生物體系中有機(jī)小分子構(gòu)建的pH值檢測熒光探針研究進(jìn)展

吳廬陵, 黃楚森, 賈能勤

(1.上海師范大學(xué) 生命與環(huán)境科學(xué)學(xué)院,資源化學(xué)教育部重點(diǎn)實(shí)驗(yàn)室,上海 200234;2.上海師范大學(xué) 生命與環(huán)境科學(xué)學(xué)院,上海市高校分子影像探針與傳感器重點(diǎn)實(shí)驗(yàn)室,上海 200234)

生物體系中有機(jī)小分子構(gòu)建的pH值檢測熒光探針研究進(jìn)展

吳廬陵1,2, 黃楚森1,2, 賈能勤1,2

(1.上海師范大學(xué) 生命與環(huán)境科學(xué)學(xué)院,資源化學(xué)教育部重點(diǎn)實(shí)驗(yàn)室,上海 200234;2.上海師范大學(xué) 生命與環(huán)境科學(xué)學(xué)院,上海市高校分子影像探針與傳感器重點(diǎn)實(shí)驗(yàn)室,上海 200234)

由于傳統(tǒng)的pH計不能很好地應(yīng)用在微環(huán)境(如亞細(xì)胞器)和復(fù)雜生物體系中的pH值動態(tài)和精確定量檢測,因此迫切需要研究可監(jiān)測復(fù)雜多變生物體系中pH值變化且具有高靈敏度的pH值響應(yīng)分子探針.就近年來用于生物體系(主要是活細(xì)胞內(nèi))pH值檢測的小分子熒光探針作簡要綜述,同時對目前基于有機(jī)小分子構(gòu)建的熒光探針用于生物體系pH值檢測研究及其未來的發(fā)展趨勢進(jìn)行展望.

pH值; 比色/熒光; 檢測; 熒光探針

生物體系微環(huán)境化學(xué)反應(yīng)的進(jìn)行或完成與pH值密切相關(guān),同樣,組織、細(xì)胞或酶的許多生理過程如細(xì)胞增殖或凋亡、離子運(yùn)輸、內(nèi)吞作用、多藥物耐藥性或肌肉收縮等活動也與pH值緊密相關(guān).pH值的改變還會通過間隙連接和信號通路的變化影響到突觸傳遞、神經(jīng)元興奮性和細(xì)胞間耦合等神經(jīng)系統(tǒng)活動.細(xì)胞內(nèi)的pH值對細(xì)胞內(nèi)穩(wěn)態(tài)和許多生理過程有著重要的影響[1].例如細(xì)胞內(nèi)酸性亞細(xì)胞器的溶酶體,其pH值的改變可能影響著蛋白質(zhì)降解,細(xì)胞膜修復(fù)甚至是細(xì)胞死亡[2-3].與此同時,細(xì)胞內(nèi)pH值不正常會引發(fā)如癌癥和阿爾茨海默等一系列疾病,有報道指出細(xì)胞酸化是細(xì)胞凋亡的早期特征[4].例如腫瘤細(xì)胞內(nèi)的pH值(6.7～7.1)要比細(xì)胞外的pH值(7.4)低,而正常細(xì)胞內(nèi)的pH值是7.2,普遍低于其細(xì)胞外的pH(7.4).在一定程度上,腫瘤細(xì)胞及其微環(huán)境的pH值異常促進(jìn)腫瘤細(xì)胞增值、新陳代謝、細(xì)胞遷移和侵襲進(jìn)程[4].因此,實(shí)現(xiàn)對生物體系尤其如細(xì)胞內(nèi)溶酶體等微環(huán)境的pH變化的動態(tài)可視化和定量檢測能夠更深入地理解受pH值影響的信號轉(zhuǎn)導(dǎo)和相關(guān)生理進(jìn)程,以便依此設(shè)計出更好的藥物和實(shí)現(xiàn)精準(zhǔn)醫(yī)學(xué)中的個體化治療.

1 熒光分析法用于pH值變化的檢測

很多生理進(jìn)程都以細(xì)胞為單位發(fā)生,因此對于生物體系中pH值的檢測通常都以細(xì)胞內(nèi)外的pH值變化為監(jiān)測對象.在正常生理?xiàng)l件下,根據(jù)pH值不同可以把生物體系中的pH值分為細(xì)胞外pH值和細(xì)胞內(nèi)pH值.后者又可以劃分為兩個部分:酸性細(xì)胞器(pH值為4.5～6.0)及細(xì)胞質(zhì)(pH值為6.8～7.4).細(xì)胞內(nèi)外酸性或堿性太強(qiáng),都會引起細(xì)胞功能障礙.由于傳統(tǒng)的pH值計不能很好地應(yīng)用于細(xì)胞微環(huán)境中pH值的檢測,因此開發(fā)出用于監(jiān)測復(fù)雜多變生物體系微環(huán)境中pH值變化的分子探針對于理解pH調(diào)控的信號轉(zhuǎn)導(dǎo)和各種生理進(jìn)程顯得極為迫切.

熒光探針檢測法是基于熒光信號變化的pH值測定方法,基于有機(jī)小分子構(gòu)建的pH值熒光探針是用一些有機(jī)小分子的熒光特性來指示目標(biāo)介質(zhì)酸堿度的變化.用熒光探針測定pH值是一種非侵入性的方法,既不會破壞樣品,同時具有靈敏度高、選擇性好、細(xì)胞毒性低、細(xì)胞膜透過性好及測試方法簡單等特點(diǎn).尤其是相比于綠色熒光蛋白等大分子構(gòu)建的pH值探針,有機(jī)小分子熒光探針對生物體系的擾動更小.因此,基于有機(jī)熒光小分子探針的pH值檢測法利用熒光信號的可視化讀出可以實(shí)時監(jiān)測細(xì)胞內(nèi)pH值的變化和動態(tài)分布,從而極大地推動了生物體系內(nèi)pH值相關(guān)的信號轉(zhuǎn)導(dǎo)和各類生理進(jìn)程的研究.基于此,用于檢測亞細(xì)胞器[5-6]、細(xì)胞質(zhì)[7]和整個細(xì)胞[8]pH值變化的熒光探針的設(shè)計與開發(fā)研究在近年來備受關(guān)注.熒光素、萘酰亞胺、花菁和BODIPY等染料在小分子pH值探針的構(gòu)建方面得到很大程度的應(yīng)用,這些pH值探針可以通過染料的熒光或顏色的變化實(shí)現(xiàn)對pH值的高選擇性動態(tài)檢測[9-11].

1.1 熒光素類

圖1 熒光素的質(zhì)子化和去質(zhì)子化過程[11]

熒光素具有較長發(fā)射波長、高量子產(chǎn)率[12]的特點(diǎn),是最早的pH值熒光探針之一,其熒光性質(zhì)隨pH值變化而變化.當(dāng)pH≤5時,熒光素是熒光很弱的中性分子,伴隨著螺環(huán)結(jié)構(gòu)存在;當(dāng)pH≥6.5時,其為帶2個負(fù)電荷的形式,熒光發(fā)射強(qiáng)度較大[13](圖1).然而熒光素分子容易從細(xì)胞內(nèi)流失這一缺陷限制了其在細(xì)胞參數(shù)表征方面的應(yīng)用[13].為了解決這一問題,人們開展了多種熒光素衍生物的pH值探針[14-17].

圖2 熒光素與單壁碳納米管非共價結(jié)合形成的高水溶性pH值熒光探針[18]

Nakayama-Ratchford等[18]設(shè)計了熒光素-聚乙二醇以π-π堆積方式形成功能化的單壁碳納米管Fluor-PEG/SWNTs.Fluor-PEG/SWNTs是分子開關(guān)“off-on”型的高水溶性pH值熒光探針,熒光素和單壁碳納米管的連接效果依賴pH值的變化;pH值增大,其連接效果差,即Fluor-PEG/SWNTs在高pH值條件下不穩(wěn)定,探針的熒光強(qiáng)度會相應(yīng)減弱.另外,該探針可被哺乳細(xì)胞吞噬,可以用于細(xì)胞標(biāo)記,同時拉曼分析結(jié)果表明熒光素成功地和單壁碳納米管連接.

1.2 羅丹明類

Yu[19]等以羅丹明B為基礎(chǔ)通過“click reaction”得到了可開環(huán)的螺環(huán)pH值探針RhP和RhPA (圖3),其pKa值分別是4.79和5.23.這兩個探針具有高靈敏度、選擇性好的特點(diǎn),且為長波長熒光發(fā)射;RhPA較低的細(xì)胞毒性和優(yōu)越的光穩(wěn)定性可用于活細(xì)胞內(nèi)酸性細(xì)胞器溶酶體的可視化熒光成像.

圖3 RhP和RhPA的pH值響應(yīng)機(jī)理[19]

1.3 BODIPY類

圖4 基于PET機(jī)理的熒光分子傳感器1a

BODIPY類熒光染料光穩(wěn)定性好,熒光量子產(chǎn)率高,摩爾消光系數(shù)高,然而其母體結(jié)構(gòu)難以引入活性基團(tuán),極大地限制了其在生物學(xué)領(lǐng)域中的應(yīng)用.Hall[20]等以BODIPY為骨架設(shè)計合成了off-on型熒光探針1a(圖4),該探針是典型的光誘導(dǎo)電子轉(zhuǎn)移(PET)機(jī)理探針,且可以被近紅外光區(qū)域波長的光激發(fā).通過對其在不同極性溶劑中的光譜性質(zhì)進(jìn)行研究發(fā)現(xiàn),熒光強(qiáng)度隨溶劑極性增大而增大;在PBS緩沖溶液中,其pKa值為6.9,熒光強(qiáng)度隨pH值的減小而增大,在響應(yīng)的pH值檢測范圍(pH值工作窗口)內(nèi)熒光強(qiáng)度增加了近3倍.該探針被用來對HeLa細(xì)胞進(jìn)行熒光成像,主要分布在細(xì)胞質(zhì)中,具有良好的成像效果.

1.4 萘酰亞胺類

圖5 化合物5和7的結(jié)構(gòu)[21]

萘酰亞胺類熒光染料色澤鮮艷、熒光量子產(chǎn)率較大,是一類極其重要的功能染料.2008年Bojinov[21]等設(shè)計合成兩種基于萘酰亞胺的具有pH值響應(yīng)的分子傳感器.化合物5和7(圖5)熒光發(fā)射是黃綠色,在N,N-二甲基甲酰胺和水/N,N-二甲基甲酰胺(1∶4,v/v)體系中分別研究了兩個化合物的光物理性質(zhì),表明化合物5的熒光發(fā)射對pH值不敏感,而化合物7在較寬的pH值范圍內(nèi)熒光變化明顯.這一現(xiàn)象是由于5的哌啶氨基質(zhì)子化更弱,而7可以是很好的“off-on”型熒光pH值探針,其PET機(jī)理如圖6所示.

圖6 pH探針7的PET機(jī)理[21]

1.5 花菁和半花菁類

圖7 基于花菁染料的pH探針結(jié)構(gòu)

花菁和半花菁類染料在最大吸收波長處有高的摩爾消光系數(shù)以及良好的水溶性,同樣具有低的細(xì)胞毒性,并且已廣泛應(yīng)用在比色、熒光傳感中[22-24].申請公布號為CN 104086536 A的中國發(fā)明專利公開了一種用于檢測pH值的熒光探針及其制備方法與專用檢測試劑盒.該發(fā)明提供的用于檢測pH值的熒光探針的結(jié)構(gòu)如圖7所示.該發(fā)明提供的用于檢測pH值的試劑盒,包括上述化合物和溶劑.上述專利公布的技術(shù)方案中,制得的熒光探針及其專用檢測試劑盒對溶液的pH值具有良好的響應(yīng)性,能夠?qū)崿F(xiàn)對溶酶體的標(biāo)記和細(xì)胞溶酶體pH值的測定,但其pH值檢測范圍較窄,且僅限于細(xì)胞溶酶體pH值的檢測,適用范圍較小,有很大的局限性.

圖8 CPH檢測pH值的原理及其熒光/比色雙模態(tài)成像

早期的pH值探針設(shè)計中,大多數(shù)pH值熒光探針都具有發(fā)射波長單一的特點(diǎn)[10-11,25-26],在實(shí)際復(fù)雜生物體系檢測中染料聚集和長時間光激發(fā)很容易引起熒光的淬滅,從而影響該類探針工作的穩(wěn)定性.因此,用這些單發(fā)射波長探針檢測pH值,尤其是定量檢測需要前期校準(zhǔn).比值型檢測機(jī)理可以克服這類問題.近年來,一些課題組設(shè)計制備出了pH值響應(yīng)并可用于細(xì)胞內(nèi)pH值檢測以及活細(xì)胞成像的比值型pH值熒光探針[8,10,22,27-32].然而,這些比值型探針專注于細(xì)胞能pH值變化的檢測,而且多不能夠?qū)?shí)現(xiàn)對pH值比色檢測.大多數(shù)的花菁和半菁類pH值探針通過菁染料吲哚環(huán)的N原子實(shí)現(xiàn)熒光的“off-on”變化,雖然可以將其與聚集態(tài)誘導(dǎo)發(fā)光染料[33-34]、羅丹明染料[35]、喹啉[29]和苯并噻唑[36]連接作為FRET的受體或供體,這樣構(gòu)建出比值型熒光pH值探針,但是上述連接需要巧妙或復(fù)雜的分子結(jié)構(gòu)設(shè)計.另外,花菁和半花菁類染料可裸眼觀察實(shí)現(xiàn)比色方式檢測pH值的研究鮮有報道.所以,本課題組[12]最近設(shè)計、合成了一種新型基于半菁染料的比值型分子探針(CPH),該探針可很好地實(shí)現(xiàn)比色和熒光雙模態(tài)檢測pH值(圖8).它主要由兩部分構(gòu)成,一部分是連接在吲哚半花菁氮原子位置的芐基,另一部分是作為pH值受體的粉羥基.與此前報道的吲哚半花菁類pH值探針不同,CPH具有如下特性:1)在吲哚環(huán)的氮原子位置引入芐基可以提高這個吲哚半花菁染料的穩(wěn)定性,從而抑制了OH-對CPH的親核加成.使得CPH對于質(zhì)子的檢測原理與曾經(jīng)報道的OH-對C=N親核加成這一普遍機(jī)理不同;2)苯酚基團(tuán)對pH值變化的響應(yīng)是形成酚和酚鹽兩種結(jié)構(gòu)形式,且這兩種結(jié)構(gòu)具有可逆性.基于這種結(jié)構(gòu)-性質(zhì)的關(guān)系,CPH可以實(shí)現(xiàn)比色/熒光雙模態(tài)檢測pH值的變化;3)CPH可以作為活細(xì)胞內(nèi)溶酶體探針且對溶酶體內(nèi)pH值變化可以通過雙波長的熒光信號進(jìn)行檢測;4)CPH同樣可以固載制成pH值試紙,該試紙對酸性及堿性蒸汽具有高敏度的比色變化,并且可以裸眼觀察.

2 展 望

綜上所述,迄今為止基于有機(jī)小分子的pH值熒光探針對于推動在生物醫(yī)學(xué)領(lǐng)域研究發(fā)揮著重要作用,相關(guān)報道已經(jīng)很多,尤其是單光子pH值熒光探針.為了更好地在生物體系中實(shí)現(xiàn)對pH值變化實(shí)時、高靈敏度、原位和便捷地檢測,今后的相關(guān)研究工作可以集中在如下幾個方面:1)開發(fā)雙光子探針,以降低生物自發(fā)熒光背景干擾,進(jìn)一步提高探針檢測的靈敏度;2)相比于開關(guān)型小分子熒光探針,比值型熒光探針在檢測時通過熒光光譜圖兩個熒光峰強(qiáng)度變化對同一物質(zhì)進(jìn)行檢測,使其具備了內(nèi)在的自校準(zhǔn)功能,因而在生物體系中對于pH值的定量檢測結(jié)果更為準(zhǔn)確和可靠.因此,發(fā)展有機(jī)小分子pH值熒光探針仍然是未來研究的一個重要方向;3)開發(fā)出穩(wěn)定的近紅外pH值熒光探針,有利于穿透更深的生物組織,從而可以實(shí)現(xiàn)活體內(nèi)微環(huán)境的pH值變化檢測;4)發(fā)展基于小分子探針的pH值傳感器,并將其與手機(jī)應(yīng)用軟件結(jié)合,制作出可穿戴式的pH值設(shè)備,實(shí)現(xiàn)對人體等生物體系中微環(huán)境pH值的快速動態(tài)檢測.

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(責(zé)任編輯：包震宇,郁 慧)

Progress of fluorescent probes based on small organic molecules for pH value detection in biological systems

WU Luling1,2, HUANG Chusen1,2, JIA Nengqin1,2

(1.Key Laboratory of Resource Chemistry,Ministry of Education,College of Life and Environmental Sciences,Shanghai Normal University,Shanghai 200234,China;2.Shanghai Municipal Education Committee Key Laboratory of Molecular Imaging Probes and Sensors,College of Life and Environmental Sciences,Shanghai Normal University,Shanghai 200234,China)

As traditional pH meters cannot work well for the real-time determination and quantitative detection of pH value changes in microenvironments (such as subcellular organelles) and complex biological systems,molecular pH-sensitive probes for monitoring pH changes in complicated and changeable biological systems are urgently needed.This review focuses on the current progress in small molecular fluorescence probes for the detection of pH value changes in biological systems (mainly in monitoring the pH fluctuation in the live cell).Future development of fluorescent probes for pH value detection in the biological systems was also discussed.

pH value; colorimetric/fluorescence; detection; fluorescent probe

2016-09-22

國家自然科學(xué)基金面上項(xiàng)目(21373138,21672150);國家自然科學(xué)基金青年科學(xué)基金(21302125);教育部博士點(diǎn)新教師基金(20133127120005);上海市晨光計劃(14CG42)

黃楚森,中國上海市徐匯區(qū)桂林路100號,上海師范大學(xué)生命與環(huán)境科學(xué)學(xué)院,郵編:200234,E-mail:huangcs@shnu.edu.cn;賈能勤,中國上海市徐匯區(qū)桂林路100號,上海師范大學(xué)生命與環(huán)境科學(xué)學(xué)院,郵編:200234,E-mail:nqjia@shnu.edu.cn

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1000-5137(2016)06-0742-06