of the present invention is shown;

[0050] Figure 7 Photos showing the reaction of the enzyme reagent with uric acid solutions of different concentrations in Comparative Example 7 of the present invention;

[0051] Figure 8 A schematic diagram of a standard curve between absorbance at 550 nm and creatinine concentration in Example 8 of the present invention is shown;

[0052] Fig. 9 Photos showing the reaction of the enzyme reagent with creatinine solutions of different concentrations in Comparative Example 8 of the present invention;

[0053] Fig.10 A schematic diagram showing the relationship between the absorbance measured at 550 nm and the creatinine concentration in Comparative Example 8 of the present invention. DETAILED DESCRIPTION

[0054] The following specific embodiments illustrate the implementation of the present invention, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Although the description of the present invention will be introduced in conjunction with the preferred embodiment, this does not mean that the features of this invention are limited to this implementation. On the contrary, the purpose of introducing the invention in conjunction with the implementation is to cover other options or modifications that may extend based on the claims of the present invention. In order to provide a deep understanding of the present invention, the following description will include many specific details. The present invention can also be implemented without using these details. In addition, in order to avoid confusion or blurring the focus of the present invention, some specific details will be omitted in the description. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0055] It should be noted that in this specification, similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further defined and explained in the subsequent drawings.

[0056] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0057] In the present application, the term "alkyl" refers to a saturated hydrocarbon chain, such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), and the like.

[0058] The term "aryl" refers to an aromatic group containing a benzene ring or a fused ring system, such as phenyl (C6H5-), naphthyl (C 10 H7-) etc.

[0059] The term "heterocyclic group" refers to a cyclic structure containing at least one heteroatom (eg, nitrogen, oxygen, sulfur), such as pyridyl (C5H4N-), furyl (C4H3O-), and the like.

[0060] The term "carbonyl" refers to a group containing a carbon-oxygen double bond, such as aldehyde (-CHO), keto (-CO-), and the like.

[0061] The term "sulfonic acid group" refers to a group containing a sulfur and oxygen double bond, such as sulfonic acid group (-SO3H).

[0062] The term "carboxylic acid derivative group" includes an ester group (-COOR) and an acyl group (-COR), and specifically also includes an amide group (-CONH2), an acyl chloride group (-COCl) and the like.

[0063] The term "amino" refers to a group in which a nitrogen atom is bonded to a hydrogen atom, such as amino (-NH2).

[0064] The term "amino substituent" refers to an amino group in which at least one of the two hydrogen atoms attached to the nitrogen is replaced by another group, such as dimethylamino ((CH3)2N-).

[0065] The term "ether group" refers to a group containing a carbon-oxygen-carbon bond, such as methoxy (CH3O-), ethoxy (CH3CH2O-), and the like.

[0066] The term "thiol group" refers to a group containing a sulfur-hydrogen bond, such as a mercapto group (-SH).

[0067] The term "alkynyl" refers to a straight or branched chain group containing a carbon-carbon triple bond, such as ethynyl (HC≡C-).

[0068] The term "alkenyl" refers to a straight or branched chain group containing a carbon-carbon double bond, such as vinyl (H2C=CH-).

[0069] The term "keto" refers to a group containing a carbon-oxygen double bond.

[0070] The term "ester group" refers to a group containing a carbon-oxygen single bond and an oxygen-carbon single bond, such as methyl formate (HCOOCH3).

[0071] The term "fatty alcohol group" refers to a class of long carbon chain alcohol structures, usually derived from natural fats and oils, whose structure contains a hydroxyl (-OH) group and a saturated or unsaturated alkyl chain.

[0072] The term "fatty acid radical" is a long chain carboxylic acid structure having one or more carbon chain structures and having a carboxyl group (-COOH) at the end.

[0073] The term "alkylsulfonate" refers to a class of organic sulfonic acid structures containing an alkyl chain and a sulfonic acid group (-SO3H) in the structure.

[0074] The term "alkyl ether group" is a structure in which an alkyl group is linked through an ether bond (-O-).

[0075] The term "alkylamino" comprises an alkyl group directly attached to one or more amino groups (-NH2).

[0076] The term "sodium alkyl sulfate group" refers to a structure consisting of the sodium salt of an alkyl sulfate.

[0077] The term "sulfonyl group" refers to a structure consisting of a sulfur atom and two oxygen atoms connected by a double bond (chemical structure: -SO2-).

[0078] The term "sulfate group" refers to a structure in which a sulfur atom is double-bonded to two oxygen atoms, and each oxygen atom is single-bonded to another oxygen atom, and the general chemical structure is -O-SO2-O-.

[0079] In the embodiments of the present invention, a "derivative group" refers to a new group obtained by replacing one or more atoms or groups of atoms from a certain group (called a "prototype" group). This replacement usually involves a specific functional group (such as a hydroxyl, carboxyl, amino, etc.), but the skeleton structure of the prototype group is maintained. The formation of a derivative group can change the physical or chemical properties of the prototype group, so that the derivative group has a different application or function from the prototype compound group. For example, an alkane derivative group refers to a new group obtained by replacing the hydrogen on the alkyl carbon with a group, and the replaced group is, for example, a halogen, a hydroxyl, an amino, etc.

[0080] At present, in the system of biochemical detection using color reaction, there is an indicator system coupled with hydrogen peroxide (H2O2), in which the indicator to be detected will generate H2O2 during the enzymatic reaction. The principle used by the system is as follows: the indicator to be detected generates H2O2 under the action of the enzyme, and H2O2 reacts with the color substrate (such as 4-aminoantipyrine and / or phenol compounds) to generate a colored substance. By detecting the absorbance of this colored substance, the concentration of the original indicator to be detected can be reflected.

[0081] However, this biochemical detection color reaction has certain problems. Taking glucose detection as an example, glucose generates H2O2 under the action of enzymes, and H2O2 reacts with 4-aminoantipyrine (4-AAP) and phenol to produce red quinone compounds. By measuring the absorbance of the red quinone compounds produced by the reaction, the color intensity of the quinone compounds is proportional to the original glucose concentration, and the concentration of glucose can be determined. The reaction principle and reaction formula are as follows:

[0082]

[0083]

[0084] From the reaction principle and reaction formula, we can know that the amount of quinone compounds generated depends on the amount of glucose to a certain extent. At the same time, 1 part of 4-AAP and phenol reacts with 2 parts of H2O2 to generate 1 part of quinone compounds. Under multiple influencing factors, the concentration of quinone compounds generated may be relatively low, resulting in a lighter color, which is even less obvious in microfluidics. Especially in low-concentration detection, the quinone compounds obtained are even more trace, and the color intensity is even difficult to be detected by the instrument. Therefore, in this case, the reliability of detection using the biochemical detection color reaction is low.

[0085] In order to solve the above problems, the first aspect of the present invention discloses a biochemical detection reagent, which has a molecule with a structure as shown in formula (1):

[0086]

[0087] After the molecule reacts with singlet oxygen, the biochemical detection reagent changes color and emits afterglow.

[0088] wherein R1 is one of hydrogen, halogen, carboxyl, sulfonic acid, keto, aldehyde, nitro, carbonyldiimidazole, sulfonyl, sulfate, cyano and phenyl,

[0089] R2 is one of hydrogen, halogen, carboxyl, sulfonic acid, keto, aldehyde, nitro, carbonyldiimidazole, sulfonyl, sulfate, cyano, phenyl, amino and amino substituents.

[0090] When the biochemical detection reagent contains this molecule (hereinafter collectively referred to as K-SOO molecule), the molecule can react with singlet oxygen to generate a molecule with a diketone structure, thereby causing the color of the biochemical detection reagent to change and emit afterglow. Taking glucose detection as an example, the reaction principle and reaction formula are as follows:

[0091]

[0092] From the reaction principle and reaction formula, it can be seen that glucose will generate H2O2 during the enzymatic reaction, and H2O2 will be decomposed into singlet oxygen ( 1 O2), K-SOO molecules can react with singlet oxygen, and after the reaction, a substance with a diketone structure is generated, which can change the color of the biochemical detection reagent and emit afterglow light at the same time. It can be seen from the reaction formula that 1 part of glucose can generate 2 parts of H2O2, and each O of the 2 parts of H2O2 can further react to singlet oxygen, and then react with K-SOO molecules to generate a substance with a diketone structure. It can be seen from this that compared to the reaction of generating color-developing substances in the prior art, under the molecular structure and reaction mechanism of the present application, even if the concentration of the biochemical indicator to be measured is low, the amount of the substance with a diketone structure generated is also large, and it can also have a more obvious color change, and the concentration of the biochemical indicator can be accurately obtained by measuring the absorbance.

[0093] According to the Beer-Lambert Law, absorbance (A) is proportional to the concentration (c) of the substance, the optical path length (l) of the solution, and the molar absorptivity (ε) of the specific substance. In a microfluidic system, the optical path length (l) is relatively short due to the size limitation of the device, so increasing the absorbance (A) is essentially increasing the concentration (c) or changing the properties of the substance (increasing ε) to achieve improved sensitivity. Therefore, the present application utilizes the chemical conversion of chromogenic molecules to increase absorbance, thereby improving the sensitivity of microfluidic devices with a smaller optical path length during the detection process. The present application selects specific chromogenic molecules, and under the catalysis of substrates or enzymes, the chromogenic molecules undergo a chemical reaction, which is further converted into molecules with darker colors. This process increases the absorbance of the sample, thereby improving the sensitivity of the detection.

[0094] The present application cleverly solves the problem of optical path limitation in microfluidic devices through a chemical conversion mechanism, significantly enhances the detection sensitivity of the system by increasing the absorbance of the color-developing molecule, and opens up a new way for the development and application of microfluidic detection technology, which is conducive to detecting target molecules at low concentrations and is of great significance for early disease diagnosis. At the same time, the biochemical detection reagent of the present application can also emit afterglow light, which can not only detect the concentration of the absorbance reaction indicator, but also detect afterglow. The detection under the two mechanisms can be performed separately or simultaneously. Compared with the detection reagent that can only emit afterglow light, the reagent has a more stable and lasting color change, which is convenient for capturing signals and improves the accuracy of detection; compared with the detection reagent that only has color changes, the detection reagent can emit afterglow light, with good signal stability and higher accuracy. Especially at high concentrations, detection using the afterglow mechanism is more accurate. Therefore, it is possible to choose which mechanism to use for detection according to actual conditions, and the flexibility is also higher. The application of this reagent and this molecule also has the prospect of expanding to fields such as environmental monitoring and food safety assessment, and has a high application value.

[0095] In the structure of formula (1), R1 is one of hydrogen, halogen, carboxyl, sulfonic acid, ketone, aldehyde, nitro, carbonyl diimidazole, sulfonyl, sulfate, cyano and phenyl; R2 is one of hydrogen, halogen, carboxyl, sulfonic acid, ketone, aldehyde, nitro, carbonyl diimidazole, sulfonyl, sulfate, cyano, phenyl, amino and amino substituents. The inventors found that the K-SOO molecule has a triangular structure. The heterocyclic group as the "vertex" of the triangle changes during the reaction to generate intermediates and diketone structures, resulting in afterglow and changes in its own color, which can be said to be the "functional area" of the entire molecule. The benzene rings on the other two "points" of the triangle are relatively stable before and after the reaction, and the benzene ring structure is not affected or changed, so that the molecule has a stable main skeleton structure and also makes the molecule more modifiable. The selectivity of R1 and R2 connected to the two benzene rings is more. Specifically, R1 and R2 can be relatively stable groups or elements, such as hydrogen, halogen, phenyl, etc., which usually have little effect on the stability of the main body of the molecule, and are relatively stable in the reaction. Hydrogen and phenyl usually do not significantly change the electron density distribution of the main body of the molecule; R1 and R2 can also be independently selected from carboxyl, sulfonic acid, ketone, aldehyde, nitro, carbonyl diimidazole, sulfonyl, sulfate and cyano, which have electron-withdrawing properties, and can further change the electron cloud distribution of the molecular structure, so that the luminescence or self-color change performance of the biochemical detection reagent can be further adjusted. In addition, R2 can also be an amino group or an amino substituent, wherein the amino substituent refers to a group in which at least one of the two H connected to N in the amino group is replaced by other groups, for example, alkyl, aryl, nitrogen-containing heterocyclic, halogen, hydroxyl, etc. are replaced. The inventors found that if R2 is designed as an amino substituent, the performance of luminescence or self-color change can also be further optimized.

[0096] The biochemical detection reagent has a molecule having a structure as shown in formula (2):

[0097]

[0098] R3 and R4 are each independently selected from an alkyl group, an alkane derivative group, an aryl group, a heterocyclic group, a halogen, a carbonyl group, a sulfonic acid group, a carboxylic acid derivative group, an amino group, an ether group, a thiol group, an alkynyl group, an alkenyl group, a keto group and an ester group.

[0099] In the structure of formula (2), the benzene ring in the lower right corner is directly connected to N, and N is connected to the other two structures (R3 and R4). N has a triple bond structure, which makes the bond energy between molecules very high, so it is not easy to participate in chemical reactions. At the same time, N has a relatively large electronegativity and has an electron-withdrawing effect. The conjugated structure can be further modified and changed. Specifically, for example, when N is connected to an alkyl group, it can be an alkylamine structure, such as a methylamine structure; when N is connected to an aryl group, it can be an arylamine structure, such as an aniline structure; when N is connected to a heterocyclic group, it can be, for example, a pyridineamine structure; when N is connected to a halogen, it can be a haloamine structure, such as a chloramine structure; when N is connected to a carbonyl group, it can be a benzenesulfonamide structure; when N is connected to a carboxylic acid derivative group, it can be an acyl chloride amine structure; when N is connected to an amino group, it can be an ethylenediamine structure; when N is connected to an ether group, it can be a methoxyamine structure; when N is connected to a thiol, it can be a methylthiolamine structure; when N is connected to an alkynyl group, it can be an acetyleneamine structure; when N is connected to a ketone group, it can be an acetoneamine structure; when N is connected to an ester group, it can be an ethyl acetate amine structure. A biochemical detection reagent having such a molecular structure can have a more obvious self-color change, and the reliability of the detection result is enhanced.

[0100] In certain embodiments of the present invention, specifically, R1 is halogen or hydrogen, and R2 is amino or amino substituent or halogen. Under this structure, the molecular structure is easier to obtain, and the color change is more obvious, which is convenient for use.

[0101] In certain specific embodiments of the present invention, specifically, the alkane derivative group is one of a fatty alcohol group, a fatty acid group, an alkyl sulfonic acid group, an alkyl ether group, an alkyl amine group and an alkyl sodium sulfate group, R1 is a halogen or hydrogen, and R3 and R4 are each independently selected from an alkyl group or an alkane derivative group having 1 to 5 carbon atoms. Alkyl and alkane derivative groups generally provide chemical stability, are not easily involved in reactions, and provide a more stable support for the molecular structure. In addition, the inventors have found that in biochemical reagents, short-chain alkyl or alkane derivative groups can make the performance of biochemical reagents more stable and reliable.

[0102] In a specific embodiment of the present invention, the structure of the molecule is shown in one of formula (3) to formula (6):

[0103]

[0104] In formula (6), Bu represents a butyl group, including normal-butyl (n-Bu), isobutyl (i-Bu), and tert-butyl (t-Bu).

[0105] The above molecular structure can make the biochemical detection reagent have obvious afterglow luminescence and color change phenomenon, fast response speed and strong structural stability. There are also differences in the color changes of these molecules, which can be selected according to actual conditions.

[0106] Preferably, the structure of the molecule is as shown in formula (3):

[0107]

[0108] This molecule has excellent color-changing and afterglow effects, and is particularly suitable for use in biochemical detection reagents. In particular, when the concentration of the biochemical index to be detected is very low, it can also have a more obvious color change, which is conducive to the detection of absorbance by instruments. It has high sensitivity and can obtain accurate detection results regardless of whether the concentration is low or high.

[0109] In a specific embodiment of the present invention, the biochemical detection reagent also includes a horseradish peroxidase solution, the biochemical detection reagent is used to detect the concentration of biochemical indicators, the biochemical indicators generate hydrogen peroxide after the enzymatic reaction, the hydrogen peroxide decomposes to generate singlet oxygen under the action of the horseradish peroxidase solution, the biochemical detection reagent reacts with the singlet oxygen to detect the concentration of the biochemical indicator, the concentration of the horseradish peroxidase solution is 2-10KU / L, within this concentration range, the horseradish peroxidase solution can give full play to its role, and can ensure the effective use of the biochemical detection reagent.

[0110] In a specific embodiment of the present invention, the biochemical detection reagent is used to detect the concentration of at least one biochemical indicator among glucose, total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, creatinine and uric acid.

[0111] In a specific embodiment of the present invention, the biochemical detection reagent can detect at least one of 0.1-20mmol / L glucose, 0-12.9mmol / L total cholesterol, 0.05-11.29mmol / L triglycerides, 0.01-3.9mmol / L high-density lipoprotein cholesterol, 0-26.160mmol / L low-density lipoprotein cholesterol, 0-30mmol / L creatinine and 0-1000μmol / L uric acid. The detection range of the biochemical detection reagent is wider than that of existing reagents, and in particular, it can also effectively detect at low concentrations.

[0112] The second aspect of the present invention discloses a method for preparing a biochemical detection reagent, comprising the following steps:

[0113] A molecular compound having a structure as shown in formula (a) and a molecular compound having a structure as shown in formula (c) are mixed in a solvent and refluxed to obtain a molecular compound having a structure as shown in formula (1):

[0114]

[0115]

[0116] That is, the preparation route is specifically as follows:

[0117]

[0118] In another specific embodiment of the present invention, the preparation method comprises the following steps:

[0119] A molecular compound having a structure as shown in formula (b) and a molecular compound having a structure as shown in formula (c) are mixed in a solvent and refluxed to obtain a molecular compound having a structure as shown in formula (2):

[0120]

[0121] That is, the preparation route is specifically as follows:

[0122]

[0123] Specifically, the solvent used is one or more of benzene, toluene, xylene, ethylbenzene, isopropylbenzene, styrene, chlorobenzene, nitrobenzene, dichlorobenzene and trichlorobenzene, which are suitable for the synthesis of the molecular structure of the present invention, are relatively stable and easy to remove. In addition, the reflux time is 5-10 hours.

[0124] Through the above method, the desired molecular structure can be synthesized conveniently and efficiently, and the molecular yield is high, which can reach more than 78%, and is suitable for large-scale production.

[0125] Specifically, the steps include:

[0126] A molecular compound having a structure as shown in formula (d) and a molecular compound having a structure as shown in formula (c) are mixed in a solvent and refluxed to obtain a molecular compound having a structure as shown in formula (3):

[0127]

[0128] The solvent is toluene and the reflux time is 5 h.

[0129] The third aspect of the present invention discloses a method for detecting the concentration of a biochemical indicator using the biochemical detection reagent in any of the above embodiments, comprising the following steps:

[0130] Preparation of samples to be tested: preparing samples of biochemical indicators to be tested, wherein the biochemical indicators to be tested are at least one of glucose, total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, creatinine and uric acid;

[0131] Preparation of biochemical detection reagents: Prepare biochemical detection reagents, which include molecules having a structure as shown in formula (1) and horseradish peroxidase solution:

[0132]

[0133] Measurement and calculation: In the microfluidic device, a microfluidic pump is used to transport the biochemical index sample to be detected and the biochemical detection reagent to the mixing chamber of the microfluidic chip, and the absorbance is measured by an optical detection device. The concentration of the biochemical index to be detected is converted according to the absorbance by Lambert-Beer law, so that the concentration of the biochemical index to be detected can be obtained. Specifically, the optical detection device includes a spectrometer and a photometer.

[0134] By using the biochemical detection reagent of the present invention and the above method, the biochemical index to be detected can be sensitively responded to, and the concentration result can be accurately obtained regardless of whether it is in a low concentration or high concentration environment.

[0135] In another specific embodiment of the present invention, the biochemical detection reagent has a molecule having a structure as shown in formula (2):

[0136]

[0137] Preferably, the biochemical detection reagent has a molecule having a structure as shown in formula (3):

[0138]

[0139] Specifically, the measurement and calculation steps are performed at a temperature of 36.5-37.2°C, which matches the temperature of the human body, and the test results are more accurate.

[0140] Specifically, the step of preparing the sample to be tested also includes adding an enzyme to allow the indicator to be tested to react and generate hydrogen peroxide.

[0141] Specifically, in the measurement and calculation steps, the absorbance is measured within a wavelength range of 450-600 nm. The molecular structure of the present invention changes color before and after the reaction, specifically, the absorption spectrum within a wavelength range of 450-600 nm changes before and after the reaction. More specifically, by replacing the detection filter of a spectrometer or a photometer, the measurement can be performed at a specific wavelength within a wavelength range of 450-600 nm.

[0142] The fourth aspect of the present invention discloses a biochemical detection kit, including biochemical detection reagents. The biochemical detection kit includes at least one of a glucose reagent detection kit, a total cholesterol reagent detection kit, a triglyceride reagent detection kit, a high-density lipoprotein cholesterol reagent detection kit, a low-density lipoprotein cholesterol reagent detection kit, a creatinine reagent detection kit and a uric acid reagent detection kit. The biochemical detection kit can be used to obtain test results conveniently and reliably.

[0143] The following will be described in conjunction with more specific implementation methods.

[0144] Embodiment 1:

[0145] This embodiment uses a biochemical detection reagent containing K-SOO molecules to detect the biochemical index of glucose.

[0146] The K-SOO molecule was prepared as follows:

[0147]

[0148] A mixture of the compound having the molecular structure 1 shown above (1 mmol) and the compound having the molecular structure 2 (1.5 mmol) in 5 mL of toluene was refluxed for 5 h, the reaction mixture was cooled, washed with water, 10% HCl, washed with water again, and dried over MgSO4, the solvent was concentrated in vacuo, and the residue was recrystallized from ethanol to obtain a compound having the molecular structure of K-SOO with a yield of 78%.

[0149] The detection principle of this embodiment is that glucose oxidase (GOD) catalyzes the reaction of β-D-glucose with oxygen to generate D-glucose acid and hydrogen peroxide (H2O2), and hydrogen peroxide decomposes under the action of horseradish peroxidase (HRP) to generate singlet oxygen, which reacts with the K-SOO molecular structure to produce yellow ketone compounds. The reaction process is as follows:

[0150]

[0151] The specific operations are as follows:

[0152] First, prepare two liquid storage chambers. One liquid storage chamber contains glucose oxidase GOD horseradish peroxidase (HRP) and a substrate solution containing K-SOO molecules, which is referred to as reagent R1. The other liquid storage chamber contains the sample glucose to be tested, which is referred to as reagent R2. Prepare R2 reagents with different concentration gradients, and the theoretical concentrations are 0.10mmol / L, 0.30mmol / L, 0.50mmol / L, 1.00mmol / L, 5.00mmol / L, and 20.00mmol / L, respectively. Then, use a microfluidic pump to deliver the two reagents to the mixing chamber of the microfluidic chip at a precise flow rate. The entire experimental process is carried out at a constant temperature (37°C), and then the absorbance is measured by the spectrometer at the outlet of the microfluidic system. The concentration is converted according to the absorbance using the Lambert-Beer law. The results are shown in Table 1 below, and the color of the solution after the reaction in the chamber of the microfluidic device is photographed, see. Figure 1 , cut out the color blocks for comparison, the results are shown in Figure 2 .

[0153] Comparative Example 1:

[0154] This comparative example is aimed at glucose, a biochemical index in organisms, which is usually called blood sugar. This comparative example is tested using a traditional method.

[0155] In the traditional biochemical index detection of Glu, glucose oxidase (GOD) catalyzes the reaction of β-D-glucose with oxygen to generate D-gluconic acid and hydrogen peroxide (H2O2). The generated hydrogen peroxide then reacts with 4-aminoantipyrine (4-AAP) and phenol, and this reaction is catalyzed by peroxidase (such as horseradish peroxidase, HRP). The generated quinone compound is a red substance, and its color intensity is proportional to the original glucose concentration. The concentration of glucose can be determined by measuring the absorbance of the red quinone compound produced by the reaction. The absorbance is proportional to the concentration of quinone compounds in the solution, so the glucose content in the sample can be quantitatively analyzed by the standard curve method or by directly comparing with a standard solution of known concentration. The detection principle is as follows:

[0156]

[0157] The specific operation process is as follows:

[0158] GOD, 4-aminoantipyrine (4-AAP), phenol, and HRP were prepared in a phosphate buffer with a pH of 7.4 to form reagent R1. Reagent R2 was a β-D-glucose solution with theoretical concentrations of 0.10mmol / L, 0.30mmol / L, 0.50mmol / L, 1.00mmol / L, 5.00mmol / L, and 20.00mmol / L, respectively. Before the microfluidic system was started, all liquids were pumped to the corresponding liquid storage chambers by a pump. The microfluidic pump was started to allow the β-D-glucose solution and the GOD mixed solution to enter the mixing chamber at a predetermined flow rate, where β-D-glucose reacted with oxygen to generate D-gluconic acid and H2O2. Subsequently, H2O2 reacted with the 4-AAP and phenol mixed solution under the catalysis of HRP to form red quinone compounds. The final mixture flowed into the detection chamber and was detected by the spectral detector, and the absorbance of the quinone compounds was measured at a wavelength of 500nm. The results are shown in Table 1. The color of the solution after the reaction in the chamber of the microfluidic device was photographed and the color blocks were cut out for comparison. The results are shown in Table 1. Figure 2 .

[0159] The glucose oxidase (GOD) catalyzes the reaction of β-D-glucose and oxygen to generate hydrogen peroxide. When participating in the subsequent reaction, the ratio of 4-aminoantipyrine (4-AAP) and phenol to hydrogen peroxide is 1:2. Therefore, the concentration of the generated quinone compounds is relatively low, the color is lighter, and it is less obvious in microfluidics.

[0160] Table 1.

[0161]

[0162] Embodiment 2:

[0163] This embodiment uses a biochemical detection reagent containing K-SOO molecules to detect the biochemical index of total cholesterol (TC). The preparation method of K-SOO molecules is the same as that of Example 1.

[0164] The biochemical detection principle of this embodiment is as follows: first, cholesterol esterase (CE) catalyzes the hydrolysis of cholesterol ester into free cholesterol and fatty acids. Then, cholesterol oxidase (COD) catalyzes the reaction of free cholesterol with oxygen to generate cholesterol-4-ketone and hydrogen peroxide (H2O2). The generated hydrogen peroxide can be decomposed into singlet oxygen by horseradish peroxidase (HRP), and the singlet oxygen and K-SOO molecules react to generate ketone compounds to develop color.

[0165] In the experiment, referring to Example 1, the microfluidic device was preheated to 37°C, and all reagent solutions were prepared, and the mixed enzyme solution containing CE and COD was introduced into the reaction chamber of the microfluidic system. The cholesterol sample to be tested was introduced into the same reaction chamber by a microfluidic pump and mixed with the enzyme solution. In the mixing chamber, CE first hydrolyzed cholesterol esters into free cholesterol and fatty acids. Subsequently, COD oxidized free cholesterol into cholesterol-4-ketone and H2O2. The formed H2O2 reacted with K-SOO under the catalysis of HRP to produce color-changing ketone compounds, wherein the theoretical concentrations of the samples to be tested were 0.015mmol / L, 0.020mmol / L, 0.100mmol / L, 1.000mmol / L, and 5.000mmol / L, respectively. Finally, the absorbance of the solution after the reaction was measured, and the concentration was converted according to the method in Example 1, and the solution after the reaction was photographed, and some color blocks with uniform colors at different concentrations were intercepted for comparison. The results are shown in Figure 3 .

[0166] Comparative Example 2:

[0167] This comparative example is aimed at the biochemical detection index total cholesterol (TC), and mainly adopts the traditional method to detect it through enzymatic reaction.

[0168] The detection principle is that, first, cholesterol esterase (CE) catalyzes the hydrolysis of cholesterol esters into free cholesterol and fatty acids. Then, cholesterol oxidase (COD) catalyzes the reaction of free cholesterol with oxygen to generate cholesterol-4-ketone and hydrogen peroxide (H2O2). The generated hydrogen peroxide can be detected by a color reaction catalyzed by horseradish peroxidase (HRP). In this color reaction, HRP catalyzes the reaction of hydrogen peroxide with a certain color substrate (such as 4-aminoantipyrine, phenol or other color reagents) to produce a dark-colored product, the absorbance of which can be measured by a spectrophotometer. Common color reagents include 4-aminoantipyrine (4-AAP) and phenol or other similar compounds, which react with hydrogen peroxide under the catalysis of HRP to generate pigments, thereby changing the color of the solution. Finally, by measuring the absorbance of the color product, the total cholesterol concentration in the sample can be quantitatively analyzed according to the standard curve.

[0169] In the experiment, first, cholesterol esterase (CE) and cholesterol oxidase (COD) were pre-mixed in phosphate buffer solution (PBS, pH 7.4) to ensure the activity of the enzymes. Then, the mixed enzyme solution containing CE and COD was introduced into the reaction chamber of the microfluidic system, and the cholesterol sample to be tested was introduced into the same reaction chamber through a microfluidic pump to mix with the enzyme solution. In the mixing chamber, CE first hydrolyzed cholesterol esters into free cholesterol and fatty acids, and then COD oxidized free cholesterol into cholesterol-4-ketone and H2O2. The formed H2O2 reacted with the chromogenic substrate 4-aminoantipyrine under the catalysis of HRP to produce a dark-colored product. The mixture flowed into the detection chamber, which was equipped with a spectral detection component to measure the absorbance of the reaction product, and record the absorbance values ​​measured for each sample and the calculated cholesterol concentration. The solution after the reaction was photographed, and some color blocks with uniform colors at different concentrations were intercepted for comparison. The results are shown in Figure 3 .

[0170] Embodiment 3:

[0171] This embodiment uses a biochemical detection reagent containing K-SOO molecules to detect the biochemical detection indicator triglycerides (TG). The preparation method of K-SOO molecules is as in Example 1.

[0172] The detection principle in this embodiment is:

[0173] First, triglycerides are hydrolyzed under the action of specific lipases (such as fatty acid lipase) to produce glycerol and free fatty acids. Then, glycerol is converted into glycerol-3-phosphate (Glycerol-3-phosphate) under the action of glycerol kinase (GK). Then, glycerol-3-phosphate is further converted into dihydroacetyl phosphate under the action of glycerol-3-phosphate oxidase (GPO), and hydrogen peroxide (H2O2) is produced at the same time. Hydrogen peroxide produces singlet oxygen under the action of horseradish peroxidase (HRP), and singlet oxygen and K-SOO molecules react to develop color.

[0174] The specific operations are as follows:

[0175] Take 20 μL of serum sample, remove impurities such as blood cells by centrifugation, and take the supernatant as the analysis sample. Prepare a specific lipase (such as pancreatic lipase) to contain 1 mg of enzyme protein per milliliter. Mix glycerol kinase and glycerol phosphate oxidase to a final concentration of 0.5 U / mL and 1 U / mL, respectively. Prepare a 0.01 U / mL horseradish peroxidase (HRP) solution. The treated serum sample is introduced into the microfluidic chip through a microinjection pump at a flow rate of 0.5 μL / min. In the reaction area of ​​the chip, first add the lipase solution and mix with the serum sample, react at 37°C for 15 minutes to completely hydrolyze triglycerides. Then add glycerol kinase and glycerol phosphate oxidase solutions, also react at 37°C for 15 minutes to generate dihydroacetyl phosphate and hydrogen peroxide. Add HRP and K-SOO molecules to the mixture and react at room temperature for 5 minutes, so that hydrogen peroxide catalyzes the generation of singlet oxygen and reacts with K-SOO molecules to produce color changes. The intensity of the color generated is detected by a photometer located in the detection zone downstream of the chip, which is equipped with a detection filter for a specific wavelength (e.g., 450 nm) suitable for the K-SOO color development product. Based on the detected light absorption value, the triglyceride concentration in the sample is calculated by a pre-set standard curve and compared with the detection results of the traditional method.

[0176] Comparative Example 3:

[0177] This comparative example is a traditional biochemical detection method for triglycerides.

[0178] Here’s how it works:

[0179] Traditional biochemical detection of triglycerides usually uses an enzymatic color reaction, which is similar to the principle of enzymatic detection of glucose. First, triglycerides are hydrolyzed by a specific lipase (such as fatty acid lipase) to produce glycerol and free fatty acids. Then, glycerol is converted into glycerol-3-phosphate (Glycerol-3-phosphate) under the action of glycerol kinase (GK). Next, glycerol-3-phosphate is further converted into dihydroacetyl phosphate under the action of glycerol-3-phosphate oxidase (GPO), and hydrogen peroxide (H2O2) is produced at the same time. Hydrogen peroxide then reacts with certain substrates (such as 4-aminoantipyrine or certain phenolic compounds) under the action of horseradish peroxidase (HRP) to develop a color. The color intensity produced by this reaction is proportional to the concentration of triglycerides (TG).

[0180] Specifically, the microfluidic chip is injected with a pretreated serum sample at a flow rate of 0.2 μL / min through a microinjection pump. Lipase is added to the microfluidic chip and mixed with the sample, and reacted at 37°C for 10 minutes to hydrolyze triglycerides to generate glycerol and free fatty acids. Glycerol is converted into glycerol-3-phosphate under the action of glycerol kinase (GK), and this step reacts at 37°C for 10 minutes. Glycerol-3-phosphate generates dihydroacetyl phosphate and hydrogen peroxide under the action of glycerol-3-phosphate oxidase (GPO), and continues to react at 37°C for 10 minutes. HRP and a chromogenic substrate (such as 4-aminoantipyrine) are added to the reaction system, and a color reaction occurs within 5 minutes at room temperature. The absorbance value is measured using a spectral detector integrated downstream of the microfluidic chip, and the concentration of triglycerides is calculated by comparing it with the standard curve. The results show that compared with the results of Example 3, Example 3 can be detected in an extremely low concentration range of 0.05mmol / L to 0.11mmol / L, while the detection range in Comparative Example 3 is 0.11mmol / L to 11.29mmol / L. Therefore, in comparison, the detection limit in Example 3 is lower and the sensitivity is higher.

[0181] Embodiment 4:

[0182] This embodiment uses a biochemical detection reagent containing K-SOO molecules to detect the biochemical detection indicator high-density lipoprotein cholesterol (HDL-C). The preparation method of K-SOO molecules is as in Example 1.

[0183] Here’s how it works:

[0184] Special reagents or precipitants (such as phosphotungstic acid or manganese sulfate) are used to precipitate or inhibit non-HDL particles (low-density lipoprotein cholesterol LDL-C, very low-density lipoprotein cholesterol VLDL-C, etc.). In the separated sample, the cholesterol esters carried by the HDL particles are first hydrolyzed by cholesterol esterase into free cholesterol and fatty acids. Then, free cholesterol is oxidized to produce cholesterol-4-ene-3-one and hydrogen peroxide under the action of cholesterol oxidase. Hydrogen peroxide produces singlet oxygen under the action of horseradish peroxidase (HRP), and singlet oxygen and K-SOO molecules react to develop color.

[0185] The specific operations are as follows:

[0186] First, serum samples containing HDL-C were mixed with phosphotungstic acid reagent at a ratio of 1:10 (sample: reagent) and allowed to stand at room temperature (about 25°C) for 10 minutes to precipitate non-HDL particles. Subsequently, the supernatant, i.e., the solution containing HDL particles, was separated by centrifugation at 2000g centrifugal force for 5 minutes using a filter device on a microfluidic chip. This supernatant was mixed with cholesterol esterase solution (concentration of 1U / mL) at a ratio of 1:5 and incubated at 37°C for 15 minutes to hydrolyze cholesterol esters into free cholesterol and fatty acids. Next, cholesterol oxidase (concentration of 0.5U / mL) and horseradish peroxidase (HRP, concentration of 0.1U / mL) were added at a mixing ratio of 1:2 and continued to incubate at 37°C for 10 minutes to oxidize free cholesterol to cholesterol-4-ene-3-one and hydrogen peroxide. Finally, K-SOO colorimetric reagent (concentration of 1mM) was added at a mixing ratio of 1:1 and reacted at room temperature for 5 minutes to produce a color reaction. The absorbance was measured at a wavelength of 500 nm using an optical detection system on a microfluidic chip, and the actual concentration of high-density lipoprotein cholesterol was calculated. The linear relationship between the actual concentration and the theoretical concentration can be found in Figure 4 , its linear relationship is y = 0.821x + 0.5764, and the correlation R 2 =0.9697. The specific values ​​of the theoretical concentration and the measured concentration are shown in Table 2 below.

[0187] Table 2:

[0188]

[0189] Comparative Example 4:

[0190] This comparative example is a traditional biochemical detection method for high-density lipoprotein cholesterol (HDL-C). The principle is: use special reagents or precipitants (such as phosphotungstic acid or manganese sulfate) to precipitate or inhibit non-HDL particles (low-density lipoprotein cholesterol LDL-C, very low-density lipoprotein cholesterol VLDL-C, etc.). In the separated sample, the cholesterol ester carried by the HDL particles is first hydrolyzed into free cholesterol and fatty acids by cholesterol esterase. Then, free cholesterol is oxidized to generate cholesterol-4-ene-3-one and hydrogen peroxide under the action of cholesterol oxidase. Finally, the generated hydrogen peroxide reacts with a pigment precursor (such as 4-aminoantipyrine or other dyes) under the catalysis of peroxidase to generate a colored product. By measuring the color intensity of the reaction product (usually expressed as absorbance), the concentration of HDL-C in the sample can be quantitatively obtained. The intensity of this color is proportional to the level of high-density lipoprotein cholesterol.

[0191] The serum sample volume was 20 μL, and 5 μL of phosphotungstic acid or manganese sulfate was added as a precipitant. The sample and precipitant were mixed and incubated at room temperature for 10 minutes to precipitate non-HDL particles. The sample was centrifuged for 10 minutes at 10,000 rpm to separate HDL particles. The cholesterol esterase activity was set to 1 U / mL, the cholesterol oxidase activity was 1 U / mL, the peroxidase activity was 0.5 U / mL, and the 4-aminoantipyrine concentration was set to 3 mM. The channel width of the microfluidic chip was set to 100 μm and the depth was set to 50 μm. The separated HDL particle solution was introduced into the microfluidic chip at a flow rate of 0.1 μL / min using a microinjection pump. Cholesterol esterase was added and reacted at 37°C for 10 minutes to hydrolyze cholesterol esters into free cholesterol and fatty acids. Cholesterol oxidase was added and incubated at 37°C for another 10 minutes to oxidize free cholesterol to generate cholesterol-4-ene-3-one and hydrogen peroxide. Peroxidase and 4-aminoantipyrine were added and incubated at room temperature for 5 minutes to form a colored product. The absorbance of the generated colored product was measured using an integrated spectral detector, and the absorbance was converted into concentration. It was found that the detection limit according to the traditional method was 0.13 mmol / L.

[0192] Embodiment 5:

[0193] This embodiment uses a biochemical detection reagent containing K-SOO molecules to detect the biochemical detection indicator low-density lipoprotein cholesterol (LDL-C). The preparation method of K-SOO molecules is as in Example 1.

[0194] First, a specialized reagent or antibody is used to selectively bind to LDL particles, which protects the cholesterol in LDL from being destroyed by enzyme reactions while precipitating or inhibiting cholesterol in non-LDL particles (such as HDL, VLDL, and cholesterol esters). After pretreatment, the cholesterol esters in LDL particles are hydrolyzed by specific cholesterol esterases to generate free cholesterol and fatty acids. Subsequently, the free cholesterol generated is oxidized to cholesterol-4-ene-3-one and hydrogen peroxide under the action of cholesterol oxidase. Hydrogen peroxide generates singlet oxygen under the action of horseradish peroxidase (HRP), and singlet oxygen and K-SOO molecules react to develop color.

[0195] Before the experiment, LDL-specific binding reagents or antibodies were prepared to selectively bind to LDL particles to protect cholesterol in LDL and precipitate or inhibit cholesterol in non-LDL particles. The pretreated LDL sample was then introduced through a specific channel of the microfluidic system. This channel was connected to a channel containing a specific cholesterol esterase, so that the cholesterol ester in the LDL particles was hydrolyzed in a specific reaction area in the microfluidic chip to generate free cholesterol and fatty acids. Thereafter, free cholesterol was converted into cholesterol-4-ene-3-one and hydrogen peroxide under the action of cholesterol oxidase. Hydrogen peroxide continued to flow through the area containing horseradish peroxidase and reacted with K-SOO molecules under the action of HRP to develop color. The generated pigment was detected by a spectrometer in the detection area, and its absorbance was proportional to the LDL cholesterol concentration. The accurate concentration of LDL cholesterol in the experimental sample was calculated. The results are shown in Table 3 below.

[0196] Table 3:

[0197]

[0198] Comparative Example 5:

[0199] This comparative example is a traditional biochemical detection method for low-density lipoprotein cholesterol (LDL-C). The principle is: first, a special reagent or antibody is used to selectively bind to LDL particles, which can protect the cholesterol in LDL from being destroyed by enzyme reactions, while precipitating or inhibiting cholesterol in non-LDL particles (such as HDL, VLDL and cholesterol esters). After pretreatment, the cholesterol ester in the LDL particles is hydrolyzed by a specific cholesterol esterase to generate free cholesterol and fatty acids. Subsequently, the free cholesterol generated is oxidized to cholesterol-4-ene-3-one and hydrogen peroxide under the action of cholesterol oxidase. The generated hydrogen peroxide acts on the pigment precursor under the catalysis of peroxidase to generate a color change, the intensity of which is proportional to the amount of hydrogen peroxide generated in the reaction, and thus proportional to the amount of LDL-C. The concentration of LDL-C in the sample can be quantitatively analyzed by measuring the absorbance of the final product with a spectrophotometer.

[0200] The specific operation is: First, mix the serum sample with a special LDL-C selective binding reagent, usually using a ratio of 1:50 (sample: reagent), and incubate at 25°C for 30 minutes. The reagent contains antibodies that can specifically bind to LDL, thereby protecting the cholesterol in LDL from being destroyed. At the same time, the reagent will precipitate or inhibit cholesterol in non-LDL particles. After incubation, use the separation unit on the microfluidic chip to centrifuge at a force of 3000g for 10 minutes, remove the precipitate, and collect the supernatant containing LDL particles. Cholesterol esterase is then added at a concentration of 2U / mL and a ratio of 1:3, mixed with the supernatant, and incubated at 37°C for 40 minutes to hydrolyze cholesterol esters in LDL particles. After that, cholesterol oxidase was added at a concentration of 1U / mL, a ratio of 1:2, and peroxidase (such as horseradish peroxidase HRP) at a concentration of 0.5U / mL, a ratio of 1:2, and incubation was continued at 37°C for 20 minutes. The free cholesterol produced by oxidation was cholesterol-4-ene-3-one and hydrogen peroxide. Finally, pigment precursors such as 4-aminoantipyrine (4-AAP) and phenolic compounds were added at a ratio of 1:1 and a concentration of 2mM. The reaction was carried out at room temperature for 15 minutes. The color intensity formed was proportional to the amount of hydrogen peroxide generated. The optical detection unit integrated in the microfluidic chip was used to measure the absorbance at a wavelength of 450nm, and the concentration of LDL-C was calculated based on this. The results showed that the detection limit of the traditional method was 0.26mmol / L.

[0201] Embodiment 6:

[0202] This embodiment uses a biochemical detection reagent containing K-SOO molecules to detect the biochemical detection indicator creatinine Cr. The preparation method of K-SOO molecules is as in Example 1.

[0203] Here’s how it works:

[0204] The biochemical detection of creatinine (Cr) is mainly carried out by enzymatic methods. Specific enzymes (such as creatinase) are used to catalyze the reaction of creatinine and water to generate sarcosine and hydrogen peroxide. This step is similar to the process of glucose being catalyzed by glucose oxidase (GOD) in glucose detection. The generated hydrogen peroxide can be detected by a color development reaction catalyzed by horseradish peroxidase (HRP). Hydrogen peroxide produces singlet oxygen under the action of horseradish peroxidase (HRP), and singlet oxygen and K-SOO molecules react to develop color.

[0205] The specific operation process is as follows:

[0206] First, creatinase and horseradish peroxidase (HRP) are prepared in buffer solutions at appropriate concentrations to ensure enzyme activity and prevent spontaneous reactions. Next, a microfluidic pump is used to transport the creatinase solution and HRP solution to the reaction area of ​​the microfluidic chip. At the intersection of these channels, the creatinine sample to be tested is introduced and mixed with the enzyme solution. Creatinase catalyzes the reaction of creatinine with water to produce sarcosine and hydrogen peroxide. This reaction is carried out efficiently under the catalysis of creatinase. Subsequently, the generated hydrogen peroxide combines with HRP to promote the color development reaction of K-SOO molecules, produces singlet oxygen, and reacts with the color development substrate to produce a color change, which is detected by the light detector designed in the detection chamber downstream of the microfluidic chip. By measuring the absorbance of the color development product, the concentration of creatinine can be quantitatively analyzed. Results can be found in Figure 5 , its linear relationship is y = 0.293x + 0.034, and the correlation R 2 =0.9999.

[0207] Comparative Example 6:

[0208] The detection principle of this comparative example is that the biochemical detection of creatinine (Cr) is mainly carried out by enzymatic method. A specific enzyme (such as creatinase) is used to catalyze the reaction of creatinine and water to generate sarcosine and hydrogen peroxide. This step is similar to the process in which glucose is catalyzed by glucose oxidase (GOD) in glucose detection. The generated hydrogen peroxide can be detected by a color development reaction catalyzed by horseradish peroxidase (HRP). In this process, hydrogen peroxide reacts with a color developing substrate (such as 4-aminoantipyrine or a phenol compound) to produce a color change under the action of HRP. This step is the same as the principle of glucose detection. Finally, the absorbance of the generated pigment is measured by a spectrophotometer to quantitatively analyze the creatinine content in the sample. The absorbance is proportional to the creatinine concentration.

[0209] The steps for performing a creatinine detection experiment on a microfluidic platform begin with preparing a reaction mixture, including creatinase and horseradish peroxidase (HRP) and a corresponding chromogenic substrate (such as 4-aminoantipyrine or a phenolic compound). First, creatinase and HRP are pre-diluted in phosphate buffered saline (PBS) at appropriate pH and concentration conditions. Subsequently, these reagents are introduced into the reaction area through different input channels of the microfluidic system. The creatinine sample to be tested is also introduced through another channel and merged with the enzyme reagent in the mixing area of ​​the chip. Under the action of creatinase, creatinine reacts with water to produce sarcosine and hydrogen peroxide. The generated hydrogen peroxide then reacts with the chromogenic substrate under the catalysis of HRP to produce a color-changing substance. The product of this color reaction flows through the detection area, which is equipped with an optical sensor, such as a spectrophotometer, to measure the light absorbance passing through the area. The measured value of absorbance is directly related to the creatinine concentration, and the reading can be converted to a specific creatinine concentration through a pre-set standard curve. During the experiment, it was necessary to record the absorbance value of each sample and calculate the creatinine concentration. The results showed that the detection limit of the traditional method was 0.0177mmol / L.

[0210] Embodiment 7:

[0211] This embodiment uses a biochemical detection reagent containing K-SOO molecules to detect the biochemical detection indicator uric acid UA. The preparation method of K-SOO molecules is the same as in Example 1.

[0212] The principle is: Urate oxidase can catalyze the oxidation of uric acid to produce allantoin, hydrogen peroxide (H2O2) and carbon dioxide (CO2). Hydrogen peroxide produces singlet oxygen under the action of horseradish peroxidase (HRP), and singlet oxygen and K-SOO molecules react to develop color.

[0213] The operation mode is as follows: prepare a series of uric acid standard solutions for calibration and construction of standard curves, dissolve uric acid (168.11 g / moL) in PBS buffer (pH = 7.4) and dilute it into 1000, 500, 250, 125, and 62.5 μmol / L uric acid standard solutions in sequence. Use a microsyringe to introduce the urine sample to be tested and the enzyme reagent mixture into the corresponding inlet of the microfluidic chip respectively. Start the microfluidic pump and set the flow rate to 2 μL / min to ensure that the sample and the reagent are mixed. Pass the mixed solution through the preheated reaction area at 37°C, and urate oxidase converts uric acid into allantoin, hydrogen peroxide and carbon dioxide. Under the action of HRP, hydrogen peroxide reacts with K-SOO to generate a color product with specific absorption characteristics. Set different reaction zone lengths to adjust the reaction time and ensure the integrity of the reaction. Subsequently, the mixed solution flows into the detection area, and a micro-spectrophotometer is embedded in this area to measure the absorbance and obtain absorbance data.

[0214] The results are shown in Table 4 below, and the standard curve of uric acid is shown in Figure 6 , its linear relationship is y = 0.00664x + 0.00127, and the correlation R 2 =0.9987.

[0215] Table 4:

[0216]

[0217] Comparative Example 7:

[0218] The biochemical detection of uric acid (UA) usually adopts the enzymatic method, which is an analytical method based on enzyme reaction and can accurately determine the uric acid content in body fluids. The principle of enzymatic detection of uric acid mainly involves the catalytic reaction of urate oxidase and peroxidase. Urate oxidase can catalyze the oxidation of uric acid to produce allantoin, hydrogen peroxide (H2O2) and carbon dioxide (CO2). Under the action of peroxidase, the generated hydrogen peroxide reacts with specific chromogens 4-aminoantipyrine and 2,4,6-tribromo-3-hydroxybenzoic acid to produce colored products. The reaction principle is as follows:

[0219]

[0220] The specific operation is as follows: prepare a series of uric acid standard solutions for calibration and construction of a standard curve, dissolve uric acid (168.11 g / moL) in PBS buffer (pH = 7.4) and dilute it into 1000, 500, 250, 125, and 62.5 μmol / L uric acid standard solutions in sequence. Prepare an enzyme reagent mixture, including urate oxidase, peroxidase, 4-aminoantipyrine, and 2,4,6-tribromo-3-hydroxybenzoic acid. Introduce the urine sample to be tested and the enzyme reagent mixture into different inlets of the microfluidic chip through a micro-syringe. Turn on the microfluidic pump, control the flow rate to 2 μL / min, and mix the sample to be tested and the enzyme reagent mixture. The mixture flows through the reaction area at a constant temperature of 37°C, and uric acid is catalyzed by urate oxidase to generate allantoin and hydrogen peroxide. Hydrogen peroxide reacts with 4-aminoantipyrine and 2,4,6-tribromo-3-hydroxybenzoic acid under the action of peroxidase to generate a color product. The reaction zone is set to different lengths to adjust the reaction time to ensure the completeness of the reaction. After the reaction is completed, the mixed solution flows to the detection area. In the detection area, a micro-spectrophotometer is equipped to measure the absorbance of the solution flowing through and record the absorbance data.

[0221] See the color photograph of this comparative example. Figure 7, the absorbance test results are shown in Table 5. Generally speaking, as the concentration of uric acid increases, the color development increases accordingly, but it can be seen from the table that when the concentration is lower than 125 μmol / L, the linearity is poor, that is, the sensitivity is not enough, but when the concentration is higher than 125 μmol / L, it has a good linear relationship.

[0222] Table 5:

[0223]

[0224] Embodiment 8:

[0225] This embodiment uses a biochemical detection reagent containing K-SOO molecules to detect the biochemical detection indicator creatinine. The preparation method of K-SOO molecules is as in Example 1.

[0226] To perform biochemical detection of creatinine in a microfluidic chip, first prepare a creatinine sample using a mixture of enzyme reagents of creatinase and horseradish peroxidase (HRP) and a colorimetric substrate, K-SOO. Two groups of samples were injected into the sample inlet of the microfluidic chip, and the pump was started to mix the sample and reagent. At 37°C, creatinase catalyzed the oxidation of creatinine to produce hydrogen peroxide, which reacted with K-SOO under the action of HRP. After the color reaction, a specific excitation light source was used to illuminate the reaction area to produce singlet oxygen. Singlet oxygen reacted with K-SOO molecules, causing the molecular structure to change and emit afterglow light. The afterglow luminescence signal was detected by the built-in photomultiplier tube (PMT) of the microfluidic chip. Since the afterglow luminescence intensity is proportional to the creatinine concentration, its concentration can be calculated by the afterglow luminescence intensity of the sample. The results show that in the high concentration area, K-SOO molecules can detect the concentration of creatinine due to the afterglow phenomenon. The linear relationship is y=0.333x-0.1142, and the correlation R 2 =0.9978. For standard curve, see Figure 8 .

[0227] Comparative Example 8:

[0228] Perform biochemical detection of creatinine in a microfluidic chip. Prepare creatinine samples using a mixture of enzyme reagents of creatinase and horseradish peroxidase (HRP), and a chromogenic substrate, K-SOO. Inject the sample into the sample inlet of the microfluidic chip, and start the pump to mix the sample and reagents. At 37°C, creatinase catalyzes the oxidation of creatinine to produce hydrogen peroxide, which reacts with K-SOO under the action of HRP. However, due to the high creatinine concentration, the chromogenic reaction cannot provide sufficient color change to distinguish the samples. See Fig. 9 It can be seen that when the creatinine concentration is 25mM, 50mM, and 100mM, the color is almost the same, see Fig.10 , after the creatinine concentration reaches a certain value, the absorbance tends to be flat.

[0229] Although the present invention has been illustrated and described with reference to certain preferred embodiments of the present invention, it should be understood by those skilled in the art that the above is a further detailed description of the present invention in conjunction with specific embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. Those skilled in the art may make various changes in form and details, including making several simple deductions or substitutions, without departing from the spirit and scope of the present invention.

Claims

1. A biochemical detection reagent, characterized in that: The biochemical detection reagent includes a molecule having a structure as shown in formula (3): After the molecule reacts with singlet oxygen, the color of the biochemical detection reagent changes and an afterglow is emitted.

2. A biochemical detection reagent as claimed in claim 1, characterized in that: The biochemical detection reagent also includes a horseradish peroxidase solution, which is used to detect the concentration of a biochemical indicator. The biochemical indicator generates hydrogen peroxide after an enzymatic reaction, and the hydrogen peroxide decomposes under the action of the horseradish peroxidase solution to generate the singlet oxygen. The biochemical detection reagent detects the concentration of the biochemical indicator after reacting with the singlet oxygen, and the concentration range of the horseradish peroxidase solution is 2-10 KU / L.

3. A biochemical detection reagent as claimed in claim 2, characterized in that: The biochemical detection reagent is used to detect the concentration of at least one biochemical index among glucose, total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, creatinine and uric acid.

4. A biochemical detection reagent as claimed in claim 3, characterized in that: The biochemical detection reagent can detect at least one of 0.1-20mmol / L glucose, 0-12.9mmol / L total cholesterol, 0.05-11.29mmol / L triglycerides, 0.01-3.9mmol / L high-density lipoprotein cholesterol, 0-26.160mmol / L low-density lipoprotein cholesterol, 0-30mmol / L creatinine and 0-1000μmol / L uric acid.

5. A method for preparing a biochemical detection reagent according to any one of claims 1 to 4, characterized in that: The steps include: A molecular compound having a structure as shown in formula (d) and a molecular compound having a structure as shown in formula (c) are mixed in a solvent and refluxed to obtain a molecular compound having a structure as shown in formula (3): The solvent is toluene and the reflux time is 5 h.

6. A method for detecting the concentration of biochemical indicators using the biochemical detection reagent according to any one of claims 1 to 4, characterized in that: The steps include: Preparation of samples to be tested: preparing samples of biochemical indicators to be tested, wherein the biochemical indicators to be tested are at least one of glucose, total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, creatinine and uric acid; Preparation of biochemical test reagents: Prepare biochemical test reagents; Measurement and calculation: In the microfluidic device, a microfluidic pump is used to transport the biochemical indicator sample to be detected and the biochemical detection reagent to the mixing chamber of the microfluidic chip, and the absorbance is measured by an optical detection device. The concentration of the biochemical indicator is converted according to the absorbance using the Lambert-Beer law to obtain the concentration of the biochemical indicator to be detected.

7. The method according to claim 6, characterized in that The test sample preparation step also includes adding enzymes to make the biochemical indicators to be detected react to generate hydrogen peroxide.

8. The method according to claim 6, characterized in that In the measuring and calculating steps, the absorbance is measured within a wavelength range of 450-600 nm.

9. A biochemical detection kit, characterized in that: The biochemical detection kit includes the biochemical detection reagent according to any one of claims 1 to 4, and the biochemical detection kit includes at least one of a glucose reagent detection kit, a total cholesterol reagent detection kit, a triglyceride reagent detection kit, a high-density lipoprotein cholesterol reagent detection kit, a low-density lipoprotein cholesterol reagent detection kit, a creatinine reagent detection kit and a uric acid reagent detection kit.

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