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Fluorescence chemical method for detecting ALP (Alkaline Phosphatase) and application thereof

A chemical method, phosphatase technology, applied in the field of enzyme activity detection, can solve the problems of time-consuming and toxic operation steps

Active Publication Date: 2019-04-05
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In recent years, relevant researchers have also explored some fluorescent probes for ALP detection, such as conjugated polyelectrolytes, small molecule organic probes, metal nanoclusters, nanosheets, quantum dots, etc., but the operation steps are time-consuming and involve toxic or expensive reagents

Method used

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  • Fluorescence chemical method for detecting ALP (Alkaline Phosphatase) and application thereof
  • Fluorescence chemical method for detecting ALP (Alkaline Phosphatase) and application thereof
  • Fluorescence chemical method for detecting ALP (Alkaline Phosphatase) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Embodiment 1 Principle and feasibility verification of the inventive method

[0079] The detection schematic diagram of alkaline phosphatase (Alkaline phosphatase) of the present invention is as figure 1 As shown, in order to verify the feasibility of the present invention, the inventor detects three important links of the experiment, and the specific process is as follows:

[0080] 1. Terminal deoxynucleotidyl transferase (TdT) can catalyze the addition reaction at the 3'-OH end of ssDNA in a template-free manner. The specific implementation process is as follows:

[0081] (1) A 14nt primer (Primer) ssDNA-2 (5'-ACCCCCCACCCCCA-3') was designed and synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0082] (2) Mix primer ssDNA-2 containing 2.5μM, 1×TdT buffer (20mM Tris-HAc, 50mM KAc, 10mMMg(Ac) 2 , pH 7.9), 250 μM CoCl 2 , 0.75mM dTTPs and TdT (addition amount: 0U, 0.5U, 1U, 2U, 5U, even if the concentration of TdT in the system is 0U / mL, 25U / mL, 50U / mL, 100U...

Embodiment 2

[0097] The optimization of embodiment 2 experimental system

[0098] 1. Condition optimization for the best ratio of dTTPs to ssDNA-p

[0099] In order to achieve the best state of the detection system, we optimize the ratio of dTTPs / ssDNA-p, the steps are as follows:

[0100] According to the method in embodiment 1.3 (feasibility verification) (wherein the consumption of ALP is 2unit in the step (2), the final concentration of TdT is 250U / mL in the step (3), HgCl in the step (4) 2 The final concentration is 2.5μM), and the dTTPs / ssDNA-p molar ratio in the reaction system is set to 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, 10000:1, 12000 :1, 14000:1, and measure the fluorescence intensity signal of the system to be tested.

[0101] The result is as image 3 Shown: by image 3 It can be seen that with the increase of the dTTPs / ssDNA-p ratio, the fluorescence signal enhancement of the detection system increases exponentially. In order to avoid data fluctuations and ob...

Embodiment 3

[0113] Example 3 Alkaline phosphatase activity detection

[0114] The specific implementation process of alkaline phosphatase activity detection is as follows:

[0115] (1) ssDNA-p containing 2.5 μ M primer probe, 1 × ALP buffer, ALP (final concentration of ALP 0 ~ 2500mU / mL; final concentration of ALP selected in this embodiment are: 0mU / mL, 0.025mU / mL, 0.05mU / mL, 0.25mU / mL, 0.5mU / mL, 2.5mU / mL, 5mU / mL, 50mU / mL, 250mU / mL, 500mU / mL, 2500mU / mL) samples were incubated at 37°C For 80 minutes, incubate at 90°C for 10 minutes to inactivate ALP, and finally cool the sample to room temperature for the next reaction.

[0116] (2) Add the sample obtained in step (1) to 5unit TdT (that is, the final concentration is 250U / mL), 1×TdTbuffer, and the final concentration is 250μM CoCl 2 , with a final concentration of 0.75mM dTTPs, mixed and incubated at 37°C for 70min, and then incubated at 72°C for 10min.

[0117] (3) 2.5μM HgCl 2 (final concentration) and the sample obtained in step (...

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Abstract

The invention discloses a fluorescence chemical method for detecting ALP (Alkaline Phosphatase) and application thereof. The fluorescence chemical method comprises the following steps of: firstly, designing a single-chain DNA (Deoxyribonucleic Acid) of which a 3' terminal is phosphorylated as a DNA probe, dephosphorylating the 3' terminal of the DNA probe and exposing a 3'-OH terminal when the ALPexists; secondly, performing catalytic addition on dTTPs (Deoxythymidine Triphosphates) at a 3'-OH terminal of the DNA probe by TdT (Terminal Deoxynucleotidyl Transferase), thus extending to generatea poly-T tail ssDNA, forming a dual-chain DNA mediated by T-HgII-T structure by the poly-T tail ssDNA and Hg ions, and drawing a detecting curve according to a detected fluorescence intensity signaland the concentration of the ALP after adding nucleic acid dye; finally, replacing the ALP with a to-be-detected sample, repeating the steps, and comparing a detecting result with the detecting curve,thus calculating the content of the ALP in the sample. The fluorescence chemical method disclosed by the invention is simple and convenient, is high in sensitivity and can be used for detecting the ALP or screening ALP inhibitors.

Description

technical field [0001] The invention belongs to the technical field of enzyme activity detection, in particular to a fluorescent chemical method and application for detecting alkaline phosphatase. Background technique [0002] Alkaline phosphatase (ALP) is the most important hydrolase in the phosphatase family, because of its wide distribution, it can be detected from bacteria to eukaryotes. ALP can not only hydrolyze and remove phosphate groups in proteins but also hydrolyze and remove phosphate groups in non-proteins. Because it can decompose organic phosphate compounds, it plays an important regulatory function in the biogeochemical cycle of phosphorus. In addition, ALP is also an exoenzyme of osteoblasts. Its main physiological function is to provide phosphoric acid for the deposition of hydroxyapatite by hydrolyzing phosphate and hydrolyzing pyrophosphate to release its inhibition on bone salt formation during osteogenesis. effect. At the same time, it is found clini...

Claims

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Application Information

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IPC IPC(8): C12Q1/44
CPCC12Q1/44G01N2333/916
Inventor 邢曦雯王媛媛姚盛中刘奋勇
Owner JINAN UNIVERSITY
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