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Zhaoyin Wang and Zhihui Dai* Received 00th January 2012, Accepted 00th January 2012 DOI: 10.1039/x0xx00000x www.rsc.org/
Carbon materials in nanoscale exhibit diverse outstanding properties, rendering them extremely suitable for the fabricating of electrochemical biosensors. Over the past two decades, advances in this area have continuously emerged. In this review, we attempt to survey the current developments of electrochemical biosensors based on six types of carbon nanomaterials (CNs), i.e., graphene, carbon nanotubes, carbon dots, carbon nanofibers, nanodiamonds and buckminsterfullerene. For each material, representative samples are introduced to expound the different roles of the CNs in electrochemical bioanalytical strategies. In addition, remaining challenges and perspectives for future developments are also briefly discussed.
1. Introduction Carbon, an element in nature, has been recognised by humans for a long time. With the continuous progress in nanotechnology, the understanding of carbon materials has reached a new level from the macroscopic scale to the nanoscale. Nowadays, diverse allotropes of carbon nanomaterials (CNs) from zero-dimensional (0-D) to threedimensional (3-D) have been found and applied in different fields including nano-electronics and high-frequency electronics,1, 2 energy storage and conversion,3, 4 field emission display,5 theranostic,6 biological and chemical sensors.7, 8 Biosensors are closely related to modern life from diagnosis of diseases to detection of biological agents in the environment.9 Among the various biosensors, electrochemical biosensors are promising due to their high sensitivity, high signal-to-noise-ratio, relative simplicity and rapid response time.10 Owing to the wide potential window, chemical inertness and low cost, carbon has historically been considered as an important electrode material for electrochemical biosensors. For instance, pyrolytic graphite electrode (PGE) and glassy carbon electrode (GCE) are two of the most widely used electrodes.11, 12 In recent decades, with the discovery of the salient properties of CNs, the roles of carbon materials in electrochemical biosensors have gradually expanded from electrode materials to building blocks.13-16 Although prominent characteristics of the different CNs vary, their common features render them extremely attractive for the construction of electrochemical biosensors as detailed below. 1) Electrochemical activity.17, 18
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Certain CNs and their derivatives can exhibit electrochemical responses due to their intrinsic or endowed electrochemical activity. 2) Electrical conductivity.19 The high electrical conductivity of CNs ensures that they serve as an ideal electron transfer medium in electrochemical biosensors. 3) Large surface area.20 Because of the large surface area of CNs, additional required components can be assembled in electrochemical biosensors. 4) Ease of functionalisation.21-23 CNs can be readily modified, which will enhance and enrich the functions of CNs in electrochemical biosensors. 5) Biocompatibility.24, 25 The biocompatibility of CNs facilitates applications of CN-based devices in biological fields. Because of these properties, CNs can act either as nanoprobes, relying on their dominant electrochemical properties, or as nanocarriers relying on their other multiple properties. By utilizing CNs in electroanalytical assays, analytical performances can be substantially improved. The field of electrochemical biosensors is vast and diverse, and in this review we emphasizes the advantages and development of electrochemical biosensors based on several CNs. Compared with graphene (GR), carbon nanotubes (CNTs) and carbon dots (CDs), carbon nanofibers (CNFs), nanodiamonds (NDs) and buckminsterfullerene (C60) are typically overlooked (Fig. 1). To provide a comprehensive overview, electrochemical biosensors based on these CNs are also briefly introduced in this review. For each CN, only selected examples can be displayed and compared due to space limitations. In addition, it should be noted that distinct routes for CN preparation may greatly affect their properties and
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applications. For more information on CN preparations and other applications, readers may consult the previous reviews and references therein.17, 26-31
2. GR-based electrochemical biosensors GR is a single-atom-thick planar sheet of sp2-bonded carbon atoms that are perfectly arranged in a honeycomb lattice. Since the first mechanical exfoliation of graphene in 2004,32 studies on GR have been dramatically increased, which has stimulated research interest in bioanalytical applications. In particular, GR possesses nearly all of the aforementioned outstanding properties of CNs, making it extremely adequate for electrochemical biosensors. To date, several kinds of GR have been synthesised and utilized in electrochemical analytical protocols. Pristine graphene, obtained by the mechanical cleavage of graphite, has some exceptional properties.19, 33, 34 However, this type of graphene has a low throughput, small size, and hydrophobicity in particular, which limits its applications in electrochemical biosensors. Graphene oxide (GO) substantially increases the hydrophilicity of the graphene layers, but GO is an electrical insulator, which reduces conductivity by many orders of magnitude.35, 36 By eliminating the oxygen-containing groups of GO, reduced graphene oxide (RGO) achieves a unique combination of excellent conductivity, large surface area, high electrochemical activity and ease of functionalization. Thus, compared with pristine graphene and GO, RGO has certain overwhelming advantages for the applications in electrochemical analytical assays. 2.1. Uses of GR as a nanoprobe GR-related materials can be used as electrochemical nanoprobes for electrochemical biosensors in the following two aspects. There is an abundance of oxygen-containing groups on the surface of GO. Through the electrochemical reduction of GO, well-defined signals from the oxygen-containing groups will be obtained. Thus, GO can be directly used as a label for electrochemical biosensors.37, 38 In an impressive example, thrombin aptamer (TBA) was immobilized on the electrode surface by physical adsorption and was further sequentially bound with thrombin and GO.38 According to the electrochemical signal of GO reduction, thrombin measurement was achieved (Fig. 2A). Previous investigations have indicated that GR and its derivatives possess intrinsic electrocatalytic
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activity.39, 40 In particuler, decorating GR with foreign atoms (e.g. potassium (K), nitrogen (N) and sulfur (S)) may further enhance its electrocatalytic activities.41, 42 Based on the high electrocatalytic activity of K-modified GR, Li et al. reported a sensitive and stable amperometric nitrite (NO2-) sensor.43 In another study, a novel N-, S-doped GR was synthesized and implemented for the fabrication of a sensor for use as a highperformance electrocatalyst.44 Good performances in the + + simultaneous determination of Cd2 and Pb2 demonstrated that the N-, S-doped GR had improved electrocatalytic properties, which should be ascribed to the synergistic effects of the dual dopants. 2.2. Uses of GR as a nanocarrier Besides its electrochemical properties, GR possesses other outstanding characteristics, such as a large specific surface area, high conductivity, and an ease of modification. Using GR as a nanocarrier, redox probes,45, 46 recognition components47 or target molecules48 may be assembled into electrochemical biosensors, resulting in improved analytical performances. Redox enzymes can be readily immobilized onto an electrode with the aid of GR, and direct electron transfer (DET) of these enzymes will be promoted in GR-based nanostructures. Fundamental studies have provided certain basic guidance for the construction of high-performance GR-based enzymatic electrodes.49, 50 In a glucose biosensor, GCE was modified by electrochemically reduced carboxyl graphene (ERCGR), which was further used as a substrate for assembly of glucose oxidase (GOx) (Fig. 2B).51 Because of the participation of GR, immobilization of GOx was realized via self-assembly under mild conditions and the DET of GOx was efficiently achieved, leading to better analytical results. Similarly, assembly of GOx with GR was utilized in an on-line microdialysis system, which can monitor the extracellular concentration of glucose in vivo.52 Co-immobilization of a bi-enzyme (cholesterol oxidase and catalase) by GR was investigated to detect cholesterol.53 In addition to redox enzymes, other proteins have also been employed in GR-based electrochemical biosensors.54, 55 An interesting example was provided by Song et al. that chitosanRGO and concanavalin A constituted a sensing layer with net charges, which exhibited a pH-switchable response to negatively charged redox probes.54 According to the principle that oxidation of glucose by GOx or hydrolyzation of urea by urease resultes in a pH decrease or increase, respectively,
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Fig. 1 Schematic illustration of certain allotropes of CNs.
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MINIREVIEW glucose and urea were detected in one sample without separation. GR-related materials, with partial planar structures and abundant groups on the surface, specifically GO and RGO, can serve as suitable platforms for the immobilization of antibodies for electrochemical biosensing.56-58 Eissa et al. reported a GRbased voltammetric competitive immunosensor for the determination of okadaic acid (OA) in shellfish.59 In this assay, the okadaic acid antibody was first appended on the carboxyphenyl modified GR electrode via carbodiimide chemistry. After competition between OA and a fixed concentration of the OA-ovalbumin conjugate for the immobilized antibodies, different electrochemical signals were obtained to reflect the amount of OA. Another practical electrochemical immunosensor for vascular endothelial growth factor receptor 2 (VEGFR2) was described by our group (Fig. 2C).60 In this sensor, graphene was employed to enhance the surface area, accelerate electron transfer and form an interface with sites for antibody immobilization, which provided the foundation for excellent analytical performances. Thus, this sensor could detect the total concentrations of VEGFR2 protein in cell lysates and could discriminate the changes in VEGFR2 expression induced by different inhibitors. Benefiting from the presence of various groups as well as πconjugated domains, anchoring of the DNA probe onto GR can be realized by either covalent grafting or noncovalent π-π stacking, both of which have been well exploited in GR-based electrochemical biosensors.61, 62 To compare the effects of different conjugation approaches, two impedimetric examples for DNA detection are presented in this review.63, 64 If probe DNA is immobilized on the surface of RGO by noncovalent adsorption, then hybridization with target DNA will release the probe DNA from the electrode surface, causing a decrease in the electrochemical signals. In contrast, if probe DNA is covalently bound to RGO, then hybridization will attract more DNA onto the electrode surface, leading to a larger signal. In both of the approaches, RGO helps acquire distinguished
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Nanoscale DOI: 10.1039/C5NR00585J signals before and after hybridization, which can be attributed to the large surface-to-volume ratio and the high conductivity of RGO. Aptamers and DNAzymes are two kinds of functional DNA. The aptamer can bind to a specific target molecule, while DNAzymes have the ability to perform a chemical reaction. Integrating the aptamer and DNAzyme into GR-based biosensors may broaden the sensing range from DNA to other target molecules.65, 66 In one study, TBA was adsorbed onto hydrazine treated GO through the strong noncovalent binding of GR with the nucleobases.67 The binding between TBA and thrombin would greatly disturb the interaction between TBA and GR, resulting in different electrochemical signals. Our group designed another GO-based analytical assay for target DNA or protein (Fig. 2D).68 In this assay, capture DNA (single stranded DNA (ssDNA) or an aptamer) can bind to target DNA or protein, causing the release of the capture DNA from the GO surface and the anchoring of [PMo12O40]3- (PMo12). Depending on the electrochemical activity of the adherent PMo12, the target DNA or protein can be easily analysed. Liang et al. decorated a L-histidine-dependent DNAzyme onto a GR-based electrochemical biosensor, which can be used to detect Lhistidine with an expanded linear range as well as excellent sensitivity and selectivity against other amino acids.66 Many previous studies have demonstrated that GR equipped with metal nanomaterials (MNs) can enhance performances in nonenzymatic electrochemical biosensors.69-72 Guo et al. created a one-step microwave-assisted heating procedure to form a new Pt nanoparticles (Pt NPs) ensemble-on-GR hybrid nanosheet, which has proven to be a good material for hydrogen peroxide (H2O2) and trinitrotoluene detection.73 The experimental results of this work confirmed that GR played a key role in enhancing the activity of Pt NPs. To date, numerous electrochemical bioanalytical assays have been developed based on GR-related materials coupled with MNs, such as Ni,74 Cu,75 Sn,76 Zn,77 Ce-Pt.78 Specially, integrating Fe3O4 nanoparticles into a GR-based biosensor may display novel properties by applying an external magnetic field.79, 80
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Fig. 2 GR-based electrochemical assays using GR as a nanoprobe or a nanocarrier. (A) Illustration of the utilization of GO nanoplatelets as inherently electroactive labels for the detection of thrombin. (Reproduced from ref. 38 with permission from The Royal Society of Chemistry.) (B) Scheme of the DET of the ERCGRGOx/GCE and the electrocatalysis of oxygen and glucose. (Reproduced from ref. 51 with permission from Copyright 2012 Elsevier.) (C) Schematic representation of the GR-based electrochemical biosensing platform for VEGFR2 protein. (Reproduced from ref. 60 with permission from Copyright 2014 Nature Publishing Group.) (D) Schematic illustration of the label-free electrochemical biosensing platform for DNA and protein analysis based on GO. (Reproduced from ref. 68 with permission from The Royal Society of Chemistry.)
3. CNT-based electrochemical biosensors CNTs, firstly discovered by Iijima in 1991,81 can be considered as a graphite sheet rolled up into a nanoscale-tube. CNTs can be divided into two main groups: single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs). Owing to their unique structural, electronic and chemical properties, CNTs have attracted increasing research interest in electrochemistry,82 and many CNT-based electrochemical biosensors have been reported.83 When considering the electrochemistry of CNTs, two aspects, i.e., their structural heterogeneity and composition heterogeneity, should be given special attention.16 Structural heterogeneity indicates that the walls of the tubes have distinct properties from the ends of the tubes. Because of this phenomenon, the rate of electrode transfer may be significantly affected by changing the orientation and arrangement of CNTs on the electrode surface. Composition heterogeneity indicates that the CNTs used for biosensor construction are often impure because of the introduction of impurities during CNTs preparation. However, the unique properties of CNTs still ensure their suitability for certain electrochemical biosensors. For example, because of their stiffness and ballistic conductivity, CNTs are an ideal material for the development of microelectrodes, which are valuable in cell and tissue sensors.84-87 Besides, biosensors containing degradable CNTs have been considered as a future direction of CNT-related researches.88
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3.1. Uses of CNTs as nanoprobes The pivotal roles of defect sites and oxygen-containing groups in the electrochemical performances of CNTs have been well studied,89-91 which explains the mechanism of electrocatalysis of CNT-related nanomaterials. To date, several electrochemical biosensors have been developed by solely and directly using the electrocatalytic activity of CNTs, thus they can be considered as nanoprobes for electrochemical biosensors. At the early stage, Ye et al. designed a rapid DNA biosensor based on the electrocatalytic activity of MWCNTs toward guanine or adenine residues of ssDNA.92 Relying on the remarkable catalytic property of acid-pretreated MWCNTs, homocysteine has also been analyzed using electrochemical methods.93 Subsequently, the excellent electrocatalytic ability of CNTs toward H2O2 was verified and employed in fabricating biosensors. Wu et al. confirmed the electrocatalytical decomposition of H2O2 by chitosan-grafted MWCNTs and have provided an example of the construction of an amperometric H2O2 biosensor.94 Compared with non-doped CNTs, N-doped CNTs reveal higher electrocatalytic activity for the oxygen reduction reaction (ORR) and redox of H2O2.95, 96 Taking the advantage of N-doped CNTs, Xu et al. reported a H2O2 biosensor at a low potential by simply dropping and drying Ndoped CNTs on the surface of a GCE.97 3.2. Uses of CNTs as nanocarriers
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MINIREVIEW Due to the advantages of CNTs (e.g. large specific surface, good biocompatibility, modifiable sidewall and excellent conductivity), loading other materials onto their surface of CNTs may create novel nanomaterials or nanostructures with enhanced properties. In addition, various methods have been exploited to realize the immobilization process.98 Therefore, CNT-based composites become essential scaffolds for electrochemical biosensors. Among the multiple materials used along with CNTs, redox enzymes, antibodies, peptides and other proteins, DNA and MNs are the most productive materials. CNTs can provide direct electrical wiring between the electrode and the active centers of the redox enzymes, promoting the DET of the enzyme by using CNTs as a mediator.99 The paradigm of this electrical communication was displayed by Gooding,100 and a typical biosensor was fabricated by Willner’s group (Fig. 3A).101 In this sensor, SWCNTs and an amino derivative of flavin adenine dinucleotide (FAD-NH2) were covalently and sequentially decorated onto an Au electrode. After reconstituting apo-glucose oxidase on the FAD-NH2 that was linked to the ends of SWCNTs, construction of this glucose biosensor was completed. Since this development, various enzymes have been applied to fabricate biosensors based on CNTs mediated DET.102-104 To further enhance this communication, Kafi et al. developed a network by crosslinking CNTs and Hb on a thiol-modified surface and exploited this network for sensing H2O2.105 By virtue of the CNT-mediated communication between Hb and the electrode, this network exhibits high conductivity and electrocatalytic activity towards H2O2. Antibodies have also been attempted to bind with CNTs to design electrochemical immunosensors. Large surface area and different groups on the surface of CNTs may facilitate the binding between antibodies and CNTs. Meanwhile, CNTrelated materials can resist unspecific adsorption of antibodies to a certain extent.106 Han et al. fabricated vertically aligned MWCNTs by chemical vapour deposition and functionalized them in alkaline solution to produce oxygen-containing groups, which can be used as handles to combine microcystin-LR (MCLR) to the surface of MWCNTs.107 By measuring the change of the electron transfer resistance before and after the conjugation of antibodies, quantification of MC-LR was achieved. By considering the effects of pH and the net charge, Puertas et al. achieved the oriented binding of the antibodies, which greatly improved the performances of immunosensor.108 Peptides and other types of proteins have been further implemented in CNT-based electrochemical biosensors. Mahmoud et al. developed a protease biosensor using a
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Nanoscale DOI: 10.1039/C5NR00585J peptide/SWCNT/gold nanoparticle-modified electrode.109 In this work, they emphasized that significant improvement of sensitivity was achieved by the participation of SWCNTs. Recently, our group described a label-free malondialdehyde biosensor with human complement factor H linked MWCNTs (Fig. 3B).110 The MWCNTs displayed an excellent electronic conductivity and large surface area, both of which were of great value in this sensor. DNA is another type of biomolecules applied in CNT-based electrochemical biosensors. DNA can be immobilized on the surface of CNTs in both covalent and non-covalent modes.111, 112 Zhu et al. demonstrated a new impedimetric DNA biosensor by binding probe ssDNA with dendrimers modified MWCNTs.113 Electrochemical impedance spectroscopy measurements were obtained to determine hybridization events between surface-confined probe ssDNA with target DNA. Another interesting development is the use of aptamers.114, 115 Miodek et al. described a MWCNT-based electrochemical biosensor with the aid of the human cellular prions PrPC aptamer (Fig. 3C).116 In this study, DNA aptamers were linked to dendrimers modified MWCNTs to construct bioreceptors, which can sense the variation of the electrochemical signal caused by PrPC. MNs can be wrapped or synthesized on the surface of CNTs, facilitating the construction of CNT-based nonenzymatic biosensors. Synergistic effects can be found in the composites of CNTs and MNs due to the high conductivity of CNTs and the catalytic activity of MNs. Yang et al. developed a H2O2 biosensor with remarkably improved sensitivity because of the synergistic electrocatalytic activity of Pt NPs and CNTs.117 In another biosensor, a high density of Pd nanoparticles (Pd NPs) was grown on the surface of SWCNTs, and the Pd-SWCNTs nanostructure can be used as a new type of catalyst to fabricate a nonenzymatic glucose biosensor.118 To date, some other MNs (Mn,119 Mo,120 Sn,121 Fe-Pt,122 etc.) have been incorporated with CNTs in electrochemical bioanalytical assays. Magnetic nanoparticles (MNPs) have also been employed in CNT-based biosensors.123 This work showed an alluring model in which a mixture of biofunctionalized MNPs with CNTs can implement magnetoswitchable bioelectrocatalysis in an external magnetic field. Another elegant application was published by our group (Fig. 3D).124 Using manganous pyrophosphate as one superoxide dismutase minic, a novel superoxide anion biosensor was fabricated. In this assay, the electrochemical response was enhanced because of the large surface area and good electronic conductivity of the MWCNTs. More importantly, the biosensor can be used to monitor living cells directly adhered to the modified electrode surface.
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Fig. 3 CNT-based electrochemical assays using CNTs as nanocarriers. (A) Assembly of the SWCNT electrically contacted glucose oxidase electrode. (Reproduced from ref. 101 with permission from Copyright 2004 WILEY-VCH.) (B) Schematic diagram of the construction of a label-free biosensor based on MWCNTs. (Reproduced from ref. 110 with permission from The Royal Society of Chemistry.) (C) Schematic representation of biosensor based on MWCNT-polyamidoamine dendrimer-ferrocene-biotin-strepatavidin and an aptamer. (Reproduced from ref. 116 with permission from Copyright 2013 American Chemical Society.) (D) Schematic illustration of a novel superoxide anion biosensor based on MWCNTs. (Reproduced from ref. 124 with permission from Copyright 2014 American Chemical Society.)
4. CD-based electrochemical biosensors CDs, one kind of 0-D CNs, refer to small carbon nanoparticles.30 Since CDs were firstly discovered by Xu et al. in 2004,125 continuous research efforts have been devoted to these popular materials. Due to the quantum confinement and edge effects, CDs can exhibit outstanding optical and electrooptical properties, similar to conventional quantum dots (QDs). However, certain superior biological properties of CDs cause CDs to outperform QDs in bio-related fields.31 In recent years, the electrochemical properties of CDs have attracted considerable interest and representative achievements have indicated the great potential of CD-based electrochemical biosensors. Up to now, two kinds of CDs, i.e., carbon quantum dots (CQDs) and graphene quantum dots (GQDs), have been widely used in diverse fields.126 Because the precursors for CQDs differ from those for GQDs, there are noticeable differences between them.127 CQDs, which can be obtained from various
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kinds of carbon materials, are typically quasi-spherical nanoparticles composed of amorphous to nanocrystalline cores with diameters less than 10 nm.30 However, GQDs are typically synthesized from GR-based materials or smaller graphene-like polycyclic aromatic hydrocarbon molecules. Thus GQDs can be regarded as small pieces of GR with lateral dimensions of less than 20 nm with single, double, and few (
Nanoscale Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: Z. Dai and Z. Wang, Nanoscale, 2015, DOI: 10.1039/C5NR00585J.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
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Zhaoyin Wang and Zhihui Dai* Received 00th January 2012, Accepted 00th January 2012 DOI: 10.1039/x0xx00000x www.rsc.org/
Carbon materials in nanoscale exhibit diverse outstanding properties, rendering them extremely suitable for the fabricating of electrochemical biosensors. Over the past two decades, advances in this area have continuously emerged. In this review, we attempt to survey the current developments of electrochemical biosensors based on six types of carbon nanomaterials (CNs), i.e., graphene, carbon nanotubes, carbon dots, carbon nanofibers, nanodiamonds and buckminsterfullerene. For each material, representative samples are introduced to expound the different roles of the CNs in electrochemical bioanalytical strategies. In addition, remaining challenges and perspectives for future developments are also briefly discussed.
1. Introduction Carbon, an element in nature, has been recognised by humans for a long time. With the continuous progress in nanotechnology, the understanding of carbon materials has reached a new level from the macroscopic scale to the nanoscale. Nowadays, diverse allotropes of carbon nanomaterials (CNs) from zero-dimensional (0-D) to threedimensional (3-D) have been found and applied in different fields including nano-electronics and high-frequency electronics,1, 2 energy storage and conversion,3, 4 field emission display,5 theranostic,6 biological and chemical sensors.7, 8 Biosensors are closely related to modern life from diagnosis of diseases to detection of biological agents in the environment.9 Among the various biosensors, electrochemical biosensors are promising due to their high sensitivity, high signal-to-noise-ratio, relative simplicity and rapid response time.10 Owing to the wide potential window, chemical inertness and low cost, carbon has historically been considered as an important electrode material for electrochemical biosensors. For instance, pyrolytic graphite electrode (PGE) and glassy carbon electrode (GCE) are two of the most widely used electrodes.11, 12 In recent decades, with the discovery of the salient properties of CNs, the roles of carbon materials in electrochemical biosensors have gradually expanded from electrode materials to building blocks.13-16 Although prominent characteristics of the different CNs vary, their common features render them extremely attractive for the construction of electrochemical biosensors as detailed below. 1) Electrochemical activity.17, 18
1 | J. Name., 2015, 00, 1-3
Certain CNs and their derivatives can exhibit electrochemical responses due to their intrinsic or endowed electrochemical activity. 2) Electrical conductivity.19 The high electrical conductivity of CNs ensures that they serve as an ideal electron transfer medium in electrochemical biosensors. 3) Large surface area.20 Because of the large surface area of CNs, additional required components can be assembled in electrochemical biosensors. 4) Ease of functionalisation.21-23 CNs can be readily modified, which will enhance and enrich the functions of CNs in electrochemical biosensors. 5) Biocompatibility.24, 25 The biocompatibility of CNs facilitates applications of CN-based devices in biological fields. Because of these properties, CNs can act either as nanoprobes, relying on their dominant electrochemical properties, or as nanocarriers relying on their other multiple properties. By utilizing CNs in electroanalytical assays, analytical performances can be substantially improved. The field of electrochemical biosensors is vast and diverse, and in this review we emphasizes the advantages and development of electrochemical biosensors based on several CNs. Compared with graphene (GR), carbon nanotubes (CNTs) and carbon dots (CDs), carbon nanofibers (CNFs), nanodiamonds (NDs) and buckminsterfullerene (C60) are typically overlooked (Fig. 1). To provide a comprehensive overview, electrochemical biosensors based on these CNs are also briefly introduced in this review. For each CN, only selected examples can be displayed and compared due to space limitations. In addition, it should be noted that distinct routes for CN preparation may greatly affect their properties and
This journal is © The Royal Society of Chemistry 2015
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Carbon nanomaterials-based electrochemical biosensors: An overview
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applications. For more information on CN preparations and other applications, readers may consult the previous reviews and references therein.17, 26-31
2. GR-based electrochemical biosensors GR is a single-atom-thick planar sheet of sp2-bonded carbon atoms that are perfectly arranged in a honeycomb lattice. Since the first mechanical exfoliation of graphene in 2004,32 studies on GR have been dramatically increased, which has stimulated research interest in bioanalytical applications. In particular, GR possesses nearly all of the aforementioned outstanding properties of CNs, making it extremely adequate for electrochemical biosensors. To date, several kinds of GR have been synthesised and utilized in electrochemical analytical protocols. Pristine graphene, obtained by the mechanical cleavage of graphite, has some exceptional properties.19, 33, 34 However, this type of graphene has a low throughput, small size, and hydrophobicity in particular, which limits its applications in electrochemical biosensors. Graphene oxide (GO) substantially increases the hydrophilicity of the graphene layers, but GO is an electrical insulator, which reduces conductivity by many orders of magnitude.35, 36 By eliminating the oxygen-containing groups of GO, reduced graphene oxide (RGO) achieves a unique combination of excellent conductivity, large surface area, high electrochemical activity and ease of functionalization. Thus, compared with pristine graphene and GO, RGO has certain overwhelming advantages for the applications in electrochemical analytical assays. 2.1. Uses of GR as a nanoprobe GR-related materials can be used as electrochemical nanoprobes for electrochemical biosensors in the following two aspects. There is an abundance of oxygen-containing groups on the surface of GO. Through the electrochemical reduction of GO, well-defined signals from the oxygen-containing groups will be obtained. Thus, GO can be directly used as a label for electrochemical biosensors.37, 38 In an impressive example, thrombin aptamer (TBA) was immobilized on the electrode surface by physical adsorption and was further sequentially bound with thrombin and GO.38 According to the electrochemical signal of GO reduction, thrombin measurement was achieved (Fig. 2A). Previous investigations have indicated that GR and its derivatives possess intrinsic electrocatalytic
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activity.39, 40 In particuler, decorating GR with foreign atoms (e.g. potassium (K), nitrogen (N) and sulfur (S)) may further enhance its electrocatalytic activities.41, 42 Based on the high electrocatalytic activity of K-modified GR, Li et al. reported a sensitive and stable amperometric nitrite (NO2-) sensor.43 In another study, a novel N-, S-doped GR was synthesized and implemented for the fabrication of a sensor for use as a highperformance electrocatalyst.44 Good performances in the + + simultaneous determination of Cd2 and Pb2 demonstrated that the N-, S-doped GR had improved electrocatalytic properties, which should be ascribed to the synergistic effects of the dual dopants. 2.2. Uses of GR as a nanocarrier Besides its electrochemical properties, GR possesses other outstanding characteristics, such as a large specific surface area, high conductivity, and an ease of modification. Using GR as a nanocarrier, redox probes,45, 46 recognition components47 or target molecules48 may be assembled into electrochemical biosensors, resulting in improved analytical performances. Redox enzymes can be readily immobilized onto an electrode with the aid of GR, and direct electron transfer (DET) of these enzymes will be promoted in GR-based nanostructures. Fundamental studies have provided certain basic guidance for the construction of high-performance GR-based enzymatic electrodes.49, 50 In a glucose biosensor, GCE was modified by electrochemically reduced carboxyl graphene (ERCGR), which was further used as a substrate for assembly of glucose oxidase (GOx) (Fig. 2B).51 Because of the participation of GR, immobilization of GOx was realized via self-assembly under mild conditions and the DET of GOx was efficiently achieved, leading to better analytical results. Similarly, assembly of GOx with GR was utilized in an on-line microdialysis system, which can monitor the extracellular concentration of glucose in vivo.52 Co-immobilization of a bi-enzyme (cholesterol oxidase and catalase) by GR was investigated to detect cholesterol.53 In addition to redox enzymes, other proteins have also been employed in GR-based electrochemical biosensors.54, 55 An interesting example was provided by Song et al. that chitosanRGO and concanavalin A constituted a sensing layer with net charges, which exhibited a pH-switchable response to negatively charged redox probes.54 According to the principle that oxidation of glucose by GOx or hydrolyzation of urea by urease resultes in a pH decrease or increase, respectively,
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Fig. 1 Schematic illustration of certain allotropes of CNs.
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MINIREVIEW glucose and urea were detected in one sample without separation. GR-related materials, with partial planar structures and abundant groups on the surface, specifically GO and RGO, can serve as suitable platforms for the immobilization of antibodies for electrochemical biosensing.56-58 Eissa et al. reported a GRbased voltammetric competitive immunosensor for the determination of okadaic acid (OA) in shellfish.59 In this assay, the okadaic acid antibody was first appended on the carboxyphenyl modified GR electrode via carbodiimide chemistry. After competition between OA and a fixed concentration of the OA-ovalbumin conjugate for the immobilized antibodies, different electrochemical signals were obtained to reflect the amount of OA. Another practical electrochemical immunosensor for vascular endothelial growth factor receptor 2 (VEGFR2) was described by our group (Fig. 2C).60 In this sensor, graphene was employed to enhance the surface area, accelerate electron transfer and form an interface with sites for antibody immobilization, which provided the foundation for excellent analytical performances. Thus, this sensor could detect the total concentrations of VEGFR2 protein in cell lysates and could discriminate the changes in VEGFR2 expression induced by different inhibitors. Benefiting from the presence of various groups as well as πconjugated domains, anchoring of the DNA probe onto GR can be realized by either covalent grafting or noncovalent π-π stacking, both of which have been well exploited in GR-based electrochemical biosensors.61, 62 To compare the effects of different conjugation approaches, two impedimetric examples for DNA detection are presented in this review.63, 64 If probe DNA is immobilized on the surface of RGO by noncovalent adsorption, then hybridization with target DNA will release the probe DNA from the electrode surface, causing a decrease in the electrochemical signals. In contrast, if probe DNA is covalently bound to RGO, then hybridization will attract more DNA onto the electrode surface, leading to a larger signal. In both of the approaches, RGO helps acquire distinguished
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Nanoscale DOI: 10.1039/C5NR00585J signals before and after hybridization, which can be attributed to the large surface-to-volume ratio and the high conductivity of RGO. Aptamers and DNAzymes are two kinds of functional DNA. The aptamer can bind to a specific target molecule, while DNAzymes have the ability to perform a chemical reaction. Integrating the aptamer and DNAzyme into GR-based biosensors may broaden the sensing range from DNA to other target molecules.65, 66 In one study, TBA was adsorbed onto hydrazine treated GO through the strong noncovalent binding of GR with the nucleobases.67 The binding between TBA and thrombin would greatly disturb the interaction between TBA and GR, resulting in different electrochemical signals. Our group designed another GO-based analytical assay for target DNA or protein (Fig. 2D).68 In this assay, capture DNA (single stranded DNA (ssDNA) or an aptamer) can bind to target DNA or protein, causing the release of the capture DNA from the GO surface and the anchoring of [PMo12O40]3- (PMo12). Depending on the electrochemical activity of the adherent PMo12, the target DNA or protein can be easily analysed. Liang et al. decorated a L-histidine-dependent DNAzyme onto a GR-based electrochemical biosensor, which can be used to detect Lhistidine with an expanded linear range as well as excellent sensitivity and selectivity against other amino acids.66 Many previous studies have demonstrated that GR equipped with metal nanomaterials (MNs) can enhance performances in nonenzymatic electrochemical biosensors.69-72 Guo et al. created a one-step microwave-assisted heating procedure to form a new Pt nanoparticles (Pt NPs) ensemble-on-GR hybrid nanosheet, which has proven to be a good material for hydrogen peroxide (H2O2) and trinitrotoluene detection.73 The experimental results of this work confirmed that GR played a key role in enhancing the activity of Pt NPs. To date, numerous electrochemical bioanalytical assays have been developed based on GR-related materials coupled with MNs, such as Ni,74 Cu,75 Sn,76 Zn,77 Ce-Pt.78 Specially, integrating Fe3O4 nanoparticles into a GR-based biosensor may display novel properties by applying an external magnetic field.79, 80
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Fig. 2 GR-based electrochemical assays using GR as a nanoprobe or a nanocarrier. (A) Illustration of the utilization of GO nanoplatelets as inherently electroactive labels for the detection of thrombin. (Reproduced from ref. 38 with permission from The Royal Society of Chemistry.) (B) Scheme of the DET of the ERCGRGOx/GCE and the electrocatalysis of oxygen and glucose. (Reproduced from ref. 51 with permission from Copyright 2012 Elsevier.) (C) Schematic representation of the GR-based electrochemical biosensing platform for VEGFR2 protein. (Reproduced from ref. 60 with permission from Copyright 2014 Nature Publishing Group.) (D) Schematic illustration of the label-free electrochemical biosensing platform for DNA and protein analysis based on GO. (Reproduced from ref. 68 with permission from The Royal Society of Chemistry.)
3. CNT-based electrochemical biosensors CNTs, firstly discovered by Iijima in 1991,81 can be considered as a graphite sheet rolled up into a nanoscale-tube. CNTs can be divided into two main groups: single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs). Owing to their unique structural, electronic and chemical properties, CNTs have attracted increasing research interest in electrochemistry,82 and many CNT-based electrochemical biosensors have been reported.83 When considering the electrochemistry of CNTs, two aspects, i.e., their structural heterogeneity and composition heterogeneity, should be given special attention.16 Structural heterogeneity indicates that the walls of the tubes have distinct properties from the ends of the tubes. Because of this phenomenon, the rate of electrode transfer may be significantly affected by changing the orientation and arrangement of CNTs on the electrode surface. Composition heterogeneity indicates that the CNTs used for biosensor construction are often impure because of the introduction of impurities during CNTs preparation. However, the unique properties of CNTs still ensure their suitability for certain electrochemical biosensors. For example, because of their stiffness and ballistic conductivity, CNTs are an ideal material for the development of microelectrodes, which are valuable in cell and tissue sensors.84-87 Besides, biosensors containing degradable CNTs have been considered as a future direction of CNT-related researches.88
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3.1. Uses of CNTs as nanoprobes The pivotal roles of defect sites and oxygen-containing groups in the electrochemical performances of CNTs have been well studied,89-91 which explains the mechanism of electrocatalysis of CNT-related nanomaterials. To date, several electrochemical biosensors have been developed by solely and directly using the electrocatalytic activity of CNTs, thus they can be considered as nanoprobes for electrochemical biosensors. At the early stage, Ye et al. designed a rapid DNA biosensor based on the electrocatalytic activity of MWCNTs toward guanine or adenine residues of ssDNA.92 Relying on the remarkable catalytic property of acid-pretreated MWCNTs, homocysteine has also been analyzed using electrochemical methods.93 Subsequently, the excellent electrocatalytic ability of CNTs toward H2O2 was verified and employed in fabricating biosensors. Wu et al. confirmed the electrocatalytical decomposition of H2O2 by chitosan-grafted MWCNTs and have provided an example of the construction of an amperometric H2O2 biosensor.94 Compared with non-doped CNTs, N-doped CNTs reveal higher electrocatalytic activity for the oxygen reduction reaction (ORR) and redox of H2O2.95, 96 Taking the advantage of N-doped CNTs, Xu et al. reported a H2O2 biosensor at a low potential by simply dropping and drying Ndoped CNTs on the surface of a GCE.97 3.2. Uses of CNTs as nanocarriers
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MINIREVIEW Due to the advantages of CNTs (e.g. large specific surface, good biocompatibility, modifiable sidewall and excellent conductivity), loading other materials onto their surface of CNTs may create novel nanomaterials or nanostructures with enhanced properties. In addition, various methods have been exploited to realize the immobilization process.98 Therefore, CNT-based composites become essential scaffolds for electrochemical biosensors. Among the multiple materials used along with CNTs, redox enzymes, antibodies, peptides and other proteins, DNA and MNs are the most productive materials. CNTs can provide direct electrical wiring between the electrode and the active centers of the redox enzymes, promoting the DET of the enzyme by using CNTs as a mediator.99 The paradigm of this electrical communication was displayed by Gooding,100 and a typical biosensor was fabricated by Willner’s group (Fig. 3A).101 In this sensor, SWCNTs and an amino derivative of flavin adenine dinucleotide (FAD-NH2) were covalently and sequentially decorated onto an Au electrode. After reconstituting apo-glucose oxidase on the FAD-NH2 that was linked to the ends of SWCNTs, construction of this glucose biosensor was completed. Since this development, various enzymes have been applied to fabricate biosensors based on CNTs mediated DET.102-104 To further enhance this communication, Kafi et al. developed a network by crosslinking CNTs and Hb on a thiol-modified surface and exploited this network for sensing H2O2.105 By virtue of the CNT-mediated communication between Hb and the electrode, this network exhibits high conductivity and electrocatalytic activity towards H2O2. Antibodies have also been attempted to bind with CNTs to design electrochemical immunosensors. Large surface area and different groups on the surface of CNTs may facilitate the binding between antibodies and CNTs. Meanwhile, CNTrelated materials can resist unspecific adsorption of antibodies to a certain extent.106 Han et al. fabricated vertically aligned MWCNTs by chemical vapour deposition and functionalized them in alkaline solution to produce oxygen-containing groups, which can be used as handles to combine microcystin-LR (MCLR) to the surface of MWCNTs.107 By measuring the change of the electron transfer resistance before and after the conjugation of antibodies, quantification of MC-LR was achieved. By considering the effects of pH and the net charge, Puertas et al. achieved the oriented binding of the antibodies, which greatly improved the performances of immunosensor.108 Peptides and other types of proteins have been further implemented in CNT-based electrochemical biosensors. Mahmoud et al. developed a protease biosensor using a
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Nanoscale DOI: 10.1039/C5NR00585J peptide/SWCNT/gold nanoparticle-modified electrode.109 In this work, they emphasized that significant improvement of sensitivity was achieved by the participation of SWCNTs. Recently, our group described a label-free malondialdehyde biosensor with human complement factor H linked MWCNTs (Fig. 3B).110 The MWCNTs displayed an excellent electronic conductivity and large surface area, both of which were of great value in this sensor. DNA is another type of biomolecules applied in CNT-based electrochemical biosensors. DNA can be immobilized on the surface of CNTs in both covalent and non-covalent modes.111, 112 Zhu et al. demonstrated a new impedimetric DNA biosensor by binding probe ssDNA with dendrimers modified MWCNTs.113 Electrochemical impedance spectroscopy measurements were obtained to determine hybridization events between surface-confined probe ssDNA with target DNA. Another interesting development is the use of aptamers.114, 115 Miodek et al. described a MWCNT-based electrochemical biosensor with the aid of the human cellular prions PrPC aptamer (Fig. 3C).116 In this study, DNA aptamers were linked to dendrimers modified MWCNTs to construct bioreceptors, which can sense the variation of the electrochemical signal caused by PrPC. MNs can be wrapped or synthesized on the surface of CNTs, facilitating the construction of CNT-based nonenzymatic biosensors. Synergistic effects can be found in the composites of CNTs and MNs due to the high conductivity of CNTs and the catalytic activity of MNs. Yang et al. developed a H2O2 biosensor with remarkably improved sensitivity because of the synergistic electrocatalytic activity of Pt NPs and CNTs.117 In another biosensor, a high density of Pd nanoparticles (Pd NPs) was grown on the surface of SWCNTs, and the Pd-SWCNTs nanostructure can be used as a new type of catalyst to fabricate a nonenzymatic glucose biosensor.118 To date, some other MNs (Mn,119 Mo,120 Sn,121 Fe-Pt,122 etc.) have been incorporated with CNTs in electrochemical bioanalytical assays. Magnetic nanoparticles (MNPs) have also been employed in CNT-based biosensors.123 This work showed an alluring model in which a mixture of biofunctionalized MNPs with CNTs can implement magnetoswitchable bioelectrocatalysis in an external magnetic field. Another elegant application was published by our group (Fig. 3D).124 Using manganous pyrophosphate as one superoxide dismutase minic, a novel superoxide anion biosensor was fabricated. In this assay, the electrochemical response was enhanced because of the large surface area and good electronic conductivity of the MWCNTs. More importantly, the biosensor can be used to monitor living cells directly adhered to the modified electrode surface.
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Fig. 3 CNT-based electrochemical assays using CNTs as nanocarriers. (A) Assembly of the SWCNT electrically contacted glucose oxidase electrode. (Reproduced from ref. 101 with permission from Copyright 2004 WILEY-VCH.) (B) Schematic diagram of the construction of a label-free biosensor based on MWCNTs. (Reproduced from ref. 110 with permission from The Royal Society of Chemistry.) (C) Schematic representation of biosensor based on MWCNT-polyamidoamine dendrimer-ferrocene-biotin-strepatavidin and an aptamer. (Reproduced from ref. 116 with permission from Copyright 2013 American Chemical Society.) (D) Schematic illustration of a novel superoxide anion biosensor based on MWCNTs. (Reproduced from ref. 124 with permission from Copyright 2014 American Chemical Society.)
4. CD-based electrochemical biosensors CDs, one kind of 0-D CNs, refer to small carbon nanoparticles.30 Since CDs were firstly discovered by Xu et al. in 2004,125 continuous research efforts have been devoted to these popular materials. Due to the quantum confinement and edge effects, CDs can exhibit outstanding optical and electrooptical properties, similar to conventional quantum dots (QDs). However, certain superior biological properties of CDs cause CDs to outperform QDs in bio-related fields.31 In recent years, the electrochemical properties of CDs have attracted considerable interest and representative achievements have indicated the great potential of CD-based electrochemical biosensors. Up to now, two kinds of CDs, i.e., carbon quantum dots (CQDs) and graphene quantum dots (GQDs), have been widely used in diverse fields.126 Because the precursors for CQDs differ from those for GQDs, there are noticeable differences between them.127 CQDs, which can be obtained from various
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kinds of carbon materials, are typically quasi-spherical nanoparticles composed of amorphous to nanocrystalline cores with diameters less than 10 nm.30 However, GQDs are typically synthesized from GR-based materials or smaller graphene-like polycyclic aromatic hydrocarbon molecules. Thus GQDs can be regarded as small pieces of GR with lateral dimensions of less than 20 nm with single, double, and few (
