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A method for measuring alkaline phosphatase based on a path selector using alkaline phosphatase as a switch, a kit and its application
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A technology of path selection and determination method, applied in the direction of determination/inspection of microorganisms, biochemical equipment and methods, instruments, etc., can solve problems such as limited selectivity, and achieve the effect of high selectivity, simple design and fast readout speed
Active Publication Date: 2022-04-26
HUAZHONG UNIV OF SCI & TECH
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However, the selectivity of λexo to 5’-PO4 and 5’-OH is limited, and even in complex signal amplification designs, the LOD of these methods is only 0.01U / L to 40U / L
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Embodiment 1
[0033] The screening of embodiment 1 track 2
[0034] Add 5 μL probe (2.5 pmol), 5 μL complementary strands (2.5 pmol) with 1nt, 2nt, 3nt, 4nt, 5nt, 6nt, 7nt, 8nt and 10nt protruding 1nt, 2nt, 3nt, 4nt, 5nt, 6nt, 7nt, 8nt and 10nt at the 5' end, 2.5 μL 10×CutSMart buffer and 2.5 μL 10×λexo buffer (commercially available λexo buffer composition: 67mM Glycine-KOH, 2.5mM MgCl 2 , 50 μg / ml BSA, pH 9.4, 25° C.) and 35 μL water. Heat up at 85°C and anneal at 55°C, add 2.5×10 when it cools down to 37°C -4 U of alkaline phosphatase and 1 U of λexo. The adsorption plate was then placed in a microplate reader for fluorescence measurement. Fluorescence intensity was recorded every 30 seconds for 20 minutes with a gain level of 60. The excitation and absorption wavelengths were set to 485nm and 582nm, respectively. The sequences of the probes and complementary strands are shown in Table 1 below.
[0035] Test results such as figure 2 As shown, the results show that for the backgro...
Embodiment 2
[0040] Embodiment 2 performance detection and application
[0041] 1. Sensitivity:
[0042] Dissolve DNAdry powder (i.e., DNA strand) in 100 μL of TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8.0), measure the concentration on a NanoDrop 2000 UV–vis spectrophotometer (ThermoFisher Scientific), and dilute the DNA solution with water to 5 μmol / L, stored at 4°C.
[0043] The DNA solution prepared in the previous step (probes with 5' end protruding 1-nt, 2-nt, 3-nt, 4-nt, 5-nt, 6-nt, 7-nt, 8-nt and 10-nt Complementary strand) was further diluted to 0.5 μmol / L. Add 35 μL of deionized water, 2.5 μL of CutSmart buffer and 2.5 μL of λexo buffer into ten 600 μL EP tubes, add 5 μL of probes, and then add 0-10 nt complementary strands (the amount of complementary strands added) Add 5 μL to 5 μmol / L, that is, 25 pmol. 0-nt refers to 0 bases protruding from the 5' end of the complementary strand, that is, no protruding). Heating at 85°C and annealing at 55°C, and adding to a 400 μL ELI...
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Abstract
The invention belongs to the technical field of biological analysis and detection, and in particular relates to an alkaline phosphataseassay method based on a path selector with alkaline phosphatase as a switch, a kit and an application thereof. The present invention designs double-stranded deoxyribonucleic acid with two tracks. Track 1 uses 5’-FAM and 3’-BHQ-1 as fluorescent probes, and there is a 2-nt overhang at the end of 5’-FAM. Track 2 is an ssDNA with a 5’‑PO4, most of which is complementary to the probe, with a 0‑10nt overhang at the 5’‑PO4 end. In the absence of alkaline phosphatase, λexo selectively binds to the 5’‑PO4 end and digests the complementary strand, resulting in a low fluorescent signal. When alkaline phosphatase is present in the sample, 5'‑PO4 in track 2 is converted to 5'‑hydroxyl; λexo selects to bind to the 5'‑FAM end and digests the probe to leave FAM and BHQ‑1 and emit strong fluorescenceSignal.
Description
technical field [0001] The invention belongs to the technical field of biological analysis and detection, and in particular relates to an alkaline phosphataseassay method based on a path selector with alkaline phosphatase as a switch, a kit and an application thereof. Background technique [0002] Alkaline phosphatase is an enzyme that catalyzes the dephosphorylation reaction. It widely exists in various tissues such as liver, intestine, kidney, bone and placenta. Abnormal levels of alkaline phosphatase have been shown to be highly correlated with a variety of diseases, including bone-related diseases, liver dysfunction, prostatecancer, breast cancer, and diabetes. [0003] So far, many methods have been developed to quantify the enzyme activity of alkaline phosphatase, such as colorimetry, surface-enhanced Raman scattering, chromatography, and electrochemical methods. However, these methods with their own characteristics inevitably suffer from some limitations, such as ...
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