纳米材料用于构建新型电化学生物传感器的研究进展

陈桂芳，梁志强，李根喜,

1.南京大学生物化学系，医药生物技术国家重点实验室，南京 210093；

2.上海大学生命科学学院，上海 200444

纳米材料用于构建新型电化学生物传感器的研究进展

陈桂芳1，梁志强2，李根喜1,2

1.南京大学生物化学系，医药生物技术国家重点实验室，南京 210093；

2.上海大学生命科学学院，上海 200444

生物传感器是目前生命科学及临床医学测试方法研究中最为活跃的领域之一，电化学生物传感器是其中一个重要分支，已广泛用于临床、工业、环境和农业分析等领域。进入21世纪，纳米科技的迅猛发展为新型生物传感器的研制提供了难得的机遇。本文简要介绍和总结近年来基于蛋白质和纳米材料发展新型电化学生物传感器的研究进展，着重探讨纳米材料在构建新型电化学生物传感器中的应用。

蛋白质；纳米材料；电化学生物传感器；酶传感器；免疫传感器

0 引 言

生物传感器一词已经被广泛用来描述一类用以监控生命体系或与之相关联的生物基元的器件。其完整的概念可以认为是由作为识别元件的固定化的生物敏感材料 (包括酶、抗体、微生物、细胞、组织、核酸等生物活性物质)，以及作为物理换能器的适当的信号转换器 (如氧电极、光敏管、场效应管、压电晶体等)所组成的体系。当待测物质与分子识别元件发生特异性的结合后，所产生的复合物信号 (如光、热、电等)通过信号转换器转变成光、电等可输出信号，从而达到对待测物质进行分析检测的目的。从识别元件来看，蛋白质 (主要包括酶、抗体)相比其他材料而言，具有功能的多样性、对特定底物识别的专一性，以及分子本身的可操控性等不可比拟的优点，因此成为目前最为常用的识别元件。而从信号转换来看，电化学技术由于具备灵敏、快速、成本低廉、检测装置轻便、低能耗且易于微型化和集成化等优点，已成为生物传感器领域最为活跃的检测手段[1,2]。

进入21世纪，随着纳米技术的飞速发展，纳米材料在医学成像、疾病诊断、药物传输、基因治疗等多个领域显示了巨大的优势。对于生物传感领域而言，纳米材料在光学性能、电学性能、磁学性能、力学性能和化学活性等方面表现出的独特性能使其成为很好的换能器元件；另一方面，生物传感器中的分子运作本身就是基于纳米层次，纳米材料的参与可以将其优良的性能很好地整合到分子运作中，从而改进甚至革新分子运作的体系。鉴于以上特点，纳米材料在生物传感器中的应用引起了越来越多科研工作者的兴趣。本文从纳米材料的角度综述了近年来基于蛋白质与纳米材料发展新型电化学生物传感器方面的研究进展。

1 金属纳米材料

金属纳米材料良好的电子传递性能使其成为电化学生物传感器中最为常用的纳米材料之一，其中尤以纳米金的应用最为广泛。纳米金制备简单、性状稳定、生物相容性良好，而且易于进行表面化学修饰，因此，利用纳米金与生物分子进行组装并介导电子传递，是构建电化学生物传感器的良好方案。

Willner课题组[3]于2003年在Science上发表文章介绍了纳米金的经典应用。他们将葡萄糖氧化酶 (glucose oxidase，GOD)的辅基FAD修饰到纳米金表面，再通过双巯基化合物的连接将FAD修饰的纳米金固定到金电极上。当引入脱辅基的GOD后，由于FAD与酶的特异结合，完整的GOD可以最大限度地保持其原有结构和活性而组装到电极表面，从而催化葡萄糖的氧化。通过监测催化过程中FAD经纳米金介导传递到电极表面的电化学信号，可以对底物葡萄糖进行定量分析。此后他们又利用纳米金与聚阴离子组成的“微杆”介导得到了溶液中GOD的电化学响应，从而实现葡萄糖的高灵敏检测 (图1)[4]。结果显示，纳米金的使用一方面使得GOD与电极间的电子传递速率提高了25倍，另一方面，“微杆”结构所提供的巨大的比表面积使得即使在溶液状态下也能获得GOD的电化学信号 (图2)。近年来，基于纳米金与GOD所构建的各种葡萄糖生物传感器层出不穷，并发展出了一系列优秀的实验设计方案。比如，Sun等发展了GOD在纳米金表面的共价固定[5]以及基于层层组装技术 (layer-by-layer，LBL)的多层设计[6]，Ju等[7]发展了掺入GOD和纳米金颗粒的碳糊电极，Chen等[8]发展了壳聚糖和纳米金的联合运用，还有许多实验方案不在此一一列举[9～16]。

图1 葡萄糖氧化酶存在条件下，通过聚苯胺-纳米金微杆(A)和聚苯胺-聚苯乙烯磺酸盐微杆(B)介导的电化学催化氧化葡萄糖的示意图[4]Fig.1 Bioelectrocatalytic oxidation of glucose in the presence of GOx mediated by Polyaniline/Au-nanoparticles composite micro-rods(A)and polyaniline/polystyrene sulfonate composite micro-rods(B)[4]

图2 葡萄糖氧化酶存在下通过微杆介导的葡萄糖的电化学催化氧化[4](A)聚苯胺-纳米金微杆介导的不同浓度葡萄糖氧化的循环伏安图；(B)聚苯胺-聚苯乙烯磺酸盐微杆介导的不同浓度葡萄糖氧化的循环伏安图。a:0 mmol/L;b:10 mmol/L;c:60 mmol/LFig.2 Bioelectrocatalytic oxidation of glucose by solubilized GOx,1 mg mL-1,and mediated by the microstructured redox-active rod assemblies[4](A) Cyclicvoltammogramsrecorded in thepresenceof the microstructured assemblies composed of PAn/Au-NPs and different concentrations of glucose; (B)Cyclic voltammograms recorded in the presence of the microstructured assemblies composed of PAn/PSS and different concentrations of glucose.a:0 mmol/L;b:10 mmol/L;c:60 mmol/L

除了基于GOD的葡萄糖传感器外，在基于辣根过氧化物酶 (horseradish peroxidase，HRP)的H2O2传感器[17～28]、基于次黄嘌呤氧化酶 (xanthine oxidase，XOD)的次黄嘌呤/黄嘌呤传感器[29～31]、基于乙醇脱氢酶 (alcohol dehydrogenase，ADH)的乙醇传感器[32]、基于乳酸脱氢酶（lactate dehydrogenase，LDH）的乳酸传感器[33]、基于乙酰胆碱酯酶的含磷杀虫剂传感器[34,35]、基于胆固醇氧化酶的胆固醇传感器[36]，以及基于酪氨酸酶的酚类传感器[37,38]等设计中，纳米金的应用同样取得了很好的效果。此外，生理条件下并不作为酶的一些氧化还原蛋白，比如血红蛋白 (hemoglobin，Hb)、肌红蛋白 (myoglobin，Mb)以及细胞色素c(cytochrome c，cyt c)，经过与纳米金在电极表面共修饰组装后也可表现出一定的酶活性，因而可用于构建检测H2O2、NO等底物的生物传感器[39～45]。

纳米金的另一个主要应用是参与构建基于抗原-抗体识别的电化学免疫传感器。图3显示的是一个典型的纳米金参与的免疫传感器的示意图。相比经典的酶联免疫传感器——ELISA，纳米金更为稳定，在复杂的检测环境不易降解和变性。更重要的是，通过与其他纳米材料联用，可以实现多靶标的检测。基于纳米金的这些优良特性，近年来，涌现出众多基于纳米金的免疫传感器[46～64]。比如，蒋健晖等[65]以人免疫球蛋白G(h IgG)为模型分析物，使固定化抗体与分析目标物h IgG及纳米金标记的h IgG抗体结合于电极表面，通过金沉积使电极表面的纳米金颗粒直径增大，并通过金增强表面吸附伏安分析实现了h IgG的高灵敏定量免疫检测。袁若[66]等通过LBL组装将多层辣根过氧化物酶/纳米金及L-半胱氨酸等材料组装到电极表面，实现对甲胎蛋白抗体的固定，研制出用于检测甲胎蛋白抗原(alpha-fetoprotein，AFP)的免疫传感器。信号标记对于免疫传感器而言是烦琐的一步，Yu等[67]使用纳米金发展了无标记的IgE检测，并有望成为通用的免疫传感设计。

图3 (A)一抗通过静电作用或共价连接固定到碳电极表面；(B)目标蛋白（抗原）被固定的一抗识别；(C)金属纳米颗粒标记的二抗结合抗原形成“三明治”结构，从而可被电化学手段定量检测到[46]Fig.3 (A)Primary antibodies are immobilized on the carbon-electrode surfaces through electrostatic attraction or covalent-binding reactions;(B)Target protein(antigen)is recognized by the primary antibody immobilized on the surface;(C)Metal nanoparticle-labeled secondary antibody binds to the antigen through its unique epitope,forming a sandwich-type complex on the surface.The accumulation of metal nanoparticles on the surface can be quantified using electrochemical-measurement techniques[46]

综合来看，纳米金用于构建以蛋白质为识别元件的电化学生物传感器主要具备以下几个优势：1)作为金属材料，纳米金是电子的良导体，可以最大限度地促进电子传递，从而实现低电位下的电分析，以避免高电位中的噪音和干扰。2)作为纳米材料，其大的比表面积十分有利于蛋白质以及电化学信号探针等分子在电极表面的大量固定，从而有效地提高信号强度并实现信号放大。3)纳米金具有良好的生物相容性，即使进行在体实验也未发现对细胞有明显的毒副作用；在分子层面，这种良好的生物相容性还可为蛋白质提供良好的微环境，从而保持蛋白质的生物活性。4)纳米金可以很方便的通过静电作用或共价作用进行表面的化学修饰，从而为多样化的蛋白质表面固定方案打下基础。

纳米银和纳米铂是另外两种在电化学生物传感器构建中较为常用的金属纳米材料[68～75]。由于与纳米金的性质相似，基于这两种纳米材料的传感器设计与基于纳米金的传感器设计有异曲同工之妙，在此不一一赘述。

2 碳纳米管

自从1991年首次被报道以来，碳纳米管 (carbon nanotubes，CNTs)可以说是被研究得最多的纳米材料。对其理化性质的深入了解，以及对其功能的不断开拓，为其在生物传感器构建中的广泛应用打下了基础。与纳米金一样， CNTs同样也具备极好的电子传递能力、蛋白质的高负载能力以及良好的生物相容性，而且，由于CNTs本身的物质基础就是碳，因此其功能化将更为方便和多样。此外，由于CNTs为一维纳米材料，意味着CNTs在电极表面的组装将呈现网络状，因此，非常有利于蛋白质的固定。

Davis等[76]精细地描绘了GOD在单壁碳纳米管 (single-walled nanotubes，SWNTs)上的组装 (图4)。他们通过氧化SWNTs，使之表面带上充裕的活性基团，一方面有助于SWNTs在溶液中的分散，另一方面也给GOD的组装提供了便利条件。结果显示，即使不加入EDC、NHS等共价连接的活化剂，GOD也能在SWNTs表面很强地吸附并保持活性。他们将此吸附有GOD的SWNTS组装到玻碳电极上，得到了良好的GOD响应，其信号强度是没有SWNTs参与时的10倍，显示出SWCNTs在酶负载和能量转换上都具有良好的效果。

图4 (A)葡萄糖氧化酶修饰的单壁碳纳米管的原子力显微镜照片；(B)葡萄糖氧化酶-单壁碳纳米管共修饰的玻碳电极在无（曲线c）或有（曲线b）二茂铁一元酸，以及加入50 mmol/L葡萄糖（曲线a）后的循环伏安图[76]Fig.4 (A)Amplitude AFM image of a glucose oxidase-modified SWNT.Scale bar=200 nm.(B)Voltammetric response of a GOX-SWNT-modified GC electrode in the absence(curve c)and presence(curve b)of 0.5 mmol/L ferrocenemonocarboxylicacid (FMCA). Thecatalyticresponse(curvea) isobserved on theaddition of 50 mmol/L glucose[76]

除了在电极表面的平铺组装外，CNTs还可以通过化学气相沉积 (chemical vapor deposition，CVD)直接在电极表面垂直生长。Fang等[77]运用这种方法在电极表面生长CNTs，并利用 (4-羧苯基)重氮盐在CNTs表面的电化学还原对CNTs进行了功能化，从而共价连接Hb，结果发现Hb具有良好的电化学响应，并可用于构建H2O2传感器（图5）。Wisitsoraat等[78]也利用这种方法在金覆盖的SiO2/Si基底上生长了CNTs，然后通过电化学聚合在CNTs表面固定聚苯胺 (polyaniline，PANI)和胆固醇氧化酶，利用循环伏安法对底物胆固醇进行了检测，取得了良好的结果。

目前，基于以上两种方式，利用CNTs构建酶传感器[79～97]和免疫传感器[98～106]的报道数量众多，在此不一一列举。需要提出的是，由于CNTs难以具备纳米金那样良好的形态分布，因此对有序的表面组装提出了挑战。另外，大多数蛋白质的尺寸都属于零维的纳米级，因此在一维的CNTs表面组装相对而言缺少灵活性。出于这些考虑，将CNTs与零维的纳米颗粒，如纳米金、纳米铂等联合运用，在一定程度上可以克服两者在某些方面的缺陷，因而也是传感器构建中的良好策略[107～113]。

图5 (A)碳纳米管阵列的扫描电子显微镜照片；(B)血红蛋白-碳纳米管阵列修饰电极在加入不同浓度过氧化氢前后的循环伏安图[77]Fig.5 (A)SEM images of ACNTs;(B)Cyclic voltammograms of the Hb-ACNTs electrode in PBS(pH 7.0)before(a)and after the addition of 160,360,560,760,960 mmol/L H2O2(curve b to f).Scan rate:40 mV/s[77]

3 纳米氧化物

除了具备纳米材料共有的一些性质外，纳米氧化物还依材料的不同具备一些特殊的效应，比如纳米Fe3O4的磁效应、纳米TiO2的光电效应等。而这些效应在新型生物传感器的构建中可以产生一些意想不到的效果。在这一部分，我们将着重就纳米氧化物特殊效应的应用进行总结和回顾。

磁性纳米颗粒现在已被广泛用于分离富集、靶向运输等方面的研究，但在生物传感领域，磁效应的应用还不广泛。Willner等[114]利用纳米Fe3O4首次实现了电化学催化的磁控制，如图6所示，他们首先合成了能够稳定分散于有机溶剂中的磁性纳米Fe3O4，并构建了一个双层的电解液，上层为有机溶剂甲苯，下层为水溶液。当磁场处于上方时，Fe3O4分散在甲苯中，下方的GOD可以催化葡萄糖的氧化并得到电信号；但当磁场转移到电极下方，Fe3O4将被吸引下来覆盖住电极表面，从而阻碍GOD与电极的电子传递，失去了电驱动的GOD便无法进行催化，由此，通过转换磁场方向即可非常方便地控制酶催化反应进程。

纳米TiO2是另一种具有特殊效应 (光电效应)的纳米材料，由于具有极强的紫外线屏蔽能力和很高的表面活性，纳米TiO2已经被大量用于污水处理、消毒杀菌，以及在化妆品和涂料中防紫外线侵蚀。作者所在实验室曾利用纳米TiO2这种特有的性质构建了高灵敏的H2O2传感器 (图7)[115]。一般来讲，蛋白质在紫外线照射下很容易变性，但是我们利用纳米TiO2与Hb共修饰后发现，一定时间的紫外线照射后，Hb不仅没有失活，其催化活性反而大大提高，电化学检测H2O2的灵敏度提高了3倍，检测限降低了两个数量级。这一方面得益于TiO2对紫外线的屏蔽保护了Hb，另一方面，TiO2在紫外线激发下产生的激发态活性物质促进了Hb催化性能的提高。作者所在实验室在后续的研究中发现纳米ZnO也具有类似的效用[116]。

图6 (A)磁性纳米颗粒远离电极从而激活二茂铁介导的葡萄糖的电化学催化氧化；(B)电极表面被疏水的磁性纳米颗粒覆盖从而阻碍了电化学催化的物质扩散过程[114]Fig.6(A)The magnetic nanoparticles are retracted from the electrode surface,which is activated toward the ferrocene-mediated bioelectrocatalytic oxidation of glucose;(B)The electrode surface is blocked by the hydrophobic magnetic nanoparticles toward the diffusional bioelectrocatalytic process,while the surface-confined ferrocene is electrochemically active[114]

图7 二氧化钛纳米颗粒与血红蛋白共修饰电极的示意图[115]Fig.7 Scheme of TiO2nanoparticle/Hb co-modified electrode[115]

4 量子点

量子点作为荧光标记物，已经被广泛用于荧光示踪。在电化学生物传感器领域，量子点同样有着特殊的用途。以金属硫/硒/碲化物 (Zn/Cd/Pb-S/Se/Te)等为代表的量子点，一方面是很好的生物标记材料，另一方面，其中的金属离子Zn2+、Cd2+、Pb2+可用于阳极溶出伏安法检测，从而提供电化学信号。

Wang等[117]利用三种量子点 (ZnS、CdS以及PbS)分别标记三种抗体，并利用磁性纳米颗粒进行分离富集，通过溶出伏安法检测Zn2+、Cd2+、Pb2+在不同电位下的峰电流，可以同时检测三种抗原的存在 (图8)。其他一些课题组也利用量子点的这种性质进行了一些传感设计[118～120]。

图8 基于不同无机纳米颗粒的多蛋白的电检测原理[117](A)引入抗体修饰的磁珠；(B)抗原结合到磁珠表面的抗体上；(C)纳米颗粒标记的二抗被磁珠捕获；(D)对纳米颗粒进行电化学溶出分析Fig.8 Multi-protein electrical-detection protocol based on different inorganic nanoparticle(NP)tracers[117](A)Introduction of antibody-modified magnetic beads;(B)Binding of antigens to antibodies on the magnetic beads;(C)Capture of the NP-labeled secondary antibodies;(D)Dissolution of NPs and electrochemical stripping detection

总的来说，基于量子点的电化学传感设计具有以下一些优点：1)由于近年来量子点相关技术的日趋成熟，用于电化学检测的量子点的制备以及功能化比较方便，有商品化的产品可供选择；2)不同的量子点可以提供不同的电化学信号，这为多组分的分析提供了条件；3)溶出伏安法是基于金属离子的氧化还原分析，其理论检测限可达到飞摩尔水平，这为痕量检测打下了基础。

5 复合纳米材料

不同的纳米材料各自具备一定的特性，在电化学生物传感器的设计中使用单一的材料难以充分发挥纳米材料的性能，因此，同时使用多种纳米材料成为一个解决方案。一种思路是首先合成两种或多种纳米材料，然后在传感器的构建中同时或在不同阶段分别运用，比如图6所介绍的量子点与磁性纳米颗粒的运用；另一种思路则是在纳米材料的合成阶段将不同的材料进行组装，即合成复合纳米材料，将不同纳米材料的特性整合到一个纳米复合体中。一个很好的例子是CNTs与金属纳米颗粒复合的材料，另一个例子则是合成核/壳结构的纳米颗粒，而且这种做法目前更为常见。

Yu等[121]合成了MgFe2O4@SiO2核/壳纳米颗粒，由SiO2外壳提供良好的酪氨酸酶组装界面，由磁性的MgFe2O4内核实现蛋白在电极表面的磁控富集。由此构建的电化学生物传感体系可以对酶底物苯酚实现良好的检测。这种核/壳纳米颗粒一方面保持了磁性纳米颗粒的磁效应，另一方面又为蛋白质提供了良好的组装界面和微环境，因而成功地综合了两种纳米材料的优良特性。Zhu等[122]则充分利用AgCl和PANI的特性合成了AgCl@PANI纳米颗粒，一方面AgCl外壳克服了PANI在水相中难以分散的缺点，并为后续的电化学信号获取提供了良好的电子传递界面，另一方面，PANI为传感器在碱性条件下的使用提供了条件，并有效地防止了抗坏血酸的氧化，他们利用此复合纳米材料构建了用于检测多巴胺的电化学生物传感器。此外， Calvo、Zeng等课题组[123～127]也开展了一系列基于核/壳纳米颗粒的传感器研究工作。

6 展 望

纳米科技与电化学技术的结合和相互渗透，为基于蛋白质构建生物传感器提供了重大的创新机遇和诱人的市场前景，各种具有优良性能的纳米材料在生物传感器构建中的应用，不仅大大提升了生物传感器的性能，还拓宽了生物传感器的适用范围，为其在临床检测、食品安全、环境监测、医疗卫生等领域的应用开辟了新的道路。当然，新技术出现的同时也面临着新的挑战，比如如何实现原位分析、如何构建稳定专一的电化学生物传感芯片、如何建立健全的评价纳米材料效用及安全性的标准体系，等等。随着蛋白质科学，以及纳米技术、电化学技术、分子识别技术、表面固定技术等相关技术的不断发展，我们相信这些问题会逐步得到解决，电化学生物传感器也会获得更大的发展，展示更为广阔的应用前景。

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This work was supported by grants from The National Science Fund for Distinguished Young Scholars(20925520)and Shanghai Science and Technology Committee(09DZ2271800)

Progress of Electrochemical Biosensors Fabricated with Nanomaterials

CHEN Guifang1,LIANG Zhiqiang2,LI Genxi1,2
1.Department of Biochemistry and National Key Laboratory of Pharmaceutical Biotechnology,Nanjing University,Nanjing 210093,China;
2.Laboratory of Biosensing Technology,School of Life Science,Shanghai University,Shanghai 200444,China

Feb 2,2010 Accepted：Apr 28,2010

LI Genxi,Tel：+86(25)83593596,E-mail：genxili@nju.edu.cn

Biosensorshave

more and more research interestsdue to the rapidlyincreased requirement for measurements in life sciences.Protein-based electrochemical biosensors,which are particular focused because of practical advantages,have been widely used in clinic,industry,environment,and agriculture.Due to the rapid development of nano Sci-Tech,many kinds of novel biosensors are fabricated.In this paper,we summarize the progress of protein-based electrochemical biosensors,with the emphasis on the discussion ofthe application ofnanomaterials to the developmentofelectrochemicalbiosensors fabricated with protein and nanomaterials.

Protein;Nanomaterial;Electrochemical biosensor;Enzyme biosensor;Immunosensor

2010-02-02；接受日期：2010-04-28

国家杰出青年科学基金项目(20925520)，上海市科学技术委员会项目(09DZ2271800)

李根喜，电话：(025)83593596，E-mail：genxili@nju.edu.cn

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