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A fluorescent probe for detecting alkaline phosphatase and its preparation method

A fluorescent probe, phosphatase technology, applied in fluorescence/phosphorescence, chemical instruments and methods, luminescent materials, etc., can solve problems such as impracticality in the biological field, achieve short response time, easy naked-eye identification, and good biocompatibility Effect

Inactive Publication Date: 2019-02-26
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There have been some recent reports on the recognition of ALP by AIE molecules, but the luminescence of these probes are in the green and blue fluorescence range, which is not practical in the biological field

Method used

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  • A fluorescent probe for detecting alkaline phosphatase and its preparation method
  • A fluorescent probe for detecting alkaline phosphatase and its preparation method
  • A fluorescent probe for detecting alkaline phosphatase and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Synthesis of fluorescent probe PN (synthetic route see figure 1 ):

[0025] Slowly add o-hydroxynaphthaldehyde (0.17g, 1.0mmol) into 20mL pyridine solution dissolved with phosphorus oxychloride (0.2mL, 2.0mmol), stir and react at room temperature in a round bottom flask for 4 hours; then pour the reaction solution into In a round-bottomed flask with 50 mL of ice water, continue to stir for 12 hours, filter, and separate by silica gel column chromatography to obtain compound 2-phosphonaphthaldehyde (0.25 g).

[0026] 1-aminonaphthalene (0.14g, 1.0mmol) and 2-phosphonaphthyl formaldehyde (0.25g, 1.0mmol) were heated to reflux in 20mL of methanol for 0.5 hours, cooled, filtered, washed, and separated by silica gel column chromatography to obtain the target compound PN (0.37g).

[0027] Characterized by electrospray mass spectrometry, it can be known that compound PN: experimental value m / z=375.3[M+H] + , theoretical value m / z=375.3[M+H] + (attached figure 2 ). NMR ...

Embodiment 2

[0029] The fluorescent probe PN can first realize the fluorescent response to alkaline phosphatase (ALP) in the solution in HEPES (pH 7.0) buffer solution, as shown in the attached image 3 . Add 2.0U / mL protein ALP, GDP, ACP, PDE, GAL, AChE, GoX and Trysin to 10μmol·L respectively -1 PN in HEPES (pH 7.0) buffer solution. attached by image 3 It can be seen that the addition of ALP makes the fluorescence of the solution at 523, 550 and 600 nm greatly enhanced (λ ex =480nm), the color of the solution gradually changed to orange. The addition of other common protein molecules GDP, ACP, PDE, GAL, AChE, GoX and Trysin basically does not affect the fluorescence intensity of the fluorescent probe PN, indicating that the probe PN has selective recognition of ALP in HEPES (pH 7.0) buffer solution Function, other common proteins will basically not interfere with the recognition of ALP. attached Figure 4 Fluorescence titration diagram of probe PN for different concentrations of A...

Embodiment 3

[0031] Probe PN can realize the sensing of endogenous ALP in cells. Figure 5 Middle a is a blank experiment without adding probes. Figure 5 In b, 10μmol·L -1 Probe PN and SW480cells were incubated in cell culture medium at 37°C for 0.5 hours. It can be clearly seen under the fluorescence microscope that the cells gradually emit orange fluorescence after adding the probe PN. Figure 5 where c and d are respectively Figure 5 Corresponding bright-field pictures of a and b. This experiment shows that the probe PN has a good fluorescence recognition function for endogenous ALP in cells.

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Abstract

The invention provides a fluorescent probe for assaying alkaline phosphatase and a preparation method thereof. The probe is 2-phosphorylnaphthaldehyde acetal aminonaphthalene, and the molecular formula is C21H14NO5P. The preparation method is as follows: o-hydroxynaphthaldehyde and phosphorus oxychloride are added into a reactor and dissolved with pyridine, and after a period of time of agitation under room temperature, reaction solution I poured into ice water to continue reaction, so that 2-phosphorylnaphthaldehyde; the 2-phosphorylnaphthaldehyde and 1-aminonaphthalene are heated in organic alcohol to reflux, and thereby the target compound is obtained. The probe has a selective fluorescent recognition function on alkaline phosphatase in buffer solution with the pH of 7.0, and can emit orange fluorescence, and the detection limit is 0.002U / mL. The probe has stable chemical properties and good biocompatibility, is easy to synthesize, and can be used in the detection of alkaline phosphatase in cells.

Description

technical field [0001] The invention relates to the detection technology of alkaline phosphatase, in particular to a fluorescent probe for detecting alkaline phosphatase and a preparation method thereof. Background technique [0002] Alkaline phosphatase (ALP) belongs to a kind of hydrolase, which has the function of promoting the dephosphorylation of different substrates such as nucleotides, proteins, and sugars. The normal concentration of ALP in human serum is in the range of 40-150mU / mL. When the concentration is abnormal, it is often the precursor of some diseases, such as hepatitis, prostate cancer, osteoporosis and bone cancer. In medical diagnosis, it is usually considered as an important biological signal molecule, and the activity of ALP represents the activity of cells to some extent. Therefore, real-time monitoring of ALP concentration is of great significance for distinguishing normal cells from abnormal cells such as hyperplasia. [0003] Some literatures ha...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07F9/12C09K11/06G01N21/64
CPCC07F9/12C09K11/06C09K2211/1011C09K2211/1014G01N21/6428G01N21/6486
Inventor 刘斌刘慕瑶
Owner SHANXI UNIV
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