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Ratio fluorescence probe for detecting hydroxylamine as well as synthesis method and application thereof

A technology of fluorescent probes and synthesis methods, applied in the field of organic small molecule fluorescent probes, can solve the problems of lack of fluorescent probes, and achieve the effects of easy-to-obtain raw materials, simple and easy synthesis process, and high specificity

Inactive Publication Date: 2018-06-29
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Aiming at the current lack of fluorescent probes for detecting intracellular hydroxylamine, the present invention provides a fluorescent probe for detecting intracellular hydroxylamine, which has good selectivity and high sensitivity

Method used

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  • Ratio fluorescence probe for detecting hydroxylamine as well as synthesis method and application thereof
  • Ratio fluorescence probe for detecting hydroxylamine as well as synthesis method and application thereof
  • Ratio fluorescence probe for detecting hydroxylamine as well as synthesis method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1 Synthesis of Fluorescent Probes

[0033] Add raw material 1 (1 mmol) and raw material 2 (1 mmol) into 5 mL of concentrated sulfuric acid, heat the reaction at 90 °C for 4 hours, and cool to room temperature. The reaction solution was poured into 10 mL of ice water, and 2 mL of perchloric acid was slowly added dropwise to precipitate a red solid, which was separated and purified to obtain the pure compound 1 (yield 52%) of the fluorescent probe. 1 H NMR spectrum see figure 1 .

[0034] Dissolve Rhodamine B (1 mmol) and 1 mL of phosphorus oxychloride in 15 mL of dichloroethane and heat to reflux. After reacting for 3 h, spin to remove dichloroethane, and add 15 mL of dichloromethane to dissolve. Subsequently, the solution was slowly added dropwise to a dichloromethane solution of compound 1 (containing 0.2 mL triethylamine) at 0 °C, and stirred at 25 °C for 4 h. After the reaction, the reaction solution was spin-dried, and the pure fluorescent probe (yield 2...

Embodiment 2

[0035] The response of embodiment 2 fluorescent probes to hydroxylamine

[0036] Dissolve the probe compound obtained in Example 1 with ethanol, add PBS buffer to prepare a 5 μM probe buffer solution (containing 20% ​​ethanol, pH = 7.4), which is the probe working solution.

[0037] Hydroxylamine was supplied in aqueous solution with hydroxylamine hydrochloride; added to the above needle buffer solution to increase the concentration of hydroxylamine from 5 μM to 200 μM, a total of 40, adding an equal amount of buffer as a control; then perform fluorescence detection (λ Ex = 540nm), calculate the fluorescence intensity in each system; by analyzing the relationship between the ratio of the fluorescence intensity at 590 nm and 637nm and the concentration of hydroxylamine, evaluate the response performance of the probe to hydroxylamine (see image 3 and Figure 4 ). figure 2 It shows that with the increase of hydroxylamine concentration, the fluorescence intensity at 590 nm of ...

Embodiment 3

[0038] Embodiment 3 Fluorescent probe specificity analysis to hydroxylamine

[0039] Take 5 mL of the probe working solution in Example 2, and then add 50 μL of probe, Hcys, Na 2 S, Na 2 SO 3 , Cys, GSH, Vc, Zn 2+ , Fe 2+ , Fe 3+ , Mg 2+ , arginine, ammonia water, ammonium chloride and other related analytes, the numbers are 1-14 in sequence, and the number 15 is hydroxylamine. Fluorescence detection is then performed (λ Ex = 540nm), calculate the fluorescence intensity in each system, and evaluate the interference of the different substances on the fluorescent probe solution (see Figure 5 ). It can be seen from the figure that when Hcys and Na are added to the probe solution 2 S, Na 2 SO 3 , Cys, GSH, VC, Zn 2+ , Fe 2+ , Fe 3+ , Mg 2+ , arginine, ammonia water, ammonium chloride and other related analytes, only hydroxylamine can cause significant fluorescence changes in the solution, while the fluorescence of the solution does not change when other small molec...

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Abstract

The invention provides a fluorescence probe for detecting hydroxylamine. The fluorescence probe is a rhodamine derivative containing biotin groups; benzoic acid groups are hydroxylamine response sites; a fluorescence peak value of the probe before the probe is reacted with hydroxylamine is about 637nm; the peak value of the probe after the probe is reacted with hydroxylamine is about 590nm; the hydroxylamine can be qualitatively detected through fluorescence intensity ratio change under two wavelengths; a linear relation is formed between the fluorescence intensity ratio under two wavelengthsand the concentration of hydroxylamine, so that the concentration of hydroxylamine can be quantitatively detected. The fluorescence probe is high in specificity, high in sensitivity and excellent in fluorescence emission spectrum characteristics (550-750nm).

Description

technical field [0001] The invention belongs to the field of small organic molecule fluorescent probes, in particular to a fluorescent probe for detecting hydroxylamine in living cells and a synthesis method thereof. Background technique [0002] Hydroxylamine (NH 2 OH) is an oxidation state of ammonia that is widely used in industry and medicine. In biological systems, hydroxylamine acts as a nitric oxide donor and participates in many biological processes. During cellular metabolism, hydroxylamine is an intermediate in the conversion of L-arginine to nitric oxide. This process involves the hydrolysis of arginine oxime to L-citrulline and hydroxylamine. Subsequently, hydroxylamine is converted to nitric oxide and peroxy radicals in the presence of hydrogen peroxide and catalyzed by catalase. At the same time, studies have shown that hydroxylamine can also be converted into nitric oxide under the attack of peroxyl radicals. Thus, in inflammatory cells, hydroxylamine rea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D311/82C09K11/06G01N21/64
Inventor 林伟英董宝利张楠孔秀琪王超宋文辉
Owner UNIV OF JINAN
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