图1 (a)(ⅰ)用3种不同的苯二胺异构体制备红绿蓝三色CDs,(ⅱ)m-CDs、o-CDs和p-CDs在日光下(左)和λ=365 nm紫外线照射下(右)的照片[
Published:01 August 2021,
Received:30 April 2021,
Revised:17 May 2021
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Recently, the fluorescence biosensor based on carbon dots(CDs) and aptamer is more and more attractive. Comparing with traditional fluorescence materials like fluorescence dye, semiconductor quantum dots and rare earth phosphors, CDs have attracted considerable attention in the field of fluorescence biosensor due to their excellent photoluminescence, low toxicity and low-cost prepare process. The fluorescence biosensor based on CDs and aptamer has extensive application prospect in the field of environment monitoring, bioimaging and biomedicine. This review will summarize the structural composition, fabrication methods, sensing mechanism and application area of fluorescence biosensor based on CDs and aptamer.
carbon dots;
aptamer;
fluorescence biosensor;
sensing mechanism
随着科学技术的发展,传统的检测方式难以满足人们在环境监测、临床诊断、生物检测方面的信息获取需求,如何制备与发展新型传感器以求对生物分子及金属离子实现快速灵敏检测是亟待解决的问题。近年来,基于荧光的传感技术因其灵敏度高、响应时间短、成本低廉、检测方便等优点受到了广泛关注。研究者们设计开发了多种荧光传感材料,其中常用的荧光基元包括有机荧光染料[
荧光生物传感器有两个关键组成部分:目标识别和信号转导模块。目标识别依靠的是探针与目标物质的特异性结合能力,如离子对结合[
碳点是一种尺寸小于10 nm的新型零维碳基纳米材料,在2004年由Xu等首次通过物理方式从电弧放电产生的烟灰中纯化得到[
由于合成方式与前体材料的多样化,碳点材料具有多种结构特征,因此碳点是一大类材料的统称。虽然目前关于碳点的分类仍有争论,但人们逐渐接受将碳点依据结构特征分为石墨烯量子点(Graphene quantum dots,GQDs)、碳量子点(Carbon quantum dots,CQDs)以及碳化聚合物点(Carbonized polymer dots,CPDs)[
碳点优异的荧光性质是其最引人入胜之处,例如较强的光吸收、高荧光量子产率、可调的荧光发射以及抗光猝灭性等。不同种类碳点的荧光吸收与荧光发射行为有着较大的差别,这些差别主要源于不同的前体原料与合成方法,它们会导致碳点具有不同的碳核结构和表面基团[
图1 (a)(ⅰ)用3种不同的苯二胺异构体制备红绿蓝三色CDs,(ⅱ)m-CDs、o-CDs和p-CDs在日光下(左)和λ=365 nm紫外线照射下(右)的照片[
Fig.1 (a)(i)Preparation of the RGB PL CDs from three different phenylenediamine isomers. (ⅱ)Photographs of m-CDs, o-CDs, and p-CDs dispersed in ethanol in daylight(left), and under λ=365 nm UV irradiation(right)[
Ellington和 Tuerk 在1990年分别独立地创建了体外指数富集配体的系统进化技术[
适配体对靶标的特异性识别作用只是传感过程中的目标识别部分,如何将探针与靶标结合前后的变化通过可测信号转导出来同样是传感过程的关键问题。近年来,纳米材料与适配体偶联构建生物传感器引起了研究者们极大的兴趣。由于纳米材料特有的光学、电学、磁性和催化特性,将适配体与纳米材料偶联之后,可以通过检测传感器与靶标结合之后纳米粒子的光、电、磁等特性变化来实现靶标的可测传感,常见的纳米材料有金纳米颗粒[
构建荧光生物传感器的荧光探针应具有灵敏度高、选择性好、操作方便、不易受电磁作用干扰、能够对靶标进行原位实时无损分析的特点。碳点相对于有机荧光分子、半导体量子点及稀土荧光材料等传统荧光材料,具有荧光性质稳定、斯托克斯位移大、生物相容性好、毒性低、表面易修饰等优势,是理想的荧光传感器构筑基元[
标记法是将碳点与适配体之间通过共价键进行连接,连接过后,碳点与适配体成为一个整体(CDs-Apt),这种方法的好处在于传感过程明晰,靶标与适配体的作用将直接影响到碳点荧光信号的变化,且传感器不易受外界复杂环境的影响。应该注意的是,单一的由碳点与适配体共价键连接所构成的荧光生物传感器难以实现信号的有效转导,还需要其他物质的配合。其中一种方式是利用金银纳米粒子[
图2 标记法构建荧光生物传感器。 (a)基于AuNPs和CDs-Apt的检测系统构建方法[
Fig.2 Construction of fluorescence biosensor by labeling method. (a)Construct method of detection system based on AuNPs and CDs-Apt[
相较于标记法,免标记的方法更加方便,且成本更低[
图3 免标记法构建的荧光生物传感器。 (a)检测啶虫脒的荧光生物传感器示意图[
Fig.3 Fabrication of fluorescence biosensor by free-labeling method. (a)Schematic illustration of fluorescent biosensor for acetamiprid detection[
基于碳点与适配体的荧光生物传感器的构建方式已经在上文进行了详细介绍,不难发现,无论是使用标记法还是免标记法构建的荧光生物传感器,在信号转导时大部分会涉及到荧光的猝灭与恢复过程。目前基于碳点与适配体构建的荧光生物传感器的传感机理主要有以下四种:荧光共振能量转移(Fluorescent resonance energy transfer,FRET)、内滤效应(Internal filter effect,IFE)、光致电荷转移(Photo-induced electron transfer,PET)和聚集诱导猝灭效应(Aggregation-caused quenching,ACQ)。
荧光共振能量转移过程是福斯特共振能量转移(Förster resonance energy transfer)的一种形式,它指的是能量供体与受体之间的非辐射能量转移过程[
图4 荧光生物传感器的传感机理。 (a)基于FRET效应检测AK的荧光传感方案示意图[
Fig.4 Sensing mechanism of fluorescent biosensors. (a)Schematic illustration of a fluorescence aptasensor for AK detection based on FRET[
内滤效应是指当荧光体与其他吸光物质共存时,由于吸光物质对于激发光或发射光的吸收而导致荧光体荧光减弱的现象[
光致电荷转移是指在光的作用下,电子在供体与受体之间传递的现象[
荧光体在稀溶液中具有较高荧光强度,但在浓溶液或者固态下荧光强度降低甚至消失的现象称为聚集诱导猝灭效应[
碳点优异的光学性质结合适配体出色的识别能力完美地构建了纳米传感器的目标识别模块和信号转导模块。时至今日,基于碳点与适配体构建的荧光生物传感器在金属离子、有机分子、核酸、蛋白质乃至细胞的灵敏传感领域取得了丰硕成果,极大地丰富了人类在环境监测、生物检测以及疾病诊疗过程中的检测手段。
金属离子在自然界中广泛存在,无论是对环境监测还是人体健康检测而言,对它的灵敏传感都显得尤为重要。传统的检测方法为电感耦合等离子体质谱法和原子吸收/发射光谱法,但这两种检测设备都需要昂贵的设备以及复杂的检测程序,不能满足常规监测中速度快、成本低的需求[
图5 检测金属离子。(a)通过二硫化钼纳米片对CDs-Apt的荧光猝灭作用检测二价汞离子[
Fig.5 Detection of metal ions. (a)Principle for the fluorescence quenching of MoS2 nanosheets through their interaction with CDs-Apt for sensitive detection of mercury(Ⅱ)[
6.2.1 有机小分子
小分子增塑剂是一种常见的化学添加剂,可以提高聚合物的可塑性,因此在化妆品及食品的包装材料中广泛应用。但这些油溶性有机小分子很容易溶解在食物或化妆品内并通过消化道或皮肤进入人体,威胁人类身体健康,因此对小分子增塑剂的快速灵敏检测显得尤为重要。近期,Wang等以抗坏血酸为原料合成了双荧光发射碳点材料,通过与氨基修饰的核酸适配体共价偶联之后,在氧化石墨烯的存在下构建了一种双荧光发射比值传感器用于特异性检测邻苯二甲酸二丁酯(DBP)[
图6 检测有机小分子。 (a)以适配体标记的双发射碳量子点构建荧光比值传感器检测邻苯二甲酸二丁酯[
Fig.6 Detection of organic small molecule. (a)Detection dibutyl phthalate by fluorescence ratiosensor based on dual-emission carbon quantum dot labeled aptamers[
地高辛(DX)是一种心脏糖苷类药物,临床用于治疗充血性心力衰竭和心律失常。DX治疗指标狭窄(1~2 ng/mL),在浓度超过2.8 ng/mL时有较高毒性,因此对患者体液(血浆和尿液)中DX浓度的实时监控非常重要。Elmizadeh等采用硼氢化钠还原的石墨烯量子点(rGQDs)分别基于光致电荷转移及荧光共振能量转移传感机制设计了两种荧光生物传感器,均可实现对生物液体中DX浓度的快速灵敏传感[
基于碳点与适配体构建的荧光生物传感器还可用于人体尿液、血液及唾液样本中毒品甲基苯丙胺(MTA)含量的快速灵敏检测。Saberi等以葡萄叶为原料合成了一种具有蓝色荧光发射的碳点,通过化学偶联作用将适配体连接在CDs表面,形成CDs-Apt复合体,利用CoOOH纳米片对CDs-Apt的吸附作用、荧光猝灭作用(FRET),以及MTA对CDs-Apt的特异性结合作用实现对MTA浓度的线性灵敏检测[
6.2.2 生物小分子
腺苷(AD)在人生理活动中起着重要作用,它可以调节心脏心肌耗氧量和血流量,调节多巴胺等大脑神经递质的释放,同时它还有助于调节平滑肌收缩、神经传递、肾血流动力学和肾素的释放。对AD的实时监控可反映人体的生理健康状态。Wang等以一水合柠檬酸与二乙烯三胺为原料通过一步水热法制备了具有蓝色荧光发射的碳点[
图7 检测生物分子。 (a)基于CDs-Apt和NG的AD传感器示意图[
Fig.7 Detection of biomolecule. (a)Schematic illustration of AD sensor based on CDs-aptamer and NG[
类似于腺苷的检测,由碳点与适配体构建的荧光生物传感器还可用于ATP 与多巴胺的快速、灵敏及特异性检测。与传统的“turn-on”型荧光变化方式不同,Luo等基于FRET机制设计了一种可进行信号放大的“turn-off”型ATP检测器[
6.2.3 细菌分泌物
黄曲霉素与赭曲霉素通常在发霉的谷物中高度富集,如果不经去除,将会残留在二次加工的农产品之中。这两类毒素的毒性极高,对人体的危害极大,高剂量暴露之下会诱导肝癌。Guo等以柠檬酸和乙二胺为原料,采用水热法合成蓝色荧光发射碳点,在该碳点表面修饰黄曲霉素B1适配体作为荧光探针(CDs-Apt),并在腐殖酸(HAs)的配合下对黄曲霉素B1进行灵敏检测[
图8 检测细菌分泌物。 (a) AFB1传感器工作原理示意图[
Fig.8 Detection of bacterial secretion. (a)Schematic diagram showing the operating principle of the sensor for AFB1[
卡那霉素是一种常见的兽用抗生素,但卡那霉素的过度使用可能导致其残留在动物性食品中,对人体健康造成威胁。Wu等采用免标记法以金纳米颗粒(AuNPs)作为吸收体,通过内滤效应猝灭碳点的荧光,构建了一种新型卡那霉素荧光传感器[
癌胚抗原(CEA)是人体组织和胎儿细胞中常见的一种作为抗原的细胞表面糖蛋白,它能参与细胞粘附,并在细胞识别和相互作用中起调节作用。通常情况下它在血液中的含量很少,而一旦体内出现肿瘤,它就会在肿瘤细胞中过度表达并分泌到体液中,因此确定CEA在体液中的含量对肿瘤的早期预警、筛查、诊断及预后分析具有重要意义[
图9 检测蛋白质。 (a)基于NIR-CDs的荧光传感器检测靶标CEA[
Fig.9 Detection of protein. (a)NIR-CDs-based fluorescence turn-on aptasensor for detection of target CEA[
Singh等以谷氨酸为原料采用热解法一步合成具有蓝色荧光发射的碳点,并通过化学反应将恶性疟原虫谷氨酸脱氢酶(PfGDH)适配体偶联到碳点表面[
基于碳点与适配体的荧光生物传感器对细胞的识别本质大部分是基于适配体对细胞膜上的特定膜蛋白的特异性识别作用[
图10 检测细胞。 (a)CD-Apt对细菌细胞的传感过程[
Fig.10 Detection of cells. (a)Scheme of the coupling process of CD-apt complexes and bacterial cells[
碳点材料具有光学性质突出、生物相容性好、毒性低、易修饰、制备简便等优良性质,是一种非常出色的荧光纳米材料。适配体在特异性识别靶标分子方面有着优异的性能,是优异的目标识别材料。本文对基于碳点与适配体的荧光生物传感器结构组成(碳点、适配体)、构建方式(标记法、免标记法)、传感机理(FRET、IFE、PET、ACQ)和应用范围(金属离子、小分子化合物、蛋白质、细胞)进行了系统的总结。目前来看,该种类型的传感器性质很突出、应用很广泛,但仍存在着一些问题:(1)碳点的荧光发射机理不明确,这阻碍了对传感机理的探究;(2)碳点结构不明晰,合成过程的不可控性使得碳点的表面状态具有宏观相似微观不同的特点,这对探针的精细结构设计影响较大;(3)碳点荧光需要向长波长方向设计以抵抗生物体的背景荧光干扰,这是临床应用的关键;(4)适配体与靶标的特异性结合位点及作用方式需要进一步研究。这些问题的解决将极大地提高基于碳点与适配体的荧光生物传感器的灵敏性、特异性以及检测范围。
我们认为,碳点结构的精确设计是解决上述问题的关键之一,通过调节前驱体的结构与优化反应条件可以实现碳点大小与组成的精确控制,但目前仍缺少普适性的指导方法。另一个关键问题是如何通过控制合成前体与路径实现碳点的荧光发射峰位向长波长移动以及优化生物相容能力。CPDs是近年来提出的一类具有核壳结构的碳点材料,其表面的聚合物链与基团结构赋予了CPDs易修饰的性质,易于与生物体之间相容,并且CPDs可以调节荧光发射至近红外区,这在临床应用中有很大的前景。总体来看,碳点与适配体结合用于荧光传感器件有着广阔的应用前景,但精确的器件结构设计以及适配体与靶标的作用形式仍需要从理论计算和谱学表征方面进行深入的研究。
本文专家审稿意见及作者回复内容的下载地址:http://cjl.lightpublishing.cn/thesisDetails#10.37188/CJL.20210161.
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