CN114134202A - Method for measuring high-density lipoprotein cholesterol by using inorganic hybrid nanoflower - Google Patents

Method for measuring high-density lipoprotein cholesterol by using inorganic hybrid nanoflower Download PDF

Info

Publication number
CN114134202A
CN114134202A CN202111435637.XA CN202111435637A CN114134202A CN 114134202 A CN114134202 A CN 114134202A CN 202111435637 A CN202111435637 A CN 202111435637A CN 114134202 A CN114134202 A CN 114134202A
Authority
CN
China
Prior art keywords
cholesterol
density lipoprotein
reaction
lipoprotein cholesterol
hdl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202111435637.XA
Other languages
Chinese (zh)
Other versions
CN114134202B (en
Inventor
单博文
张继明
王荣光
董婷
林芊仪
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
LABNOVATION TECHNOLOGIES Inc
Original Assignee
LABNOVATION TECHNOLOGIES Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by LABNOVATION TECHNOLOGIES Inc filed Critical LABNOVATION TECHNOLOGIES Inc
Priority to CN202111435637.XA priority Critical patent/CN114134202B/en
Publication of CN114134202A publication Critical patent/CN114134202A/en
Application granted granted Critical
Publication of CN114134202B publication Critical patent/CN114134202B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/18—Multi-enzyme systems
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/03006—Cholesterol oxidase (1.1.3.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/01—Peroxidases (1.11.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01013—Sterol esterase (3.1.1.13)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/323—Arteriosclerosis, Stenosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Nanotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Manufacturing & Machinery (AREA)
  • General Physics & Mathematics (AREA)
  • Condensed Matter Physics & Semiconductors (AREA)
  • Medical Informatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Inorganic Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a method for measuring high-density lipoprotein cholesterol by inorganic hybridization nanometer flower, wherein a dry chemical test paper strip of a kit adopted by the method is provided with three layers, namely a diffusion layer, a blood filtration layer and a reaction reagent layer; the diffusion layer adopts a silk net film; the blood filtering layer adopts an asymmetric polyether sulfone (PES) membrane, is used for filtering blood cells, and can be used for directly detecting a whole blood sample. Compared with a magnesium phosphotungstate precipitation method, the method adopts a selective inhibition method, does not need direct precipitation, selects a surfactant with strong specificity, selectively dissociates HDL-C, selectively inhibits non-HDL-C, and provides a basis for timely, rapid and accurate determination of HDL-C clinically; compared with the traditional dry chemical method, the method has the advantages that the detection samples of various enzyme-inorganic hybrid nano flowers can directly carry out in-situ reaction on the surfaces of the nano flowers, the diffusion and the decomposition of intermediate products are reduced, the detection result is more sensitive and accurate, the method is high in accuracy, the reaction time is short, and the interference resistance is strong.

Description

Method for measuring high-density lipoprotein cholesterol by using inorganic hybrid nanoflower
Technical Field
The invention relates to the technical field of high-density lipoprotein cholesterol, in particular to a method for determining high-density lipoprotein cholesterol by using inorganic hybrid nanoflower.
Background
High density lipoproteins bind to cholesterol, i.e., high density lipoprotein cholesterol (HDL-C), and low density lipoproteins bind to cholesterol to form low density lipoprotein cholesterol (LDL-C). The decrease in HDL-C and its major apolipoprotein is closely associated with the incidence of atherosclerotic cardiovascular disease. HDL-C has effects in regulating reverse cholesterol transport, resisting oxidation, resisting thrombosis, resisting inflammation and affecting vascular endothelial function, and has positive effect on coronary heart disease.
Current studies have demonstrated that high density lipoprotein cholesterol levels to some extent determine the probability of atherosclerosis and coronary heart disease development. The adult therapist group of the national cholesterol education program of the United states believes that low HDL-C levels are an independent risk factor for inducing coronary heart disease, and therefore, it is of great clinical significance to detect HDL-C content. HDL-C content determination is now routinely performed in clinical laboratories. The suggestion by the American Cholesterol education program (NCEP) for the determination of HDL-C indicates that the accuracy of the determination of HDL-C is very important because (i) HDL-C is negatively associated with Coronary Heart Disease (CHD) in a narrow range; ② unlike other lipid assays, the CHD risk increase limits are at the low end of the HDL-C concentration range, and small errors in the assay can have a relatively large effect.
HDL can transport peripheral tissues such as cholesterol in blood vessel walls to liver for catabolism, namely reverse cholesterol transport, can reduce the deposition of cholesterol on blood vessel walls, and plays a role in resisting atherosclerosis. Because the content of cholesterol in HDL is relatively stable, the level of HDL in blood is often indirectly known by measuring the amount of cholesterol contained therein. HDL-C levels are also significantly affected by genetic factors. In the case of severe malnutrition, serum TC is significantly reduced and HDL-C is also reduced. The HDL-C level is also low in obese people. Smoking can lower HDL-C. Disease states such as diabetes, hepatitis and liver cirrhosis may be accompanied by low HDL-C. Patients with hypertriglyceridemia are often associated with low HDL-C. While exercise and small alcohol consumption can raise HDL-C. A large body of epidemiological data suggests that serum HDL-C levels are inversely correlated with risk of ASCVD. HDL-C detection methods are various, including ultracentrifugation, electrophoresis, chromatography, precipitation, homogenization, etc. At present, the liquid phase detection of HDL-C mainly adopts a homogeneous phase method.
The method for directly measuring HDL-C in the clinical laboratory in China at present can be divided into 3 generations:
generation 1 is chemical precipitation, commonly used precipitating agents are polyanions such as heparin, dextran sulfate or polymers such as polyethylene glycol or detergents with divalent cations such as (Mg)2+、Ca2+) Etc. work together. The specific principle is that the precipitation reagent can selectively gather the apoB-containing lipoprotein with positive charge, and HDL is dissolved in the solution. non-HDL aggregates are separated by centrifugation and HDL-C can be determined by cholesterol analysis of the supernatant. The Chinese medical society's test division recommends the use of magnesium phosphotungstate precipitation as a method for determining high density lipoprotein cholesterol, and as a reference standard for the detection of lipoproteins and apolipoproteins, whichIs a useful and effective criterion for evaluating other detection methods. However, the results of the detection have some errors, mainly because HDL can be classified into two types according to whether it contains apoE component, and HDL-C produced by HDL containing apoE component accounts for 10% of the total HDL-C. The precipitation method can precipitate HDL containing apoE components, but HDL containing no apoE components cannot be precipitated, so that the concentration of HDL-C is reduced, and simultaneously, lipoprotein of apoB cannot be completely precipitated, so that the supernatant has certain turbidity, and the accuracy of the result is influenced; meanwhile, the sample is large in dosage, so that a plurality of link errors are easy to generate, and particularly, the sample needs to be subjected to centrifugal precipitation in advance, so that the full automation of the whole detection process cannot be realized, and the use of the method is limited to a certain extent.
In the 2 nd generation, a magnetic bead separation method is used, wherein reagents, magnetic beads and the like are added into serum, the serum is mixed and oscillated in a vortex mixer, then the serum is transferred into a magnetic test tube and placed in a Hitachi analyzer, the magnetic beads can be combined with particles containing apoB, the apoB can be magnetically attracted and separated in a magnetic field, and HDL-C can be detected by an enzyme solution after remaining in a supernatant. However, the magnetic bead method is susceptible to interference, which affects the accuracy of the detection result. The magnetic bead method omits a centrifugation step, but a special device is needed, the invention and the reagent are not suitable for popularization and application, and the accuracy is not ideal, so the application range is limited.
The 3 rd generation is a homogeneous phase determination method, a homogeneous phase method for directly determining HDL-C without precipitation is not needed, the sample dosage is less, precipitation treatment is not needed, the method can be used for determination of an automatic biochemical analyzer, and the method is simple, convenient and quick, reduces the pretreatment, reduces the cost, is easy to automate, attracts the attention at home and abroad, is widely applied to clinical routine analysis, is used for clinical automatic determination, and basically achieves the analysis target in the aspects of accuracy and precision, so the method is rapidly adopted by clinical laboratories in short years.
The methods all need to be provided with large-scale biochemical instruments, are more suitable for batch detection of a large number of samples, and require special training of operators. But more and more medical staff and consumers tend to use more economical and easier measurement methods and, in addition, in order to make such tests more practical for consumers and physicians, the equipment used for the tests must be small and mobile. As a result of the demand, the reagent and the matching instrument which can realize bedside detection have low price, small volume and simple and convenient operation and can better meet the clinical and user demands. At present, a dry chemical commercial kit for testing blood fat exists, but a natural enzyme is required to be used for carrying out catalytic reaction on lipoprotein in a dry chemical test strip, and the molecular structure of the enzyme is easy to degrade, so that the catalytic activity of the enzyme is lost. In addition, the experimental result of the natural enzyme catalytic reaction has poor repeatability and complex purification process, and the application of the method is also greatly limited.
The enzyme is a biocatalyst in chemical reactions, has the advantages of high catalytic efficiency, high selectivity, strong substrate specificity, high water solubility, low toxicity, mild reaction conditions, wide sources and the like, and is widely applied. In general, most enzymes catalyze reactions specifically under mild experimental conditions. However, most enzymes have proteins as their main components, and the proteins are easily affected by high temperature, strong acid or alkali conditions, and organic solvents. Therefore, under severe conditions, the molecular structure of the enzyme is easily degraded, thereby losing the catalytic activity of the enzyme. In addition, the experimental result of the enzyme catalytic reaction has poor repeatability and complex purification process, and the application of the enzyme catalytic reaction in the fields of catalysis, biosensors and the like is also greatly limited.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for determining high-density lipoprotein cholesterol by using inorganic hybrid nanoflowers, which overcomes the problems of long testing time, large error, complex testing method, low detection sensitivity and the like in the prior art, and the kit can simply, rapidly and highly sensitively detect the concentration of the high-density lipoprotein cholesterol. The determination method can overcome the defects of the existing dry chemical commodity kit in high density lipoprotein cholesterol content, and the method can be used for determining the high density lipoprotein cholesterol content in whole blood, serum or plasma on a dry biochemical analyzer by monitoring the change of absorbance.
In order to achieve the purpose, the invention provides a kit adopted by a method for determining high-density lipoprotein cholesterol by using inorganic hybrid nano flowers, a dry chemical test strip of the kit is provided with three layers, wherein,
a first layer: a diffusion layer;
a second layer: a blood filtration layer;
and a third layer: a reaction reagent layer;
the diffusion layer adopts a silk net film; the blood filtering layer adopts an asymmetric polyether sulfone (PES) membrane, is used for filtering blood cells, and can be used for directly detecting a whole blood sample.
Preferably, the reactive agent layer includes: selecting a PIPES buffer solution: 100-200mmol/L, enzyme-Zn3(PO4)21-4g/L, B-6610-20 g/L, F-8810-20 g/L, MADB 6.3.3 g/L, 4-AA 5.6g/L, 0.01g/L potassium ferrocyanide, 1-10ml/L surfactant and 1-10mmol/L stabilizer, preparing a reaction solution, uniformly coating the reaction solution on the surface of a reaction film, and then drying, preferably drying at 37 ℃ for 4 hours.
Preferably, the selected PIPES buffer has a pH of 7.0 to 8.
Preferably, the stabilizer is selected from one of sucrose and trehalose.
Preferably, the enzyme-Zn3(PO4)2The nanoflower includes: selecting
Figure BDA0003377970510000041
Then stirring for a period of time, and centrifuging to obtain multi-enzyme-Zn3(PO4)2Hybridizing the nanometer flowers, and performing freeze-drying and collection.
Preferably, the pH of the PBS buffer solution is 7.0-8.0.
The invention also provides a method for measuring high-density lipoprotein cholesterol by using the inorganic hybrid nanoflower, which comprises the following steps:
step one, synthesizing various enzymes-Zn3(PO4)2Nano flower: selection of Zn3(PO4)2·4H2O is an inorganic component, cholesterol esterase, cholesterol oxidase and peroxidase are organic components, and in the presence of Phosphate Buffer Solution (PBS), a one-step coprecipitation method is adopted to synthesize a plurality of enzymes-Zn3(PO4)2The hybridized nanometer flower is characterized in that the nanometer flower,
first, the protein molecule is reacted with Zn2+Forming a complex by coordination of amide groups in the protein backbone;
then, protein and Zn2+The interaction between them forms micron-sized particles with nanometer-scale features and shaped like petals;
finally, the formation of fully hybrid nanoflowers by anisotropic growth, during which the protein induces Zn3(PO4)2The nucleation of the crystal forms petals, and the protein self-assembles and combines the nanoparticles and the petals like 'glue';
and step two, performing a color development reaction: formed multiple enzymes-Zn3(PO4)2The enzymatic active sites of the hybridized nanoflower are close to each other in the nanomaterial, so that the reaction can carry out cascade reaction in situ; reacting analytes in a sample with a plurality of enzyme-Zn 3(PO4)2 hybridized nanoflowers, catalyzing cholesterol ester by cholesterol esterase to form free cholesterol and fatty acid, oxidizing the free cholesterol into cholestenone and hydrogen peroxide under the action of cholesterol oxidase, and generating a colored dye by the hydrogen peroxide and a colorless dye under the action of peroxidase;
step three, determining the concentration of the high-density lipoprotein cholesterol: the quinoneimine generated by the color reaction has an absorption peak at the wavelength of 500 nm-600 nm, the change of the absorbance value measured at the wavelength of 500 nm-600 nm is proportional to the color depth, and the concentration of the high-density lipoprotein cholesterol is determined by the reflected light photometry of an instrument.
By adopting the technical scheme of the invention, the invention has the following beneficial effects: 1. compared with liquid phase biochemical reaction, the method adopts a dry chemical method, so that the prepared instrument has small volume, simple and convenient operation and short detection time, can be operated without professional inspectors, and better meets the requirements of clinical clients.
2. Compared with a magnesium phosphotungstate precipitation method, the method adopts a selective inhibition method, does not need direct precipitation, selects a surfactant with strong specificity, selectively dissociates HDL-C, selectively inhibits non-HDL-C, and provides a foundation for timely, rapid and accurate determination of HDL-C clinically.
3. Compared with the traditional dry chemical method test, the method adopts a self-assembly method to synthesize various enzyme-inorganic hybrid nanoflowers, the detection sample can directly carry out in-situ reaction on the surface of the nanoflowers, the diffusion and the decomposition of intermediate products are reduced, the detection result is more sensitive and accurate, and the method has high accuracy, quick reaction time and strong interference resistance.
Detailed Description
The present invention will be further described with reference to the following examples.
The invention provides a kit for determining high-density lipoprotein cholesterol by inorganic hybridization nanometer flower, a dry chemical test paper strip of the kit is provided with three layers, wherein,
a first layer: a diffusion layer;
a second layer: a blood filtration layer;
and a third layer: a reaction reagent layer;
the diffusion layer adopts a silk net film; the blood filtering layer adopts an asymmetric polyether sulfone (PES) membrane, is used for filtering blood cells, and can be used for directly detecting a whole blood sample.
The reactive agent layer includes: selecting a PIPES buffer solution: 100-200mmol/L, enzyme-Zn3(PO4)21-4g/L, B-6610-20 g/L, F-8810-20 g/L, MADB 6.3.3 g/L, 4-AA 5.6g/L, 0.01g/L potassium ferrocyanide, 1-10ml/L surfactant and 1-10mmol/L stabilizer, preparing a reaction solution, uniformly coating the reaction solution on the surface of a reaction film, and drying the reaction film at 37 ℃ for 4 hours.
The pH of the selected PIPES buffer solution is 7.0-8.
The stabilizer is selected from one of sucrose and trehalose.
The enzyme-Zn3(PO4)2The nanoflower includes: selecting
Figure BDA0003377970510000061
Then stirring for 3 hours, and obtaining the multi-enzyme-Zn by centrifugation3(PO4)2Hybridizing the nanometer flowers, and performing freeze-drying and collection.
The pH of the PBS buffer solution is 7.0-8.
The invention also provides a method for measuring high-density lipoprotein cholesterol by using the inorganic hybrid nanoflower, which comprises the following steps:
step one, synthesizing various enzymes-Zn3(PO4)2Nano flower: selection of Zn3(PO4)2·4H2O is an inorganic component, cholesterol esterase, cholesterol oxidase and peroxidase are organic components, and in the presence of Phosphate Buffer Solution (PBS), a one-step coprecipitation method is adopted to synthesize a plurality of enzymes-Zn3(PO4)2The hybridized nanometer flower is characterized in that the nanometer flower,
first, the protein molecule is reacted with Zn2+Complexes are formed by coordination of amide groups in the protein backbone, and these complexes become Zn3(PO4)2Nucleation sites for primary crystals;
then, protein and Zn2+The interaction between them forms micron-sized particles with nanometer-scale features and shaped like petals;
finally, the formation of fully hybrid nanoflowers by anisotropic growth, during which the protein induces Zn3(PO4)2The nucleation of the crystal forms petals, and the protein self-assembles and combines the nanoparticles and the petals like 'glue';
and step two, performing a color development reaction: the enzymatic active sites of the formed multiple enzyme-Zn 3(PO4)2 hybrid nanoflowers are close to each other in the nanomaterial, so that the reaction can be carried out in situ to carry out a cascade reaction; reacting analytes in a sample with a plurality of enzyme-Zn 3(PO4)2 hybridized nanoflowers, catalyzing cholesterol ester by cholesterol esterase to form free cholesterol and fatty acid, oxidizing the free cholesterol into cholestenone and hydrogen peroxide under the action of cholesterol oxidase, and generating a colored dye by the hydrogen peroxide and a colorless dye under the action of peroxidase;
step three, determining the concentration of the high-density lipoprotein cholesterol: the quinoneimine generated by the color reaction has an absorption peak at the wavelength of 500 nm-600 nm, the change of the absorbance value measured at the wavelength of 500 nm-600 nm is proportional to the color depth, and the concentration of the high-density lipoprotein cholesterol is determined by the reflected light photometry of an instrument.
Among the various surfactants screened by the invention, F-68 has a better selective dissociation inhibition effect on LDL, and B-66 has a better selective dissociation effect on HDL.
Figure BDA0003377970510000071
Figure BDA0003377970510000072
Figure BDA0003377970510000073
When an enzyme is used as a protein component of the hybrid nanoflower, it shows enhanced enzymatic activity and stability compared to the free enzyme. The immobilized enzyme has a nano-scale structure of a nano material and a high specific surface area, is beneficial to reducing mass transfer limitation of enzyme and substrate in an enzyme catalysis reaction, keeps the catalytic activity of the enzyme and enhances the stability of the enzyme, and is a nano biocatalyst different from the traditional nano enzyme.
The dry test strip prepared by the formula is used for testing an internal reference substance on a dry biochemical analyzer, and the performance indexes of the reagent are evaluated:
1. accuracy:
taking reference substances No. 2 and No. 5, respectively and repeatedly measuring the two reference substances for 3 times by using a dry biochemical analyzer, and respectively calculating the relative measurement deviation B of the samples with two different concentrations, wherein the relative deviation is less than or equal to 10 percent, and the following table 1:
TABLE 1
Figure BDA0003377970510000081
From the above table 1, it can be seen that the relative deviation is within the range of-10% to 10%, and the accuracy of the kit meets the requirements.
2、CV：
Taking reference samples No. 3 and No. 4 as samples, measuring each concentration sample for 10 times, respectively calculating the average value, the standard deviation and the variation coefficient, wherein the measurement result is less than or equal to 10 percent, and the following table 2:
TABLE 2
Figure BDA0003377970510000082
Figure BDA0003377970510000091
As can be seen from the table 2, CV values are all less than or equal to 10%, and the accuracy of the kit meets the requirement.
3. Linearity:
the high concentration samples (H) near the upper limit of the linear region are diluted with the low concentration samples (L) near the lower limit of the linear region, mixed into 6 diluted concentrations (Xi), and the samples of each concentration are repeatedly examined 3 times to calculate an average value. And (4) performing linear fitting by taking the dilution concentration (X) as an independent variable and the concentration mean (Y) of the detection result as a dependent variable to obtain a linear regression equation. Calculating the correlation coefficient (r) of the linear regression, wherein r is more than or equal to 0.9900, and the following table 3:
TABLE 3
Figure BDA0003377970510000092
From the above Table 3, it can be seen that the linear correlation coefficient r is not less than 0.9900, and meets the performance requirements.
As can be seen from tables 1 to 3 above: the performance evaluation experiment result of the kit shows that the precision accuracy and the linear range evaluation experiment of the product both meet the design requirements and can meet the clinical actual requirements.
By adopting the technical scheme of the invention, the invention has the following beneficial effects: 1. compared with liquid phase biochemical reaction, the method adopts a dry chemical method, so that the prepared instrument has small volume, simple and convenient operation and short detection time, can be operated without professional inspectors, and better meets the requirements of clinical clients.
2. Compared with a magnesium phosphotungstate precipitation method, the method adopts a selective inhibition method, does not need direct precipitation, selects a surfactant with strong specificity, selectively dissociates HDL-C, selectively inhibits non-HDL-C, and provides a foundation for timely, rapid and accurate determination of HDL-C clinically.
3. Compared with the traditional dry chemical method test, the method adopts a self-assembly method to synthesize various enzyme-inorganic hybrid nanoflowers, the detection sample can directly carry out in-situ reaction on the surface of the nanoflowers, the diffusion and the decomposition of intermediate products are reduced, the detection result is more sensitive and accurate, and the method has high accuracy, quick reaction time and strong interference resistance.
The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications and equivalents of the present invention, which are made by the present specification and directly/indirectly applied to other related technical fields within the spirit of the present invention are included in the scope of the present invention.

Claims (7)

1. A kit for measuring high-density lipoprotein cholesterol by inorganic hybridization nanometer flower is characterized in that a dry chemical test paper strip of the kit is provided with three layers, wherein,
a first layer: a diffusion layer;
a second layer: a blood filtration layer;
and a third layer: a reaction reagent layer;
the diffusion layer adopts a silk net film; the blood filtering layer adopts an asymmetric polyether sulfone (PES) membrane, is used for filtering blood cells, and can be used for directly detecting a whole blood sample.
2. The kit for measuring high density lipoprotein cholesterol by using inorganic hybrid nanoflower according to claim 1, wherein the preparation of the reaction reagent layer comprises: selecting a PIPES buffer solution: 100-200mmol/L enzyme-Zn3(PO4)21-4g/L, B-6610-20 g/L, F-8810-20 g/L, MADB 6.3.3 g/L of nanoflower, 5.6g/L of 4-AA, 0.01g/L of potassium ferrocyanide, 1-10ml/L of surfactant and 1-10mmol/L of stabilizer, preparing a reaction solution, uniformly coating the reaction solution on the surface of a reaction film, and drying.
3. The kit for measuring high density lipoprotein cholesterol by inorganic hybrid nanoflower according to claim 6, wherein the pH of the selected PIPES buffer is 7.0-8.
4. The kit for measuring high density lipoprotein cholesterol by using inorganic hybrid nanoflower according to claim 6, wherein the stabilizer is one selected from sucrose and trehalose.
5. The kit for assaying high density lipoprotein cholesterol by inorganic hybrid nanoflower according to claim 6, wherein the enzyme-Zn3(PO4)2Preparing the nanoflower: selecting
Figure FDA0003377970500000011
Then stirring for a period of time, and centrifuging to obtain the multi-enzyme-Zn 3(PO4)2 hybrid nanoflower, and freeze-drying and collecting.
6. The kit for performing high density lipoprotein cholesterol assay of inorganic hybrid nanoflower according to claim 6, wherein the pH of the PBS buffer solution is 7.0-8.
7. A method for measuring high-density lipoprotein cholesterol by inorganic hybrid nano flowers is characterized by comprising the following steps:
step one, synthesizing various enzymes-Zn3(PO4)2Nano flower: selection of Zn3(PO4)2·4H2O is an inorganic component, cholesterol esterase, cholesterol oxidase and peroxidase are organic components, and in the presence of phosphate buffer solution, a one-step coprecipitation method is adopted to synthesize various enzymes-Zn3(PO4)2The hybridized nanometer flower is characterized in that the nanometer flower,
first, the protein molecule is reacted with Zn2+Forming a complex by coordination of amide groups in the protein backbone;
then, protein and Zn2+The interaction between them forms micron-sized particles with nanometer-scale features and shaped like petals;
finally, forming complete hybrid nanoflower through anisotropic growth;
and step two, performing a color development reaction: the enzymatic active sites of the formed multiple enzyme-Zn 3(PO4)2 hybrid nanoflowers are close to each other in the nanomaterial, so that the reaction can be carried out in situ to carry out a cascade reaction; reacting analytes in a sample with a plurality of enzyme-Zn 3(PO4)2 hybridized nanoflowers, catalyzing cholesterol ester by cholesterol esterase to form free cholesterol and fatty acid, oxidizing the free cholesterol into cholestenone and hydrogen peroxide under the action of cholesterol oxidase, and generating a colored dye by the hydrogen peroxide and a colorless dye under the action of peroxidase;
step three, determining the concentration of the high-density lipoprotein cholesterol: the quinoneimine generated by the color reaction has an absorption peak at the wavelength of 500 nm-600 nm, the change of the absorbance value measured at the wavelength of 500 nm-600 nm is proportional to the color depth, and the concentration of the high-density lipoprotein cholesterol is determined by the reflected light photometry of an instrument.
CN202111435637.XA 2021-11-26 2021-11-26 Method for measuring high-density lipoprotein cholesterol by using inorganic hybrid nanoflower Active CN114134202B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111435637.XA CN114134202B (en) 2021-11-26 2021-11-26 Method for measuring high-density lipoprotein cholesterol by using inorganic hybrid nanoflower

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111435637.XA CN114134202B (en) 2021-11-26 2021-11-26 Method for measuring high-density lipoprotein cholesterol by using inorganic hybrid nanoflower

Publications (2)

Publication Number Publication Date
CN114134202A true CN114134202A (en) 2022-03-04
CN114134202B CN114134202B (en) 2022-12-13

Family

ID=80389115

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111435637.XA Active CN114134202B (en) 2021-11-26 2021-11-26 Method for measuring high-density lipoprotein cholesterol by using inorganic hybrid nanoflower

Country Status (1)

Country Link
CN (1) CN114134202B (en)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007295935A (en) * 2007-08-03 2007-11-15 Dai Ichi Pure Chem Co Ltd Quantitative determination method of cholesterol
WO2008087895A1 (en) * 2007-01-15 2008-07-24 Toyo Boseki Kabushiki Kaisha Method for determination of high-density lipoprotein cholesterol, and reagent for the method
US20090246807A1 (en) * 2008-04-01 2009-10-01 Guojing Sun Method for quantitative measurements of HDL-C and LDL-C
CN104437280A (en) * 2014-11-03 2015-03-25 西北工业大学 Preparation method of organic/inorganic hybridized magnetic nano flower
CN106383116A (en) * 2016-10-21 2017-02-08 北京世纪沃德生物科技有限公司 Kit for detecting high density lipoprotein cholesterol
CN107449748A (en) * 2017-08-10 2017-12-08 威特曼生物科技（南京）有限公司 HDL-C detection kit and its application method
CN109709320A (en) * 2019-01-18 2019-05-03 吉林大学 Based on protein-inorganic hybrid nano flower acetylcholine detection kit and preparation method thereof
CN109884042A (en) * 2019-03-08 2019-06-14 武汉璟泓万方堂医药科技股份有限公司 A kind of test paper and its preparation method and application for measuring high-density lipoprotein cholesterol

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008087895A1 (en) * 2007-01-15 2008-07-24 Toyo Boseki Kabushiki Kaisha Method for determination of high-density lipoprotein cholesterol, and reagent for the method
JP2007295935A (en) * 2007-08-03 2007-11-15 Dai Ichi Pure Chem Co Ltd Quantitative determination method of cholesterol
US20090246807A1 (en) * 2008-04-01 2009-10-01 Guojing Sun Method for quantitative measurements of HDL-C and LDL-C
CN104437280A (en) * 2014-11-03 2015-03-25 西北工业大学 Preparation method of organic/inorganic hybridized magnetic nano flower
CN106383116A (en) * 2016-10-21 2017-02-08 北京世纪沃德生物科技有限公司 Kit for detecting high density lipoprotein cholesterol
CN107449748A (en) * 2017-08-10 2017-12-08 威特曼生物科技（南京）有限公司 HDL-C detection kit and its application method
CN109709320A (en) * 2019-01-18 2019-05-03 吉林大学 Based on protein-inorganic hybrid nano flower acetylcholine detection kit and preparation method thereof
CN109884042A (en) * 2019-03-08 2019-06-14 武汉璟泓万方堂医药科技股份有限公司 A kind of test paper and its preparation method and application for measuring high-density lipoprotein cholesterol

Also Published As

Publication number Publication date
CN114134202B (en) 2022-12-13

Similar Documents

Publication Publication Date Title
EP0252747B1 (en) Use of phenols and anilines to increase the rate of peroxidase catalyzed oxidation of leuco dyes
CN106383116A (en) Kit for detecting high density lipoprotein cholesterol
CN1756834A (en) Dry chemistry, lateral flow-reconstituted chromatographic enzyme-driven assays
EP2108961B1 (en) Dry analytical element for measurement of high density lipoprotein cholesterol
CN106018829A (en) Testing reagent for serum amyloid protein A and preparation method of testing reagent
Mascini et al. Determination of free and total cholesterol in human bile samples using an enzyme electrode
CN106442355A (en) Determination reagent for heart-type fatty acid binding protein and preparation method of determination reagent
CN108949902A (en) A kind of serum small and dense low-density lipoprotein cholesterin detection reagent box
CN114134202B (en) Method for measuring high-density lipoprotein cholesterol by using inorganic hybrid nanoflower
JP2000116400A (en) Quantitative analysis of cholesterol in lipoprotein
US20090130696A1 (en) HDL Cholesterol Sensor Using Selective Surfactant
JP3677452B2 (en) Procedures and diagnostic products for the determination of triglycerides in lipoproteins
KR100449216B1 (en) Measurement method of test substance by chemiluminescence amount control
CN114813875B (en) Method for detecting 1, 5-anhydroglucitol based on optical addressing potential sensor
CN109916892A (en) A kind of active dry chemistry reagent piece of quantitative determination alanine aminotransferase and preparation method thereof
Sharma et al. The turbid specimen as an analytical medium: hemoglobin determination as a model
CN110160974A (en) A kind of kit detecting sdLDL-C
CN113308513B (en) Small and dense low-density lipoprotein cholesterol detection kit and detection method thereof
CN114134203B (en) Method for measuring high-density lipoprotein cholesterol by using nano enzyme
CN108037281A (en) A kind of cardic fatty acid binding protein immunologic function test reagent and its preparation and detection method
CN101221129B (en) Sulfated bile acid enzyme fluorescence capillary analytical method and enzyme fluorescence quantitative reagent kit
MX2008012641A (en) Lipovrotein senso.
JPS5845562A (en) Method of measuring beta-lipoprotein fraction in body fluid and measurement reagent
US6013465A (en) Method for detection and determination of enzymatic reactions in urine
CN114891854B (en) Reagent for measuring small dense low density lipoprotein cholesterol and kit thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant