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A compound and an alkaline phosphatase activity fluorescence detection method adopting the compound

A phosphatase activity and compound technology, applied in the field of new compounds, can solve the problems of high background and non-specific binding, and achieve the effect of improving accuracy and sensitivity

Active Publication Date: 2014-10-22
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Tetraphenylethylene is also an emerging fluorescent dye in biochemistry. Although it has high molar extinction coefficient and quantum yield, and has important applications in the field of biomacromolecule detection, it has high background and non-specific binding. , then FRET can solve this error very well and exclude the influence of other factors

Method used

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  • A compound and an alkaline phosphatase activity fluorescence detection method adopting the compound
  • A compound and an alkaline phosphatase activity fluorescence detection method adopting the compound
  • A compound and an alkaline phosphatase activity fluorescence detection method adopting the compound

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Preparation of 2-(3,6-diphosphoryloxy-9H-oxanthene-9-yl)-4(5)-((4-(1,2-diphenyl-2-(p Tolyl) vinyl) benzyl) carbamoyl) benzoic acid

[0045]

[0046] Firstly, methyl benzophenone (1.96g, 10mmol) and zinc powder (1.95g, 30mmol) were added into a dry three-necked flask, a magnet was put into it, and then it was evacuated, followed by nitrogen gas. Use a syringe to pipette the newly distilled THF solution (50 mL) into a three-necked flask, and stir the solution, and the solution becomes a gray suspension at this time. Pipette (1.7mL, 15mmol) TiCl with a syringe 4 , and gradually added dropwise to the three-neck flask at -60°C, with the TiCl 4 The solution reacted violently, and a yellow solid appeared on the wall of the flask. After the dropwise addition, it reacted at room temperature, and the color of the solution became bright black. Then, the temperature of the oil bath was raised to 78° C. and refluxed overnight. After the reaction was completed, the m...

Embodiment 2

[0054] Embodiment 2: Real-time detection of alkaline phosphatase activity by FRET fluorescent probe:

[0055] Dissolve the sample of alkaline phosphatase activity to be tested in a microcentrifuge tube with a concentration of 50 mM and a boric acid buffer solution with a pH value of 8.4 to obtain a sample solution (22.5 mg / mL) of alkaline phosphatase activity to be tested, and take 20 μL to be tested The sample solution of alkaline phosphatase activity was put into 278 μL of 50 mM pH8.4 boric acid buffer, and 2 μL of the compound (i) prepared in Example 1 was added with a concentration of 7.5 mM. The final concentration of the fluorescent probe in the reaction solution was 50 μM. Use a microplate reader to immediately detect the fluorescence intensity in the sieve plate, and analyze the relationship between the fluorescence intensity and time as follows: figure 1 , figure 2 shown.

[0056] Experiments have shown that the fluorescence intensity increases gradually with the p...

Embodiment 3

[0057] Embodiment 3: FRET fluorescent probe concentration is to the detection of alkaline phosphatase activity:

[0058] In order to obtain the effect of probe concentration on the detection results, a set of gradient experiments was designed according to the application model of fluorescent probes, and three parallel experimental groups were taken. Add an appropriate amount of boric acid buffer solution (concentration 50mM, pH=8.4) in a microcentrifuge tube, and then add 20 μL of enzyme solution (alkaline phosphatase sample is dissolved in a boric acid buffer solution with a concentration of 50 mM and a pH value of 8.4 to obtain a concentration of 22.5 mg / mL enzyme solution), and finally add an appropriate amount of probe to make the final concentration of 0μM, 25μM, 50μM, 75μM, 100μM, 150μM, 200μM, take 200μL after 2h of reaction and record the experimental data with a fluorescent microplate reader, λ ex / λ em = 350 / 530nm.

[0059] The effect of probe concentration on the...

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Abstract

The invention discloses a compound shown as a formula (i) and a preparing method thereof. The compound can be adopted as a fluorescence probe and applied for an alkaline phosphatase activity fluorescence detection method. By utilization of a fluorescence resonance energy transfer theory, and by adopting a tetraphenylethylene derivative and 5(6)-carboxyl fluoresce as a donor and a receptor for an FRET system, an "OFF-ON"FRET probe adopting a phosphate group as a recognition group is designed and synthesized. Equipment needed for preparation of the fluorescence probe is simple. Operation processes are simple. The sources of raw materials are commercialized and easily available. The probe has high specificity for the alkaline phosphatase. When the fluorescence probe is applied for alkaline phosphatase activity fluorescence detection, the accuracy and the sensitivity are largely improved than those of basic methods used in the prior art for detection the alkaline phosphatase activity.

Description

(1) Technical field [0001] The invention relates to a novel compound, which can be used as a FRET fluorescent probe and applied to a fluorescent detection method for alkaline phosphatase activity. (2) Background technology [0002] Alkaline phosphatase can be derived from the bone, liver, placenta and intestine of different mammals. As a common analytical enzyme, it can catalyze the hydrolysis of various phosphate compounds and has substrate specificity. Alkaline phosphatase can be used as an important biological indicator in clinical diagnosis. It can be detected in many diseases such as bone diseases, liver dysfunction, ovarian cancer, breast cancer, prostate cancer, leukemia and diabetes. Abnormal levels to make a preliminary diagnosis. At present, many methods have been used for the detection of alkaline phosphatase, and the fluorescence detection method is popular because of its fast and sensitive transduction signal. [0003] Fluorescence resonance energy transfer (F...

Claims

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Application Information

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IPC IPC(8): C07F9/655C09K11/06G01N21/64
Inventor 朱勍李雪峰
Owner ZHEJIANG UNIV OF TECH
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