本文關(guān)鍵詞： Snap-tag 近紅外熒光染料 熒光探針 熒光成像 熒光壽命成像　出處：《大連理工大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：隨著光學(xué)顯微成像技術(shù)的迅速發(fā)展,熒光染料和熒光探針在生物醫(yī)學(xué)領(lǐng)域應(yīng)用越來越廣泛;然而大多數(shù)熒光探針亞細(xì)胞器靶向性不穩(wěn)定,發(fā)射波長較短,易受細(xì)胞自發(fā)熒光干擾。因此,開發(fā)具有亞細(xì)胞器穩(wěn)定靶向和近紅外熒光探針成為了研究熱點(diǎn)。本文設(shè)計合成Snap-tag融合蛋白靶向和亞細(xì)胞器穩(wěn)定靶向的熒光探針,及一系列近紅外熒光染料。研究了其化學(xué)和光物理性質(zhì),探索其在熒光成像及檢測方面的應(yīng)用。1、從O6-鳥嘌呤出發(fā),設(shè)計合成了 Snap-tag靶向通用中間體PYBG和ADBG。通過PYBG和ADBG分別與羅丹明、萘酰亞胺、氟硼吡咯的Click反應(yīng)和Sonogashira偶聯(lián),得到 PYBG-TMR、PYBG-BODIPY、ADBG-Np-N、ADBG-Np-N+、ADBG-Np-OH,PYBG-SF、PYBG-NO、PYBG-ClO 和 PYBG-Vis。熒光染料 PYBG-TMR、PYBG-BODIPY和ADBG-Np-OH都高效特異性標(biāo)記表達(dá)在細(xì)胞核、線粒體、細(xì)胞質(zhì)基質(zhì)中的Snap-tag蛋白。熒光探針PYBG-NO在活細(xì)胞內(nèi)成功響應(yīng)信號分子NO;熒光探針PYBG-ClO在活細(xì)胞內(nèi)成功響應(yīng)次氯酸;熒光探針PYBG-Vis成功探測細(xì)胞內(nèi)不同亞細(xì)胞器的粘度差異。2、設(shè)計合成了能夠與蛋白反應(yīng)的粘度探針Cl-N-Vis和M-Vis-A,比率型氧氣敏感探針M-O-A。其中Cl-N-Vis和M-Vis-A的熒光壽命隨著粘度變化呈現(xiàn)線性關(guān)系。探針Cl-N-Vis定位在脂滴中,通過熒光壽命成像方法展示了肝細(xì)胞(HL7702細(xì)胞)的脂滴粘度。探針M-Vis-A通過醛基與蛋白質(zhì)上氨基的化學(xué)反應(yīng)鍵合在線粒體上,并通過熒光壽命成像原位指示SMMC7721細(xì)胞病理狀態(tài)下線粒體的粘度變化。探針M-O-A通過芐氯蛋白質(zhì)上巰基的化學(xué)反應(yīng)鍵合在線粒體上,并通過熒光比率成像原位的指示細(xì)胞內(nèi)氧氣濃度變化。3、結(jié)合不對稱羅丹明和菁類染料,設(shè)計合成近紅外熒光染料N-NIR、S-NIR和K-NIR,近紅外pH熒光探針pH-A-NIR和pH-B-NIR;通過預(yù)留溴原子,得到衍生平臺染料Br-N-NIR。在甲醇中N-NIR和S-NIR分別有0.4、0.21的熒光量子產(chǎn)率和10萬以上的摩爾消光系數(shù);熒光染料K-NIR有接近800nm的最大吸收波長。通過Br-N-NIR得到可用于Snap-tag標(biāo)記的近紅外底物PYBG-NIR,并實(shí)現(xiàn)PYBG-NIR特異性標(biāo)記細(xì)胞核、線粒體、細(xì)胞質(zhì)基質(zhì)中的Snap-tag蛋白。pH響應(yīng)探針pH-A-NIR(pKa=7.2)和pH-B-NIR(pKa=6.1)有接近中性pKa和較高熒光量子產(chǎn)率。pH探針成功實(shí)現(xiàn)探測活細(xì)胞內(nèi)pH變化。
[Abstract]:With the rapid development of optical microscopic imaging technology, fluorescent dyes and fluorescent probes are more and more widely used in the field of biomedicine. However, most fluorescent probes have unstable suborganelles, short emission wavelengths and are susceptible to cellular autofluorescence interference. The development of suborganele-stable and near-infrared fluorescent probes has become a hot topic. In this paper, we designed and synthesized Snap-tag fusion protein targeting and suborganelle stable targeting fluorescent probes. And a series of near infrared fluorescent dyes. The chemical and photophysical properties of the dyes were studied, and their applications in fluorescence imaging and detection were explored, starting from O6-guanine. PYBG and ADBG, the universal intermediates of Snap-tag targeting, were designed and synthesized with Rhodamine and naphthalimide by PYBG and ADBG, respectively. The Click reaction of Fluoropyrrole was coupled with Sonogashira to obtain PYBG-BODIPYN ADBG-Np-N. ADBG-Np-N ADBG-Np-OHG PYBG-SFN PYBG-NO. PYBG-ClO and PYBG-Vis. fluorescent dye PYBG-TMR. Both PYBG-BODIPY and ADBG-Np-OH are highly specific and expressed in nuclei and mitochondria. Snap-tag protein in cytoplasmic matrix. Fluorescent probe PYBG-NO successfully responded to signal molecule no in living cells. Fluorescence probe PYBG-ClO successfully responded to hypochloric acid in living cells. Fluorescence probe PYBG-Vis was successfully used to detect the viscosity difference of different suborganelles in cells. The viscosity probes Cl-N-Vis and M-Vis-A were designed and synthesized to react with protein. Ratio oxygen sensitive probe M-O-A.The fluorescence lifetime of Cl-N-Vis and M-Vis-A showed a linear relationship with the change of viscosity, and the probe Cl-N-Vis was located in the lipid droplet. The lipid droplet viscosity of hepatocyte HL-7702 was demonstrated by fluorescence lifetime imaging. The probe M-Vis-A was bonded to mitochondria by chemical reaction of aldehyde group with amino group on protein. Fluorescence lifetime imaging in situ indicated the changes of mitochondrial viscosity in SMMC7721 cells. The probe M-O-A was bound to mitochondria by the chemical reaction of mercapto group on benzyl chloride protein. Near-infrared fluorescent dyes N-NIRS-NIR and K-NIR were designed and synthesized by fluorescence ratio imaging in situ indicating the change of oxygen concentration in the cell. Combined with asymmetric Rhodamine and cyanine dyes, N-NIRS-NIR and K-NIR were synthesized. Near infrared pH fluorescence probe pH-A-NIR and pH -B-NIR; By reserving bromine atoms, the derivative platform dye Br-N-NIR was obtained. The N-NIR and S-NIR in methanol were 0.4 and 0.4 respectively. The fluorescence quantum yield and molar extinction coefficient above 100,000; The fluorescence dye K-NIR has a maximum absorption wavelength of nearly 800nm. The near infrared substrate PYBG-NIR, which can be used for Snap-tag labeling, was obtained by Br-N-NIR. The specific labeling of nucleus and mitochondria by PYBG-NIR was realized. Snap-tag protein in cytoplasmic matrix. PH-A-NIRP pKa7. 2) and pH-B-NIRI pKaV6. 1). Close to neutral pKa and high fluorescence quantum yield. Ph probe was successfully used to detect the pH changes in living cells.
【學(xué)位授予單位】：大連理工大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：O657.3

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