石墨烯及其衍生物在生化傳感器中的作用

王 鵬，賈蕓芳，徐紅梅，王中榮，范清杰*

（1.天津市蘭力科化學(xué)電子高技術(shù)有限公司，天津300384）（2.南開大學(xué)電子信息與光學(xué)工程學(xué)院，天津300071）

0 引言

石墨烯是一種二維碳材料，與零維碳材料-富勒烯[1]、一維碳納米管[2]、三維碳材料(金剛石、石墨)一起被統(tǒng)稱為碳的同素異形體。與一維和三維碳材料相比，石墨烯不僅出現(xiàn)得晚，而且其存在性曾是歷史上備受爭議的話題[3-5]，直到2004年，Geim和Novoselov采用機(jī)械剝離方法發(fā)現(xiàn)了穩(wěn)定、獨(dú)立存在的單層石墨烯[6]，石墨烯的存在才真正得到認(rèn)可。它的存在被解釋為：從三維結(jié)構(gòu)中剝離出來的二維結(jié)構(gòu)，由于石墨烯表面具有輕微的褶皺，使其具有自發(fā)的穩(wěn)定性；石墨烯在三維方向上的彎曲(側(cè)面觀察大約10 nm)導(dǎo)致彈性能增加的同時(shí)抑制了熱振動(dòng)，當(dāng)處于較高溫度時(shí)，可以使自由能降到最低限，從而使其能夠自由、穩(wěn)定地存在[7-8]。

石墨烯目前已在高速器件[9-12]、儲(chǔ)能器件[13-14]、柔性器件[15-16]、生化傳感器[17]等多方面開展了應(yīng)用研究，表現(xiàn)出很好的前景；該文主要結(jié)合生化傳感器發(fā)展趨勢，對石墨烯及其衍生物在生化傳感器中的作用進(jìn)行綜述分析。

1 生化傳感器的簡介

生化傳感器是指能夠響應(yīng)生物、化學(xué)量，并按一定規(guī)律將其轉(zhuǎn)換成可用信號(光信號、電信號等)的器件或裝置。在結(jié)構(gòu)上主要由兩部分組成：1）感受器，通常為含有生化活性物質(zhì)、具有特異性識(shí)別能力的敏感膜；2）轉(zhuǎn)換器，將敏感膜感知的生化信息轉(zhuǎn)換為可測量的光信號或電信號[18]。提高生化傳感器靈敏性的研究主要集中在感受器、轉(zhuǎn)換器以及二者銜接三個(gè)方面。

納米材料及其與導(dǎo)電聚合物、生物探針相結(jié)合是感受器研究的主流趨勢，如：TiO2[19]、Co3O4[20]、CuO[21]和Cu2O[22]等納米金屬氧化物作為感受器，可提高對H2O2的靈敏度；導(dǎo)電聚合物與碳納米管結(jié)合可提高氨氣的靈敏度[23]，導(dǎo)電聚合物與石墨烯結(jié)合，可提高對DNA的靈敏度[24]；單鏈DNA與納米金粒子結(jié)合[25]，酶和碳納米管結(jié)合[26]，適配子與石墨烯相結(jié)合[27]，分別可提高對互補(bǔ)DNA、葡萄糖以及待測細(xì)胞的靈敏度。

利用新型電子材料來提高轉(zhuǎn)換器的信號強(qiáng)度則是轉(zhuǎn)換器研究的方向，如，碳納米管、石墨烯為溝道材料的場效應(yīng)晶體管 （Field Effect Transistor，F(xiàn)ET），可提高對DNA[28-29]、蛋白質(zhì)[30-31]以及葡萄糖[32-33]的響應(yīng)強(qiáng)度；在熒光共振能量轉(zhuǎn)移(fluorescence resonance energy transfer，F(xiàn)RET)型生化傳感器中，采用量子點(diǎn)取代熒光標(biāo)記物，可實(shí)現(xiàn)將H2O2[34]和氰離子[35]濃度等生化信息轉(zhuǎn)化為熒光信號，通過對熒光強(qiáng)度的測量實(shí)現(xiàn)定量檢測。

在構(gòu)建感受器與轉(zhuǎn)換器間敏感界面的研究中，則主要圍繞促進(jìn)感受器與轉(zhuǎn)換器之間電荷傳輸速率和提高轉(zhuǎn)換器表面感受器的固定效率兩方面展開。CNTs、碳量子點(diǎn)、納米碳纖維、納米金剛石、富勒烯和石墨烯等新型碳材料可作為探針載體，提高探針在玻碳電極 （Glassy Carbon Electrode，GCE）上的固定效率[36]；應(yīng)用石墨烯、CNTs可以實(shí)現(xiàn)促進(jìn)葡萄糖氧化酶活性中心[37-38]，DNA探針[39-40]與GCE之間的電子傳遞。

由此可見，促進(jìn)感受器與轉(zhuǎn)換器之間的電荷傳輸、提高感受器在轉(zhuǎn)換器上的固定效率、提高轉(zhuǎn)換器的信號強(qiáng)度是生化傳感器發(fā)展的重要需求，新型電子材料石墨烯的出現(xiàn)，使同時(shí)滿足生化傳感器的這三個(gè)需求成為可能。

2 石墨烯結(jié)構(gòu)與電學(xué)特性

石墨烯具有獨(dú)特的結(jié)構(gòu)和優(yōu)良的電學(xué)特性，為改善生化傳感器靈敏性奠定了優(yōu)良的物質(zhì)基礎(chǔ)。

單層石墨烯由碳原子依靠共價(jià)鍵相互連接形成完美的六邊形結(jié)構(gòu)，其厚度僅有0.335 nm，約為頭發(fā)半徑的20萬分之一，是目前已知最薄的晶體材料；具有較高的比表面積[41]，理論上可達(dá)到2630 m2/g，即使在實(shí)際制備過程中發(fā)生團(tuán)聚或產(chǎn)生結(jié)構(gòu)缺陷，石墨烯的比表面積也約為700 m2/g，仍遠(yuǎn)高于大部分納米材料，比如：碳納米管[42]以及Al2O3和CeO2等金屬納米粒子[43]；這種高比表面積的特性，使其更容易與其他材料充分接觸，對各種原子或者分子具有較強(qiáng)的吸附能力[44]。

石墨烯中每個(gè)碳原子有4個(gè)價(jià)電子，其中3個(gè)價(jià)電子以sp2雜化方式形成σ鍵，最后一個(gè)p軌道價(jià)電子形成π鍵，π鍵電子是可自由移動(dòng)的載流子，濃度約為1013cm-2，石墨烯具有極低的電阻率，約為 1.59×10-6Ω·cm[45]，載流子遷移率約為1.5×105cm2/V·s[46]；石墨烯是一種零帶隙半導(dǎo)體或者半金屬，三維能帶結(jié)構(gòu)如圖1所示，其中導(dǎo)帶和價(jià)帶相交于狄拉克點(diǎn)，研究顯示狄拉克點(diǎn)的位置可被外加偏壓調(diào)節(jié)，表現(xiàn)出雙極特性[47]。

圖1 石墨烯在三維狀態(tài)下的能帶結(jié)構(gòu)圖[47]Fig.1 The energy band structure of graphene in three dimensions

GO攜帶大量含氧基團(tuán)，分子空間結(jié)構(gòu)如圖2所示。根據(jù)Lerf-Klinowski模型[48]和Rourke-Wilson模型[49]，GO具有與石墨烯相似的片層結(jié)構(gòu)，GO的片層內(nèi)部，仍保留了石墨烯完美的六邊形結(jié)構(gòu)，這使得GO具有一定的導(dǎo)電能力，GO片層之間存在通過π-π鍵共軛作用相互粘附，如圖2b所示[50]，這說明GO片層之間仍存在載流子輸運(yùn)通道；更重要的是，GO片層邊緣存在大量含氧基團(tuán)，如羥基（-COOH）和環(huán)氧基（-O=O-），為生物功能化提供了活性位點(diǎn)。

圖2 GO 結(jié)構(gòu)的 Lerf-Klinowski模型[48]（a）和 Rourke-Wilson 模型[49]（b）Fig.2 Lerf-Klinowski model(a)and Rourke-Wilson model(b)of GO structure

從GO的結(jié)構(gòu)可見，GO上含氧基團(tuán)為功能基團(tuán)（如ssDNA、抗體等）的固定提供了活性位點(diǎn)。例如，當(dāng)功能基團(tuán)存在氨基（-NH2）時(shí)，可與GO邊緣-COOH形成共價(jià)鍵，從而將功能基團(tuán)嫁接在GO上。但是，由于含氧基團(tuán)的引入，π鍵電子減少，從而使GO表現(xiàn)出較大的表面電阻Rs（與本征石墨烯相比）[51]，這不利于GO表面功能基團(tuán)與GO片層之間的載流子傳輸，因而不利于增強(qiáng)傳感器輸出信號。還原GO（reduced GO，rGO）可使GO上部分含氧基團(tuán)脫落，rGO的Rs可以下降幾個(gè)到十幾個(gè)數(shù)量級[52]，但是，rGO上仍有殘留含氧基團(tuán)和結(jié)構(gòu)缺陷，使其Rs仍明顯高于石墨烯[53]。

由此可見，rGO的石墨烯形態(tài)，既保證了功能基團(tuán)在石墨烯傳感器表面的固定，又能使功能基團(tuán)與GO之間載流子輸運(yùn)具有較低的電阻。

3 石墨烯在生化傳感器中的作用

3.1 增強(qiáng)生物探針與轉(zhuǎn)換器之間的電子傳輸

采用循環(huán)伏安法和交流阻抗法，對比分析聚苯胺、還原氧化石墨烯 (reduced graphene oxide，rGO)和DNA探針修飾的GCE型DNA傳感器，結(jié)果表明修飾rGO的GCE響應(yīng)電流大、阻抗小，證明了rGO具有促進(jìn)DNA探針與電極之間電子傳輸?shù)淖饔肹54]。rGO、石墨修飾和空GCE三種情況的對比實(shí)驗(yàn)顯示[55]，rGO修飾的GCE對不同堿基均具有最大的響應(yīng)電流，證明了rGO有助于加快電極與DNA堿基的電子轉(zhuǎn)移。

蛋白質(zhì)型生化傳感器中氧化還原蛋白的電子轉(zhuǎn)移是生化響應(yīng)的核心，石墨烯對氧化還原蛋白具有不可逆的吸附性能以及良好的電子傳遞能力，是促進(jìn)蛋白質(zhì)電子轉(zhuǎn)移的理想材料[56]。GO對蛋白質(zhì)電荷轉(zhuǎn)移性能影響的研究顯示[57]，只有GO與蛋白質(zhì)混合物修飾的GCE才可以清晰地觀察到氧化還原峰，而未加入GO的GCE則相對較小。在殼聚糖GCE型血紅蛋白質(zhì)傳感器研究中發(fā)現(xiàn)，石墨烯具有促進(jìn)蛋白質(zhì)與電極間的電子轉(zhuǎn)移的作用[58]；基于rGO和ZnO雜化物的谷胱甘肽生化傳感器研究顯示[59]，rGO可促進(jìn)谷胱甘肽與電極間電荷傳輸，使得rGO修飾的電極光電流成倍增長。

葡萄糖電化學(xué)傳感器中，利用石墨烯特殊的電子傳輸性質(zhì)可實(shí)現(xiàn)葡萄糖氧化酶與電極之間的直接電子轉(zhuǎn)移。例如，GO修飾的Pt電極可促進(jìn)葡萄糖氧化酶與電極間電子轉(zhuǎn)移[60]；對石墨烯和納米銀復(fù)合物修飾的葡萄糖氧化酶GCE研究表明[61]，石墨烯的加入可促進(jìn)葡萄糖氧化酶和GCE間的電荷轉(zhuǎn)移；同時(shí)，采用滴涂法制備石墨烯-葡萄糖氧化酶GCE傳感器，也顯示增強(qiáng)氧化還原峰的特性，實(shí)驗(yàn)計(jì)算顯示葡萄糖氧化酶與電極間的電子轉(zhuǎn)移速率約為2.68 s-1[62]。

3.2 增強(qiáng)生物探針固定量

由于石墨烯具有大比表面積，通過石墨烯的修飾的電極可以獲得更高的比表面積，進(jìn)而提高探針在其表面的負(fù)載量[63]。Lin等[64]通過石墨烯與DNA之間的π-π鍵相互作用，實(shí)現(xiàn)GCE表面DNA功能化，有效地提高了電極表面DNA探針的固定量。利用石墨烯和免疫功能化的納米碳球，基于雙重信號放大策略制備了用于甲胎蛋白(Alpha fetal protein，AFP)檢測的免疫傳感器[65]，由于石墨烯的大比表面積有效提高了抗體在傳感器表面的固定量，與未修飾石墨烯的免疫傳感器進(jìn)行對比，該免疫傳感器的檢測信號強(qiáng)度增大了7倍。

殼聚糖、葡萄糖氧化酶/殼聚糖、殼聚糖/石墨烯、葡萄糖氧化酶/殼聚糖/石墨烯混合物修飾GCE的電化學(xué)研究顯示，含有石墨烯的混合物修飾電極的響應(yīng)電流較大，葡萄糖氧化酶在含有石墨烯的GCE表面的負(fù)載量可達(dá)到1.12×10-9mol/cm2[66]。

Fe2O3和rGO修飾GCE的亞硝酸鹽生化傳感器[67]，與 MnO2、Au/ZnO/MWCNTs 等材料修飾的GCE對比顯示，由于石墨烯提高電極表面Fe2O3負(fù)載量，其對亞硝酸鹽具有催化活性，從而改善了傳感器對亞硝酸鹽檢測靈敏性，使得該傳感器對亞硝酸鹽檢測響應(yīng)電流明顯較大。

GO上羧基基團(tuán)與DNA探針3'末端的氨基發(fā)生酰胺反應(yīng)，將DNA探針在光尋址電位傳感器 （LightAddressable Potentiometric Sensor，LAPS），表面，制備出用于DNA檢測的石墨烯-LAPS(graphene modified LAPS，G-LAPS)生化傳感器[68]。該傳感器對互補(bǔ)DNA的響應(yīng)電流明顯大于未修飾GO的LAPS傳感器，結(jié)合XPS表面分析表明GO的修飾作用，增加了LAPS表面DNA探針的固定量，從而增強(qiáng)了LAPS對DNA的響應(yīng)強(qiáng)度。

羧基化GO（GO-COOH）對GCE修飾作用的研究也表明[69]，GO-COOH中-COOH基團(tuán)可以以共價(jià)鍵方式連接ssDNA探針，增加了GCE上DNA探針固定量，與未修飾GO-COOH的GCE相比，其交流阻抗譜曲線半徑明顯減小。

3.3 直接作為轉(zhuǎn)換器

石墨烯及其衍生物作為轉(zhuǎn)換器，主要是將被測物信息轉(zhuǎn)換為可測量的光信號或電信號，作為轉(zhuǎn)換器典型的應(yīng)用主要在FRET型和石墨烯場效應(yīng)晶體管(graphene field effect transistor，GFET)型生化傳感器中。

FRET型石墨烯生化傳感器是以GO熒光特性為基礎(chǔ)。在GO制備過程中，其表面和邊沿?cái)y帶大量含氧基團(tuán)，破壞了石墨烯的大π鍵，使其具有光致發(fā)光的特性，可在較寬的范圍內(nèi)(近紅外到紫外)發(fā)出熒光[70-71]。GO作為能量受體[72]，通過π-π作用與其接觸的熒光基團(tuán)發(fā)生能量轉(zhuǎn)移導(dǎo)致熒光猝滅，實(shí)現(xiàn)生化量與光學(xué)量的轉(zhuǎn)換。

基于GO熒光特性結(jié)構(gòu)的多巴胺生化傳感器[73]，多巴胺以π-π鍵吸附到GO表面，使GO熒光猝滅，且隨著多巴胺濃度逐漸增大，GO熒光強(qiáng)度降低?；贕O熒光特性的輪狀病毒檢測的FRET免疫傳感器[74]，通過在GO表面修飾輪狀病毒的一級抗體，用于捕獲感染了輪狀病毒的細(xì)胞，用修飾納米金的二級抗體與輪狀病毒再次結(jié)合形成三明治結(jié)構(gòu)，納米金可以猝滅GO熒光，從而實(shí)現(xiàn)對輪狀病毒的檢測。GO熒光猝滅原理和凝血酶適體相結(jié)合的凝血酶FRET生化傳感器[75]， 最低檢測限為31.3 pmol/L，相對于基于CNTs的FRET低兩個(gè)數(shù)量級。

GFET型生化傳感器以石墨烯作為FET的導(dǎo)電溝道，通過離子、分子的吸附或脫落影響石墨烯電荷密度和類型[76-78]，從而實(shí)現(xiàn)通過GFET輸出電信號的變化檢測生化量檢測。將DNA探針固定于石墨烯表面形成DNA-GFET傳感器[79]，DNA探針與其互補(bǔ)DNA雜交形成石墨烯摻雜效應(yīng)，使得GFET溝道載流子濃度發(fā)生變化，從而使源漏電流發(fā)生改變。蛋白質(zhì)GFET傳感器[80]，則利用蛋白質(zhì)吸附導(dǎo)致GFET響應(yīng)輸出改變，實(shí)現(xiàn)蛋白質(zhì)濃度的直接電學(xué)檢測。葡萄糖-GFET生化傳感器[81]則通過固定在石墨烯上的葡萄糖氧化酶催化葡萄糖氧化產(chǎn)生H2O2并吸附在石墨烯表面，使GFET溝道電導(dǎo)發(fā)生變化，實(shí)現(xiàn)對葡萄糖的快速檢測。

以離子選擇透過性膜修飾GFET形成多離子（Na+、K+、Ca2+、H+）GFET 傳感器陣列[82]，實(shí)現(xiàn)對多離子高效并行檢測?；趹腋尉┑腇ET型生化傳感器用于三種腫瘤標(biāo)記物檢測(ANXA2，VEGF和ENO1)[83]，該傳感器避免了來自基底晶界與散射對石墨烯的影響，提升了GFET導(dǎo)電溝道內(nèi)載流子傳輸速率，更加高效的實(shí)現(xiàn)生化量與電學(xué)量的轉(zhuǎn)換，最低檢測限可達(dá)0.1 pg/mL。

4 結(jié)論

石墨烯具有極高的電子遷移率，即可有效的提高感受器與轉(zhuǎn)換器間電荷傳輸速率，又可用于研制GFET型生化傳感器；同時(shí)，石墨烯片層結(jié)構(gòu)具有極大比表面積可提高轉(zhuǎn)換器表面生物探針的固定量，有助于提高生化傳感器的響應(yīng)強(qiáng)度和響應(yīng)范圍；最后，石墨烯衍生物富含羧基、羥基等含氧基團(tuán)，可作為多種生物探針的固定位點(diǎn)，使其成為多種生化響應(yīng)機(jī)制的載體。綜上所述，石墨烯及其衍生物在構(gòu)建高靈敏度、多功能型生化傳感器方面具有極大的潛力。

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