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端粒酶活性檢測(cè)方法研究進(jìn)展

2015-02-02 13:40郭林燕陽(yáng)明輝
化學(xué)傳感器 2015年3期
關(guān)鍵詞:端粒酶端粒探針

郭林燕,陽(yáng)明輝

(中南大學(xué)化學(xué)化工學(xué)院,湖南長(zhǎng)沙410083)

端粒酶活性檢測(cè)方法研究進(jìn)展

郭林燕,陽(yáng)明輝*

(中南大學(xué)化學(xué)化工學(xué)院,湖南長(zhǎng)沙410083)

人體端粒酶是一種核糖核蛋白復(fù)合物,在生物體內(nèi),以其自身的RNA為模板,通過(guò)催化添加端粒重復(fù)序列TTAGGG至染色體末端。由于在絕大多數(shù)癌癥中端粒酶活性與惡性腫瘤之間存在著較大的關(guān)聯(lián),其在腫瘤的發(fā)生、發(fā)展過(guò)程中起著重要作用,因而對(duì)端粒酶活性的檢測(cè)可為癌癥的診斷、預(yù)測(cè)以及臨床治療提供重要依據(jù)。該文總結(jié)了近幾年來(lái)針對(duì)端粒酶活性幾種不同的檢測(cè)方法,重點(diǎn)強(qiáng)調(diào)了幾種機(jī)體內(nèi)原位檢測(cè)方法,并適當(dāng)?shù)倪M(jìn)行了分析比較。

端粒酶;G-四聯(lián)體;惡性腫瘤;分析檢測(cè)

0 引言

端粒,是一種核蛋白,由短的富含鳥嘌呤的串聯(lián)重復(fù)序列組成,在人類細(xì)胞中真核染色體的末端形成帽狀結(jié)構(gòu),以抑制不必要的降解、重組或端到端的融合[1]。在一些體細(xì)胞中,DNA復(fù)制循環(huán)后,因DNA聚合酶的不完全復(fù)制而使得端粒逐漸縮短,當(dāng)長(zhǎng)度達(dá)到一個(gè)臨界值時(shí),細(xì)胞便會(huì)停止分裂到達(dá)衰老階段[2]。端粒作為維持染色體復(fù)制過(guò)程完整性必不可少的組成,由于其縮短或加帽蛋白缺乏會(huì)造成不利的后果,包括形成染色體異常,細(xì)胞衰老和細(xì)胞凋亡[3-5]。

端粒酶是一種核糖核蛋白逆轉(zhuǎn)錄酶,由一個(gè)基本的催化亞基和RNA模板以及端粒相關(guān)蛋白一起維持端粒長(zhǎng)度和功能[6-8]。通過(guò)其逆轉(zhuǎn)錄酶活性及其內(nèi)在的RNA作為模板催化添加端粒重復(fù)序列(TTAGGG)n至染色體DNA的3’端[9],由于其富含鳥嘌呤(Guanine,G)序列,在生理?xiàng)l件下,端粒DNA形成了一種分子間和分子內(nèi)的鳥嘌呤四聯(lián)結(jié)構(gòu)。研究發(fā)現(xiàn),在超過(guò)85%的人類惡性腫瘤中端粒酶具有異常高的活性,如乳腺癌,結(jié)腸癌,肺癌,前列腺癌,卵巢癌,胃癌和皮膚癌等。而在正常體細(xì)胞中,端粒酶的表達(dá)是被抑制或成缺失狀態(tài)[10-12]。由于癌細(xì)胞中過(guò)度表達(dá)的端粒酶會(huì)維持端粒的長(zhǎng)度,使得細(xì)胞無(wú)限制地增生而不會(huì)衰老,因此科學(xué)家認(rèn)為端粒酶在腫瘤的形成過(guò)程中起著關(guān)鍵作用。端粒酶的這種特性使得其在癌癥早期診斷、預(yù)測(cè)和治療領(lǐng)域成為一種重要的生物標(biāo)志物[13-16]。除此之外,端粒酶和端粒的結(jié)構(gòu)在研究其它疾病、基因調(diào)節(jié)、細(xì)胞/機(jī)體老化和哺乳類動(dòng)物的克隆方面也起到了至關(guān)重要的作用[17-18]。鑒于此,高靈敏度的檢測(cè)端粒酶活性方法對(duì)于疾病機(jī)理研究和臨床診斷顯得尤為重要[19]。

1 端粒酶活性的傳統(tǒng)檢測(cè)方法

近年來(lái),已經(jīng)開發(fā)出多種測(cè)定端粒酶活性的方法[5,20-21],而 Kim開發(fā)的以聚合酶聯(lián)反應(yīng)(Polymerase Chain Reaction,PCR)為基礎(chǔ)的端粒重復(fù)序列擴(kuò)增法 (Telomeric Repeat Amplification Protocol,TRAP)因其超高的靈敏度已被發(fā)展為端粒酶活性的標(biāo)準(zhǔn)分析方法[22]。

1.1 TRAP分析法的原理

TRAP法是一個(gè)單管反應(yīng),首先,從細(xì)胞或組織中提取端粒酶合成端粒重復(fù)序列,接著從外源添加端粒鏈引物,然后將這些延伸后的產(chǎn)品作為模板進(jìn)行PCR擴(kuò)增,隨后進(jìn)行聚丙烯酰胺凝膠電泳鑒定,最后使用密度測(cè)定法進(jìn)行定量[23],從而實(shí)現(xiàn)端粒酶活性的檢測(cè)。

1.2 TRAP分析法的不足

TRAP分析法能夠?qū)崿F(xiàn)高通量高靈敏度的檢測(cè)[24-25],無(wú)疑是一個(gè)功能強(qiáng)大的測(cè)定方法,但保留著PCR放大技術(shù)帶來(lái)的缺點(diǎn)。此外,TRAP法需要使用昂貴的設(shè)備和試劑,相當(dāng)耗時(shí)[26]。再加上抑制端粒酶活性已被提議作為人類癌癥治療的潛在方法,而TRAP在篩選有效的端粒酶抑制劑如G-四聯(lián)體配體時(shí)易受PCR-派生產(chǎn)物的影響,因此,該方法存在很多局限性。

1.3 TRAP分析法的發(fā)展

為了克服TRAP分析法的上述缺點(diǎn),不斷有新的改善過(guò)的PCR步驟被提出來(lái)。包括采用TRAP法結(jié)合PCR后雜交協(xié)議的化學(xué)發(fā)光探針、熒光染料或生物素化的引物來(lái)測(cè)量PCR擴(kuò)增雙鏈DNA的數(shù)量[27-29]。例如2010年,Plaxco等利用引物修飾金納米粒子來(lái)檢測(cè)高濃度細(xì)胞溶解物中端粒酶的活性,這種方法雖然比傳統(tǒng)的TRAP法在特異性和靈敏性方面有所提高,但是這種以凝膠為基礎(chǔ)的方法仍然耗時(shí)且需要豐富的專業(yè)知識(shí)[30]。

2 端粒酶活性的最新檢測(cè)方法

為替代傳統(tǒng)的TRAP分析法,研究者們已經(jīng)開發(fā)了許多PCR-free分析方法并將之應(yīng)用到端粒酶活性的檢測(cè)中,例如光學(xué)傳感器、表面等離子共振、電致化學(xué)發(fā)光、電化學(xué)檢測(cè)和指數(shù)等溫?cái)U(kuò)增分析等[31-35]。

2.1 紫外吸收法

納米金由于其自身較高的消光系數(shù)和粒徑依賴型的光學(xué)性能在構(gòu)建生物傳感平臺(tái)方面顯示出極大的應(yīng)用潛力,被廣泛應(yīng)用到眾多領(lǐng)域的檢測(cè)[36-39]。基于納米金的分析方法主要優(yōu)點(diǎn)就是分子識(shí)別行為可以轉(zhuǎn)換成顏色改變,繼而可以通過(guò)肉眼觀察或者利用紫外可見(jiàn)光譜簡(jiǎn)單測(cè)定出來(lái)。因此,該文簡(jiǎn)要列舉了兩個(gè)例子來(lái)說(shuō)明其在端粒酶活性檢測(cè)方向的應(yīng)用。

Willner等[40]開發(fā)了一種利用端粒酶產(chǎn)生的端粒-氯化血紅素/G-四聯(lián)體催化氧化L-半胱氨酸誘導(dǎo)納米金聚集的方法來(lái)定量分析端粒酶活性,其反應(yīng)原理如圖1。端粒是富含鳥嘌呤核苷的折疊核酸鏈,在K+和氯化血紅素的存在下,端粒單元自組裝成氯化血紅素/G-四聯(lián)體結(jié)構(gòu),表現(xiàn)出辣根過(guò)氧化物酶催化活性[41]。端粒-氯化血紅素/G-四聯(lián)體結(jié)構(gòu)能催化硫醇類物質(zhì) (例如L-半胱氨酸)氧化成二硫化物(例如胱氨酸)[42]。L-半胱氨酸能促進(jìn)Au納米顆粒的聚集,伴隨著紫外吸收的變化,從紅色(單個(gè)金納米粒子)變?yōu)樗{(lán)色(團(tuán)聚的金納米粒子),該過(guò)程被用來(lái)定量分析端粒酶活性。從293T腫瘤細(xì)胞中提取端粒酶,當(dāng)存在脫氧核糖核苷三磷酸混合物(dNTPs)和合適的引物探針時(shí),所述調(diào)聚反應(yīng)將催化端粒-氯化血紅素/G-四聯(lián)體鏈的生成,用以控制L-半胱氨酸介導(dǎo)的納米金的聚集。因此,聚合的程度由端粒酶的濃度控制。該方法檢測(cè)限能檢測(cè)到27細(xì)胞/ μL中端粒酶活性。并且此方法是靈敏度高,檢測(cè)時(shí)間相對(duì)較短(3h),以及無(wú)需進(jìn)行復(fù)雜的處理和昂貴的儀器。

此外,Xia等[43]設(shè)計(jì)了一個(gè)擁有四個(gè)檢測(cè)色狀態(tài)(如藍(lán)色、紫色、紅色和沉淀)的雙功能納米金(GNP)探針,用于雙向直接、準(zhǔn)確簡(jiǎn)單的檢測(cè)尿液中端粒酶活性,并成功地將其應(yīng)用于膀胱癌的無(wú)痛診斷,反應(yīng)機(jī)理如圖2。根據(jù)數(shù)軸理論,首先定義紅色GNP探針為原點(diǎn),它代表尿提取物中端粒酶無(wú)活性。正方向?qū)?yīng)于相對(duì)高濃度活性端粒酶,在該系統(tǒng)中GNP探針組裝明顯,產(chǎn)生沉淀。負(fù)方向?qū)?yīng)于相對(duì)低濃度(紫色)和無(wú)端粒酶存在(藍(lán)色)的樣品。四個(gè)檢測(cè)狀態(tài)可以通過(guò)肉眼或紫外-可見(jiàn)光譜來(lái)區(qū)分。沉淀和紫色狀態(tài)代表膀胱癌患者,而藍(lán)色和紅色狀態(tài)代表正常個(gè)體。相比于傳統(tǒng)比色法,具有優(yōu)良的辨別能力,并且準(zhǔn)確簡(jiǎn)潔,避免大量的操作和復(fù)雜的儀器。此外,該系統(tǒng)穩(wěn)定性極強(qiáng),探針保存一個(gè)月后依然可以區(qū)分。因此,這種具有良好的精度、成本、選擇性、穩(wěn)定性和適用性的檢測(cè)系統(tǒng)在端粒酶活性和膀胱癌甚至其它癌癥的診斷方面具有廣闊的應(yīng)用前景。

圖1 基于氯化血紅素/端粒-G四聯(lián)體控制的L-半胱氨酸介導(dǎo)納米金團(tuán)聚檢測(cè)端粒酶活性機(jī)理圖Fig.1 Analysis of telomerase activity by following the hemin/telomere-G-quadruplex-controlled L-cysteine mediated aggregation of Au NPs

2.2 等溫?cái)U(kuò)增法

2013年,Weizmann等[44]開發(fā)了一種新穎的指 數(shù)等溫?cái)U(kuò)增 端粒重復(fù)序 列(exponential isothermal amplification of telomere repeat,EXPIATR)分析法,這是一種簡(jiǎn)單、靈敏、可靠的檢測(cè)細(xì)胞提取物中端粒酶活性的方法。

如圖3所示,此法基于DNA等溫?cái)U(kuò)增這樣一個(gè)策略,通過(guò)鏈置換擴(kuò)增激發(fā),使用限制性內(nèi)切酶刻痕于一個(gè)識(shí)別位點(diǎn),并利用聚合酶多次復(fù)制和重置目標(biāo),實(shí)現(xiàn)25 min的超快檢測(cè)。這種方法不僅具有TRAP的優(yōu)越性,還在此基礎(chǔ)上有了一定的提升。它放棄了昂貴的熱循環(huán)協(xié)議,使該法更加廉價(jià)且在臨床上更加通用,此外其檢測(cè)時(shí)間相較于其他方法大大縮短。由于高通量、等溫、即用型等特點(diǎn),EXPIATR在未來(lái)臨床試驗(yàn)使用中顯示出了巨大潛力。

此后,Zhang等[45]提出了一種利用端粒誘導(dǎo)兩級(jí)等溫?cái)U(kuò)增介導(dǎo)化學(xué)發(fā)光的分析方法來(lái)檢測(cè)單細(xì)胞水平的端粒酶活性。如圖4,存在端粒酶時(shí),端粒重復(fù)序列 (TTAGGG)n被加到端粒酶基底引物的3’端,這可以轉(zhuǎn)化成鏈置換擴(kuò)增的模板[46-47],用來(lái)生成短寡核苷酸 、DNA 酶和(TTAGGG)n中的端粒重復(fù)序列。短寡核苷酸可以充當(dāng)一個(gè)新的觸發(fā)點(diǎn)來(lái)結(jié)合游離的端粒酶基底引物,從而啟動(dòng)一個(gè)恒溫指數(shù)擴(kuò)增反應(yīng)[48-50],生成大量的催化DNA酶。無(wú)論是DNA酶還是富含G的端粒重復(fù)單元都可以與血紅素相互作用,形成催化血紅素-G-四聯(lián)體納米結(jié)構(gòu),在魯米諾與過(guò)氧化氫共存時(shí),可以催化產(chǎn)生化學(xué)發(fā)光信號(hào)。而在缺乏端粒酶時(shí),兩階段等溫?cái)U(kuò)增不能啟動(dòng),所以觀察不到化學(xué)發(fā)光信號(hào)。

圖2 (a)膀胱癌的無(wú)痛臨床估計(jì)數(shù)軸理論;(b)端粒酶提取尿樣;(c)端粒酶檢測(cè)原理圖Fig.2 (a)The number axis theory for clinical estimate of bladder cancer without pain.(b)Telomerase extraction from urine samples.(c)The scheme for telomerase detection

圖3 EXPIATR測(cè)定的原理圖Fig.3 Schematic diagram of the EXPIATR assay

該方法與TRAP相比,更簡(jiǎn)單、更靈敏、更快速,而且不需要熱循環(huán)、洗滌和分離步驟[27]。而與EXPIATR分析相比,該方法有一個(gè)突出的優(yōu)點(diǎn)就是成本低,它不需要使用昂貴的熒光標(biāo)記核苷酸[44]。由于兩階段等溫?cái)U(kuò)增的擴(kuò)增效率高、靈敏度高以及化學(xué)發(fā)光測(cè)定法的動(dòng)態(tài)范圍寬等優(yōu)點(diǎn),該方法能夠靈敏地檢測(cè)到從單一的HeLa癌細(xì)胞中提取的端粒酶活性,而不需要任何標(biāo)記的DNA探針。上述方法可進(jìn)一步用于篩選抗癌藥物,并且可能提供一種有前途的方法來(lái)發(fā)現(xiàn)新的抗癌藥物。

圖4 端粒誘導(dǎo)兩級(jí)等溫?cái)U(kuò)增介導(dǎo)化學(xué)發(fā)光檢測(cè)端粒酶活性原理圖Fig.4 Schematic illustration for the detection of telomerase activity using telomeres-induced two-stage isothermal amplification-mediated chemiluminescence assay

2.3 電化學(xué)分析法

電化學(xué)法由于其自身的高靈敏度、操作簡(jiǎn)單、反應(yīng)迅速和成本低廉等優(yōu)點(diǎn)在分析端粒酶活性方面正越來(lái)越備受關(guān)注[51-54]。

Ozsoz等[31]開發(fā)了一種基于鳥嘌呤氧化信號(hào)來(lái)檢測(cè)端粒酶活性的免標(biāo)記電化學(xué)分析法,如圖5所示,這種分析技術(shù)利用一次性電極——碳石墨電極作為電化學(xué)傳感器。通過(guò)以端粒重復(fù)序列擴(kuò)增為基礎(chǔ)的PCR分析法,將鳥嘌呤的氧化信號(hào)作為端粒酶活性的量度來(lái)進(jìn)行電化學(xué)檢測(cè)。此分析法是第一個(gè)免標(biāo)記的電化學(xué)分析法,相較于其他檢測(cè)方法,如表面等離子共振和石英晶體微天平等具有快速,簡(jiǎn)單,廉價(jià)而且無(wú)放射性等優(yōu)點(diǎn)。并且無(wú)需標(biāo)記,使其適用于臨床樣品的定量測(cè)定。然而這種方法也存在PCR分析法的固有局限性,為用于常規(guī)分析,有必要開發(fā)一種不依賴于易產(chǎn)生誤差的PCR的分析方法。

研究表明,生物傳感器中使用電化學(xué)阻抗法(Electrochemical Impedance Spectroscopy,EIS)有著諸多優(yōu)點(diǎn),如高靈敏度、高精密度、檢測(cè)速度快以及可實(shí)現(xiàn)實(shí)時(shí)監(jiān)測(cè)等[55-58],Lin等[59]開發(fā)了一種基于免標(biāo)記的電化學(xué)阻抗法來(lái)檢測(cè)端粒酶活性,反應(yīng)原理如圖6所示。該方法檢出限能夠測(cè)定1000 HeLa細(xì)胞中端粒酶的活性。首先將硫醇化的DNA引物固定到金表面上,然后在dNTPs的存在下與端粒酶一起孵育進(jìn)行延伸反應(yīng)。隨著端粒酶活性的增強(qiáng),延長(zhǎng)的DNA引物可阻斷Au電極表面Fe(CN)63-/Fe(CN)64-電子的轉(zhuǎn)移,導(dǎo)致阻抗逐漸增大。該方法沒(méi)有涉及PCR和任何信號(hào)放大技術(shù),因此不存在和這些技術(shù)相關(guān)的缺陷。同時(shí)還發(fā)現(xiàn),阻抗值與103~105范圍內(nèi)的HeLa細(xì)胞中端粒酶活性呈對(duì)數(shù)線性相關(guān)。由于其簡(jiǎn)單的和方便的操作,在癌癥的臨床診斷方面顯示出潛在的應(yīng)用。

Cunci等[60]利用免標(biāo)記電化學(xué)阻抗法也制備了生物傳感芯片實(shí)現(xiàn)原位檢測(cè)癌細(xì)胞中端粒酶的活性。首先將能與端粒酶特異性結(jié)合的單鏈DNA探針修飾在金電極陣列表面,接著利用EIS實(shí)現(xiàn)端粒酶活性檢測(cè)。

圖5 分別來(lái)自端粒酶陽(yáng)性細(xì)胞提取的熱滅活的陰性(A)、中度(B)和強(qiáng)端粒酶活性(C)的PCR產(chǎn)物的鳥嘌呤氧化信號(hào)示意圖Fig.5 Schematic illustration of guanine oxidation signals of PCR products from(A)heat inactivated negative controls,telomerase positive cell extracts with(B)moderate,and(C)strong telomerase activity

圖6 基于EIS方法的端粒酶活性分析原理圖Fig.6 Illustration for the protocol of telomerase activity analysis based on EIS method

為了進(jìn)一步提高電化學(xué)檢測(cè)的靈敏度,通常會(huì)考慮降低背景或放大信號(hào),因而核酸外切酶III被用來(lái)減少背景信號(hào)[61-62],但蛋白酶本身具有檢測(cè)相對(duì)昂貴和復(fù)雜的缺點(diǎn)。因此,仍然急需合適的方法來(lái)放大信號(hào)。雜交鏈反應(yīng)(Hybridization Chain Reaction,HCR)是一種無(wú)酶、室溫線性放大的方法,它操作簡(jiǎn)單,而且只使用了DNA單鏈,成本效益高[63],兩個(gè)DNA發(fā)夾探針可以穩(wěn)定地共存于雜交溶液中。但引入的引發(fā)劑會(huì)觸發(fā)兩個(gè)探針的交替雜交形成有缺口的雙螺旋。因此,它已被廣泛應(yīng)用在DNA、miRNA、小分子,蛋白質(zhì)和細(xì)胞的放大檢測(cè)[64-67],并已取得了一系列令人滿意的結(jié)果。然而,HCR尚未用于端粒酶的檢測(cè)。對(duì)此,Zhang等[68]開發(fā)了一種基于金納米粒子觸發(fā)模擬HCR來(lái)產(chǎn)生無(wú)酶雙信號(hào)擴(kuò)增以檢測(cè)端粒酶活性的電化學(xué)傳感器。如圖7,在該檢測(cè)方法中,使用了AuNPs和兩個(gè)發(fā)夾探針。AuNPs作為初始放大元件,不僅在電極上與端粒重復(fù)序列雜交以擴(kuò)增信號(hào),而且還啟動(dòng)了隨后的二次擴(kuò)增,利用兩個(gè)發(fā)夾探針模擬HCR[69]。如果細(xì)胞提取物的端粒酶活性呈陽(yáng)性,AuNPs就可以被捕獲到電極上,從而觸發(fā)兩個(gè)發(fā)夾探針產(chǎn)生有缺口雙螺旋交替雜交反應(yīng),導(dǎo)致大量的三氯化六銨合釕通過(guò)靜電相互作用被插入到雙螺旋DNA的長(zhǎng)鏈,從而在合適的電位產(chǎn)生電化學(xué)信號(hào)。用這種方法,不僅可以將檢測(cè)限降低至兩個(gè)HeLa細(xì)胞,能實(shí)現(xiàn)10~10000個(gè)細(xì)胞范圍內(nèi)的端粒酶活性檢測(cè),同時(shí)還能成功地評(píng)估不同細(xì)胞內(nèi)端粒酶的活性。該方法具有較高的靈敏性,而且使用了兩步信號(hào)擴(kuò)增,操作簡(jiǎn)單,可用于臨床細(xì)胞提取物中端粒酶活性的檢測(cè)。

圖7 基于SNAs AuNPs觸發(fā)模擬HCR雙信號(hào)擴(kuò)增的電化學(xué)檢測(cè)端粒酶活性原理圖Fig.7 Schematic illustration of SNAs AuNPs triggered mimic-HCR dual signal amplification electrochemical assay for telomerase activity detection

另一方面,均相電化學(xué)是一種免固定化的方法,其中,探針DNA與靶DNA之間的雜交,以及酶的識(shí)別都發(fā)生在溶液相,而不是在電極的表面上。因此具有簡(jiǎn)單,響應(yīng)快速,識(shí)別效率高的優(yōu)點(diǎn)[70-74]。利用這些優(yōu)點(diǎn),已經(jīng)開發(fā)了許多均相電化學(xué)法用于各種目標(biāo),如DNA,小生物分子和金屬離子的檢測(cè)[71,73-74]。 Li等提出了一種基于T7核酸外切酶輔助目標(biāo)循環(huán)擴(kuò)增的均相電化學(xué)檢測(cè)法,實(shí)現(xiàn)端粒酶活性的簡(jiǎn)單、快速、高靈敏度的測(cè)定[75]。如圖8所示,在此方法中,設(shè)計(jì)了一個(gè)5’端亞甲基藍(lán)(MB)標(biāo)記的發(fā)夾探針,它可以與端粒酶反應(yīng)產(chǎn)物雜交,然后被T7核酸外切酶切割,釋放大量的MB標(biāo)記的單核苷酸以引起電化學(xué)信號(hào)的顯著增強(qiáng)。充分利用了T7啟動(dòng)輔助目標(biāo)循環(huán)的高擴(kuò)增效率優(yōu)勢(shì),該測(cè)定法能夠檢測(cè)單細(xì)胞水平的端粒酶活性,這是優(yōu)于一般檢測(cè)方法的。此外,測(cè)定是在均勻的溶液中進(jìn)行,無(wú)需復(fù)雜的修飾或固定過(guò)程,其具有操作簡(jiǎn)單,反應(yīng)快速和識(shí)別效率高的優(yōu)點(diǎn),這對(duì)基于端粒酶的癌癥早期診斷具有極大的臨床應(yīng)用前景。

圖8 基于T7核酸外切酶輔助目標(biāo)回收擴(kuò)增的電化學(xué)均相分析法檢測(cè)端粒酶活性的原理圖Fig.8 Principle of the homogeneous electrochemical strategy for the detection of telomerase activity based on T7 exonuclease-aided target recycling amplification

2.4 電化學(xué)發(fā)光檢測(cè)法

電化學(xué)發(fā)光(ECL)作為一個(gè)高靈敏度的檢測(cè)技術(shù),集合了電化學(xué)方法與化學(xué)發(fā)光法兩者的優(yōu)點(diǎn),已在藥物分析,臨床診斷,環(huán)境和食品分析,免疫測(cè)定法以及DNA檢測(cè)方面引起了相當(dāng)大的關(guān)注[76-82]。

魯米諾因其電化學(xué)發(fā)光性能在生物傳感檢測(cè)方面應(yīng)用廣泛[83-84],而早期已有報(bào)道顯示納米金能夠增強(qiáng)魯米諾的電化學(xué)發(fā)光強(qiáng)度[85-86]。鑒于魯米諾與納米金結(jié)合形成的復(fù)合物的特殊性質(zhì),Xu等研究了一種可見(jiàn)電化學(xué)發(fā)光分析方法來(lái)分析端粒酶的活性[87]。 利用端粒酶反應(yīng)后生成的G-四聯(lián)體脫氧核酶和魯米諾在微陣列芯片上共同修飾納米金,作為催化放大信號(hào)來(lái)提高魯米諾-雙氧水系統(tǒng)的化學(xué)發(fā)光信號(hào),從而實(shí)現(xiàn)端粒酶活性的多通路檢測(cè)。該方法通過(guò)一種簡(jiǎn)便靈敏的視覺(jué)方式來(lái)達(dá)到檢測(cè)313~10000范圍內(nèi)的HL-60癌細(xì)胞的目的。

此后,Xu等又設(shè)計(jì)了一種新穎的雙電位ECL信號(hào)檢測(cè)方法來(lái)檢測(cè)癌細(xì)胞中端粒酶的活性[88]。首先將CdS量子點(diǎn)修飾在玻碳電極表面,接著將巰基修飾的端粒酶引物經(jīng)由Cd—S作用固定到CdS量子點(diǎn)上,這種引物在端粒酶和dNTPs的存在下可以被延伸,而延伸部分則與魯米諾-納米金標(biāo)記的捕獲DNA發(fā)生雜交,從而導(dǎo)致硫化鎘量子點(diǎn)的ECL發(fā)光強(qiáng)度增強(qiáng),這種增強(qiáng)的發(fā)光強(qiáng)度來(lái)源于兩處,一處是硫化鎘量子點(diǎn)被納米金表面等離子共振誘導(dǎo)產(chǎn)生的-1.25 V,另一處則是來(lái)自于魯米諾的一個(gè)新的電致化學(xué)發(fā)光信號(hào)-0.45 V。這雙電位信號(hào)檢測(cè)方法可以檢測(cè)100~9000 cell范圍內(nèi)的HL-60細(xì)胞提取的端粒酶活性。這種方法不同于ECL比例感測(cè)方法,兩個(gè)激發(fā)電位的ECL強(qiáng)度之比可用于靈敏地檢測(cè)目標(biāo)DNA的濃度[89-90]。

2.5 熒光法

Majerska等[91]設(shè)計(jì)了一種新穎的不需要使用放射性材料和高純度端粒酶樣品的免PCR端粒酶分析法—一種基于等溫循環(huán)鏈置換聚合反應(yīng)的熒光分析法,反應(yīng)原理如圖9。該方法以端粒酶引物在端粒酶存在時(shí)的延伸反應(yīng)、發(fā)夾熒光探針的固有信號(hào)轉(zhuǎn)導(dǎo)機(jī)制和聚合酶的鏈置換特性為基礎(chǔ),采用放大熒光法來(lái)檢測(cè)癌細(xì)胞中端粒酶活性。此處的發(fā)夾熒光探針不僅可以用作熒光信號(hào)載體,還可成為端粒延伸反應(yīng)的模板。當(dāng)端粒酶存在時(shí),發(fā)夾探針的“莖”被打開,從而根據(jù)熒光的增強(qiáng)來(lái)確定端粒酶活性。用此方法可檢測(cè)到HeLa細(xì)胞中的提取的端粒酶活性相當(dāng)于 40~ 1000個(gè)細(xì)胞。打開的探針退火后,引發(fā)聚合反應(yīng),該方法能檢出從細(xì)胞提取的端粒酶活性當(dāng)量下降至4個(gè)HeLa細(xì)胞。這表明目前熒光戰(zhàn)略在癌癥的早期診斷生化分析方面具有巨大潛力。

眾所周知SYBR Gree(SG)是用于DNA染色的敏感熒光染料。利用SG與單、雙鏈DNA的不同相互作用可鑒別單鏈DNA和雙鏈DNA的結(jié)構(gòu)[84]。大量的研究表明,在K+存在時(shí)富含鳥嘌呤的單鏈DNA可以折疊成一個(gè)G-四聯(lián)體結(jié)構(gòu)[92-94]。 也有其他報(bào)告表明,SG可以插入這些G-四聯(lián)體結(jié)構(gòu)中使熒光強(qiáng)度顯著增加[95]。基于上述特點(diǎn),Chung等[96]開發(fā)了一種利用SG將端粒酶活性轉(zhuǎn)換成熒光信號(hào)的免PCR分析法。該法是將TS寡核苷酸引物與腫瘤細(xì)胞中的端粒酶提取物在dNTPs的存在下一起孵育,接著TTAGGG重復(fù)單元在TS引物的3’端連續(xù)合成,以形成一個(gè)更長(zhǎng)的單鏈DNA分子。在K+存在下,被端粒酶延伸的端粒重復(fù)單元能形成G-四聯(lián)體。此時(shí)加入SG會(huì)導(dǎo)致熒光強(qiáng)度的急劇增強(qiáng)。然而,在細(xì)胞提取物中沒(méi)有端粒酶活性或有端粒酶抑制劑的存在下,TS引物是不伸長(zhǎng)的,因此,加入的SG不能使熒光強(qiáng)度發(fā)生變化。

圖9 端粒酶活性的熒光放大分析Fig.9 Fluorescence amplification assay of telomerase activity

近來(lái),隨著納米技術(shù)的不斷成熟,基于納米材料的探針在生物相關(guān)領(lǐng)域的應(yīng)用逐漸增加。由于納米材料特殊的物理和化學(xué)性能,許多熒光檢測(cè)法均采用納米粒子作為熒光探針來(lái)實(shí)現(xiàn)端粒酶的檢測(cè),例如,介孔二氧化硅納米粒子(Mesoporous Silica Nanoparticle,MSN)具有獨(dú)特的孔隙結(jié)構(gòu),生物相容性,且易于功能化等優(yōu)點(diǎn)[97-98]。此外,MSN還擁有較大的孔體積和表面積,可以負(fù)載大量的分子[99]。

圖10 基于MSN探針的細(xì)胞內(nèi)端粒酶分析示意圖Fig.10 Schematic illustration of MSN probe-based intracellular analysis of telomerase

因此,Ju等[100]利用MSN負(fù)載熒光素作為熒光探針實(shí)現(xiàn)細(xì)胞內(nèi)端粒酶活性原位“開-關(guān)”的成像技術(shù)。如圖10,用MSN組裝特殊設(shè)計(jì)的DNA作為探針的生物門。當(dāng)存在端粒酶時(shí),在DNA的3’端原位合成了端粒重復(fù)序列,致使DNA從MSN表面脫離釋放負(fù)載在MSN孔隙中的熒光素,從而觸發(fā)熒光的釋放,實(shí)現(xiàn)了原位檢測(cè)細(xì)胞內(nèi)端粒酶活性的目的。當(dāng)前,大多數(shù)檢測(cè)方法都使用細(xì)胞提取物來(lái)分析端粒酶活性,因此很難在原位檢測(cè)和提供單細(xì)胞水平的端粒酶信息。該方法的問(wèn)世正彌補(bǔ)了這一缺陷。因MSN無(wú)毒、無(wú)干擾、特異性強(qiáng),可以用來(lái)作為細(xì)胞內(nèi)傳遞工具,當(dāng)細(xì)胞吞入MSN后,細(xì)胞內(nèi)端粒酶能激活熒光,通過(guò)觀察信號(hào),即可獲得端粒酶活性的原位軌道,以監(jiān)測(cè)藥物對(duì)端粒酶活性的影響,從而探索出合適的抑制藥物。但是,該方法也存在其固有缺陷,引物DNA的表面吸附易受周圍環(huán)境影響,導(dǎo)致熒光素的非特異性釋放,以產(chǎn)生一定程度的背景。

近來(lái),Ju等[101]又針對(duì)上述不足,設(shè)計(jì)了一個(gè)智能囊泡試劑盒實(shí)現(xiàn)原位成像和檢測(cè)細(xì)胞質(zhì)內(nèi)端粒酶活性,機(jī)理如圖11所示。

圖11 (a)端粒酶觸發(fā)TSP伸長(zhǎng)引起探針的熒光恢復(fù);(b)內(nèi)化囊泡試劑盒對(duì)端粒酶的原位檢測(cè)Fig.11 (a)Telomerase-triggered TSP elongation and following fluorescence recovery of probe.(b)Internalization of vesicle kit for in situ detection of telomerase

試劑盒中含有TS引物和Cy5標(biāo)記的分子信標(biāo)(MB)功能化的納米金[102-106],通過(guò)將其聯(lián)合封裝在脂質(zhì)體內(nèi),從而實(shí)現(xiàn)細(xì)胞內(nèi)遞送[107]。之后,囊泡試劑盒轉(zhuǎn)染到細(xì)胞質(zhì)中,釋放的TS引物可以在端粒酶存在下得到一定的延伸,在DNA的3’末端產(chǎn)生端粒重復(fù)序列,它與組裝在探針表面的MB環(huán)互補(bǔ)。因此,一旦雜交,MB就會(huì)被打開,將熒光狀態(tài)從“關(guān)”切換為“開”。熒光信號(hào)強(qiáng)弱取決于端粒酶活性,從而達(dá)到了原位追蹤細(xì)胞質(zhì)內(nèi)端粒酶活性的目的。在每個(gè)HeLa細(xì)胞,BEL腫瘤細(xì)胞和QSG正常細(xì)胞內(nèi),用該方法評(píng)估得到的細(xì)胞質(zhì)內(nèi)端粒酶活性分別為 3.2×10-11,2.4×10-11,8.6×10-13IU,這充分證明該方法能夠在單細(xì)胞水平區(qū)分腫瘤細(xì)胞和正常細(xì)胞。相較于當(dāng)前存在的其他方法,這種囊泡試劑盒可以通過(guò)“一步孵化”實(shí)現(xiàn)原位檢測(cè),并且,由于納米金的高效率熒光共振能量轉(zhuǎn)移,從而具有較低的熒光背景。因此上述方法是一種成本低廉、操作簡(jiǎn)便、靈敏度高的方法,還可以用于動(dòng)態(tài)監(jiān)測(cè)細(xì)胞質(zhì)內(nèi)端粒酶活性,在癌癥的診斷、治療和端粒酶相關(guān)藥物的發(fā)現(xiàn)與篩選方面成為一種良好的分析工具。

3 結(jié)語(yǔ)

關(guān)于端粒酶活性的檢測(cè)方法還有很多未詳細(xì)列舉出來(lái),例如,Mirkin等開發(fā)出一種基于寡核苷酸功能化的金納米粒子的生物條形碼,以改善端粒酶的檢測(cè)[108]。Willner等使用Zn(II)-卟啉化合物作為熒光團(tuán)結(jié)合到G-四聯(lián)體上,以檢測(cè)端粒酶活性[109]。

然而,上述大多數(shù)策略因?yàn)門S引物的固定化、成本高、靈敏度低和操作復(fù)雜等問(wèn)題在應(yīng)用時(shí)或多或少受到了限制。此外,當(dāng)前的大多數(shù)檢測(cè)方法都使用細(xì)胞提取物來(lái)分析端粒酶活性,于臨床應(yīng)用較為不符,而能夠?qū)崿F(xiàn)原位分析和動(dòng)態(tài)監(jiān)測(cè)細(xì)胞內(nèi)端粒酶活性的檢測(cè)方法則鳳毛麟角。端粒酶活性的檢測(cè)對(duì)癌癥的早期診斷具有重要的生物學(xué)意義,對(duì)臨床上癌癥的預(yù)警和治療這一復(fù)雜科學(xué)問(wèn)題的研究起到促進(jìn)作用。因此,對(duì)端粒酶活性分析的簡(jiǎn)單、靈敏、低成本的原位實(shí)時(shí)監(jiān)測(cè)和抑制劑篩查技術(shù)仍然是當(dāng)前亟待解決的問(wèn)題。

[1]Blackburn E H,Szostak J W.The molecular structure of centromeres and telomeres[J].Annual review of biochemistry,1984,53(1):163-194.

[2]Rodier F,Campisi J.Four faces of cellular senescence [J].The Journal of cell biology,2011,192(4):547-556.

[3]Harley C B,Futcher A B,Greider C W.Telomeres shorten during ageing of human fibroblasts[J].Nature,1990, 345(6274):458-460.

[4]Hastie N D,Dempster M,Dunlop M G,et al.Telomere reduction in human colorectal carcinoma and with ageing [J].Nature,1990,346(6287):866-873.

[5]Counter C M,Avilion A A,LeFeuvre C E,et al.Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity [J].The EMBO Journal,1992,11(5):1921-1929.

[6]Bodnar A G,Ouellette M,Frolkis M,et al.Extension of life-span by introduction of telomerase into normal human cells[J].Science (New York,N.Y.),1998,279 (5349):349-352.

[7]Perry P J,Jenkins T C.Recent advances in the development of telomerase inhibitors for the treatment of cancer [J].Expert Opinion on Investigational Drugs,1999, 8(12):1981-2008.

[8]Ulaner G A,Hu J F,Vu T H,et al.Telomerase activity in human development is regulated by human telomerase reverse transcriptase (hTERT)transcription and by alternate splicing of hTERT transcripts[J].Cancer research, 1998,58(18):4168-4172.

[9]Cech T R.Life at the end of the chromosome:Telomeres and telomerase[J].Angewandte Chemie-International Edition,2000,39(1):34-43.

[10]Holt S E,Wright W E,Shay J W.Regulation of telomerase activity in immortal cell lines[J].Molecular and cellular biology,1996,16(6):2932-2939.

[11]Shay J W,Bacchetti S.A survey of telomerase activity in human cancer[J].European journal of cancer(Oxford, England:1990),1997,33(5):787-791.

[12]Greider C W.Telomerase activity,cell proliferation,and cancer[J].Proceedings of the National Academy of Sciences of the United States of America,1998,95(1):90-102.

[13]Agrawal A,Dang S,Gabrani R.Recent Patents on Anti-Telomerase Cancer Therapy[J].Recent Patents on Anti-Cancer Drug Discovery,2012,7(1):102-117.

[14]Shay J W,Zou Y,Hiyama E,et al.Telomerase and cancer [J].Human Molecular Genetics,2001,10(7):677-685.

[15]Williams S C P.No end in sight for telomerase-targeted cancer drugs[J].Nature Medicine,2013,19(1):6-6.

[16]Zhou X M,Xing D.Assays for human telomerase activity: progress and prospects[J].Chemical Society Reviews, 2012,41(13):4643-4656.

[17]Artandi S E,DePinho R A.Telomeres and telomerase in cancer[J].Carcinogenesis,2010,31(1):9-18.

[18]Cong Y S,Shay J W.Actions of human telomerase beyond telomeres[J].Cell Research,2008,18(7):725-732.

[19]De Cian A,Lacroix L,Douarre C,et al.Targeting telomeres and telomerase[J].Biochimie,2008,90(1):131-155.

[20]Nemos C,Rémy-Martin J P,Adami P,et al.Improved TRAP-silver staining versus conventional radioactive TRAP assays:quantification of telomerase activity during immortalization and in pathological human endometrium [J].Clinical Biochemistry,2003,36(8):621-628.

[21]Wen J M,Sun L B,Zhang M,et al.A non-isotopic method for the detection of telomerase activity in tumour tissues:TRAP-silver staining assay[J].Molecular Pathology,1998,51(2):110-112.

[22]Savoysky E,Akamatsu K,Tsuchiya M,et al.Detection of telomerase activity by combination of TRAP method and scintillation proximity assay(SPA)[J].Nucleic acids research,1996,24(6):1175-1176.

[23]Piatyszek M,Kim N,Weinrich S,et al.Detection of telomerase activity in human cells and tumors by a telomeric repeat amplification protocol(TRAP)[J].Method incell science,1995,17(1):1-15.

[24]Herbert B S,Hochreiter A E,Wright W E,et al.Nonradioactive detection of telomerase activity using the telomeric repeat amplification protocol[J].Nature Protocols, 2006,1(3):1583-1590.

[25]Zuo X L,Xia F,Patterson A,et al.Two-Step,PCR-Free Telomerase Detection by Using Exonuclease III-Aided Target Recycling[J].Chembiochem,2011,12(18):2745-2747.

[26]Krupp G,Kühne K,Tamm S,et al.Molecular basis of artifacts in the detection of telomerase activity and a modified primer for a more robust‘TRAP’assay[J].Nucleic acids research,1997,25(4):919-921.

[27]Herbert B-S, Hochreiter A E,Wright W E, et al. Nonradioactive detection of telomerase activity using the telomeric repeat amplification protocol[J].Nature Protocols,2006,1(3):1583-1590.

[28]Hirose M,Abe-Hashimoto J,Ogura K,et al.A rapid, useful and quantitative method to measure telomerase activity by hybridization protection assay connected with a telomeric repeat amplification protocol[J].Journal of Cancer Research and Clinical Oncology,1997,123(6): 227-344.

[29]Wu Y-Y,Hruszkewycz A M,Delgado R M,et al. Limitations on the quantitative determination of telomerase activity by the electrophoretic and ELISA based TRAP assays[J].Clinica Chimica Acta,2000,293(1-2): 199-212.

[30]Xiao Y, Dane K Y,Uzawa T,et al.Detection of Telomerase Activity in High Concentration of Cell Lysates Using Primer-Modified Gold Nanoparticles[J]. Journal of the American Chemical Society,2010,132 (43):15299-15307.

[31]Eskiocak U,Ozkan-Ariksoysal D,Ozsoz M,et al.Labelfree detection of telomerase activity using guanine electrochemical oxidation signal[J].Analytical chemistry, 2007,79(22):8807-8811.

[32]Takata M,Kerman K,Nagatani N,et al.Label-free bioelectronic immunoassay for the detection of human telomerase reverse transcriptase in urine[J].Journal of Electroanalytical Chemistry,2006,596(2):109-116.

[33]Wu L,Wang J,Ren J,et al.Ultrasensitive Telomerase Activity Detection in Circulating Tumor Cells Based on DNA Metallization and Sharp Solid-State Electrochemical Techniques[J].Advanced FunctionalMaterials, 2014,24(18):2727-2733.

[34]Sharon E,Freeman R,Riskin M,et al.Optical,Electrical and Surface Plasmon Resonance Methods for Detecting Telomerase Activity[J].Analytical Chemistry,2010,82 (20):8390-8397.

[35]Tian L,Weizmann Y.Real-Time Detection of Telomerase Activity Using the Exponential Isothermal Amplification of Telomere Repeat Assay[J].Journal of the American Chemical Society,2013,135(5):1661-1664.

[36]Liu J,Lu Y.Preparation of aptamer-linked gold nanoparticle purple aggregates for colorimetric sensing of analytes[J].Nature Protocols,2006,1(1):246-252.

[37]Zhang X,Servos M R,Liu J.Instantaneous and quantitative functionalization of gold nanoparticles with thiolated DNA using a pH-assisted and surfactant-free route[J].Journal of the American Chemical Society, 2012,134(17):7266-7269.

[38]Li H,Huang J,Lv J,et al.Nanoparticle PCR:Nanogold-Assisted PCR with Enhanced Specificity [J]. Angewandte Chemie International Edition,2005,44(32): 5100-5103.

[39]Shen H,Hu M,Yang Z,et al.Polymerase chain reaction of Au nanoparticle-bound primers[J].Chinese Science Bulletin,2005,50(18):2344-2350.

[40]Sharon E,Golub E,Niazov-Elkan A,et al.Analysis of telomerase by the telomeric hemin/G-quadruplexcontrolled aggregation of au nanoparticles in the presence of cysteine[J].Analytical Chemistry,2014,86(6):3153-3158.

[41]Freeman R,Sharon E,Teller C,et al.DNAzyme-Like Activity of Hemin-Telomeric G-Quadruplexes for the Optical Analysis of Telomerase and its Inhibitors[J]. Chembiochem,2010,11(17):2362-2367.

[42]Golub E,Freeman R,Willner I.Hemin/G-Quadruplex-Catalyzed Aerobic Oxidation of Thiols to Disulfides: Application of the Process for the Development of Sensors and Aptasensors and for Probing Acetylcholine Esterase Activity[J].Analytical Chemistry,2013,85 (24):12126-12133.

[43]Duan R,Wang B,Zhang T,et al.Sensitive and Bidirectional Detection of Urine Telomerase Based on the Four Detection-ColorStatesofDifunctionalGold Nanoparticle Probe[J].Analytical Chemistry,2014,86 (19):9781-9785.

[44]Tian L,Weizmann Y.Real-time detection of telomerase activity using the exponential isothermal amplification of telomere repeat assay[J].Journal of the American Chemical Society,2013,135(5):1661-1665.

[45]Yuefei W,Renliang H,Wei Q,et al.Kineticallycontrolled self-assembly of redox-active ferrocenediphenylalanine:from nanospheres to nanofibers[J]. Nanotechnology,2013,24(46):465603.

[46]Connolly A R,Trau M.Isothermal detection of DNA by beacon-assisted detection amplification[J].Angewandte Chemie,2010,49(15):2697-2863.

[47]Guo Q,Yang X,Wang K,et al.Sensitive fluorescence detection of nucleic acids based on isothermal circular strand-displacement polymerization reaction[J].Nucleic Acids Research,2009,37(3):1-6.

[48]Jia H,Li Z,Liu C,et al.Ultrasensitive Detection of microRNAs by Exponential Isothermal Amplification[J]. Angewandte Chemie International Edition,2010,49(32): 5498-5501.

[49]Van Ness J,Van Ness L K,Galas D J.Isothermal reactions for the amplification of oligonucleotides[J].Procceedings of the National Academy of Sciences of the U-nited States of America,2003,100(8):4504-4509.

[50]Zhang Y,Hu J,Zhang C-y.Sensitive Detection of Transcription Factors by Isothermal Exponential Amplification-Based Colorimetric Assay[J].Analytical Chemistry, 2012,84(21):9544-9549.

[51]Hou T,Liu X,Wang X,et al.DNAzyme-guided polymerization of aniline for ultrasensitive electrochemical detection of nucleic acid with bio-bar codes-initiated rolling circle amplification[J].Sensors and Actuators B:Chemical,2014,190:384-388.

[52]Liu X,Chen M,Hou T,et al.A novel electrochemical biosensor for label-free detection of uracil DNA glycosylase activity based on enzyme-catalyzed removal of uracil bases inducing strand release[J].Electrochimica Acta,2013,113:514-518.

[53]Sato S,Kondo H,Nojima T,et al.Electrochemical Telomerase Assay with Ferrocenylnaphthalene Diimide as a Tetraplex DNA-Specific Binder[J].Analytical Chemistry,2005,77(22):7304-7309.

[54]Eskiocak U,Ozkan-Ariksoysal D,Ozsoz M,et al.Label-Free Detection of Telomerase Activity Using Guanine Electrochemical Oxidation Signal[J].Analytical Chemistry,2007,79(22):8807-8811.

[55]Gao Z,Deng H,Shen W,et al.A Label-Free Biosensor for Electrochemical Detection of Femtomolar MicroRNAs [J].Analytical Chemistry,2013,85(3):1624-1630.

[56]Bonanni A,Pumera M.Graphene Platform for Hairpin-DNA-Based Impedimetric Genosensing[J].ACS Nano, 2011,5(3):2356-2361.

[57]Guo S,Wen D,Zhai Y,et al.Platinum Nanoparticle Ensemble-on-Graphene Hybrid Nanosheet:One-Pot, Rapid Synthesis,and Used as New Electrode Material for Electrochemical Sensing[J].ACS Nano,2010,4(7): 3959-3968.

[58]Basuray S,Senapati S,Aijian A,et al.Shear and AC Field Enhanced Carbon Nanotube Impedance Assay for Rapid,Sensitive,and Mismatch-Discriminating DNA Hybridization[J].ACS Nano,2009,3(7):1823-1830.

[59]Yang W,Zhu X,Liu Q,et al.Label-free detection of telomerase activity in HeLa cells using electrochemical impedance spectroscopy[J].Chemical Communications, 2011,47(11):3129-3131.

[60]Cunci L,Vargas M M,Cunci R,et al.Real-Time Detection of Telomerase Activity in Cancer Cells using a Label-Free Electrochemical Impedimetric Biosensing Microchip[J].RSC Advances,2014,4(94):52357-52365.

[61]Wu L,Wang J,Ren J,et al.Ultrasensitive Telomerase Activity Detection in Circulating Tumor Cells Based on DNA Metallization and Sharp Solid-State Electrochemical Techniques[J].Advanced Functional Materials, 2014,24(18):2727-2733.

[62]Zhang Z,Wu L,Wang J,et al.A Pt-nanoparticle electrocatalytic assay used for PCR-free sensitive telomerase detection[J].Chemical Communications,2013,49(85): 9986-9988.

[63]Dirks R M,Pierce N A.Triggered amplification by hybridization chain reaction[J].Procceedings of the National Academy of Sciences of the United States of America,2004,101(43):15275-15278.

[64]Huang J,Wu Y,Chen Y,et al.Pyrene-Excimer Probes Based on the Hybridization Chain Reaction for the Detection of Nucleic Acids in Complex Biological Fluids [J].Angewandte Chemie International Edition,2011,50 (2):401-404.

[65]Huang J,Gao X,Jia J,et al.Graphene Oxide-Based Amplified Fluorescent Biosensor for Hg2+Detection through Hybridization Chain Reactions[J].Analytical Chemistry, 2014,86(6):3209-3215.

[66]Zhang B,Liu B,Tang D,et al.DNA-Based Hybridization Chain Reaction for Amplified Bioelectronic Signal and Ultrasensitive Detection ofProteins[J].Analytical Chemistry,2012,84(12):5392-5399.

[67]Choi J,Routenberg Love K,Gong Y,et al.Immuno-Hybridization Chain Reaction for Enhancing Detection of Individual Cytokine-Secreting Human Peripheral Mononuclear Cells[J].Analytical Chemistry,2011,83 (17):6890-6895.

[68]Wang W J,Li J J,Rui K,et al.Sensitive electrochemical detection of telomerase activity using spherical nucleic acids gold nanoparticles triggered mimic-hybridization chain reaction enzyme-free dual signal amplification[J]. Analytical Chemistry,2015,87(5):3019-3026.

[69]Zhang J,Song S,Zhang L,et al.Sequence-Specific Detection of Femtomolar DNA via a Chronocoulometric DNA Sensor(CDS):Effects of Nanoparticle-Mediated Amplification and Nanoscale Control of DNA Assembly at Electrodes[J].Journal of the American Chemical Society,2006,128(26):8575-8580.

[70]Liu S,Lin Y,Wang L,et al.Exonuclease III-Aided Autocatalytic DNA Biosensing Platform for Immobilization-Free and Ultrasensitive Electrochemical Detection of Nucleic Acid and Protein[J].Analytical Chemistry, 2014,86(8):4008-4015.

[71]Luo X,Lee T M-H,Hsing I M.Immobilization-Free Sequence-Specific Electrochemical Detection of DNA Using Ferrocene-Labeled Peptide Nucleic Acid[J].Analytical Chemistry,2008,80(19):7341-7346.

[72]Wang X,Liu X,Hou T,et al.Highly sensitive homogeneous electrochemical assay for methyltransferase activity based on methylation-responsive exonuclease III-assisted signal amplification[J].Sensors and Actuators B: Chemical,2015,208:575-580.

[73]Xuan F,Luo X,Hsing I M.Ultrasensitive Solution-Phase Electrochemical Molecular Beacon-Based DNA Detection with Signal Amplification by Exonuclease III-Assisted Target Recycling[J].Analytical Chemistry,2012, 84(12):5216-5220.

[74]Xuan F,Luo X,Hsing I M.Conformation-Dependent Exonuclease III Activity Mediated by Metal Ions Reshuffling on Thymine-Rich DNA Duplexes for an Ultrasensitive ElectrochemicalMethod for Hg2+Detection[J]. Analytical Chemistry,2013,85(9):4586-4593.

[75]Liu X,Li W,Hou T,et al.Homogeneous electrochemical strategy for human telomerase activity assay at singlecell level based on t7 exonuclease-aided target recycling amplification[J].Analytical Chemistry,2015,87(7): 4030-4036.

[76]Richter M M.Electrochemiluminescence (ECL)[J]. Chemical Reviews,2004,104(6):3003-3036.

[77]Miao W J.Electrogenerated chemiluminescence and its biorelated applications[J].Chemical Reviews,2008,108 (7):2506-2553.

[78]Zhang H-R,Xu J-J,Chen H-Y.Electrochemiluminescence Ratiometry:A New Approach to DNA Biosensing [J].Analytical Chemistry,2013,85(11):5321-5325.

[79]Zhou H,Zhang Y Y,Liu J,et al.Efficient quenching of electrochemiluminescence from K-doped graphene-CdS: Eu NCs by G-quadruplex-hemin and target recyclingassisted amplification for ultrasensitive DNA biosensing [J].Chemical Communications,2013,49(22):2246-2258.

[80]Zhang H-R,Xia X-H,Xu J-J,et al.Sensitive cancer cell detection based on Au nanoparticles enhanced electrochemiluminescence of CdS nanocrystal film supplemented by magnetic separation[J].Electrochemistry Communications,2012,25:112-115.

[81]Tian C Y,Xu J J,Chen H Y.A novel aptasensor for the detection of adenosine in cancer cells by electrochemiluminescence of nitrogen doped TiO2nanotubes[J].Chemical Communications,2012,48(66):8234-8236.

[82]Wang J,Shan Y,Zhao W-W,et al.Gold Nanoparticle Enhanced Electrochemiluminescence of CdS Thin Films for Ultrasensitive Thrombin Detection[J].Analytical Chemistry,2011,83(11):4004-4011.

[83]Rose A L,Waite T D.Chemiluminescence of Luminol in the Presence of Iron(II)and Oxygen:Oxidation Mechanism and Implications for Its Analytical Use[J].Analytical Chemistry,2001,73(24):5909-5920.

[84]Chai Y,Tian D,Wang W,et al.A novel electrochemiluminescence strategy for ultrasensitive DNA assay using luminol functionalized gold nanoparticles multi-labeling and amplification of gold nanoparticles and biotin-streptavidin system[J].Chemical Communications,2010,46 (40):7560-7562.

[85]Cui H,Zou G-Z,Lin X-Q.Electrochemiluminescence of Luminol in Alkaline Solution at a Paraffin-Impregnated Graphite Electrode[J].Analytical Chemistry,2003,75 (2):324-331.

[86]Tian D,Duan C,Wang W,et al.Ultrasensitive electrochemiluminescence immunosensor based on luminol functionalized gold nanoparticle labeling[J].Biosensors and Bioelectronics,2010,25(10):2290-2295.

[87]Zhang H R,Wang Y Z,Wu M S,et al.Visual electrochemiluminescence detection oftelomerase activity based on multifunctional Au nanoparticles modified with G-quadruplex deoxyribozyme and luminol[J].Chemical Communications,2014,50(83):12575-12579.

[88]Zhang H R,Wu M S,Xu J J,et al.Signal-on dual-potential electrochemiluminescence based on luminol-gold bifunctional nanoparticles for telomerase detection[J]. Analytical Chemistry,2014,86(8):3834-3840.

[89]Branchini B R,Rosenberg J C,Ablamsky D M,et al.Sequential bioluminescence resonance energy transferfluorescence resonance energy transfer-based ratiometric protease assays with fusion proteins of firefly luciferase and red fluorescent protein[J].Analytical Biochemistry, 2011,414(2):239-245.

[90]Takeuchi M,Nagaoka Y,Yamada T,et al.Ratiometric Bioluminescence Indicators for Monitoring Cyclic Adenosine 3′,5′-Monophosphate in Live Cells Based on Luciferase-Fragment Complementation[J].Analytical Chemistry,2010,82(22):9306-9313.

[91]Majerska J,Sykorova E,Fajkus J.Non-telomeric activities of telomerase[J].Molecular Biosystems,2011,7(4): 1013-1023.

[92]Hu K,Huang Y,Zhao S,et al.Ultrasensitive detection of potassium ions based on target induced DNA conformational switch enhanced fluorescence polarization[J].Analyst,2012,137(12):2770-2773.

[93]Qin H,Ren J,Wang J,et al.G-Quadruplex-Modulated Fluorescence Detection of Potassium in the Presence of a 3500-Fold Excess of Sodium Ions[J].Analytical Chemistry,2010,82(19):8356-8360.

[94]Huang C-C,Chang H-T.Aptamer-based fluorescence sensor for rapid detection of potassium ions in urine[J]. Chemical Communications,2008,12:1461-1463.

[95]Xu H,Gao S,Yang Q,et al.Amplified Fluorescent Recognition of G-Quadruplex Folding with a Cationic Conjugated Polymer and DNA Intercalator[J].ACS Applied Materials&Interfaces,2010,2(11):3211-3216.

[96]Quach Q H,Jung J,Kim H,et al.A simple,fast and highly sensitive assay for the detection of telomerase activity [J].Chemical Communications,2013,49(59):6596-6598.

[97]Climent E,Martínez-Má?ez R,Sancenón F,et al.Controlled Delivery Using Oligonucleotide-Capped Mesoporous Silica Nanoparticles[J].Angewandte Chemie, 2010,122(40):7439-7441.

[98]Niu D,Ma Z,Li Y,et al.Synthesis of Core-Shell Structured Dual-Mesoporous Silica Spheres with Tunable Pore Size and Controllable Shell Thickness[J].Journal of the American Chemical Society,2010,132(43):15144-15147.

[99]Slowing I,Trewyn B G,Lin V S Y.Effect of Surface Functionalization of MCM-41-Type Mesoporous Silica Nanoparticles on the Endocytosis by Human Cancer Cells[J].Journal of the American Chemical Society, 2006,128(46):14792-14793.

[100]Qian R,Ding L,Ju H.Switchable fluorescent imaging of intracellular telomerase activity using telomerase-responsive mesoporous silica nanoparticle[J].Journal of the American Chemical Society,2013,135(36):13282-13285.

[101]Qian R,Ding L,Yan L,et al.Smart vesicle kit for in situ monitoring of intracellular telomerase activity using a telomerase-responsive probe[J].Analitical Chemistry, 2014,86(17):8642-8648.

[102]Llevot A,Astruc D.Applications of vectorized gold nanoparticles to the diagnosis and therapy of cancer [J].Chemical Society Reviews,2012,41(1):242-257.

[103]Duncan B,Kim C,Rotello V M.Gold nanoparticle platforms as drug and biomacromolecule delivery systems [J].Journal of Controlled Release,2010,148(1):122-127.

[104]Li F,Zhang H,Dever B,et al.Thermal Stability of DNA Functionalized Gold Nanoparticles[J].Bioconjugate Chemistry,2013,24(11):1790-1797.

[105]Giljohann D A,Seferos D S,Patel P C,et al.Oligonucleotide Loading Determines Cellular Uptake of DNAModified Gold Nanoparticles[J].Nano Letters,2007,7 (12):3818-3821.

[106]Seferos D S,Giljohann D A,Hill H D,et al.Nano-Flares:Probes for Transfection and mRNA Detection in Living Cells[J].Journal of the American Chemical Society,2007,129(50):15477-15479.

[107]Dalby B,Cates S,Harris A,et al.Advanced transfection with Lipofectamine 2000 reagent:primary neurons, siRNA,and high-throughput applications[J].Methods, 2004,33(2):95-103.

[108]Zheng G,Daniel W L,Mirkin C A.A New Approach to Amplified Telomerase Detection with Polyvalent Oligonucleotide Nanoparticle Conjugates[J].Journal of the American Chemical Society,2008,130(30):9644-9645.

[109]Zhang Z,Sharon E,Freeman R,et al.Fluorescence Detection of DNA,Adenosine-5′-Triphosphate(ATP), and Telomerase Activity by Zinc(II)-Protoporphyrin IX/ G-Quadruplex Labels[J].Analytical Chemistry,2012, 84(11):4789-4797.

Recent research on monitoring telomerase activity

Guo Lin-yan,Yang Ming-hui*
(College of Chemistry and Chemical Engineering,Central South University,Changsha 410083,China)

Human telomerase is a ribonucleoprotein complex,which functions as a telomere terminal transferase by catalytic adding telomere repeat TTAGGG to the end of chromosome using its RNA as the template.Due to a very strong association between telomerase activity and malignancy in nearly all types of cancer,telomerase plays a key role in the tumor occurrence as well as development.Monitoring telomerase activity is believed to be important for cancer diagnosis,prediction and provides an important basis for clinical treatment.This review summarizes recent development for detection of telomerase activity,with emphasis placed on in situ detection.

telomerase;G-quadruplex;in situ;analysis detection

*通信聯(lián)系人,E-mail:yangminghui@csu.edu.cn,Tel:0731-88836356

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