本文關(guān)鍵詞：熒光傳感器體系的構(gòu)建及其在幾種重要生命物質(zhì)檢測中的應(yīng)用　出處：《華南理工大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 熒光傳感器 硫離子 γ-谷酰胺轉(zhuǎn)肽酶 堿性磷酸酶

【摘要】：某些生命物質(zhì)在生物體中起著重要作用,其含量的變化對生命活動具有重大的影響;因此,對這類物質(zhì)的檢測、監(jiān)控和實時成像,在生理學(xué)和病理學(xué)研究中極具意義。在本文中,針對幾種代表性的生命物質(zhì),選擇合適的機理,設(shè)計并制備了四種熒光傳感器,實現(xiàn)了對硫離子(S2-)、γ-谷酰胺轉(zhuǎn)肽酶(GGT)及堿性磷酸酶(ALP)的便利、靈敏檢測和細(xì)胞(或活體)成像。首先,針對硫離子,我們制備了利用配體分子改性的碳點(CD-S)作為探針前驅(qū)體,與銅離子(Cu2+)形成絡(luò)合物并淬滅CD-S的熒光,作為檢測S2-的熒光增強型探針。由于S2-與Cu2+之間有很高的結(jié)合能力,會奪取探針中的銅離子而生成穩(wěn)定的CuS(Ksp=3.63×10-36);由此脫除了Cu2+的碳點的熒光得以恢復(fù),該探針對硫離子的檢測下限達(dá)0.78μM。該CD-S/Cu2+探針具有良好的水溶性、生物相容性、選擇性和靈敏度,可以成功用于自來水中硫離子濃度的檢測和活細(xì)胞中硫離子的檢測、成像。其次,我們設(shè)計并合成了檢測γ-谷酰胺轉(zhuǎn)肽酶(GGT)的熒光探針Glu-TPE;作為四苯乙烯衍生物,探針Glu-TPE具有聚集誘導(dǎo)發(fā)光(AIE)效應(yīng);分子中的兩個γ-谷酰胺基團使得探針Glu-TPE具有良好的水溶性與生物相容性;由于GGT催化探針Glu-TPE分子中γ-谷酰胺基團的水解,疏水性的酶解產(chǎn)物聚集誘發(fā)AIE效應(yīng)而發(fā)出藍(lán)色熒光,從而實現(xiàn)對GGT的增強型熒光檢測�；贕GT酶促反應(yīng)的專一性和高效性,探針Glu-TPE具有良好的選擇性和靈敏性;能夠用于對人血清樣品中GGT含量的檢測和A2780細(xì)胞中Qg源性GGT的檢測、成像。針對GGT的檢測,我們還制備了另一種基于分子內(nèi)電荷轉(zhuǎn)移(ICT)機理的熒光探針PEG-NA-Glu;當(dāng)探針分子PEG-NA-Glu中γ-谷酰胺基團被GGT催化水解后,在這種ICT發(fā)光機理的熒光團中,電子供體的電子云密度發(fā)生變化,進(jìn)而導(dǎo)致熒光發(fā)射波長的變化,由此實現(xiàn)了對GGT的比率型熒光檢測。探針PEG-NA-Glu具有高靈敏度和生物相容性,其檢測下限為0.76 U/L,可用于尿液和血清中檢測GGT含量,且比ELISA Kit方法操作簡便很多;并成功地實現(xiàn)了對活細(xì)胞內(nèi)的GGT的雙色熒光成像。再者,針對堿性磷酸酶(ALP),我們利用萘二甲酰亞胺衍生物的ICT熒光機理,設(shè)計并制備了探針AO-NA-P;ALP催化探針分子AO-NA-P的脫磷酸反應(yīng),使得探針中萘環(huán)4號位上的供電子基團發(fā)生變化,其結(jié)果增強了熒光分子的“推-拉”電子效應(yīng),熒光發(fā)射波長紅移;基于此原理成功實現(xiàn)了對ALP的比率型熒光檢測。由于ALP催化水解反應(yīng)具有專一性和高效性,探針AO-NA-P具備高靈敏性和選擇性,探針對ALP的檢測下限達(dá)0.38 U/L;另外,探針分子AO-NA-P具有極好的水溶性和生物相溶性;可用于人血清樣品、活細(xì)胞、活體中的ALP檢測。我們首次實現(xiàn)了活體(斑馬魚)內(nèi)ALP的比率型熒光成像,對斑馬魚內(nèi)藥物引致器官損傷而引發(fā)的ALP含量升高進(jìn)行了成像和檢測。相關(guān)結(jié)果可為研究器官損傷而引發(fā)的生命物質(zhì)變化提供了有益的參考。
[Abstract]:Some life substances play an important role in organisms. The change of their contents has a great impact on life activities. Therefore, detection, monitoring and real-time imaging of these substances are of great significance in physiology and pathology research. In this paper, aiming at several representative life substances, we choose the appropriate mechanism to design and prepare four kinds of fluorescent sensors, realizing the convenient, sensitive detection and cell (or living) imaging of sulfur ion (S2-), gamma glutamyl trans peptidase (GGT) and alkaline phosphatase (ALP). First, for sulfur ion, we prepared the carbon dots (CD-S) modified by ligand molecules as the precursor of the probe, formed complexes with copper ions (Cu2+) and quenched the fluorescence of CD-S, which served as a fluorescence enhanced probe for detecting S2-. Because of the high binding ability between S2- and Cu2+, it will seize the copper ions in the probe and generate stable CuS (Ksp=3.63 * 10-36). Therefore, the fluorescence of the carbon dots of Cu2+ can be recovered, and the detection limit of the probe for sulfur ion is 0.78 M. The CD-S/Cu2+ probe has good water solubility, biocompatibility, selectivity and sensitivity. It can be successfully applied to the detection of sulfur ion in tap water and the detection and imaging of sulfur ions in living cells. Secondly, we design and detection of gamma glutamyl transpeptidase (GGT) synthesized Glu-TPE as fluorescence probe; four stilbene derivatives, Glu-TPE probe with aggregation induced emission (AIE) effect; two gamma molecules in the glutamine group makes Glu-TPE probe has good water solubility and biocompatiblity of; because of GGT catalyzed hydrolysis of Glu-TPE molecular probes in gamma glutamyl groups, hydrophobic enzymatic product aggregation induced AIE effect and emit blue fluorescence, so as to realize the enhanced fluorescence detection of GGT. Based on the specificity and efficiency of GGT enzymatic reaction, probe Glu-TPE has good selectivity and sensitivity. It can be used to detect GGT content in human serum samples and detect and imaging Qg derived GGT in A2780 cells. For the detection of GGT, we also made another based on intramolecular charge transfer (ICT) by fluorescence probe PEG-NA-Glu mechanism; when the probe molecule PEG-NA-Glu gamma glutamyl moiety catalyzed by GGT after hydrolysis, the fluorophore emitting mechanism in this ICT, the electron donor electron cloud density changes, leading to the change of fluorescence emission wavelength, thus achieving a ratiometric fluorescent detection of GGT. The probe PEG-NA-Glu has high sensitivity and biocompatibility. The detection limit is 0.76 U/L, which can be used for detecting GGT content in urine and serum. It is much simpler and more convenient than ELISA Kit method, and successfully realizes the two-color fluorescence imaging of GGT in living cells. Moreover, the alkaline phosphatase (ALP), we use the ICT fluorescence mechanism of naphthalene two phthalimide derivatives, design and probe AO-NA-P were prepared; phosphate removal catalyzed by ALP probe molecule AO-NA-P, makes the probe in naphthalene ring electron donating 4 position changes, the results of enhanced fluorescent molecules the "push-pull" electronic effect, fluorescence emission wavelength; based on this principle, the successful implementation of the ratiometric fluorescent ALP detection. Because the ALP catalytic hydrolysis with specificity and high efficiency, AO-NA-P probe with high sensitivity and selectivity, detection limit of ALP probe was 0.38 U/L; in addition, the probe molecule AO-NA-P has excellent water solubility and biocompatibility; can be used in human serum samples, ALP detection of viable cells, in vivo. We first realized the ratio type fluorescence imaging of ALP in vivo (zebrafish), and imaging and detecting the increase of ALP content induced by drug damage in zebrafish. The related results can provide a useful reference for the study of the changes of life substances caused by organ damage.
【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：O629.8;TP212

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