Carbon nanotubes (CNTs) have received considerable attention in the field of electrochemical sensing, due to their unique structural, electronic and chemical properties, for instance, unique tubular nanostructure, large specific surface, excellent conductivity, modifiable sidewall, high conductivity, good biocompatibility, and so on. Here, we tried to give a comprehensive review on some important aspects of the applications of CNT-based electrochemical sensors in biomedical systems, including the electrochemical nature of CNTs, the methods for dispersing CNTs in solution, the approaches to the immobilization of functional CNT sensing films on electrodes, and the extensive biomedical applications of the CNT-based electrochemical sensors. In the last section, we mainly focused on the applications of CNT-based electrochemical sensors in the analysis of various biological substances and drugs, the methods for constructing enzyme-based electrochemical biosensors and the direct electron transfer of redox proteins on CNTs. Because several crucial factors (e.g., the surface properties of carbon nanotubes, the methods for constructing carbon nanotube electrodes and the manners for electrochemical sensing applications) predominated the analytical performances of carbon nanotube electrodes, a systematical comprehension of the related knowledge was essential to the acquaintance, mastery and development of carbon nanotube-based electrochemical sensors.
Carbon nanotubes (CNTs) have become the subject of intense researches in the last decades because of their unique properties and the promising applications in any aspect of nanotechnology. Because of their unique one-dimensional nanostructures, CNTs display fascinating electronic and optical properties that are distinct from other carbonaceous materials and nanoparticles of other types. CNTs are widely used in electronic and optoelectronic, biomedical, pharmaceutical, energy, catalytic, analytical, and material fields. Particularly, the properties of small dimensions, functional surfaces, good conductivity, excellent biocompatibility, modifiable sidewall, and high reactivity make CNTs ideal candidates for constructing sensors with high performances. As an example, CNTs have been extensively employed in constructing various electrochemical sensors. Compared with the conventional scale materials and other types of nanomaterials, the special nanostructural properties make CNTs have some overwhelming advantages in fabricating electrochemical sensors, including
the large specific area producing high sensitivity;
the tubular nanostructure and the chemical stability allowing the fabrication of ultrasensitive sensors consisting of only one nanotube;
the good biocompatibility that is suitable for constructing electrochemical biosensors, especially for facilitating the electron transfer of redox proteins and enzymes;
the modifiable ends and sidewalls providing a chance for fabricating multifunctioned electrochemical sensors via the construction of functional nanostructures;
the possibility of achieving miniaturization;
the possibility of constructing ultrasensitive nanoarrays.
In fact, since the first application of CNTs in electrochemistry by Britto et al. [
The electrochemical qualities of carbon material-based electrodes are significantly dependent on the surface properties as the creation of specific surface functional groups (especially oxygen-containing groups) can considerably increase the rate of electron transfer (ET) [
Due to the simple and well-defined responses at carbon materials, the
The critical roles of defect sites and oxygen-containing groups on the electrochemical performances of CNT-based electrodes have been proved by several fundamental research works [
Cyclic voltammograms for the reduction of 1 mM ferricyanide for CNT- and C60 film-modified bppg electrodes. Also shown is the response of a bare bppg electrode and an edge plane pyrolytic graphite electrode. All scans at a rate of 100 mV/s. [Reprinted with permission from Banks et al. [
As a result of the strong intertube interactions, CNTs generally existed as highly tangled ropes and were insoluble in almost all solvents, which greatly hindered their practical applications. In addition, as-produced SWNTs, regardless of the production procedure, usually contain amorphous carbon, carbon nanoparticles and residues from the metal catalysts. To overcome these limitations and to create defect-like sites and oxygen-containing groups on CNTs that favored the electrochemical properties of CNTs, as-received CNTs were usually treated by chemical methods using oxidative, such as refluxing in dilute nitric acid or refluxing/sonication in a concentrated H2SO4/HNO3 mixture [
Decomposition of oxygen related functionalities on o-SWNTs. Transmission IR spectra of o-SWNTs following heating in vacuum to the indicated temperatures. The spectra indicate that carboxylic acid and quinone groups are thermally destroyed in vacuum above about 600 K. Other unassigned surface functionalities are also observed to disappear upon heating. Removal of these functionalities reveals the IR transition associated with a 1580 cm-1 phonon mode of nanotubes. [Adapted with permission from Kuznetsova et al. [
Since their first application in electrochemistry by Britto et al. [
The most widely used methods for fabricating CNT-based electrochemical sensors are the approaches that involve the dispersing of CNTs in a certain solvent with sonication after their purification and activation pretreatments, followed by dropping the resultant suspensions on the electrode surfaces and allowing to dry (i.e., the casting methods). Among the reported solvents, N,N-dimethylformamide (DMF) is the most extensively used polar solvent and more than half of the papers deal with CNT-based electrochemical sensors using DMF as the dispersing solvent [
To improve the solubility and stability of CNTs in their suspensions, various additives are added into solvents to assist the dispersion of CNTs, such as surfactants and polymers. The structures of these species are shown in Figure
Molecular structures of various additives for dispersing CNTs in solutions.
As a typical cationic ion exchange resin, Nafion has been used extensively for the modification of electrode surfaces and for the construction of amperometric biosensors, due to the unique ion-exchange, discriminative, and biocompatibility properties [
Since the report by Abatemarco [
Besides the Nafion dispersing system of CNTs, Wu et al. also developed another CNT dispersing system on the basis of the stable dispersion of CNTs in the aqueous solution of dihexadecyl hydrogen phosphate (DHP) (Figure
Chitosan (CHIT) is a polysaccharide biopolymer (Figure
As a positively charged polyelectrolye, polyethyleneimine (PEI) (Figure
The sol-gel and hydrogel have been widely used in recent years to immobilize biomolecules (e.g., enzymes) for constructing electrochemical biosensors because of their easy fabrication, chemical inertness, thermal stability and good biocompatibility. However, the lack of electrochemical reactivity and the poor conductivity of these materials, especially for silica sol-gel, greatly hinder their promising applications. A typical procedure for preparing CNT-based sol-gel consists of the dispersion of CNTs in solvents, the mixing of the CNT suspensions with the sols and finally the casting of the resultant mixture on the electrode surfaces. Recently, Gavalas et al. prepared CNT aqueous sol-gel composites as enzyme-friendly platforms for the development of stable biosensors [
SEM pictures of the surface of the CNTs sol-gel composite (a) without and (b) with 0.25 mg colloidal silica particles per mg of MWNTs. Bar, 5
Room-temperature ionic liquids (RTILs) are thermally stable salts, liquid at room temperature, constituted by an organic cation and either an organic or an inorganic anion. Unlike traditional inorganic molten salts such as NaCl, NaAlF6, or the eutectic mixture LiCl-KCl, room temperature ionic liquids present a high degree of asymmetry that inhibits crystallisation at room temperatures. RTILs have some marvelous properties, including nonvolatile nature or nonmeasurable vapour pressure, low melt point (even as low as
Environmental scanning electron microscopic (ESEM) images of (a) MWNT/[bmim]PF6 and (b) MCMB/[bmim]PF6, the scale bar is 1
The employement of proteins as functional biomaterials has been widely reported due to the excellent biocompatibility and the abundant derativie surface groups. As a kind of special amphiphilic biomacromolecues, proteins are proved to be capable of dispersing CNTs in water. Lin et al. firstly discovered that SWNTs are naturally protein-affinitive in an aqueous ferritin solution, resulting in significant ferritin-SWNT conjugation and the solubilization of the nanotubes [
Photos of GOx-grafted MWNTs (a) and MWNTs (b) in water (left), and cyclic voltammograms of the MWNT- (a) and GOx-grafted MWNT- (b) modified glass carbon electrode in 0.1 M phosphate buffer solution (pH 6.9) (right) [
Cyclodextrins (CDs) are crystalline, water soluble, and cyclic oligosaccharides built up of glucopyranose units (glucose units) and contain a relatively hydrophobic central cavity and hydrophilic outer surface. The soft cutting of SWNTs by CDs was firstly reported by Chen et al. [
Different from the above weak Van der Waals interactions between the dispersing agents and CNTs, conjugated organic compounds, especially organic dyes containing amino groups, can combine with CNTs via stronger
SEM images of as-received MWNTs (a) and CR-functionalized MWNTs (b) [
The applications of CNTs in electrochemical sensors by self-assembled methods were mainly achieved through the attachment of water-soluble polyelectrolytes on the sidewall of CNTs. The molecular design of strong SWNT/polyelectrolyte multilayer composites has been reported previously [
Representative SEM images of
The negatively charged carboxyl groups on CNTs produced during the acid treatments provide two approaches for the direct self-assembling immobilization of CNTs on electrode surfaces: one is the covalent bonding of CNTs to cysteamine self-assembled monolayer (SAM) modified gold electrodes via the reaction of carboxyl groups on CNTs and amino groups on cysteamine SAM in the presence of coupling reagents; the other is the attachment of CNTs to electrode surfaces via the electrostatic interactions between negatively charged carboxyl groups on CNTs and the positively charged species on the electrode surfaces. The self-assembly of CNTs on a gold electrode was reported by Liu et al. [
A schematic showing the steps involved in the fabrication of aligned shortened SWNTs arrays for direct electron transfer with enzymes such as microperoxidase MP-11. [Reprinted with permission from Gooding et al. [
CNT paste electrodes (CNTPEs) refer to the paste electrodes made up of liquid binder and CNTs. CNTPEs retain the properties of the classical carbon paste electrodes (CPEs) such as the feasibility to incorporate different substances, the low background currents, the wide potential range, the easy renewal and composite nature. CNTPEs also preserve the unique electrochemical properties of CNTs, like the ability to promote electron-transfer reactions of various species, especially for biomolecules. Compared with other methods for preparing CNT-based electrochemical sensors, CNTs in CNTPEs do not need any additional treatment (e.g., purification, modification or shortening treatment) and are suitable for all kinds of CNTs. Britto et al. [
Powder microelectrodes (PMEs) have been proved to be an effective technology for the purpose. PMEs, originated from Liu’s laboratory, are a convenient technique for both the characterization of powder materials and the subsequent electroanalytical applications [
The grinding immobilization of CNTs on the surface of conventional scale electrodes was firstly reported by Luo et al. [
SEM images of CNTs used to fabricate the CNT-modified bppg electrode, magnification (a) 10000 and (b) 33000. [Reprinted with permission from Salimi et al. [
The polymerization of various monomers in the presence of dispersed CNTs in solutions by electrochemical methods has been employed for the immobilization of CNTs on the electrode surface. In this process, CNTs were enwrapped in polymers during the electropolymerization process in the form of counter ions or dopants. Wu et al. reported a marvelous method for the dispersion and immobilization of CNTs on the electrode surface by water-soluble alizarin red S (ARS, 3,4-dihydroxy-9,10-dioxo-2- anthracenesulfonic acid, sodium salt) [
Despite the randomly dispersed CNTs, well-aligned CNTs were also used for constructing electrochemical sensors. These CNT-based sensors were generally constructed from CNT ensembles directly grown from CVD methods. Based on their different density, aligned CNT electrodes have the electrochemical properties resembling conventional scale electrodes at high density or microelectrode arrays at low density. This section will introduce the fabrication of aligned CNT electrochemical sensors made up of these two kinds.
The unmodified aligned CNT electrodes were typically constructed by two steps: the CVD synthesis and the transfer of the aligned CNT ensembles to various substrates. Sometimes, the prepared aligned CNT electrodes were further activated by electrochemical methods. Ye et al. developed an aligned CNT electrode for the selective determination of UA [
The modified aligned CNT electrochemical sensors were usually fabricated through the immobilization of functional species (e.g., biocatalysts like enzymes) by various methods. Soundarrajan et al. developed a simple pyrolytic method for large-scale production of aligned CNTs arrays perpendicular to the substrate [
At low density, the CNT ensembles possessed the electrochemical properties resembled the microelectrodes, which were denoted as CNT nanoelectrode arrays (NEAs). To make each nanotube work as an individual nanoelectrode, the spacing needs to be sufficiently larger than the diameter of the nanotubes to prevent diffusion layer overlap with the neighboring electrodes. In contrast to the conventional scale aligned CNT ensembles at high density, the size reduction of each individual electrode and the increased total number of electrodes result in improvements in both the signal-to-noise ratio and detection limits (DLs). A typical procedure for preparing CNT-based NEAs included the direct CVD growth of well-aligned CNTs with low density, the coating of the CNT layer by insulating materials (e.g., epoxy) and the removing of the protruding parts of CNTs by polishing. Sometimes, the CNT-based NEAs were further electrochemically activated by applying a potential bias 1.0 M NaOH for a certain period to obtain functional groups like carboxyl groups. The major works regarding the construction of CNT-based NEAs were done by Ren [
Fabrication scheme of the NEAs (A): (a) Ni nanoparticles electrodeposition; (b) aligned carbon nanotube growth; (c) coating of SiO2 and M-Bond; and (d) polishing to expose CNTs. Scanning electron microscope images of the NEAs (B): (a) Top view; (b)–(f) from a
Although possessing the electrochemical properties similar to edge plane pyrolytic graphite electrodes, CNTs still have some overwhelming advantages for fabricating carbon material-based electrochemical sensors, including the good conductivity, the good biocompatibility, the large specific area, the well-defined nanotube structure, the modifiable surface, the functional groups on the surface with pretreatments and the capacity of being solubilized or dispersed in solutions. Based on these unique properties, numerous works have been dealt with CNT-based electrochemical sensors, which can be mainly divided into two kinds: the nonenzymatic and the enzymatic CNT-based electrochemical sensors.
Aminothiols are of physiological importance as biological agents and metabolites. The levels of these compounds in biological matrices such as plasma and urine are valuable biomarkers in a number of clinical situations. Increased levels of HcySH and CySH are associated with risk of cardiovascular diseases. The ratio of GSH to glutathione disulfide (GSSG) indicates the redox status of cells. In addition, N-acetylcysteine is an important mucolytic agent used to reduce the viscosity of pulmonary secretions in respiratory diseases. A substantial challenge to the development of electrochemical methods for thiol detection was that the direct oxidation of thiols at solid electrodes is slow and usually large overpotentials are required [
The electrochemical behavior of CySH at CNT powder microelectrodes (CNTPMEs) was investigated by Zhao et al. [
Besides the unmodified CNTs, Pt decorated CNTs were also used for the sensitive determination of thiols. Xian et al. prepared a MWNT/Pt microparticle nanocomposite by electrodepositing Pt microparticles onto the MWNT matrix [
Electrocatalytic oxidation of carbohydrates is of great interest in the points of view ranging from medical applications of the blood glucose sensing to ecological approaches (e.g., waste-water treatment in food industry). Most previous studies on this subject involved the use of GOx, which catalyzes the oxidation of glucose to gluconolactone. However, due to the intrinsic nature of enzymes, such enzyme-based sensors suffer from the stability problem. Thus, great efforts are made to achieve direct determination of carbohydrates. Ye et al. recently reported the direct electrocatalytic oxidation of glucose in alkaline medium at well-aligned MWNT electrodes [
Besides unmodified CNTs, the composites made up of copper particles and CNTs were also used by several works to achieve the electrooxidation of carbohydrates. Male et al. reported a copper decorated CNT-based electrochemical sensor for the deterimiation of carbohydrates [
The determination of neurotransmitters and other important microbiomolecules is of considerable significance in biochemical and clinical diagnosis. Generally, these species are electroactive, providing a simple but sensitive way for their determination by electrochemical methods. However, owning to the overlapping of the oxidation potentials of these species, it is difficult to selectively determine them at bare electrodes. To overcome this obstacle, various materials have been used to modify the bare electrodes. Among these modifiers, CNTs have become the most outstanding material and most recent works on the determination of the microbiomolecules employ CNTs as the sensing materials.
The work regarding the enhanced electrochemical response of some biomolecules at CNT electrodes was firstly reported by Luo et al. [
Sensitive determination of electroactive microbiomolecules at CNT-based sensors.
Targets | Electrodes | Supporting electrolyte | Detection potential/V | Linear range/ | DL/ | Ref. |
---|---|---|---|---|---|---|
UA | SWNT/Au | 0.1 M HAc-NaAc pH 5.0 | 0.451 | 1.0 | [ | |
Thymine | MWNT-CD/CE | NaHCO3-Na2CO3 pH 10.8 | 0.87 | 5.0 | [ | |
NE | SWNT/GCE | B-R pH 5.72 | 0.261 | 6.0 | [ | |
Xanthine (Xa) | MWNT/GCE | HAc-NaAc pH 5.20 | 0.8 | 0.004 | [ | |
DA | MWNT-IE | 0.05 M PBS pH 7.4 | 0.13 | 0.1 |
[ | |
5-HT | 0.32 | 0.2 | ||||
Hypo-xanthine | MWNT-DHP/GCE | 0.1 M PBS pH 7.3 | 1.06 | 0.2 | [ | |
DA | MWNT-IE | 50 mM NaH2PO4 pH 5.0 | 0.4 | 0.1 |
[ | |
AA | 0.13 | 20 | ||||
DA | MWNT/GCE | 0.2 M PBS pH 5.0 | 0.24 | 0.00025 |
[ | |
NE | — | 0.00025 | ||||
5-HT | — | 0.00035 | ||||
DA | SWNT/CFNE | 25 mM Tris-HCl pH 7.4 | — | 0.0077 |
[ | |
EP | 0.038 | |||||
NE | 0.042 | |||||
UA | Aligned MWNTs | PBS pH 7.4 | 0.295 | 0.1 | [ | |
Dopa | SWNT/GCE | 0.1 M PBS pH 5.0 | 0.27 | 0.3 | [ | |
EP | MWNT/bppg | 0.1 M PBS pH 4.3 | 0.18 | 0.02 | [ | |
DA | MWNT/GCE | 0.2 M PBS pH 5.0 | — | 0.0002 |
[ | |
NE | 0.0002 | |||||
5-HT | 0.0003 |
The selective determination of the electroactive microbiomolecules was also performed at CNT composite modified electrodes. Compared with CNTs, the combination of CNTs with other components not only provides higher stability and better uniformity of the composite films but also endows the composite films with the unique properties of both components. A typical example is that the composite films made up of CNTs and Nafion can effectively eliminate the interference of negatively charged AA on the determination of other species due to the exclusion of AA from the electrode surface by a negatively charged Nafion film. Table
Sensitive determination of electroactive microbiomolecules at CNT composite film-modified electrodes.
Targets | Electrodes | Supporting electrolyte | Detection potential/V | Linear range/ | DL/ | Ref. |
---|---|---|---|---|---|---|
UA | CNT- | 0.2 M HAc-NaAc pH 4.5 | 0.45 | 0.2 | [ | |
DA | MWNT-Nafion/GCE | 0.1 M PBS pH 5.5 | 0.235 | 0.0025 | [ | |
XA | MWNT-DHP/GCE | 0.1 M PBS pH 5.5 | 0.77 | 0.04 |
[ | |
UA | 0.38 | 0.01 | ||||
DA | MWNT-DHP/GCE | 0.1 M PBS pH 7.0 | 0.18 | 0.011 |
[ | |
5-HT | 0.36 | 0.005 | ||||
NE | CNT- | 0.1 M PBS pH 6.0 | 0.25 | 0.5 | [ | |
DA | MWNT-CHIT/GCE | 0.1 M PBS pH 7.2 | 0.144 | 0.2 |
[ | |
AA | 2.0 | |||||
DA | MWNT-Nafion/GCE | 0.1 M PBS pH 6.0 | 0.235 | 0.0025 | [ | |
UA | MWNT-CHIT/GCE | 0.1 M PBS pH 5.5 | 0.484 | 0.75 |
[ | |
EP | 0.329 | 1.0 | ||||
DA | MWNT-Nafion/CFME | 0.05 M PBS pH 7.5 | 0.145 | 0.07 | [ | |
DA | MWNT-RTIL/GCE | 0.1 M PBS pH 7.0 | 0.16 | 0.1 | [ | |
EP | MWNT-PBCB/GCE | 0.1 M PBS pH 6.0 | 0.28 | 0.01 | [ |
Amino acids detected by electrochemical methods are generally electroactive and usually contain phenol moieties. Xu et al. prepared a MWNT film-modified electrode and coupled this electrode to an ion chromatography system for the simultaneous determination of oxidizable amino acids, including CySH, tryptophane and tyrosine [
The electrochemical determination of traditionally “nonelectroactive’’ amino acids was also performed by Deo et al. at a SWNT modified electrode that was coated by a nickel hydroxide film [
The development of sensitive analytical techniques of drugs has drawn much attention due to the promising applications in environment protection, pharmacology and biomedical studies. However, due to the chemical stability and the large bodies, the responses of these substances were generally weak at common bare electrodes. To improve the detection sensitivity, various nanomaterials are employed to fabricate modified electrodes with high performance. Among these materials, CNTs have also been widely to improve the sensitivity of some drugs. Compared with other nanomaterials, CNTs have some advantages, such as good conductivity and strong electrocatalytic activity. The enhanced adsorption of some water insoluble or conjugated drugs on the hydrophobic and
Sensitive determination of drugs at CNT-modified electrodes.
Targets | Electrodes | Supporting electrolyte | Detection potential/V | Linear range/ | DL/ | Ref. |
---|---|---|---|---|---|---|
6-Mercaptopurine | MWNT/GCE | 0.1 M PBS pH 7.0 | 0.53 | 0.2 | [ | |
Daunomycin | MWNT-DHP/GCE | 0.1 M PBS pH 5.0 | 0.008 | [ | ||
6-Benzylamino-purine | CNT/GCE | NH3-NH4Cl | 0.65 | 0.005 | [ | |
Chloramphenicol | MWNT-DHP/GCE | 0.1 M PBS pH 7.0 | 0.045 | [ | ||
Indole-3-acetic acid | MWNT-DHP/GCE | 0.1 M PBS pH 2.0 | 0.68 | 0.02 | [ | |
Vitamin B6 | MWNT-DHP/GCE | 0.1 M PBS pH 6.0 | 0.8 | 0.2 | [ | |
Thyroxine | MWNT-DHP/GCE | 0.1 M HCl | 0.8 | 5 | [ | |
8-Azaguanine | MWNT-DHP/GCE | 0.1 M PBS pH 7.0 | 0.86 | 0.01 | [ | |
Insulin | MWNT/GCE | 0.1 M PBS pH 7.4 | 0.8 | 0.014 | [ | |
Tinidazole | MWNT-DHP/GCE | 0.1 M B-R pH 10.0 | 0.01 | [ | ||
Metronidazole | MWNT-DHP/GCE | 0.1 M B-R pH 9.0 | 0.006 | [ | ||
Fluphenazin | MWNT-MPS/Au | 0.05 M HAc-NaAc pH 3.0 | 0.78 | 0.01 | [ | |
Thyroxine | SWNT-DHP/GCE | 0.1 M HCl | 0.78 | 0.02 | [ | |
Reserpine | MWNT-DHP/GCE | 0.1 M PBS pH 6.0 | 0.64 | 0.0075 | [ | |
Mitomycin c | MWNT-DHP/GCE | — | 0.79 | 0.08 | [ | |
Epirubicin | SWNT-DHP/GCE | 0.1 M PBS pH 7.0 | 0.02 | [ | ||
Brucine | MWNT/GCE | H2SO4 pH 1.0 | 0.405 | 0.2 | [ | |
Amitrole | CNTPE | 0.05 M PBS pH 7.4 | 0.9 | 0.57 | [ | |
Simvastatin | MWNT-DHP/GCE | 0.1 M H2SO4 | 1.06 | 0.05 | [ | |
Morphine | MWNT/GCE | 0.1 M PBS pH 7.0 | 0.5 | 0.2 | [ | |
Prochlorperazine | MWNT/DDT/Au | 0.05 M PBS pH 5.8 | 0.69 | 0.1 | [ | |
Melatonin | MWNT-DHP/GCE | 0.1 M PBS pH 7.5 | 0.61 | 0.02 | [ | |
Diclofenac sodium | MWNT-DHP/GCE | 0.1 M HAc-NaAc pH 5.0 | 0.69 | 0.08 | [ | |
Ofloxacin | MWNT-CR/GCE | 0.1 M PBS pH 6.0 | 0.9 | 0.009 | [ | |
Acetylspiramycin | SWNT-DHP/GCE | 0.1 M PBS pH 5.5 | 0.89 | 1.3 | [ | |
Lincomycin | MWNT-DHP/GCE | 0.1 M PBS pH 7.0 | 0.85 | 0.2 | [ |
The incorporation of enzymes in a transducer allows one to fabricate enzyme electrodes which exhibit high selectivity, high-sensitivity and long-term stable response to bioanalytes. Many different materials and methods have been used to immobilize enzymes on the electrode surfaces, such as sol-gels, carbon pastes, conductive polymers and recently nanostructured materials. Of particular interest is the use of CNTs as an electrochemical transducer, due to the excellent electron transfer rate, the well-defined nanostructure, the good biocompatibility, and the modifiable surface allowing biomaterial immobilization of CNTs.
Among all the enzyme-based biosensors, glucose biosensors are most widely studied because the diagnosis of diabetes mellitus requires an accurate monitoring of blood glucose levels. Most of the amperometric glucose biosensors are based on glucose oxidase (GOx), which catalyzes the oxidation of glucose to gluconolactone:
Electropolymerization represents an attractive and well-controlled one-step avenue for preparing amperometric enzyme electrodes. The main advantages of this immobilization avenue are the simple one-step preparation, exclusion of electroactive and surface-active interferences, control of film thickness, and localization of biocatalysts onto tiny electrode surfaces [
Blending of enzymes and some polymeric materials that are capable of forming uniform and stable films on electrode surfaces provides a convenient noncovalent approach for the immobilization of enzymes while maintaining the native properties and reactivities of enzymes. This makes the composite blending technique a potential tool for the development of new electrochemical biosensors. Leaching of the entrapped enzymes does not occur or occurs very slowly because these species usually have large bodies and possible interactions (e.g., electrostatic and hydrophobic interactions) between enzymes and the film matrices might exist. Several types of film matrices have been employed for constructing glucose electrochemical biosensors, including sol-gels, Nafion, and CHIT. However, due to their poor conductivity and film compactness, the performance of the biosensors fabricated from these species and enzymes were usually poor. This deficiency can be completely overcome by the incorporation of CNTs, which leads to the improvement of the conductivity and the diffusion process of the composite films. On the basis of the stable dispersion of CNTs in Nafion solutions, Wang et al. firstly studied the electrochemical behavior of H2O2 at CNT-based electrodes [
As a typical method, carbon pastes have been extensively used to prepare enzyme-based electrochemical biosensor because this method has some unique advantages, such as easy fabrication, low cost, wide potential window, high signal-to-noise ratio and retaining of enzyme activity. Recently, Rubianes et al. studied the electrochemical response of H2O2 at a CNTPE by the hydrodynamic voltammetric method. Similar to that at the MWNT-Nafion/GCE [
Although the noncovalent methods for fabricating CNT-based electrochemical biosensors have some advantages like easy fabrication and low cost, the poor stability and leaching of enzymes sometimes significantly influence the analytical performance of these biosensors. To overcome this limitation, great efforts have been made to covalently attach enzymes to the electrode surfaces via various methods and reagents. Xue et al. described a simple method for fabricating glucose biosensors by covalently attaching GOx onto a SWNT modified Pt electrode [
(a) TEM micrograph of SWNTs in the presence of Pt nanoparticles. Inset: AFM tapping-mode phase image (size, 1
Some other methods have also been proposed by several workers for immobilizing of GOx on CNT-based electrodes to prepare glucose electrochemical biosensors, such as physical adsorption and coelectrodeposition. Wang et al. developed a novel biosensor, comprising of electrode of gold/MWNT-GOx (Au/MWNT-GOx) [
The determination of cholesterol is of great significance in clinical diagnosis of coronary heart disease, arteriosclerosis, myocardial infarction and brain thrombosis. Among the various biosensors of cholesterol, cholesterol oxidase (ChOx)-based electrochemical biosensors are especially attractive because they offer a simple, rapid and inexpensive means of quantifying cholesterol. Similar to glucose electrochemical biosensors, most of the amperometric cholesterol biosensors are based on the ChOx enzyme, which catalyzes the oxidation of cholesterol to 4-cholesten-3-one:
Similar to the oxidases, some dehydrogenases are also employed to construct various electrochemical biosensors, including alcohol-dehydrogenase (ADH), D-fructose dehydrogenase (FDH) and glucose dehydrogenase (GDH). Generally, the response of dehydrogenase-based electrochemical biosenors is a current due to the electrooxidation of NADH that is produced in the enzymatic reaction
The direct electrochemical oxidation of NADH is becoming more and more interesting since many dehydrogenases need it as cofactors to catalyze redox reaction. However, the direct electrochemical oxidation of NADH at a bare electrode takes place at high overpotentials, for example, the reported overpotential at pH 7.0 is about 1.1 V at carbon electrodes and 1.3 V at Pt electrodes. Many efforts have been made to reduce the overpotential of NADH electrooxidation. Musameh et al. [
Based on the electrocatalytic oxidation of NADH at CNT electrodes, several dehydrogenase-based electrochemical biosensors have been constructed. Wang et al. reported a MWNT-based alcohol biosensor, based on the incorporation of alcohol-dehydrogenase/NAD+ within a three-dimensional MWNT/Teflon matrix [
(A) Linear scan voltammograms recorded at (a) GC/CHIT and (b) GC/CNT-CHIT film electrodes in 0.10 mM NADH solution. Traces a1 and b1 were recorded in a background electrolyte solution. For the clarity of presentation, traces b and b1 were shifted downward by 6
There were also some other enzymes used by several workers to prepare electrochemical biosensors, such as acetylcholinesterase, alkaline phosphatase, organophosphorus hydrolase, urease and tyrosinase. Based on the reversible Nernstian response of a SWNT modified electrode to analytes associated with solution pH change, Xu et al. constructed voltammetric urease and acetylcholinesterase biosensors by immobilizing the enzymes with a sol-gel hybrid material that was cast on the surface of the SWNT modified electrode [
The direct electron transfer of redox proteins and enzymes at electrode surface has attracted more and more attention recently. These works are of great importance for studying the biological redox process, which can help us to understand the energy transform and the material metaboly in life action. But unfortunately, it is difficult for these macrobiomolecules to achieve direct exchange electron at electrode surfaces because they usually have big and complex structures with the redox centers deeply immersing in the bodies. Furthermore, the adsorption of these species at the electrode surface often results in the change of their native configuration and the consequent denature. Earlier, mediators were employed by some groups to improve the electron contact between enzymes and electrodes. However, using mediators generally makes the biosensors complicated and leads to a slow response. The development of mediatorless biosensors, thus, becomes the goal of extensive researches. The first work regarding the direct electron transfer of proteins at CNT electrodes was reported by Davis et al. in 1997 by the physical adsorption of horse heart cytochrome c (Cyt c) on CNTs [
Myoglobin (Mb) is a small size heme protein with a molar mass of approximately 17 500, and it functions in both transport and short-term storage of oxygen. Due to its commercial availability and a well-known structure, Mb is considered to be an ideal model protein for the study of heme proteins or enzymes. Thus, the achievement of the direct electron transfer (DET) of Mb not only provides a convenient approach for fabricating Mb-based biosensors but also creates nice model systems for studying the biological functions of these proteins. Similar to other nanoscale materials, the good conductivity, the nanosized tubular structural and the functional groups make CNTs act as molecular wires to allow the electrical communication between the underlying electrodes and Mb. Recently Zhao’s group developed a simple but effective method for the immobilization and the direct electron transfer of Mb at CNT-based electrodes [
(A) CV of Mb/MWNT/GCE in the presence of H2O2: (a) 0 mM, (b) 0.4 mM, and (c) 0.8 mM; scan rate: 200 mV/s. (B) Amperometric response of H2O2 at Mb/MWNT/GCE in 0.1 M PBS (pH 4.0); each addition was 30
Hemoglobin (Hb), functioning physiologically in the storage and transport of molecular oxygen in the blood of vertebrates, has a molecular weight of 64.000 to 67.000, and comprises four polypeptide subunits (two
Cytochrome c (Cyt c) is a basic redox heme protein with 12,384 g/mol molecular weight and approximately spherical shaper with 34
Horseradish peroxidase (HRP), functioning physiologically as the biocatalysis of H2O2, has been extensively investigated, due to its commercial availability and the well-known structure. HRP has been used as a typical model for studying the structural, kinetic and thermodynamic properties of peroxidases. The first work on the direct electron transfer of HRP was reported by Zhao et al. using CNT-based powder microelectrodes (CNTPME) [
Catalase (Ct) is a redox enzyme, present in almost all aerobic organisms. It catalyses the disproportionation of hydrogen peroxide into oxygen and water without the formation of free radicals, so it serves in part to protect cells from the toxic effects of hydrogen peroxide. Catalase contains four equal subunits, and each of them has a molecular weight of 57.000 daltons and is equipped with a Fe (III)-protoporphyrin-IX. The direct electron transfer of Ct was mainly accomplished through physical adsorption approaches. Wang et al. recently studied the direct electron transfer of Ct at a gold electrode modified with SWNTs [
Microperoxidase (MP-11) is an oligopeptide consisting of eleven amino acids and a covalently linked FeIII-protoporphyrin IX heme site. The direct electron transfer between MP-11 and electrodes can be used in biosensors and biofuel cells. In biofuel cells, the MP-11 modified electrodes are used as cathode materials to catalyze the reduction of oxygen. The direct electron transfer of MP-11 has recently been studied by Dong et al. in different CNT-based electrode systems [
Glucose oxidase (GOx), from Aspergillus or Penicillium, is a homodimer with a molecular weight of about
Besides GOx, the direct electron transfer of some other oxidases like putrescine oxidase (POx) and xanthine oxidase (XOx) was also investigated. Similar to other oxidases, POx is a rather big molecule (
Based on the above discussions, it is clear that CNT electrodes can effectively facilitate the direct electron transfer of various redox proteins and enzymes. However, few works gain insights into the fundamental information of this important process. Guiseppi-Elie et al. studied the direct electron transfer between SWNTs and the redox center of adsorbed GOx [
Sequence-specific DNA detection has been a topic of significant interest due to its promising application in the diagnosis of pathogenic and genetic diseases. Electrochemical DNA biosensors are currently receiving considerable attention because of their high sensitivity, portability, low-cost and rapid response. Such devices rely on the conversion of the nucleic acid recognition processes into a useful electrical signal. Similar to other electrochemical sensors, the DNA electrochemical sensors benefit much from the use of CNTs based on the enhanced oxidation of the oxidable bases, the improved responses of the indicaters or the excellent properties of CNTs as the carrier platforms.
Fang’s group has reported several CNT-based DNA electrochemical sensors for the determination of sequence-specific DNA over the last few years [
SEM images of (a) an as-grown MWCNT array, (b) the surface of a polished MWCNT array embedded in an SiO2 matrix, (c) a high-density MWCNT nanoelectrode array (hd,
Wu et al. studied the electrochemical behaviors of DNA, guanine and adenine at a MWNT-DHP composite film-modified electrode (MWNT-DHP/GCE) [
Besides the application in DNA detection, CNTs were also used as the carrier platforms of DNA by several workers. Guo et al. examined the electrochemical characteristics of the covalent immobilization of calf thymus DNA molecules on MWNTs by EDC [
As a new type of carbonaceous materials, carbon nanotubes (CNTs) possess some unique properties that are much different from either the conventional scaled materials or other types of nanoparticles, such as the special and well-defined tubular structure of nanosizes, the excellent chemical stability, the modifiable surface and the strong electrocatalytic activity. These special properties foresee their promising applications in electroanalytical chemistry for constructing CNT-based sensors. The fact is that the modification of electrodes with CNTs has been observed to apparently improve the responses of substrates from small H2O2 molecules to huge redox proteins. Most of current works mainly focused on the fabrication of randomly dispersed CNT-based electrochemical sensors and few reports deal with the fabrications of well organized nanostructures of CNTs. As a tubular nanomaterials, the key advantages of CNTs are their small diameter and huge length to diameter ratio that allows them to be used as molecular wires for facilitating electron transfer between proteins and electrodes or to be organized in a low density manner for using as a single nanoelectrode or nanoelectrode arrays with ultrasensitivity. Since the direct growth by CVD methods inevitably surfers from the need of rigorous conditions, development of simple physical or chemical immobilization methods that can control the orientation of CNTs on electrode surfaces has become a matter of great urgency.
Ascorbic acid
Acteylcholinesterase
Alcohol-dehydrogenase
o-aminophenol
3-aminopropyltriethoxy-silane
3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonic acid, sodium salt
Anodic stripping voltammetry
Acteylthiocholine
1-butyl-3-methylimidazolium hexafluorophosphate
Basal-plane pyrolytic graphite
Ceramic-carbon nanotubes nanocomposite electrodes
Cyclodextrins
Capillary electrophoresis
Carbon fiber nanoelectrode
Chitosan
Cholesterol oxidase
Carbon nanotube paste electrodes
Carbon nanotube powder microelectrodes
Carbon nanotubes
Carbon nanotubes-intercalated graphite electrode
[tetrakis(N-methylpyridyl)porphyrinato] cobalt
Carbon paste
Carbon paste electrodes
Catalase
Cetyltrimethylammonium bromide
Cyclic voltammetry
Chemical vapour deposition
Cysteine
Cytochrome c
Dalton
Dopamine
Diaminobenzidine
Dicyclohexyl carbodiimide
Direct electron transfer
Dihydroxybenzaldehyde
Dihexadecyl hydrogen phosphate
Detection limit
N,N-dimethylformamide
Double-strand DNA
Single-strand DNA
Daunomycin
Differential pulse voltammetry
Formal potential
Ethidium bromide
Electrochemical detection
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
Electrochemical impedance spectroscopy
Redox peak separation
Epinephrine
Environmental scanning electron microscopy
Electron transfer
Flavin adenine dnucleotide
Ferrocenyl-derivatised SWNTs
D-fructose dehydrogenase
Iron-phthalocyanine
Flow injection analysis
Glassy carbon electrode
Glucose dehydrogenase
Glutaraldehyde
Glucose oxidase
Glutathione
Glutathione disulfide
Hemoglobin
Homocysteine
Highly oriented pyrolytic graphite
High performance liquid chromatography
Horseradish peroxidase
Serotonin
Infrared spectra
Layer-by-layer
Myoglobin
Methylene blue
Mesocarbon microbeads
Metallized double-stranded DNA
Multiwalled carbon nanotubes
Microperoxidase
Reduced
Norepinephrine
Nanoelectrode arrays
N-methyl-2-pyrrolidone
N-succinimidyl acrylate
Ordered carbon nanotubes
1-octyl-3-methyl imidazolium hexafluorophosphate
Organophosphorus
Poly(aminobenzenesulfonic acid)
Photoacoustic Fourier transform infrared
Polyaniline
Prussian blue
Phosphate buffer solution
Poly(3,
Poly(diallyl dimethyl ammonium) chloride
Pyrolytic graphite
Powder microelectrodes
Putrescine oxidase
Poly(o-phenylenediamine)
Polypyrrole
Piezoelectric quartz crystal impedance
Room-temperature ionic liquids
Mercapatoacetic acid
Self-assembled monolayers
Saturated calomel electrode
Sodium dodecyl sulphate
Scanning electron microscopy
Signal-to-noise ratio
Escherichia coli single-strand binding protein
N-hydroxysulfo-succinimide
Single-walled carbon nanotubes
Toluidine Blue O
Transmission electron microscopy
Thermogravimetric analysis
Tapping-mode atomic force microscopy
Tetra-phenyl-porphyrin
Uric acid
Xanthine
Xanthine oxidase
X-ray photoelectron spectroscopy
The research is supported by the National Natural Science Foundation of China (Nos. 30770549, 20805035, and 90817103).