生物熒光探針的分子動(dòng)力學(xué)研究與應(yīng)用
發(fā)布時(shí)間：2018-09-11 09:32
【摘要】：在探究生命領(lǐng)域時(shí),熒光技術(shù)被認(rèn)為是最為有效的手段之一。時(shí)間分辨熒光光譜相比于穩(wěn)態(tài)熒光光譜不易受熒光分子濃度、激發(fā)波長(zhǎng)和功率等因素的影響,近年來(lái)備受研究者們的關(guān)注。在研究不發(fā)光的生物分子和細(xì)胞時(shí),研究者們需要借助熒光探針,其中使用最廣的主要包括有機(jī)小分子熒光探針、氨基酸多肽熒光探針、熒光蛋白探針和基于新型納米發(fā)光材料的探針等。在本文中,我們將利用時(shí)間分辨熒光光譜來(lái)研究不同類型探針?lè)肿拥臒晒鈩?dòng)力學(xué),并將它們用于生物應(yīng)用研究。氨苯乙烯類的熒光分子是典型的有機(jī)小分子探針。我們合成了 o-DASPMI和p-DASPMI,并分別用穩(wěn)態(tài)熒光光譜、皮秒級(jí)的時(shí)間分辨熒光光譜和飛秒級(jí)時(shí)間分辨熒光光譜技術(shù)來(lái)表征它們的熒光性質(zhì)。o-DASPMI和p-DASPMI斯托克斯位移超過(guò)150nm,熒光壽命分別為6.6ps和12.4ps.超短的熒光壽命使得DASPMI的熒光可以用來(lái)測(cè)量TCSPC或者FLIM設(shè)備的儀器響應(yīng)函數(shù)(IRF)。在本文中,我們利用DASPMI熒光測(cè)得的IRF分別擬合了羅丹明B(RhB)、酰磺羅丹明B(SRB)和SRB與RNA適配子SRB2m復(fù)合體的熒光壽命,擬合的效果與散射光作為IRF 一致。實(shí)驗(yàn)結(jié)果證明DASPMI的熒光可以用作TCSPC或者FLIM設(shè)備的IRF檢測(cè),并有效的解決了探測(cè)器顏色效應(yīng)等問(wèn)題。在緩沖液中,游離的p-DASPMI的熒光壽命為12.4ps,與BSA結(jié)合后出現(xiàn)兩個(gè)更長(zhǎng)的熒光壽命,分別是0.9 ns和2.6 ns.通過(guò)熒光壽命的比重和DAS光譜,我們將0.9 ns和2.6 ns分別對(duì)應(yīng)到BSA的ⅢA區(qū)和ⅡA區(qū)。通過(guò)比較熒光壽命變化、壽命比重變化和參數(shù)(α2τ2+α3τ3)/α1τ1與參數(shù)α3τ3/α2τ2的變化,我們研究了 pH和Cu2+對(duì)蛋白質(zhì)結(jié)構(gòu)的影響。結(jié)果表明借助時(shí)間分辨熒光光譜,p-DASPMI即能更靈敏的定量檢測(cè)蛋白質(zhì)濃度,也能同時(shí)對(duì)游離態(tài)的探針和不同綁定位點(diǎn)處的探針進(jìn)行監(jiān)控,實(shí)現(xiàn)了對(duì)蛋白質(zhì)多個(gè)位點(diǎn)的結(jié)構(gòu)研究。pH對(duì)細(xì)胞內(nèi)的生理過(guò)程起到至關(guān)重要的作用。借助pH探針實(shí)時(shí)觀察生理環(huán)境的pH波動(dòng)是非常有意義的。我們合成了多種基于Trp-X的肽鏈,研究了其熒光性質(zhì)隨pH的變化,并嘗試將其接在多肽上,用于測(cè)量多肽周圍環(huán)境的pH值。我們首先研究Trp-Trp雙肽和它的三種衍生物(NATrp2Me、NBTrp2和Trp2Me)在不同pH下的熒光光譜,發(fā)現(xiàn)裸露的氨基是Trp-X對(duì)pH響應(yīng)的關(guān)鍵。然后我們研究Trp-X在多肽中的性質(zhì)。在多肽Trp-X-Ala-Ser中,Trp-X仍然保持著pH的響應(yīng)。相比于 Trp-Trp-Ala-Ser(WWAS),Trp-Ala-Ala-Ser(WAAS)和 Trp-Glu-Ala-Ser(WEAS)的熒光量子產(chǎn)率更高、熒光壽命更長(zhǎng),對(duì)pH的響應(yīng)更敏感。干擾實(shí)驗(yàn)中,金屬,陽(yáng)離子雖然干擾了探針?lè)�(wěn)態(tài)熒光強(qiáng)度,但對(duì)時(shí)間分辨熒光光譜幾乎沒(méi)有影響。研究結(jié)果表明,Trp-X雙肽的平均熒光壽命可以超靈敏、高選擇性的標(biāo)定多肽周圍的pH環(huán)境。roUnaG-BR作為一種新型的氧化還原探針,不僅克服GFP類熒光蛋白在厭氧環(huán)境下表達(dá)不佳的問(wèn)題,還實(shí)現(xiàn)了可隨時(shí)"點(diǎn)亮"熒光的功能。但是roUnaG-BR是一種單通道激發(fā)和發(fā)射的熒光探針,飽和氧化態(tài)和飽和還原態(tài)之間的平均熒光壽命只有0.3ns的變化,很難采用穩(wěn)態(tài)熒光強(qiáng)度或者平均熒光壽命來(lái)標(biāo)定細(xì)胞內(nèi)的氧化還原狀態(tài)。本文采用時(shí)間分辨熒光光譜研究roUnaG-BR,并通過(guò)對(duì)比UnaG-BR的熒光和結(jié)構(gòu)性質(zhì),發(fā)現(xiàn)在還原態(tài)的體系中,2.2ns熒光壽命對(duì)應(yīng)的分子構(gòu)型占主導(dǎo)地位;而在氧化態(tài)的體系中,0.2ns熒光壽命對(duì)應(yīng)的分子構(gòu)型占主導(dǎo)地位。采用參數(shù)α3/α1和α3τ3/α1τ1卻可以獲得10倍和8.1倍的動(dòng)態(tài)變化范圍,這比穩(wěn)態(tài)熒光的7倍還要大。結(jié)果表明,借助時(shí)間分辨熒光技術(shù),單激發(fā)通道和單發(fā)射通道的roUnaG-BR可以定量標(biāo)定細(xì)胞內(nèi)的氧化還原狀態(tài),并可獲得比穩(wěn)態(tài)熒光強(qiáng)度和平均熒光壽命更高的靈敏度。
[Abstract]:Time-resolved fluorescence spectroscopy is considered to be one of the most effective means to explore the field of life. Compared with steady-state fluorescence spectroscopy, time-resolved fluorescence spectroscopy is not easily affected by fluorescence molecular concentration, excitation wavelength and power. Recently, it has attracted much attention of researchers. The most widely used fluorescent probes include organic small molecule fluorescent probes, amino acid polypeptide fluorescent probes, fluorescent protein probes and probes based on novel nanoluminescent materials. O-DASPMI and p-DASPMI were synthesized and characterized by steady-state fluorescence spectroscopy, picosecond time-resolved fluorescence spectroscopy and femtosecond time-resolved fluorescence spectroscopy respectively. The fluorescence lifetime of DASPMI is 6.6 PS and 12.4 PS at 150 nm, respectively. The fluorescence lifetime of DASPMI can be used to measure the instrumental response function (IRF) of TCSPC or FLIM equipment. In this paper, the IRF measured by DASPMI fluorescence is used to fit the fluorescence lifetime of Rhodamine B (RhB), Sulfonyl Rhodamine B (SRB) and SRB-SRB-SRB2m complexes, respectively. The experimental results show that the fluorescence of DASPMI can be used as the IRF detection of TCSPC or FLIM equipment, and the color effect of detector can be effectively solved. In the buffer solution, the fluorescence lifetime of the free p-DASPMI is 12.4 ps, and two longer fluorescence lifetimes appear after binding with BSA, respectively, are 0.9 PS. NS and 2.6 ns. The ratios of fluorescence lifetime and DAS spectra of 0.9 ns and 2.6 ns correspond to the regions III A and II A of BS A, respectively. The effects of pH and Cu 2+ on the structure of protein were studied by comparing the changes of fluorescence lifetime, specific gravity of lifetime and the parameters (alpha 2_2+alpha 3 3)/alpha 1 and the parameters alpha 3_3/alpha 2. Time-resolved fluorescence spectroscopy, p-DASPMI can detect protein concentration more sensitively and quantitatively, and also can monitor the free probe and probe at different binding sites simultaneously. The structure of multiple protein sites can be studied. pH plays an important role in the physiological process of cells. We have synthesized a variety of Trp-X-based peptide chains and studied their fluorescence properties as a function of pH. We have attempted to graft them onto polypeptides to measure the pH of the environment around the peptides. We first studied the fluorescence spectra of Trp-Trp dipeptide and its three derivatives (NATrp2Me, NBTrp2 and Trp2Me) at different pHs. It was found that the exposed amino group was the key to the pH response of Trp-X. Then we studied the properties of Trp-X in polypeptides. In polypeptide Trp-X-Ala-Ser, Trp-X still maintained pH response. Compared with Trp-Trp-Ala-Ser (WWAS), Trp-Ala-Ala-Ser (WAAS) and Trp-Glu-Ala-Ser (WEAS), Trp-Ala-Ser had higher fluorescence quantum yield, longer fluorescence lifetime and more sensitive response to pH. The results show that the average fluorescence lifetime of Trp-X dipeptide can be highly sensitive and selective to calibrate the pH environment around the peptide. As a new redox probe, roUnaG-BR can not only overcome G but also overcome G. However, roUnaG-BR is a single-channel fluorescent probe. The average fluorescence lifetime between saturated oxidized and reduced states is only 0.3 ns. It is difficult to use steady-state fluorescence intensity or average fluorescence lifetime. In this paper, time-resolved fluorescence spectroscopy was used to study the fluorescence and structural properties of roUnaG-BR. It was found that the molecular configurations corresponding to the fluorescence lifetime of 2.2ns were dominant in the reduced system, and the molecular configurations corresponding to the fluorescence lifetime of 0.2ns were dominant in the oxidized system. The results show that roUnaG-BR with single excitation channel and single emission channel can quantitatively calibrate the redox state of cells and obtain stronger fluorescence than that with steady state fluorescence. The sensitivity of degree and average fluorescence lifetime is even higher.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：O657.3
本文編號(hào)：2236308
[Abstract]:Time-resolved fluorescence spectroscopy is considered to be one of the most effective means to explore the field of life. Compared with steady-state fluorescence spectroscopy, time-resolved fluorescence spectroscopy is not easily affected by fluorescence molecular concentration, excitation wavelength and power. Recently, it has attracted much attention of researchers. The most widely used fluorescent probes include organic small molecule fluorescent probes, amino acid polypeptide fluorescent probes, fluorescent protein probes and probes based on novel nanoluminescent materials. O-DASPMI and p-DASPMI were synthesized and characterized by steady-state fluorescence spectroscopy, picosecond time-resolved fluorescence spectroscopy and femtosecond time-resolved fluorescence spectroscopy respectively. The fluorescence lifetime of DASPMI is 6.6 PS and 12.4 PS at 150 nm, respectively. The fluorescence lifetime of DASPMI can be used to measure the instrumental response function (IRF) of TCSPC or FLIM equipment. In this paper, the IRF measured by DASPMI fluorescence is used to fit the fluorescence lifetime of Rhodamine B (RhB), Sulfonyl Rhodamine B (SRB) and SRB-SRB-SRB2m complexes, respectively. The experimental results show that the fluorescence of DASPMI can be used as the IRF detection of TCSPC or FLIM equipment, and the color effect of detector can be effectively solved. In the buffer solution, the fluorescence lifetime of the free p-DASPMI is 12.4 ps, and two longer fluorescence lifetimes appear after binding with BSA, respectively, are 0.9 PS. NS and 2.6 ns. The ratios of fluorescence lifetime and DAS spectra of 0.9 ns and 2.6 ns correspond to the regions III A and II A of BS A, respectively. The effects of pH and Cu 2+ on the structure of protein were studied by comparing the changes of fluorescence lifetime, specific gravity of lifetime and the parameters (alpha 2_2+alpha 3 3)/alpha 1 and the parameters alpha 3_3/alpha 2. Time-resolved fluorescence spectroscopy, p-DASPMI can detect protein concentration more sensitively and quantitatively, and also can monitor the free probe and probe at different binding sites simultaneously. The structure of multiple protein sites can be studied. pH plays an important role in the physiological process of cells. We have synthesized a variety of Trp-X-based peptide chains and studied their fluorescence properties as a function of pH. We have attempted to graft them onto polypeptides to measure the pH of the environment around the peptides. We first studied the fluorescence spectra of Trp-Trp dipeptide and its three derivatives (NATrp2Me, NBTrp2 and Trp2Me) at different pHs. It was found that the exposed amino group was the key to the pH response of Trp-X. Then we studied the properties of Trp-X in polypeptides. In polypeptide Trp-X-Ala-Ser, Trp-X still maintained pH response. Compared with Trp-Trp-Ala-Ser (WWAS), Trp-Ala-Ala-Ser (WAAS) and Trp-Glu-Ala-Ser (WEAS), Trp-Ala-Ser had higher fluorescence quantum yield, longer fluorescence lifetime and more sensitive response to pH. The results show that the average fluorescence lifetime of Trp-X dipeptide can be highly sensitive and selective to calibrate the pH environment around the peptide. As a new redox probe, roUnaG-BR can not only overcome G but also overcome G. However, roUnaG-BR is a single-channel fluorescent probe. The average fluorescence lifetime between saturated oxidized and reduced states is only 0.3 ns. It is difficult to use steady-state fluorescence intensity or average fluorescence lifetime. In this paper, time-resolved fluorescence spectroscopy was used to study the fluorescence and structural properties of roUnaG-BR. It was found that the molecular configurations corresponding to the fluorescence lifetime of 2.2ns were dominant in the reduced system, and the molecular configurations corresponding to the fluorescence lifetime of 0.2ns were dominant in the oxidized system. The results show that roUnaG-BR with single excitation channel and single emission channel can quantitatively calibrate the redox state of cells and obtain stronger fluorescence than that with steady state fluorescence. The sensitivity of degree and average fluorescence lifetime is even higher.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：O657.3
【參考文獻(xiàn)】
相關(guān)博士學(xué)位論文 前2條
1 賈夢(mèng)輝;基于超快熒光光譜的蛋白表面水合化動(dòng)力學(xué)研究[D];華東師范大學(xué);2016年
2 袁玉峰;新型銀納米材料與生物輔酶的精密光譜特性研究[D];華東師范大學(xué);2016年
，本文編號(hào)：2236308
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