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ALP activity detection kit based on photo-ATRP signal amplification strategy and use method thereof

An activity detection and signal amplification technology, applied in the field of biological analysis, can solve the problems related to diseases such as osteoblastic bone tumor, osteomalacia, leukemia reaction or lymphoma, ALP inhibition, activity below the normal range, etc. People are satisfied with the effect of selectivity, easy operation and good sensitivity

Pending Publication Date: 2022-05-10
HENAN UNIV OF CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Studies have shown that abnormal ALP activity is closely related to a variety of diseases. For example, elevated ALP activity in serum is usually associated with diseases such as biliary obstruction, osteoblastic bone tumors, osteomalacia, leukemia-like reactions, or lymphomas; on the contrary, some metabolic diseases , such as Wilson's disease, chronic myelogenous leukemia, etc. will cause pathological inhibition of ALP activity, making its activity lower than the normal range

Method used

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  • ALP activity detection kit based on photo-ATRP signal amplification strategy and use method thereof
  • ALP activity detection kit based on photo-ATRP signal amplification strategy and use method thereof
  • ALP activity detection kit based on photo-ATRP signal amplification strategy and use method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: Kit

[0045] An ALP activity detection kit based on the photo-ATRP signal amplification strategy, including the following raw materials: electrode, 3-mercaptopropionic acid (MPA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiethylene Amine Hydrochloride (EDC), N-Hydroxysuccinimide (NHS), O-Phosphoethanolamine, 2-Bromoisobutyryl Bromide (BIBB), Dimethyl Sulfoxide (DMSO), Eosin Y (EY) , Tris-(N,N-dimethylaminoethyl)amine (Me 6 TREN), ferrocenylmethyl methacrylate (FMMA), LiClO 4 .

[0046] When in use, prepare some raw materials into solutions, wherein the concentration of MPA solution is 10mM, the concentration of EDC in the EDC / NHS mixed solution is 20mM, the concentration of NHS is 5mM, the concentration of O-phosphoethanolamine solution is 10mM, and the concentration of BIBB solution is 10mM. The solution concentration is 5mM, Me 6 The concentration of TREN solution is 10mM, the concentration of FMMA solution is 10mM, LiClO 4 The solution concentration i...

Embodiment 2

[0047] Embodiment 2: ALP activity detection method

[0048] (1) MPA modification

[0049] Soak the pretreated gold electrode in 400μL 10mM MPA solution and incubate at 37℃ for 2h;

[0050] (2) Activation and modification of MPA carboxyl group phosphoethanolamine

[0051] Soak the electrode obtained in step (1) in 400 μL EDC / NHS mixed solution (EDC concentration is 20 mM, NHS concentration is 5 mM), incubate at 37 °C for 0.5 h, then soak the electrode in 400 μL 10 mM O-phosphoethanolamine solution, 37 °C Incubate for 1h;

[0052] (3) ALP dephosphorylation and BIBB modification

[0053] Drop 10 μL of the solution to be detected (including ALP) on the surface of the electrode obtained in step (2), incubate at 37°C for 1 hour, then soak the electrode in 400 μL of 10mM BIBB solution, and incubate at 37°C for 1 hour;

[0054] (4) Light-mediated ATRP reaction

[0055] Soak the electrode obtained in step (3) in the photo-ATRP reaction solution (the reaction solution is to add 3990 ...

Embodiment 3

[0062] Example 3: Feasibility Study

[0063] In this study, a series of blank control experiments were used to study the feasibility of the method of the present invention in detecting alkaline phosphatase (ALP) activity. The SWV curves of various modified electrodes can be found in figure 2 see in. Without MPA (curve b), O-phosphoethanolamine (curve c), ALP (curve d), BIBB (curve e), FMMA (curve f), EY (curve g), Me 6 In the case of TREN (curve h) and no light (curve i), there is almost no electrochemical signal response except for a weak background signal. When the electrode is modified step by step according to the method of the present invention, an obvious oxidation current signal (curve a) can be observed, the peak potential is about 0.28V, and the electroactive molecules of ferrocene in LiClO 4 The potential range in solution is consistent. The generation of oxidation current can be attributed to the electrochemical oxidation of ferrocene electroactive molecules, d...

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Abstract

The invention discloses an ALP activity detection kit based on a photo-ATRP signal amplification strategy and a use method thereof. The kit comprises the following raw materials: an electrode, MPA, EDC, NHS, O-phosphoethanolamine, BIBB, DMSO, EY, Me6TREN, FMMA and LiClO4. The photo-ATRP reaction is used as a polymerization reaction signal amplification strategy, so that the sensitivity of ALP activity detection is remarkably improved, meanwhile, the use of a transition metal catalyst is avoided, and the method has the advantages of high efficiency, convenience in operation and environmental friendliness. Under the optimal experimental conditions, the linear range of the method for ALP activity detection is 10-150mU / mL, and the limit of detection (LOD) is 2.12 mU / mL, so that the method has good sensitivity. The method provided by the invention has satisfactory selectivity, anti-interference performance, reproducibility and stability, and can obtain good experimental results in clinical serum sample and inhibition rate experiments.

Description

technical field [0001] The invention relates to an alkaline phosphatase (ALP) activity detection kit based on a light-mediated atom transfer radical polymerization (photo-ATRP) signal amplification strategy and a use method thereof, belonging to the technical field of biological analysis. Background technique [0002] Alkaline phosphatase (ALP) is a homodimeric metalloprotease widely present in prokaryotes and eukaryotes, and the zinc and magnesium atoms in its structure play a crucial role in the dephosphorylation of ALP role. Under alkaline conditions, ALP can catalyze the hydrolysis of phosphate monoester structures in proteins, nucleic acids and small molecules. Therefore, ALP plays an important role in physiological functions such as cell division, osteogenesis, mineralization, and detoxification. Studies have shown that abnormal ALP activity is closely related to a variety of diseases. For example, elevated ALP activity in serum is usually associated with diseases su...

Claims

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Application Information

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IPC IPC(8): G01N27/48C12Q1/42
CPCG01N27/48C12Q1/42
Inventor 杨怀霞郭亮张亚萍陈璐瑶张雨婷卢静郭文锋司富春
Owner HENAN UNIV OF CHINESE MEDICINE
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