Background and overview[1][2]
Phosphatase is an enzyme that hydrolyzes phosphoesters. It is widely distributed and has many types. According to the different hydrogen ion concentration required during action, it can be divided into two categories: alkaline phosphatase and acid phosphatase. Determination of phosphatase in plasma is often used in the clinical diagnosis of certain diseases such as liver disease. Alkaline phosphatase catalyzes the removal of 5′-phosphate residues from single- or double-stranded DNA and RNA molecules, that is, dephosphorylation. In 1977, bacterial alkaline phosphatase (BAP) and calf intestinal alkaline phosphatase (CIP) were isolated from bacteria and calf intestine. In genetic engineering, this enzyme is mainly used to treat vector DNA cut by restriction endonucleases to remove the 5′-phosphate residues at both ends of the vector DNA to prevent self-circulation of the vector DNA. Thereby improving its reorganization efficiency. At the same time, when labeling the 5′-OH end with the isotope 32P to prepare DNA or RNA probes, first use this enzyme to remove 5′ -phosphate group to generate a 5′-OH end, and then perform end labeling.
Structure[3-5]
Alkaline phosphatase (AP, alkaline phosphatase) is a phosphatase that exists widely in various organisms and participates in cellular phosphorus metabolism and signal peptide transduction. Its optimal pH value is above 7.0, and it can catalyzes the hydrolysis of many phosphate monoesters at a rate of Phosphate cannot be synthesized in cells. When the amount of phosphate is limited, cells must obtain the phosphate required by the organism from nucleic acids, phosphorylated sugars and proteins through hydrolysis by phosphatase. Therefore, AP comes from a wide range of sources, including bacteria, fungi, algae, invertebrates, and vertebrates. AP is a labeling enzyme used in enzyme-linked immunolabeling assays (ELISA). It interacts with alkaline phosphatase and a chromogenic reagent or a luminescent substrate after dephosphorylation to reveal the presence of the target and detection enzyme complex. . AP as a non-isotopic label is widely used in various aspects such as Southern and Northern imprint analysis and DNA sequence analysis. AP can catalyze the removal of the 5′ phosphate group of DNA, RNA, ribonucleoside triphosphate and deoxyribonucleoside triphosphate, excise the 5′ terminal phosphate group of rNTPs and dNTPs, prepare templates for 5′ end labeling, and prevent DNA fragmentation Self-ligation or self-circulation ligation of cloning vectors, dephosphorylation of proteins, and dephosphorylation of protein serine, threonine, and tyrosine residues. DNA fragments treated with AP lack the 5′ phosphate end required by ligase, making them unable to self-ligate. This feature can be used to reduce the background of vector DNA during cloning.
In addition, alkaline phosphatase (AP) is an enzyme widely present in various biological tissues. The concentration of AP is closely related to the occurrence of bone diseases, hepatitis, prostate cancer and other diseases. Hypophosphatasia (HPP) is a rare autosomal genetic disease due to the alkaline phosphatase gene. Systemic diseases caused by mutations in the alkalinephosphatase gene (ALPL). Severe HPP is usually inherited in an autosomal recessive manner, while mild HPP can be inherited in a dominant or recessive manner. Typical symptoms are hypomineralization of bones and teeth and low serum alkaline phosphatase (ALP) activity. The incidence of severe HPP is about one in 100,000, while mild HPP is more common. HPP has many clinical manifestations of varying degrees. The main diagnostic basis is serum ALP activity detection and molecular genetic testing. There is currently no reliable treatment for this disease.
In addition, alkaline phosphatase is also widely used in analysis and detection, such as the analysis and detection of L-phenylalanine. The steps are as follows: (1) Dilute the sample to be tested in TBS buffer solution to prepare The solution to be tested; (2) Add alkaline phosphatase solution to the solution to be tested, and react to prepare an enzyme reaction solution; (3) Drop the enzyme reaction solution onto the liquid crystal modified by the sodium dodecyl phosphate solution On the sensor, observe using a polarizing microscope. The invention relates to a method for detecting L-phenylalanine in the urine of patients with phenylketonuria based on liquid crystal sensor analysis. It has low detection limit, easy-to-obtain detection instruments, simple, fast and sensitive detection methods, low reagent consumption and low sample processing. It has the advantages of short time and solves the problems of complex and high cost of existing detection methods. There are also experiments that use the catalytic reaction of alkaline phosphatase to trigger the photocurrent of the C antioxidant 4020dS photoelectrode, and establish a high-throughput immunoassay method that separates the immune reaction from the photoelectrode. The CdS photoelectrode with metal oxides or hydrated metal oxides deposited on the surface basically has no photocurrent signal, and the reducing agent produced by the catalytic hydrolysis reaction of alkaline phosphatase decomposes the metal oxides or hydrated metal oxides, resulting in CdS photoelectrode A substantial increase in auroral current. Using mouse IgG as a model, alkaline phosphatase-labeled detection antibodies were used to detect the sandwich structure immunoreaction performed in a 96-well microplate. The biggest advantage of this method is to separate the immune reaction from the electrode surface, avoiding the impact of biomolecules immobilized on the electrode surface on the sensitivity and accuracy of photoelectrochemical measurement. More importantly, the introduction of microplate reactions and the separation of immune reactions and photoelectric detection have greatly improved detection efficiency.
Vitality detection 3]
A method for detecting alkaline phosphatase activity based on photoactive nanomaterial simulated enzyme. The photoactive nanomaterial simulated enzyme can be produced in situ through the catalytic reaction of alkaline phosphatase; using alkalineThe photoactive nanomaterial formed by the phosphatase catalytic reaction simulates the efficient enzyme-like catalytic activity of the enzyme to achieve signal amplification, which can conveniently, sensitively and quickly detect alkaline phosphatase activity. The technical solution is as follows:
a. Preparation of titanium dioxide nanomaterials: Add 5 mL of titanium-containing compound dropwise to 75 mL of ultrapure water, and the mixed solution is continuously stirred at room temperature. The above solution was then transferred to the reaction kettle and heated to 160°C for 24 h. The product is obtained by centrifugation, washed several times with ultrapure water, and finally dried to obtain white titanium dioxide nanoparticles;
b. Determination of alkaline phosphatase activity: 5 μmol/L alkaline phosphatase substrate and 10 μL alkaline phosphatase of different concentrations are mixed and added to a 96-well microplate, and allowed to react at room temperature for a period of time; After adding 10 μL of 1.5 mg/mL titanium dioxide nanomaterials and reacting for 15 minutes, add 100 μL of 0.2 mol/L acetic acid buffer solution with a pH of 4.0 and 20 μL of 5 mmol/L nanomaterials simulating the characteristic substrate of the enzyme, place it under a xenon lamp and use visible light (λ≥nm) After irradiation for 10 minutes, the absorption spectrum or fluorescence spectrum of the system was measured on a microplate reader.
Main reference materials
[1] Research progress on thermostable alkaline phosphatase in food biotechnology
[2] CN201811110180.3 An analysis and detection method of L-phenylalanine based on liquid crystal sensor
[3] CN201711316296.8 An immunoassay method based on alkaline phosphatase catalyzed reaction triggering CdS photocurrent
[4] Research progress on hypoalkaline phosphatasia
