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【答案】應(yīng)助回帖
鐵（Fe）是人體中含量豐富的金屬元素，在生物學(xué)和病理學(xué)方面發(fā)揮著重要作用。鐵元素在生物體中主要以亞鐵（Fe2+）和鐵（Fe3+）離子的形式存在，由于生物體內(nèi)的Fe3+容易被還原成Fe2+及在偏堿性的生理?xiàng)l件下溶解性不好；另一方面，生物體內(nèi)的Fe2+會(huì)催化芬頓反應(yīng)（Fenton reaction）產(chǎn)生自由基，可能引發(fā)一系列疾病。因此，設(shè)計(jì)一個(gè)快速、準(zhǔn)確檢測(cè)生物體內(nèi)亞鐵離子的探針具有十分重要的意義。
過(guò)氧亞硝基陰離子（Peroxynitrite anion, ONOO-）作為生物體內(nèi)活性氮（Reactive nitrogen species, RNS）分子之一，它是由超氧自由基（O2•-）和一氧化氮（NO）反應(yīng)生成。ONOO-作為很強(qiáng)的生物氧化劑，具有不穩(wěn)定性和較高的反應(yīng)活性，是一些生物體內(nèi)循環(huán)途徑的信號(hào)傳導(dǎo)分子。但這種傳導(dǎo)分子也可能對(duì)一些生物靶細(xì)胞造成不可逆轉(zhuǎn)的損害，比如引發(fā)癌癥、炎癥和心血管疾病等多種疾病。因此，設(shè)計(jì)一個(gè)合適的探針對(duì)ONOO-檢測(cè)顯得至關(guān)重要。
（一）設(shè)計(jì)合成探針K1檢測(cè)Fe2+。在苯并吡喃腈衍生物中引入氮氧化物（N-Oxide）合成熒光探針K1，探究探針K1與Fe2+發(fā)生反應(yīng)可能的機(jī)理，并對(duì)其結(jié)構(gòu)進(jìn)行表征。本文通過(guò)光物理性質(zhì)研究發(fā)現(xiàn)，該探針在PBS/DMF=1:9(0.01 M, pH 7.4)緩沖溶液中幾乎沒(méi)有熒光，加入Fe2+后，N-Oxide被還原，熒光恢復(fù)，在波長(zhǎng)685 nm處有最大發(fā)射。該探針具有選擇性好和靈敏度高（檢測(cè)極限為2.96×10-6 mM）等特點(diǎn)，被應(yīng)用于秀麗隱桿線蟲(chóng)和活體細(xì)胞中的Fe2+熒光成像。
（二）設(shè)計(jì)合成探針K2檢測(cè)ONOO-。以苯并吡喃腈和香豆素合成熒光探針K2，探究探針K2與ONOO-發(fā)生反應(yīng)可能的機(jī)理，并對(duì)其結(jié)構(gòu)進(jìn)行表征。在HEPES/DMF=1:9 (0.01 M, pH 7.4)緩沖體系下進(jìn)行光物理性質(zhì)測(cè)試，當(dāng)加入飽和當(dāng)量ONOO-時(shí)，該探針熒光光譜發(fā)生明顯藍(lán)移，并且能夠?qū)崿F(xiàn)對(duì)ONOO-的專(zhuān)一性識(shí)別，檢測(cè)極限低至6×10-7 mM。更重要的是，該探針被成功應(yīng)用于活體細(xì)胞中的ONOO- 熒光成像。
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關(guān)鍵詞：亞鐵離子；過(guò)氧亞硝基陰離子；苯并吡喃腈；熒光探針；熒光成像
Iron (Fe) is rich in human body, existing mainly as ferrous iron (Fe2+) and iron (Fe3+) ions , and plays an important role in biology and pathology.
It is very important to design a probe to detect (quickly and accurately) ferrous ions in living organisms, as Fe3+ soluble poorly under alkaline physiological conditions and tending to reduced to Fe2+,
and Fe2+( in the body) would catalyze the Fenton reaction generating free radicals which may cause a series of diseases,
It is critical to design a suitable probe for ONOO-detection, because Peroxynitrite anion (ONOO-), being one of the reactive nitrogen species (RNS) molecules and a strong biological oxidant (with high instability and reactivity) in the body, may damage (some) biological target cells and cause cancer, inflammation and cardiovascular diseases.
(1) Designing a synthetic probe K1 to detect Fe2+.
The fluorescent probe K1 was synthesized by introducing nitrogen oxide (N-Oxide) into the benzopyranonitrile derivative, its structure was characterized and the possible mechanism of the reaction of probe K1 and Fe2+ was investigated.
The probe had almost no fluorescence in the buffer solution of PBS/DMF=1:9 (0.01 M, pH 7.4), but N-Oxide was reduced and the fluorescence recovered nm by adding Fe2+, with a maximum emission at 685.
The probe was applied to Fe2+ fluorescence imaging in C. elegans and living cells and showed high selectivity and good sensitivity (detection limit 2.96×10-6 mM).
(B) Designing synthetic probe K2 to detect ONOO-.
The fluorescent probe K2 was synthesized with benzopyranonitrile and coumarin, its structure was characterized , and the possible mechanism of the reaction of probe K2 and ONOO- was investigated.
The fluorescence spectrum of the probe was significantly blue-shifted when saturated equivalent ONOO- was added, in HEPES/DMF=1:9 (0.01 M, pH 7.4) buffer, and the ONOO-specialization was realized, with a detection limit as low as 6 × 10-7 mM.
More importantly, the probe was successfully applied to ONOO-fluorescence imaging in living cells.
Key words: ferrous ion; peroxynitrite; benzopyranonitrile; fluorescent probe; fluorescence imaging |
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