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Preparation method and application of near-infrared alkaline phosphatase fluorescence probe

A fluorescent probe and phosphatase technology, applied in the field of fluorescent probes, to achieve rapid response

Inactive Publication Date: 2017-05-31
XIANGTAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But there are few near-infrared fluorescent probes for detecting ALP

Method used

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  • Preparation method and application of near-infrared alkaline phosphatase fluorescence probe
  • Preparation method and application of near-infrared alkaline phosphatase fluorescence probe
  • Preparation method and application of near-infrared alkaline phosphatase fluorescence probe

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Experimental program
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Effect test

Embodiment 1

[0028] Synthesis of fluorescent probes

[0029] Synthesis of CyOH: In a 100 mL round bottom flask, resorcinol (1.0 mmol, 110 mg) and potassium carbonate (1.0 mmol, 138 mg) were sequentially added, and then 20 mL of acetonitrile was added as a solvent. After magnetic stirring for 10 min at room temperature, a solution of IR-780 (0.5 mmol, 305 mg) dissolved in 20 mL of acetonitrile was added dropwise to the reaction solution. N 2 Under protection, heat to 50°C and continue to stir for about 4 hours. After the reaction is completed, remove the solvent with a rotary evaporator. The crude product is separated and purified by column chromatography using dichloromethane / methanol at a ratio of 20:1 (volume ratio) as eluent to obtain Blue-green solid 169 mg, yield 82%. 1 H NMR (400MHz, CDCl 3 ):δ8.02(d,1H,J=13.2Hz),7.25-7.21(m,2H),7.14(t,1H,J=7.6Hz),7.06-7.03(m,2H),6.97(s, 1H), 6.90(d, 1H, J=8.0Hz), 6.81(d, 1H, J=8.0Hz), 5.56(d, 1H, J=13.2Hz), 3.76(t, 2H, J=7.2Hz) ,2.67(t,2H,J=6.0...

Embodiment 2

[0032] Preparation of solution for interaction between fluorescent probe and ALP

[0033] A certain amount of fluorescent probe was dissolved in EtOH to obtain a concentration of 1.0 × 10 -4 mol L -1 Probe stock solution. Add 1.0mL of the spare solution of the probe into a 10mL volumetric flask, and after constant volume with buffer solution, the obtained concentration is 1.0×10 -5 mol L -1 fluorescent probe solution. ALP was formulated to the following concentrations (0, 0.01, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0 U / mL).

Embodiment 3

[0035] Measurement of Fluorescence Spectrum of Interaction of Fluorescent Probe with ALP

[0036] A buffer solution with a pH value of 8 was used as a solvent to measure the fluorescence spectrum of the fluorescent probe interacting with ALP, and the results were as follows: figure 1 . The concentration of the fluorescent probe is 10 μM, the concentration of ALP is 0, 0.01, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0 U / mL, the excitation wavelength is fixed at 595nm, and the emission wavelength range is 685-850nm. The slit width is 10.0nm / 10.0nm. From figure 1 It can be seen that with the addition of ALP, the emission peak at 703nm is greatly enhanced, and with the increase of ALP concentration, the fluorescence intensity of the probe is continuously enhanced. Such as figure 1 As shown in the inset of , the fluorescence intensity has a linear relationship with the concentration of ALP, the linear range is 0.01-2.0U / mL, and the detection limit is 0.003U / mL. The fluorescence...

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Abstract

The invention discloses a preparation method and application of a near-infrared alkaline phosphatase fluorescence probe. The near-infrared alkaline phosphatase fluorescence probe has the structural formula shown in the description. The biological near-infrared fluorescence probe based on hemicyanine is designed and synthesized with IR-780, resorcinol and phosphorus oxychloride as raw materials. The probe can be used for detecting the concentration of ALP in a solution and shows good sensitivity, the linearity range is from 0.01 U / mL to 2.0 U / mL, the detection limit is 0.003 U / mL. The probe shows very good selectivity and is not affected by other enzymes, or biological mercaptan or amino acids or metal ions. More importantly, the probe can be applied to bioimaging and used for detecting the content of the ALP in cells and tissue, which has an important significance on in-depth studies on the physiological and pathological processes of the ALP.

Description

technical field [0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a preparation method and application of a near-infrared alkaline phosphatase fluorescent probe. Background technique [0002] Alkaline phosphatase (ALP) is widely distributed in tissues such as human liver, bone, intestine, kidney and placenta. J.E. Coleman, Annu. Rev. Biophys. Biomol. Struct., 1992, 21, 441-483). Changes in the concentration of ALP in organisms are closely related to whether certain life processes are normal or not. Studies have shown that abnormal levels of ALP are related to many diseases, including breast cancer, prostate cancer, diabetes, bone disease, and abnormal liver function (document 2: J.A.Lorente, H.Valenzuela, J.Morote, A.Gelabert, Eur.J .Nucl., 1999,26,625-632; Literature 3: D.L.Robertson, G.F.Joyce, Nature, 1990,344,467-468; Literature 4: P.Colombatto, A.Randone, G.Civitico, J.M.Gorin, L.Dolci, N . Medaina, F. Oliveri, G....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C09K11/06C07F9/655G01N21/64G01N21/33
CPCC07F9/65522C09K11/06C09K2211/1029C09K2211/1088G01N21/33G01N21/6486
Inventor 李春艳李宋娇
Owner XIANGTAN UNIV
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