VIETNAM JOURNAL OF CHEMISTRY VOL, 50(5) 630-646 OCTOBER 2012
CARBON NANOTUBES-BASED BIOSENSOR: A REVIEW
Phuong Dinh Tarn
.Uivanced hislilute for Science and Technology, Hanoi University of Science and Technology Received 15 June 2012
Abstract
Carbon nanotubes (CNTs) have many distinct properties that may be exploited to develop lof^
generations of biosensor. This work reviews usmg CNTs for biosensor applications. The mam content of this review is to highlight the present researches in the area of carbon nanobutes based biosensors for practical applications. This review aims to give reference source for researcher to help them in developing new applications of CNTs based biosensors.
Keywords: Multi-walled nanotubes, single-walled nanotubes, biosensor 1. INTRODUCTION
A biosensor is a device consisting of a bio- receptor, a transducer and a signal processing system. The bio-receptor is normally a sensitive biological element (e.g. DNA sequence, antibody, enzyme) that is immobilized onto transducer to detect the analysis (e.g. complementary DNA sequences, antigens, enzyme substrate). The transducer is used to convert biochemical signal resulting from the interaction between analyte and bio-receptor mto a measurable signal. Signal processing system converts signal into a readable form such as electncal [1], optical [2] or mechanic signal [3] (fig.l). Biosensor can be also divided into several categories relied on the transducers such as optical biosensor, mechanic biosensor, electrochemical biosensor and conductivity biosensor. According to the receptor type, biosensor can be also classified as DNA biosensor, enzyme biosensor, immunosensor or cell biosensor, etc.
Generally, there are several basic cntena for good and efficient biosensing systems consisting of high sensitivity, low cost, fast response, good selectivity, simple operation (easy handling) and high reliability. To date, biosensor continues to make significant effect in everyday life with application range from pathogen virus detection to food analysis [4, 5]. This has led to intensive researches in the developing of new sensing materials for biosensor application that can reduce size, weigh, low cost as well. Materials commonly used for biosensor including conducting polymer, semiconductor metal oxides, and other nano
composite structured material. Recent development of nanotechnoiogy has made huge potential to build a biosensor with highly sensitivity, low cost, easy to use. With the extremely high surface to volume ratio and porous structure, nanomaterials such as nanowires, nanotubes, nanopaticles, nanobelts have been widely studied for biosensor applications.
Among nanomaterials, carbon nanotubes have attracted much attention. Since lijima fist discovered carbon nanotubes in 1991 [6], the CNTs has gained the attention of researchers to develop CNTs-based biosensor for vanous applications [7-13] due to their unique properties such as high surface area, good biocompatibility, chemical stability and high electncal conductivity.
Fig. 1: Schematic diagram of biosensor
The CNTs relate to the family of fullerene structures. Depending on the arrangement of the graphene cylinders, the CNTs can be divided into two structiu-e form as single-walled carbon
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nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs). The SWCNTs comprise a single graphite sheet rolled with a tube diameter of 1-2 nm; whereas the MWCNTs are concentric closed graphite with diameter of 2-50 nm and an interlayer distance approximately 0.34 nm as indicated in Fig. 2.
Fig. 2: Schematic of a single cylindrical layer of carbon nanotubes (a), HR TEM image of an individual multi walled nanotubes. The parallel iiingers have 0.34 nm separation between them and
correspond to individual layer of the coaxial cylindrical geometry (b) [14]. HR TEM image of an
individual single walled nanotube (c) [15]
This article reviews to highlight the present researches in the area of carbon nanobutes based biosensor and theu- application, (e.g., DNA sensors, en2yme sensor or immunosensor). In this paper, we will briefly describe the synthesis, purification process, and properties of CNTs. After that, we will review briefly recent advances in developing CNTs based biosensor (fabrication and application).
Finally, concluding and remarks.
2. SYNTHESIS, PURIFICATION AND PROPERTIES OF CARBON NANOTUBES
Phuong Dinh Tarn that is placed in an inert gas (helium or argon) at low pressure (500 Torr). A direct current typically around 100A driven by approximately 20V creates a high temperature discharge between the terminals.
The discharge vaporizes one of the carbon rods and forms a small rod shaped deposit on the other rod.
The nanotubes grow from the surface of these terminals. Arc discharge method produces high quality CNTs with nearly perfect structures. Both SWCNTs and MWCNTs can be grown by this approach. If SWCNTs are preferable, the anode have to be doped with metal catalyst, example, Co, Ni, Mo, Fe. If both electrodes are graphite, the product will be MWCNTs. The schematic diagram of the experimental setup for arc discharge method was presented in figure 3a.
2.1.2. Laser ablation technique
Laser vaporization approach was developed for produce CNTs in 1995 by Ting Guo et al [17]. In this work, the synthesis system including a temperature controlled furnace, a 2.5 cm diameter, 50 cm long quartz reactor tube, and laser beam source as presented in fig. 3b. In this method, graphite target is placed in tube; the tube is sealed and pumped down to < 10 Torr, oven temperature increased to HOO^C for 4-5 h. High-punty (99.99%) argon is then flowed through the tube at a rate of 0.2-2 cm.s"' at a pressure of 500 Torr. The vaporization laser is die second harmonic (532 nm) of a Nd:YAG laser providing 10 ns, 250 mj pulses at 10 Hz. A laser beam is focused onto the graphite rod target that located inside the reactor tube. Target is vaporized in high temperature argon buffer gas and performed to copper collector cooled down with coater. Produced CNTs by laser ablation method were spheroidal with lengths up 300 nm and the inner diameter varied between 1.5-3.5 nm.
2.1.Synthesis of carbon nanotubes
To date, three techniques are usually used to for produce CNTs as follow: arc-discharge technique was firet discovered by Ijima in 1991 [6]. hi 1993, chemical vapor deposition technique (CVD) was proposed by Yacaman et al [16]. And the laser - ablation technique was used in 1995 by Ting Guo et al[171.
2.1.1. Arc discharge technique
The arc discharge approach was first successfully method to synthesize CNTs. In this method, CNTs produced through arc vaporization of anode and cathode terminals made of graphite rods
Fig^ 3: Schematic of arc-discharge technique (a), the oven laser vaporization apparatus (b),
CVD system (c)
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2,7, J. Chemical vapor deposition technique Up to now, chemical vapor deposition is the most popular technique for produce CNTs. In this technique, thermal decomposition of a hydrocarbon vapor IS obtained with a metal catalyst. As compared with laser ablation technique and Arc discharge technique, CVD is simple and economic approach for produce CNTs at low temperature and ambient pressure. However, in yield and purity, CVD method beats the arc discharge and laser ablation approach.
Figure 3c shows a schematic diagram of the experimental setup used for CNTs growth by CVD method. The generalized process for CNTs growth using CVD techniques is presented as follow; A substrate material is cleaned and covered with a layer of metal catalyst particles such as cobalt, nickel, iron, or a combination by thermal evaporation. The furnace is increased to a temperature between 70O''C and 1 SOOT and hydrocarbon gas including acetylene, ethylene is slowly pumped into reactor. Materials grown over the catalyst are collected upon cooling the system to room temperature.
2.2. Punfication of carbon nanotubes
hi all of these three growth methods, CNTs are frequently contaminated with metal catalysts, amorphous carbon or graphitic nanoparticles which may have negative effects on CNTs' inherent properties. Therefore, the removal of these impunties is important in terms of final product to make CNTs based devices more efficient. Up to now, methods developed for CNTs purification, for example, chemical oxidation such as gas-phase oxidation [18-20], liquid phase oxidation with mild oxidants [21], electrochemical oxidation [22]. Or physical based purification such as filtration [23], centrifugation [24], high temperature annealing [25- 26], Microwave techniques [27]. Or combination of both methods [28]. And other techniques [29].
2.3. Properties of carbon nanotubes
The CNTs have many interesting properties for nano-device in general and nano-biosensor in particular. The CNTs can enhance the electrochemical reactivity and promote the electron fransfer reactions of biomolecules which are suitable for the electrochemical biosensors. This material can display a semiconducting material that has been doped with other nano-materials for biosensor applications. Additionally, the CNTs have high electric conductivity and specific surface, good
Carbon nanotubes-based biosensor: a review mechanical properties. Therefore, these properties make CNTs extremely attractive for a wide range of biosensors from enzyme electrode to DNA biosensor. Table 1 summarizes CNTs properties
Table I: Properties of carbon nanotube material
Specific gravity (g/cm*) HIcclrical conductivity (S/cm)
Electron mobility (cm'/(Vs))
Tlicrmal conductivity (W/(mK))
0.8 10'-10*
. 1 0 ' 6000
1.8 lO'-lO' lO'-lO' 2000
3. CARBON BIOSENSOR
NANOTUBES
Due to CNTs have unique electric properties, their large length to diameter aspect ratios provide for high surface to volume ratios and abihty to mediate fast electron-transfer, miniaturized devices for small sample volumes. Consequently, flie CNTs have been widely used in biosensor applications.
Numerous papers dealing with flie use of CNTs for biosensor fabrication have been reported [7-13]. The major problem for preparation of CNTs - based biosensor is the ftmctionalization of the CNTs. To overcome this deficiency, the dispersed CNTs m various suspensions are immobilized bio-receptor.
Subsequently, the bio-receptor immobilized Q^Ts are coated on the transducer surface by physical or chemical approaches. This section wilt focus on introduction general approaches for preparation of CNTs - based biosensor.
3.1. Dispersion of CNTs in vanous surfactants Several scholars have indicated that flie CNTs can be dissolved to form individual tubes in a solvent [30-34]. The CNT dispersion can be performed by different approaches such as mechanics, covalent and noncovalent functionallzation. Bystrzejewski et al [35] studied flie dispersion of multi-walled CNTs (MWCNTs) in sodium dodecyl sulphate (SDS) and sodiimi dodecyl benzenosulfonate (SDBS). The effects of surfactant structure and concentration on flie diameter distribution of dispersed CNTs were investigated in the present paper. Results showed that the SDBS surfactant has 26% to 45% higher dispersing power compared with SDS. Rubianes and R i \ ^ [36]
reported MWCNT dispersion in polyethilenimine in
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preparing electrochemical sensors for glucose detection. In the present study work, no change was found in the stability of dispersed CNTs after 14 days. The dispersion of single-walled CNTs (SWCNTs) in various solvents was also investigated by Ham et al. [30]. In the present shidy, the CNTs were unbundled using sanitation. Dispersion results showed that the nanotubes were found to be well dispersed by surfactants with hydrophobic alkyl chains equal to or longer than a decyl group.
Vukovic et al. [37] used amino groups to disperse MWCNTs via surface functionalization. In the present study, the MWCNT function in 1,4- phenylenediamine using N-HATU at 40°C for 4 h produced the best dispersion compared with other
3.2.Immobilization of bio-receptor on wall of CNTs The bio-receptor immobilization technique is one of the key factors for a reliable biosensor preparation. A critena for bio-receptor immobilization is not denature of bio-receptor components, maintain their structure, and not to be desorbed durmg the use of the biosensor [38], To date, flie immobilization of bio-receptor onto CNTs may be separated into none-covalent attachment, and the covalent attachment.
3.2. L Non-covalent immobilization technique The non-covalent attachment is based on the ability of the extended K system of CNTs sidewall to bind wifli bio- receptor via 7C-7i stacking interactions or through van der Walls interactions between the bio-receptor and the CNTs. Adsorption is a commonly used non-covalent method which involves the bio-receptor being physically adsorbed onto the CNTs, The schematic representation of a non covalent attachment is shown in figure 4 (a).
This approach can be performed by direct physical absorption onto the CNTs or absorption with an assistance of substances such as linking molecules, or polymer [39, 40]. Zhou et al reported a non- covalent attachment method of NAD"^ cofactor onto the CNTs for preparation of elecft-ochemical biosensor to determine glucose concentiation [41].
h this work, they demonsti^ted the confinement of NAD* cofactor onto sensor surface is based on the stiong n-% stacking interaction between the adenine subimit in the NAD* molecules and the CNTs. The results showed that the adsorption of NAD* cofactor on the CNTs is stable and very responsive toward glucose. This method is simple
Phuong Dinh Tarn and reliable, very suitable for development of biosensor. The noncovalent assembly of concanavalm A (Con A) on carbonxylated CNTs with poly (diallydemethylammonium) (PDDA) as linker was consftojcted by Xue et al [42]. They showed that due the PDDA and ConA can be integrated onto flie CNTs at pH 7. 4 as present in figure 5.
SotlicflllOn(t25W. 120minsl Canlrirugation(2Q00Tjni 1h) CNTslmiiilliiS _ DNAsl[9ii!Js__„ _ ^ ^ _
Fig 4: (a) A schematic representation of a non covalent attachment. This interaction is based on the
ability of the extended n system of CNTs to bind between DNA sequence and CNTs via n-n stacking
interaction, (b) immobilization of ammo on CNTs using covalent attachment. The CNTs are oxidized to form carboxylic group (1), in the second step, carboxylic group react with amine via an acid/base
reaction (2)
' - J - --,
. J PDDA co^urg ^ M'WCNT <^y°n uwcml;E
h
"'..^'^••Wm |wH(.t-.on4>
• I" _• [ S l o w i n g
P'--^' ^^J^^^,^^,^,^
" " n ia««r.iiiBiiiaa- _,.„.,\. ....-...=
• • • U I M H I
Fig. 5: Schematic diagram of noncovalent lectin frmctionalization of CNTs and electiochemical (I)
and optical (II) monitoring of dynamic glycan expression on living cell [42]
3.2.2. Covalent immobilization technique Covalent immobilization of bio-receptor on CNTs has been demonstrated in many studies [43- 47]. hi order to attach bio-receptor to CNTs, the first 633
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requirement is the formation of functional groups onto the CNTs. Normally, the carboxylic acid group is usually the best choice due to it could undergo a vanety of reaction and is easily formed on CNTs through oxidation treatment. This treatment also creates several functional groups including carbonyl, hydroxyl at the defect sites of the outer graphene sheet [43]. A schematic of a possible immobilization process using covalent attachment is shown in figure 5 (b). Niu et al studied immobilization of DNA sequence onto MWCNTs using covalent attachment method for DNA sensor application [44]. In this work, MWCNTs was functionalized with carboxylic acid group, and then, DNA sequences with the 5'amino group were covalently bonded to the carboxylic group of MWCNTs. The results showed that the using of covalent attachment techniques, the sensitivity of biosensor was enhanced with a detection limit of 3.81x10'" M. Wang et al developed a covalent immobilization using glutaraldehyde as an arm linker to fabricate biosensor [45]. The nanocomposite containing chitosan and MWCNTs was coated on a glassy carbon electrode Then a highly reactive dialdehyde regent of glutaraldehyde was applied as an arm linker to covalent graft the 5' amino modified DNA probe to the chitosan-MWCNTs surface though the aldehyde-ammonia condensation reaction. Park et al [46] investigated a covalent attachment method using plasma-functionalized MWCNTs for fabrication of DNA sensor. In this paper, the MWCNTs were treated with O2 plasma to create ftmctional groups such as ~ C - O, C = O and - COO. And then, the MWCNTs were chemically activated with an aqueous solution containing EDC and NHSS. Finally, the MWCNTs were immersed in solution containing amine terminated probe DNA sequence. The MWCNTs based DNA sensor was successfully operated in a target concentration range of lOpM to 100 nM and had a lower detection limit than a pnstine MWCNTs based on DNA sensor. In a simple, inexpensive, and environmental friendly method using microwave for immobilization of amino acids was developed by Zardini et al [48], hi this method, the pristine MWCNTs were sonicated with a mixture of HNO3 and H2SO4 in order to produce MWCNTs -COOH. The MWCNTs products and NaNOa were sonicated in dimethylacetamide solution. The products were then heated in an industrial microware. Finally the products were incubated in solution containing Escherichia coli bacterial for 3h at 3 7 T .
As above mentioned, each attachment approach has advantages and disadvantages. With non covalent attachment method, the immobilization is
Carbon nanotubes-based biosensor: a review not stable due to a very weak interaction between solid supports and the biomolecules. However, this method is simple, easy for use. The covalent attachment approach is more stable but expensive and complicated. Thus, the choice of the most appropriate method for bio-receptor immobilization depends on feasibility of the approach in the study process.
4. CNTs-BASED BIOSENSOR APPLICATION 4.1. CNTs based DNA biosensor
DNA correlated with the genie diseases is an important molecule for human life. Thus, the detection of DNA is plays a major role in clinical, forensic as well as pharmaceutical application and environmental analysis. Many methods were used to detect DNA sequence such as polymerase chain reaction (PCR) [49], ELISA [50], as well as DNA sensor [7-13]. The nanomaterials and its hybnd materials based DNA sensor has been developed in the last few years. Among them, the combination of CNTs with DNA sequences for DNA sensor device has gained the attention many research groups [7- 13]. In this below part, we discuss recent studies in development of DNA biosensor based on carbon nanotubes.
4.1.1. DNA biosensor based on CNTs modified electrodes
The biosensor based on CNTs modified electrodes (eiflier glassy carbon electrodes (GEC) or metal electrode (Au, Pt)) for amperomeOic or voltammetry detection was investigated by many research groups [51-55]. Such electrodes were fabricated by casting or depositing a solution of CNTs onto siuface of electrode. And then, the DNA sequences can be attached on modified electrode by covalent attachment or non-covalent method (Fig. 6).
Wang et al described a DNA biosensor based on MWCNTs [51]. In this paper, DNA probe sequences were attached by forming covalent amide bonds between carboxyl groups at the nanotubes and amino groups at the ends of the DNA stiands as illusttated in figure 7. They indicated that hybridization between DNA probes and its complementary stiands was determined by the changes in flie voltaimnetric peak of indicator of methylene blue.
The DNA biosensor based on chitosan film doped with carbon nanotubes was reported by Li et al [52]. In this report, CNTs wrapped by chitosan film were immobilized on the surface of graphite
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Fig. 6. Schematic diagram illustrating the electrode structure. Photographs of (a) electiode whose sensor has an overall size of 11 mm x 3.5 mm, (b) the effective area of the sensor, which is 1.2 mm x 1.5
mm (detailed); (c) a cartoon of device-deposited CNTs; and (d) a typical FE-SEM image of
electrode-deposited CNTs
Fig. 7. A schematic representation of immobilization DNA stiands onto MWCNTs modified gold electiodes and detection of DNA
target sequences [51 ]
electiodes. Methylene blue was employed as a DNA indicator. The hybridization was determined by using differential pulse voltammetry signal. It was found that a detection limit of 0.252 nM fish sperm DNA was achieved with a linear range of 0.5 - 10 nM. 23iang et al presented an electrochemical biosensor based on zinc oxide nanoparticles and MWCNTs [53]. The DNA probe was immobilized onto the MWCNTs / ZnO / CHFT composite film modified electiode. The hybridization events were detemiined by differential pulse voltammetiy using methylene blue as indicator. They showed that the sensor has a detection limit of 2.8 x 10''^ ML"' of DNA target sequence. In another study, Wang et al studied biosensor based on the covalent immobilization of DNA probe on chitosan- MWCNTs nanocomposite by using glutaraldehyde as an arm linker [45]. The CS-MWCNTs gel was dropped on electiode, and then immersed into glutaraldehyde solution. Finally, elecfrode was incubated in DNA solution. The hybridization was monitored by electrochemical impedance
Phuong Dinh Tam specti-oscopy using [Fe(CN)6]^"'^" as an indicating probe. The results showed that the biosensor obtained a low detection limit of 8.5x10"'" M and a detection range from 1x10"'^ M to 5x10"'" M. hi 2009, we developed a DNA sensor based on interdigitated electiode using MWCNTs for direct and label free detection of influenza virus [54]. The functionalized MWCNTs act as linkers to immobilized DNA probe sequence on sensor surface. With the current design of our sensor, we demonstiated a detection limit as low as 0.5nM of DNA target concentiation with response time about 4 min. Recently, Muti et al developed a biosensor using SWCNTs polymer as linker to immobilized DNA sequence on electiode [55]. Graphite electrode modified by SWCNTs-poly(vinylferrocenium) (PVF*) polymer, and then immersed mto the solution containing of amino-linked HBV probe. To determine hybridization of DNA sequence, DNA probe immobilized electiode was immersed into DNA target solution. The voltammetnc transduction measurements were performed by scanning from - 0.1 V to -t-1.45 V with the scan rate of 50 mV.s"'.
The hybridization signal was determined by change in the guanine oxidation signal.
4.1.2. DNA biosensor based on CNTs-FET The first carbon nanotube field effect tiansistors (CNTFET) were reported in 1998 by Tan et al [56].
These were fabricated by depositing SWCNTs on platinum or gold electiodes in which served as source and drain, connected via the nanotubes chatmel and the doped Si substiate served as the gate. To date, due to high biocompatibilities, the CNTFETs are a promising candidate for DNA sensor. Subramanian et al reported a DNA sensor based on CNTFET arrays to detect Escherichia coli 0157 [57]. hi this work, the CNTFET was fabricated by CNTs grown on 500 pm thick silicon oxide substrate and contacted by two electiodes labeled source and drain. The DNA probe was immobilized onto the CNTFET by covalent attachment. The results showed fliat DNA CNTFET can determine DNA target concentiation as low as Ipg.pL''. The carbon nanotubes network field effect ttansistors (NTNFETs) to detect DNA hybridization published Star et al [58]. The DNA probe was immobilized on CNTs by non-covalent attachment.
They found that the DNA hybridization wath complementary DNA target sequence take place on the device surface that results in reduction of NTNFETs conductance. This point was also demonstiated by Hwang et al [59]. Gui et al [60]
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reported on the sensing mechanism of electiical detection of deoxyribonucleic acid (DNA) hybridization for Au- and Cr-contacted field effect tiansistors based on single-walled carbon nanotube (SWCNT) networks. According to Gui et al, the DNA binding to CNTs occurs in the channel area, which accounts for part of the drain current change.
The different electiical characteristics DNA immobilization and hybridization were also observed in Au-and Cr-contacted CNTsFET as presented in Fig 8.
The results showed that the DNA sensing mechanism is though the charge density modification near the electrode SWCNTsjunctions.
4.2 CNTs based on Enzyme biosensor 4.2.1. Glucose biosensor
Glucose biosensor, one of the most popular biosensor has been intensively studied by scientists and firms due to its importance in clinics, environment and food industry. Several commercial
Carbon nanotubes-based biosensor: a review
<i0h-^
Fig. 8: Schematic diagram illustrates transfer curves of CNTFETs with (a) SWCNTs-Au, (b) SWCNTs- Cr, and (c) dual contact before immobilization and after immobilization and upon hybridizatitm with
DNA target sequence [60]
Table 2: DNA biosensor using CNTs
Anal vie
ILSI D \ , \
Salmonella Test DNA nbona\in Test DNA Test DNA Test DNA Influenza virus
Test DNA Thrombin E,coli0157;H7
Hepatitis C H63D
Iransduccr niKn*ckLin)dcs CN I s L-lcctrodc CNTs electiode
clcclrodc electrode electrode electrode electrode electrode CNTFET CNTFET arrays
CNTFET NTNFET
| ) > . t . , . i | . . i i """ H e l L C l i u n "
lik-cIinchcmiLal 1 lOdxlO' hleclruchcmical I IxlU"
Electiochemical Elcclrochcmical Electrochemical Electrochemical Electrochemical Electncal Electncal Electrical Electncal Electncal Electi-ical
3.5x10'-' 1.43x10"
6.2xl0''' 1.4x10"'"
1,0x10"'-' o,5.\ia"
S.SxlO"'""
0.5x10"'-
Linciirils
1.0x10'+
1.0x10"
5.0x10-'+
3.0x10' 5.0x10"'°+
1.0x10"
6.7x10'°+
8.4x10' 1.0x10"+
5x10"' 1.0x10'+
10x10"' 1.0x10"+
5x10'°
1.0x10"'+
1x10"' 7 8 9 10 61 62 63 54 45 64 57 65 58
636
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devices are widely used for in vitio monitoring of glucose. To date, the researchers have showed that the glucose measurements are based on interaction with one of the three enzymes such as glucose oxidase, hexokinase, and glucose -1-dehydrogenase.
They have been also shovm that glucose concenti-ation can be related to the amount of oxygen consumed or the amount of hydrogen peroxide produced or the decrease in pH because of the conversion of D-gluconolactone to D-gluconic acid [66]. Table 3 summarizes the glucose biosensor based on the CNTs. The CNTs - based glucose biosensor was developed first in 2003 by Wang et al [67]. The MWCNTs - based biosensor exhibits a
Phuong Dinh Tam stiong glucose response at applied potentials of 0.65 and 0.45 V versus Ag /AgCI. A high stability was achieved of 86.7% of the initial activity to glucose after four month storage. To date, glucose biosensor was studied by groups [68-76]. Lin et al reported a glucose biosensor based on CNTs nanoelectiode ensembles [68]. This paper described glucose oxidase was covalently immobilized onto the CNT NEEs via carbodiimide chemisti-y by forming amide linkages between their amine residues and carboxylic acid groups on the CNTs tips. The results obtained a linear response of the biosensor was up to 30 mM of glucose and a detection limit was 0.08 mM.
„ /Vualyli: ...
Glucose Glucose Glucose Glucose Glucose Glucose Glucose
Table 3: Glucose biosensor based on CNTs
,. .Detection metiiod -.
c lee [roc hemic a 1 electrochemical electrochemical electiochemical electrochemical electrochemical electrochemical
Detection ')7.10"'' 1.0.10"' 8.10"' 0.01.10"'
0.5.10"'
Scnsitivily 5»,') 9.32 1096 2190 20.6
Liiicaj-ily range 1 i'\ 10"'+23x10"' 0.25x10"'+3x10"' Up to 7.5x10"'
Up to 3x10"' 0.05x10"'+13x10"'
Up to 8x10"' 1x10"'+1x10"'
. reference
**—^ '
74 13 75 76 71 77 Mariana et A developed an amperometnc
biosensor based on glucose oxidase functionalized CNTs [70]. Glucose oxidase enzyme was immobilized on CNTs entrapped into chitosan matrices by covalent binding with l-ethyl-3(3- dimethylaminopropyl) carbodiimide (EDC), N- hydroxylsuccinimide (NHS). Analytical parameters were detemiined by cyclic voltammetry and electrochemical impedance spectroscopy. In another study, a biosensor based on covalent attachment of GOx on MWCNTs grown direcfly on Si with fenicyanide as redox mediator was studied by Chen et al [71]. The MWCNTs were grown direcfly on a layered stiiictiire of Co / Ti / Cr on a SiOj / Si substrate by microwave-heated chemical vapor deposition. The covalent attachment was used to immobilize GOx on carboxylated MWCNTs via carbodiimide coupling reaction. The sensor response showed a sensitivity of biosensor was 20.6 ^iA.mM~' cm ^ that depends on the acid pretieatment and GOx reaction times. A linear range was obtained of up to 8 mM and a response time of < 5 s. Wang et al reported a glucose biosensor based on immobilization of glucose oxidase by cross linking
m matrix of CNT/chitosan (CHIT)/gold nanopaticle (GNp) multilayer films through glutaraldehyde with bovine serum albumin (BSA) [72]. They showed that the eight layers of multilayer film modified GC electrode are the best for biosensor with high stability, good sensitivity, and fast response time. The biosensor based on chitosan/magnetic nanoparticles composite modified MWCNTs were also studied in [73]. In the present study, developed biosensor showed a high sensitive and good reproducibility. A wide detection linear range (1.0 xlO"''-1.0 x io"'° g mL^') for the determination of bovine serum albumin with the low detection limit of 2.8 x 10~" g.mL"' was determined. Hung et al reported glucose electiodes using SWCNTs films [74]. Glucose electiodes were produced by encapsulating glucose oxidase by Nafion binder into flie SWCNTs and vanation in current response as a function of enzyme loading amount. They showed that glucose electiode could determine a glucose concentiation ranging from 0.25 to 3.0 mM. A detection limit and sensitivity of sensor obtained around 97 pM and 32 P-A.mM^'.cm"^, respectively.
VJCVol. 50(5), 2012 4.2.2. Cholesterol biosensor
Cholesterol is a structural component of biological membranes. It is in nerve tissues, brain, skin etc. Monitoring of cholesterol concentiation in human blood is significant attended in clinical diagnosis To date, the nanomaterials based biosensors have been developed to determine cholesterol concentration [78]. In this part, the CNTs based cholesterol biosensor will be reviewed. As mentioned above, the CNTs have good electiical properties that exhibited excellent electron tiansfer capabilities for the oxidation of biomolecules [79].
Thus, the CNTs can be combined with cholesterol oxidase and the nanohybrid materials to construct a cholesterol biosensor. G. Li et al studied a CNTs modified biosensor for determination of total cholesterol in blood [80]. Sensor consists of a working electrode and a reference electiode that screen -printed on a polycarbonate substiate.
Cholesterol oxidase was immobilized on sensor surface. The results showed that biosensor can determine cholesterol concentration over range of 100-400 mg.dL'' and the average sensitivity was 0.0059 pA.mg'.dL"'. Tan and co-worker reported an amperometiic biosensor based on MWCNTs and organically modified sol-gen/chitosan hybrid composite film [81]. The hybnd composite film was used to immobilize cholesterol oxidase on the surface of electiode. The biosensor was used to determine the free cholesterol concentiation in real human blood samples with a sensitivity of 0.54 pA.mM"', a detection limit of 4.0x10'* M, the linear range of 8.0x10"* M to 4.5x10^ M and a response time of 25 s. In another study about amperometnc biosensor, Tsai et al developed a biosensor based on CNTs/chitosan/platinum/cholesterol oxidase nanobiocomposite [82]. In this work, the MWCNTs/chitosan/cholesterol oxidase solution was ultrasonicated to form a homogeneous of MWCNTs/CHIT/COx solution. And flien, this solution was casted on the surface of elecfrode. The results showed sensitivity of sensor obtained of 0.044 A.M''.cm'^, a response time of about 8 s. A biosensor based on nanobiocomposite materials was developed in [83]. The ChEt and ChOx were immobilized onto SiOa-CHIT/MWCNTs subsequently deposited on electiode. The cholesterol concentiation was monitored by using differential pulse voltammetry. This biosensor exhibited a lineariy from 10 to 500 mg.dL"' wifli a sensitivity of 3.8 pA.mM"'. In another study, flie cholesterol MWCNTs / gold nanoparticles/Chitosan composite based biosensor was developed by Gopalan et al
Carbon nanotubes-based biosensor: a review [84]. The sensor was fabricated based on the immobilization of ChOx into a cross-linked matiU of CHIT. The response was measured by cyclic voltammetry and chronoamperometry that showed a linear response to cholesterol in the range of 0.5 to 5 mM with sensitivity of 200 (lA.M'' and response time was about 7 s. Recently, M. Eguilaz et al reported a cholesterol biosensor based nanocomposite of AuNPs/poly-Cdiallyldimethyl- ammoniumchloride (PDDA)/MWCNTs [85]. In fliis work, mixture of AuNPs/PDDA/MWCNTs was casted onto elecfrode. And then, ChOx was immobilized by dropping ChOx solution onto the modified electiode surface. The obtained results in the analysis of cholesterol concentration were showed that the biosensor had a limit of detection 4.4 \iM with a slope of 2.23 ^A.mM"'. The combination between conducting polymer and CNTs to develop cholesterol biosensor was also studied by Dhand et al [86]. In this work, a nanocomposite film composed of polyaniline and MWCNTs was synthesized by clectrophoretically. The cholesterol oxidase enzyme was covalent immobilized on the composite matenal surface. The response measurement performed on the biosensor using LSV reveal a range of 1.29 to 12.93 mM for cholesterol with sensitivity of 6800 nA.mM"' and a response time of 10 s. Photometric studies for biosensor also indicated that biosensor has a shelf life of approximately 12 weeks when stored at 4''C.
4.2.3. Other enzyme biosensor
In addition to the above CNTs based biosensors for substance detection (e.g., glucose, cholesterol), oflier biosensors have also been developed to detect substance including acetycholine, urea, penicillin, creatinine, oxalate or acid uric. The research group of Lee et al reported a enzymatic acetylcholine biosensor based on CNTs thin film [87]. In this study, carboxylated SWCNTs were deposited on top of Cr/Au metal elecfrode, enzymatic was immobilized on SWCNTs - modified electrode. The results demonstrated the output signal linearly decreasing potential referenced to the Ag / AgCl reference elecfrode wifli the increasing pH valuce.
Sensor has a sensing resolution of 10 \xM of Ach and sensitivity of 19 mV/decade. An amperometnc acetylcholine biosensor based on gold nanoparticles and MWCNTs composite was developed by Hou et al [88]. They used a thiolated aqueous silica sol containing MWCNTs and choline oxidase enzyme to drop on the surface of Pt electrode. AuNPs were attached on electrode through chemisoiption and
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electrostatic atti^ction. Solution of poly (diallyldimethylammonium chloride) and AChE was deposited on the AuNPs modified elecfrode. The resuls showed a wide linear range of 0.005-0.4 mM, sensitivity of 3.395 ^lA-rnM"' and response time about 15 s. Chauhan et al reported an amperometric unc acid biosensor based on MWCNTs-gold nanoparticle composite [89]. In this study, uriccase enzyme immobilized - AuNP / MWCNTs layer was deposited on Au electiode via carbodiimide linkage.
The uric acid was monitored by oxidation of enzymically generated H2O2 at 0.4 V. The linear working range of biosensor was obtained at 0.01-0.8 mM with the detection limit was 0.01 mM. An amperometiic oxalate biosensor based on MWCNTs-polyaniline composite film was developed by Yadav et A [90]. Sensor was fabricated by covalent linkage between enzyme and electropolymenzed c-MWCNTs/PANI composite film on Pt wire. The constructed biosensor showed linear response range of 8.4-272 pM, a sensitivity approximately obtained 0.0113 pA.pM' with a detection limit of 3.0 pM.
In another paper, Yaday and co-worker published an amperometric creatinine biosensor based on MWCNTs/PANI composite film [91]. hi
Phuong Dinh Tam this work, creatinine biosensor was fabricated by using enzyme/c-MWCNT/PANI/Pt as working electixide, Ag/AgCl as reference electiode and Pt wire as auxiliary electiode connected via potentiostat. A detection of creatinine was determined as low as 0.1 ^iM with sensitivity of 40 pA.mM'.cm^"'. Chen et al successfully developed a penicillin biosensor by immobilization of MWCNTs, hematein, p-lactamase on electiode [92].
Penicillin concentiation was monitored by electiochemical approach fliat depends on catalysis of the immobilized enzyme to penicillin leading to decrease the local pH value. The results showed that the MWCNTs used as an elecfron tiansfer enhancer as well as an efficient immobilization matiix for the sensitivity enhancement. The detection limit of 50 nM was monitored by biosensor. A potentiometilc urea biosensor based on MWCNTs/silica composite was fabricated with urease immobilized on MWCNTs embedded in silica matiix deposited on die surface of electiode [93]. In this work, urease enzyme was covalently linked with the exposed free -COOH group of functionalized MWCNTs. The results obtained a good response performance to urea detection with linear range from 2.18 x 10"^- 1.07 X 10"^ M wifli response time of 10-25 s.
Table 4: Enzyme biosensor using CNT
Acetylcholine Acetylcholine Uric acid
oxalate Creatinine penicillin
urea Alcohol dehydrogenase
Tnazophos pesticide
pM electrode electrode electrode electrode electrode electrode electrode electrode electrode
^S
0.01 mM 3.0 pM 0.1 pM 50 nM
4.7 pmolL"' O.OlpM
19 mV 3.395 pA/mM
0.0113 pA.pM"' 40 pAiTiMlcm'"
23 mV/dacade/cm'
2.27 pA/mM
O.Sx 10"'-04x 10"' M 0.01 -0.8mM 8.4 - 272 pM
10-750pM 5.0nM-2.6niM 2.18 X 10"'-1.07xl0"'M 0.02-1.0 niM 0.03 - 32 pM
87 88 89 90 91 92 93 94 95
4.3.Cnts-based immimebiosensor
' Immune-biosensor is an analytical device of the biosensor family. It is used to determine antigen- antibody interaction that plays the role of direct numitoring of molecular recognition for diagnosis
application of pathogen. A variety of immunosensor has been developed using of CNTs. Pmgli and co- worker fabricated a label free elecfrochemical immunosensor for cenbuterol detection [96]. The Clenbuterol was covalent linked to MWCNTs by
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using l-(3-dimethyl aminD)-propyl)-3- ethylcarbodiimmde and N-hydroxysulfo- succmimide as cross-linkers and casted on elecfrode surface. The output signal of immunosensor was characterized by cyclic voltammefric analysis that showed a detection limit of 0.32 ng.mL" with a range of 0.8-1000 ng.mL''. A CNTFET based immunosensor for determination of salmonella was investigated by Villamizar et al [97]. In this work, anti-salmonella antibodies were adsorbed on SWCNTs and subsequently were protected with Tween 20 to prevent the non-specific binding of other bacterial or proteins. To check in the presence of bactenal, the functionalized CNTFET was immersed in a bacteria solution containing 500 CFU.mL"' of S.pyotenes for Ih at 37"C. The fig: 9 illustiated the structure of CNTFET based biosensor was investigated by Villamizar et al. The CNTFETs were electtncally characterized with back gate voltage in the range +10 V to -10 V and the bias voltage was fixed at 250 mV. The results showed that immunosensor can detect at least 100 CFU=mL'' of s.mfantis for 1 h. In another study, Villamizar et al reported an immunosensor based on SWCNTFET that could detect at least 50 CFU.mL' of Candida albicans in only Ih [98]. The immunosensor based on SWCNTs for the detection of swine influenza virus HlNl was developed by Lee et al [99]. In this study, they used self-assembled CNTs random network tin film for the electiical detection of H l N l . The resistance shifts upon virus binding were normalized with the resistance of bare chips. The detection hmit of immunosensor was obtained as 10^
fold dilution from multiple tests with about 10%
background signal. The immunosensor based on optical transducer using CNTs was studied by Yang and co-worker [100]. In fliis work, antibody-CNTs mixture was bound onto a polycarbonate film.
Staphylococcal enterotoxin B (SEB) were then detected by ELISA assay on the polycarbonate film with sensitivity at least 6 fold and a linear range of 0.1 ng.mL"' to 100 ng.mL''.
An immunosensor based on microelecfrode was reported by M. Bhattachaiya et al [101]. Gold elecfrodes were patterned in the form of resistors onto a Si/SiO: subsfrate. Antibodies was adsorbed on CNTs-coated elecfrode and then blocked by BSA. Detection of virus was monitored by using vanous dilutions of the stock virus that measured by changing of current voltage before and after the application of the antigen. Recently, Venkatanarayanan et al was published an immimosensor based elecfrochemiluminescence sensor array [102]. In this contiibution, they reported a novel protein array featiiring a patterned assembly
Carbon nanotubes-based biosensor: a review of SWCNTs formed by Inkjet printing on tiansparent ITO substrate. IgG immunoassays were performed using silica nano particles (Si N?) functionalized with the ECL luminophore [Ru(bpy)2PICH2]2+], and IgG labelled G1.5 acid terminated PAMAM dendrimers. The PAMAM is poly(amido amine), bpy is 2,2_-bipyridyl and PICH2 is (2-(4- carboxyphcnyl) imidazo [4,5-f| [1,10] phenan throline). The carboxyl terminal of [Ru(bpy)2PICH2]2+ (fluorescence lifetime ~ 682 ± 5 ns) dye was covalenfly coupled to amine groups on the 800 nm diameter silica spheres in order to produce signi ficant ECL enhancement in the presence of sodium oxalate as co-reactant in PBS at pH 7.2). The results showed that immunosensor has a wide linear dynamic range for IgG conccnti^tions between 20 pM and 300 nM. A detection limit of 1,1
± 0.1 pM IgG is obtained under optimal conditions.
( A )
"1
5tO.
-^^^.
D r a m
^ ^ ~ \
m
. ' • » ' •
.•i^mwf?
S'."A:M ner*o''
Fig 9; (A) Structiu-e of CNTFET biosensor, (B) Antigen-antibody interaction of S.infantis with a
SWCNT fiinctionalized v«fli anti-salmonella antibodies [97]
5. DISCUSSION
It IS known that nannomaterials play an important role in a biosensor construction. Among them, the CNTs have been popularly used due to have unique properties. To construct a biosensor for promising applications, immobilization of bio- receptor onto the transducer surface should be carefully considered as key step due to the important roles of the mount and bioactivity of immobilized bio-receptor with the performance of biosensors.
Generally, due to the CNTs have high surface area, high lEP, which allows the effective immobilization of the larger amoimts of the bio-receptor. This can
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increase performance of sensor with high sensitive, fast response time, stable, reliable.
Therefore, the immobilization of bio-receptor on transducer surface using CNTs as linker is carried out by various approaches. For DNA biosensor, DNA sequence can be immobilized by covalent attachment or adsorption method. For enzyme biosensor (e.g., glucose biosensor, cholesterol biosensor, urea biosensor etc), enzyme have been immobilized by physical adsorption due to its simplicity. For immunosensor, either adsorption or covalent attachment is normally used for antibodies immobilization.
To date, the majority of the current CNTs based biosensor is of the electiochemical type, due to their better sensitivity, reproducibility, as well as low cost. The elecfrochemical sensor can be subdivided into potentiometric, amperometiic and conductometric type. Among them, amperometnc sensors are the most common devices commercially available and have been widely studied in last few years. This sensor activity depends on ciuxent generated when election are exchanged either direcfly or indirectly between a biological system and an electrode. Beside, piezoelectric and optical transducers are also used for the CNTs biosensor.
6. CONCLUSIONS
Witii twenty years history, the applications in CNTs related technology have been developed fast because of their umque inherent properties. This paper reviews the development of the CNTs based biosensors (e.g., DNA biosensor, enzyme biosensor or immimosensor). The synthesis, purification process, and properties of CNTs were briefly descnbed. The preparation of CNTs based biosensors was also presented for overview. The comparative features of biosensor have been summarized in several tables for an easier overview.
As reviewed, although the CNTs have demonstrated their great potential for biosensing experimentally, but there are still challenges remained including ways to confrol morphology and device homogeneity must be addressed before inqjlementing nanotubes based on biosensor in commercial products. Construction of biosensors have a low cost, easy to use is another challenge to be addressed for flie CNTs based biosensor.
However, it is still believed fliat, wifli the increase interests and development of related technologies, the CNTs based biosensors have a promising future and will bring a huge change to biomedicine field.
Phuong Dinh Tam Acknowledgments. This work was supported by
Vietnam's National Foundation for Science and Technology Development (NAFOSTED Code:
103.02-2010.15).
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