Upload
awad-albalwi
View
268
Download
5
Tags:
Embed Size (px)
Citation preview
Project Proposal for:
Modification of Carbone Nanotube (CNTs) with metal
nanoparticles for electrochemical immunoassay of
alpha-fetoprotein
By
Awad Nasser Albalwi
Departement of Chemistry
King Saud Univerisity
Contents
Subject Page N0#
Review for modification methods of CNTsIntroductionEndohedral Functionalization Non-Covalent FunctionalizationDefect FunctionalizationCovalent Sidewall FunctionalizationThe chemical modificationCarboxylation of the terminal carbons and defect sites of CNTsHalogenation of Carbon NanotubesRadical additionsCycloadditions Carbene AdditionAddition of NitrenesNucleophilic Cyclopropanation:Physical functionalizationFunctionalization of SWNTs with oligomers and polymersApplication: biosensor for of detecting glucose:
Project Proposal for:GoldMag -Modified Carbone Nanotube (CNTs) sensing for electrochemical
immunoassay of alpha-fetoprotein1- Abstract2- Introduction
An immunosensor multi-walled carbon nanotubes ( MWCNTs) Alpha-fetoprotein (AFP): Some Latest review 3- Objective:
4-Methodology Modification Carbon Nanotube with Carboxylic Acid group : Carbon Nanotubes modified with Magnetic Nanoparticles Synthesis and bioconjugation of GoldMag-functionalized CNTs Principle the magneto-controlled electrochemical immunoassay 5- Caractrization of bio- GMCNTs
6- References:
2
General Review for modification Methods of CNTS
Introduction:
Carbon nanotubes (CNTs) (fig.1) were discovered by Iijima in 1991, and then have attracted the fancy
of many scientists worldwide. Their small size and unusual physical properties make them become a
unique material with a whole range of promising applications [1].
Large aspect ratios of CNTs with high chemical stability, thermal conductivity, and high
mechanical strength are advantageous for applications to the field emitter [2].
Carbon nanotubes were found to have great potential applications in various fields Such as
biosensors and nanobiotechnology and many others applications, due to the electrical
conductivity and stability toward chemical reaction.[2] Moreover , carbon nanotube (CNT)
consider one of the best sensing materials for electrochemical and biochemical
applications [3,4]. Moreover, the unique chemical, physical, electronic (metallic or semiconducting) and high thermal
properties of carbon nanotubes (CNTs) made them interesting materials for widespread application in the fields such as
electrochemical sensors, biosensors, supports for heterogeneous metal catalysts in organic synthesis, fuel cells,
semiconductors, batteries, random access memory cells, field effect transistor, field emission display, atomic force microscopy
probes, microelectrodes, specific adsorbents to remove organic pollutants from water and waste water and as a potential drug
carriers in cancer therapy. [5]
A major barrier for the preparation of CNTs-based biosensors is the insolubility of CNTs in
most solvents [4]. Moreover, the poor solubility of carbon nanotubes in organic solvents restricts them to be used as
drug delivery agents into living systems in drug therapy. Hence many modification approaches like physical, chemical or
combined have been exploited for their homogeneous dispersion in common solvents to improve their solubility [6]. The
reports appeared till now in the literature reveals that the modification is required to control the dispersion and such
modification introduces specific functionalities as molecular wedges onto the surface of the carbon nanotubes. Thus the
functional groups present on the surface control the lateral interactions between the bundles and separate indivisual tubes.
Hence homogeneous dispersion can be achieved by breaking the close lateral contact between them which enhances the
affinity towards solvents and other related matrices [6 ]. Due to these specific properties, many research groups explored the
development of novel methods for the modification of nanotubes and investigated the mechanistic aspects of these new class
of novel materials called chemically modified carbon nanotubes (CMCNTs).[5]
There are different strategies addressing the surface of CNTs which can be divided in two
general classes (Figure 2). a) The supramolecular approach including the self assembled
construction using non-covalent interactions b) the chemical addition of reactive molecules,
resulting in the formation of a new covalent bond and. [7 ]
The supramolecular approach comprises
1) the endohedral filling
2) the non-covalent functionalization
The chemical approach includes
3) the defect functionalization and
4) the covalent sidewall functionalization.
4
Fig.1
Endohedral Functionalization
One possibility of CNT functionalization is the filling of the inner cavity with molecules,
called endohedral functionalization, which especially is interesting for the development of
nanowires, storage applications of e.g. fuel and nanocontainers. Additionally,
metallofullerenes were encapsulated in CNTs as well as noble metals (e.g. Au, Ag, Pt, Pd)
yielding metallic nanowires. In opened MWCNTs with an inner diameter of 2-10 nm also
bio molecules such as carotene or proteins (lactamase) were channeled in, showing a
41
Fig.2
catalytic activity inside the CNTs. Even the polymerization of conductive polymers inside a
carbon nanotube was achieved.[7]
Non-Covalent Functionalization
One major drawback of CNTs is the poor solubility in organic solvents or water, due to their
strong interfacial π- π -interactions. These properties can also be utilized for a further kind of
functionalization (non-covalent) which coincidently results in an efficient debundling,.
Hereby, a broad variety of e.g. amphiphilic- or -surfactants as well as different kinds of
polymers are added to a CNT dispersion yielding significantly individualization after ultra
sonication or stirring. An important benefit of this kind of functionalization is the
reversibility and the structural integrity of the nanotube as no covalent bonds between the
addend molecules and the nanotube surface are formed. Another invaluable advantage is the
fact that besides the design of the corresponding addend molecules, no special chemical
equipment and knowledge is necessary, making this method very convenient and scalable.
First basic findings in this field exhibited that indeed π - π -interactions are fundamental for
the attaching of addends to the CNT surface, including experiments with small aromatic
molecules such as cyclohexane, cyclohexene. Further on, classical detergents such as
SDS(Sodium dodecylsulfate) or SDBS(Sodium dodecylbenzenesulfonate) have been
extensively becoming standard surfactants in carbon nanotube chemistry and processing,
due to their unlimited availability and low prices in contrast to other surfactant molecules.
As for the classical surfactants, another substance class successfully attached on the CNT
surface, yielding water soluble CNT materials are ionic liquids. Especially the detergent
6
molecules SDS and SDBS can not only interact via π-π -interactions with the CNT surface,
but their unpolar tail can additionally wrap around the nanotube. This binding motif can be
used wrapping a huge variety of synthetic- and bio-polymers as for example DNA around
nanotubes, tailoring the degree of debundling and the solubility in different solvents. The
DNA functionalized samples could be separated into nanotube batches with different
diameters, as well as by ion exchange chromatography and density ultra centrifugation .[7]
Defect Functionalization
One convenient method is the oxidation of the impurities possessing a higher reactivity than
the 1D sp2 lattice of the nanotubes. The fact that nanotubes have two fullerene like end-caps
and in reality are not perfect sp2 cylinders, additionally results in a partial oxidation of the
CNT material. Next to their end-caps nanotubes display various disorders from topological-,
rehybridization- and incomplete bonding defects right up to intramolecular junctions and
Stone-Wales defects. These defects occur preferentially on small diameter CNTs and in
general are locations with an increased reactivity which are oxidized during the purification
process. The oxidation mainly yields an opening of the sp2 carbon lattice and the generation
of carboxylic acid or other oxygen containing functionalities at the defect sites.
There are different oxidation approaches including nitric and sulfuric acid, mixtures of
hydrogen peroxide and sulfuric acid, gaseous oxygen and ozone as well as potassium
permanganate. Additionally ultra sonication steps, further shorten the oxidized nanotubes
yielding an even higher degree of introduced oxygen containing functionalities. Materials
which was treated in such a fashion, afterwards exhibit increased solubility in polar organic
solvents. [7]
Covalent Sidewall Functionalization
One of the outstanding properties of CNTs is their high aspect ratio. In the course of defect
functionalization only the ends and the defects of CNTs can be functionalized, whereby only
a small surface area of the CNT can be addressed. As a consequence, the degree of
functionalization is relatively low and the introduction of defect sites deteriorates the
electronic and mechanical properties of the nanotube. By direct covalent sidewall
functionalization, the complete CNT surface can be addressed, resulting in a very high
degree of functionalization and the preservation of the structural integrity of the CNT
framework. The addition only yields a rehybridization of sp2 into sp3 carbon atoms which
has a minor influence on the mechanic stability of the CNT. the reactivity of the 1D carbon
allotrope (SWCNT) is by far lower in comparison to the 0D representative, the fullerenes.
Although some reaction sequences were successfully transferred from fullerene to CNT
chemistry, “hot addened ” needed. Another opportunity is the activation of CNTs in
combination with subsequently mild reactions, as in the case of fluorinated ones (Figure 3).[7]
8
The chemical modification:
Fig.3
The modification protocol was generally achieved by attaching specific molecule or entity
which imparts chemical specificity to the substrate material [8]. These chemical modifications can be
easily achieved in many ways However, in this work the modification routes are mainly classified into two types namely
surface modification and bulk modification. The surface modification includes electrochemical induced method, polymer
grafting and metal nanoparticle deposition. The later includes chemical reduction of diazonium salts using hypo phosphorous
acid as a reducing agent, thermally activated covalent modification, microwave assisted modification and ball milling
modification.[2]
Here in Fig. 4 is a summarize of the covalent surface chemistry of CNTs,
Fig. 4 Surface functionalization of carbon nanotubes.[2]
Carboxylation of the terminal carbons and defect sites of CNTs:
10
One of the most common functionalization techniques is the “oxidative purification” of
nanotubes by liquid-phase or gas-phase oxidation, introducing carboxylic groups and some
other oxygen-bearing functionalities such as hydroxy, carbonyl, ester and nitro groups into
the tubes[9].
Acid treatment typically involves the use of a strong oxidizing agent . SWNTs have been
refluxed with HNO3, H2SO4, HCl or mixtures of these acids . Many groups have varied
many parameters for this treatment such as duration, concentration, and repeated cycles. It is
known that the use of these processes can cause defects and/or shortening of SWNTs.
Oxidation usually results in the formation of carboxylate groups on the surface of the tube.[2].
the Haddon group using the acid moieties for attaching long alkyl chains to SWNTs via
amide linkages or carboxylate-ammonium salt ionic interactions [10] .
Halogenation of Carbon Nanotubes
Being the strongest element, fluorine can fluorinate the sidewalls of CNTs between room
temperature and 600°C (Fig. 1). Fluorinated CNTs have been extensively characterized
by (TEM), (STM), (EELS), and (XPS), . The sidewall carbons on which fluorine atoms
attached adopt sp3 hybridization and possess tetrahedral configuration. This destroys the
Fig.5 section of an oxidized SWCNT , reflecting terminal and sidewall oxidation [9] [7]
electronic band structure of metallic or semiconducting CNTs and generates an insulating
material. Although there is controversy regarding the favorable pattern of F addition onto
the sidewall of CNTs being either 1,2-addition or 1,4-addition, . The highest degree of
functionalization was estimated to be C2F by elemental analysis. Fluorinated CNTs were
reported to have a moderate solubility ( 1 mg/mL) in
alcoholic solvents.The fluorination reaction is very useful because further substitution of F
can introduce useful functional groups. The fluorine atom can be replaced with alkyl groups
using Grignard or organolithium reagents.The alkylated CNTs are well dispersed in
organic solvents and can be completely dealkylated upon heating at 500 °C in inert
atmosphere, thus recovering pristine CNTs. Several diamines and diols were also reported to
substitute fluorine atoms on fluorinated CNTs. Infrared (IR) spectroscopy was used to
confirm the disappearance of the C–F bond stretching at 1225 cm−1 as the indication of F-
substitution on CNTs. These reacted CNTs, such as amino alkylated CNTs, can be further
modified on the free amino groups to introduce more sophisticated functionalities which
may bind biomolecules for biological applications. Chlorination or bromination reactions to
CNTs were also accomplished through electrochemical means but the products contain
significant amounts of carboxyl or hydroxyl groups. [10]
By treatment Fluorinated CNTs with hydrazine or LiBH4/LiAlH4, the majority of the
covalently bound fluorine could be removed (fig 7, b) ], restoring most of the conductivity
and spectroscopic properties of the pristine material.[9 ].
12
Radical additions:
Molecular dynamics simulations showed that there is a great probability of reaction of
radicals on the sidewalls of CNTs. Experimentally the covalent sidewall
functionalization via radical addition was achieved with diazonium salts.
Electrochemical reduction of substituted aryl diazonium salts in organic media generates an
aryl radical in situ which covalently attaches to the surface of CNTs. The formation of aryl
radicals was triggered by electron transfer between CNT and the aryl diazonium salts in a
self-catalyzed reaction. A similar reaction was later reported, utilizing water-soluble
diazonium salts, which have been shown to react selectively with metallic CNTs. These
diazonium salt functionalized CNTs are well-dispersed in DMF or aqueous solutions. In situ
electrochemical modification of individual CNTs was demonstrated by attachment of
substituted phenyl groups. Two types of coupling reactions were proposed, namely the
reductive coupling of aryl diazonium salts and the oxidative coupling of aromatic amines. In
fig.6
fig.7
both cases a radical species is produced on the surface of the nanotube, which attacks the
carbon lattice to form a covalent bond. In the former case, the reaction resulted in a C–C
bond formation at the graphitic surface, whereas in the latter, amines were directly attached
to CNTs. Reductive alkylation of SWNTs with lithium metal in liquid ammonia followed by
the addition of either alkyl iodides/sulfides or aryl iodides/sulfides is also proposed to be a
radical rocess. The reductive intercalation of lithium ion onto the nanotube surface
in ammonia or in polar aprotic solvents has been observed by TEM and AFM. The
negatively charged tubes were found to exchange electrons with long chain
aryl/alkyliodides, resulting in transient aryl/alkyl radicals. The latter were covalently added
to the graphitic surface of the nanotubes to afford modified products. A similar reaction was
reported for the functionalization of CNTs via one-
electron reduction of benzophenone by potassium. A radical anion is generated from the
reaction of a potassium atom with benzophenone molecule that results in transferring one
electron from the potassium to the benzophenone. The radical anion adds readily to
sidewalls to yield diphenylcarbinol-functionalized SWNTs. Thermal and photochemical
methods have also been applied to the successful covalent functionalization of CNTs with
radicals. Alkyl or aryl peroxides were decomposed thermally and the resulting radicals
added to the graphitic network. The reaction of CNTs with succinic or glutaric acid acyl
peroxides resulted in the addition of carboxyalkyl radicals onto the sidewalls. These acid-
functionalized CNTs can be converted to materials with new functions. Addition of
perfluoroalkyl radicals to CNTs was obtained by photo induced reactions. [10]
Tour et al 2001, reacted a series of aryl diazonium salts with purified
14
SWCNTs in an electrochemical reaction, using a bucky paper as working electrode, to
prepare a variety of functionalized SWCNTs . The corresponding reactive aryl radicals are
generated from the diazonium salts via one-electron reduction (Scheme 8, a) & latter the
authors described in similar reactions the in situ generation of the diazonium ions from the
corresponding anilines (Scheme 8, b).[9]
Marcoux et al 2004, described the covalent exohedral derivatizations of HiPco nanotubes,
through electrochemical reduction of aryldiazonium salts, After the evolution of the spectra
(area of the D-band), as a function of the number of grafted groups, led to the conclusion
that the electrochemical reduction of aryldiazonium salts into radicals gives rise to the
growth of aryl chains on the sidewalls of the nanotubes (scheme 9) [8].
Scheme18
Cycloadditions
Carbene Addition
In the course of a study on organic functionalization of CNTs, Haddon et al. discovered in
1998 that dichlorocarbene was covalently bound to soluble SWCNTs (Scheme 10) [11]. The
carbene was first generated from chloroform with potassium hydroxide [12 656], and later from
phenyl(bromodichloromethyl)mercury [11]. However, the degree of functionalization was
low: a chlorine atom amount of only 1.6 wt % was determined by XPS [.Because of impure
starting material and a large amount of amorphous carbon, the site of reaction could not be
ascertained [9].
16
scheme 9Illustrations (b) and (c) schematize two samples giving rise to D-bands that have the sameintensity (same number of covalent bonds on the sidewalls of the nanotubes)but show different amounts of bromine in the XPS spectra. This difference can be explained through the growth of aryl chains on SWNTs.[8]
Addition of Nitrenes
Sidewall functionalization of SWCNTs was achieved via the addition of reactive
alkyloxycarbonyl nitrenes obtained from alkoxycarbonyl azides. The driving force for this
reaction is the thermally induced N2-extrusion (Scheme 11). Such nitrenes attack nanotube
sidewalls in a [2+1] cycloaddition forming an aziridine ring at the tubes’ sidewalls [9].
Jiang et al 2011 reported a new approach to produce amino-CNTs by cycloaddition of
nitrenes as in the Scheme [13]
Fig.10 Functionalization of carbon nanotubes by cycloaddition reactions.
7
Scheme.12 produce amino-CNTs by cycloaddition of nitrenes
Scheme.11
Nucleophilic Cyclopropanation:
Fullerenes are known to react easily with bromomalonates to form cyclopropanated
methanofullerenes .A similar reaction was performed by Coleman et al. [14] using purified
SWCNTs and diethyl bromomalonate as addend (Scheme 13). The authors have developed a
chemical tagging technique which allows the functional groups to be visualized by
AFM.Immobilized SWCNT derivatives were transesterified with 2-(methylthio)ethanol, and
by exploiting the gold–sulfur binding interaction the cyclopropane group was “tagged” using
preformed 5 nm gold colloids [14].
Scheme 13 . Nucleophilic Cyclopropanation
18
Physical functionalization
Functionalization of CNTs using covalent method can provide useful functional groups onto
the CNT surface. However, these methods have two major drawbacks: firstly, during the
functionalization reaction, especially along with damaging ultrasonication process, a large
number of defects are inevitably created on the CNT sidewalls, and in some extreme cases,
CNTs are fragmented into smaller pieces. These damaging effects result in severe
degradation in mechanical properties of CNTs as well as disruption of p electron system in
nanotubes. The disruption of p electrons is detrimental to transport properties of CNTs
because defect sites scatter electrons and phonons that are responsible for the electrical and
thermal conductions of CNTs, respectively. Secondly, concentrated acids or strong oxidants
are often used for CNT functionalization, which are environmentally unfriendly. Therefore,
many efforts have been put forward to developing methods that are convenient to use, of
low cost and less damage to CNT structure. Non-covalent functionalization is an alternative
method for tuning the interfacial properties of nanotubes. The suspension of CNTs in the
presence of polymers, such as poly(phenylene vinylene) [15] or polystyrene [16], lead to the
wrapping of polymer around the CNTs to form supermolecular complexes of CNTs. This is
a typical example of non-covalent functionalization of CNTs (Fig. 13A). The polymer
wrapping process is achieved through the van der Waals interactions and π-π stacking
between CNTs and polymer chains containing aromatic rings. [17]
Functionalization of SWNTs with oligomers and polymers
SWNTs are considered to be the ideal reinforcing fibers due to their exceptional mechanical,
electronic and thermal properties, low density and high aspect ratio. However, the
incorporation of SWNTs into the polymer matrix is often problematic due to the chemical
inertness of SWNTs. The covalent functionalization of SWNTs is a valuable route towards
the development of high-performance composites. It provides homogeneously dispersed
SWNTs incorporated in the polymer and a strong interfacial bonding between the polymer
and SWNTs. Bekyarova et al 2004reported appling chemically functionalized SWNTs to
prepare a number of SWNT-polymer composite materials. In one approach, poly (m-
aminobenzene sulphonic acid), PABS, a conducting water soluble polymer, was covalently
attached to chemically functionalized SWNTs (Scheme 1).[18]
In another study Sen et al 2004 & co-workers have prepared SWNT-reinforced
polyurethane (PU) and polystyrene (PS) membranes.[ 19]
20
SChem14 CNTs functionalization using non-covalent method a)polymer wrapping ,b)surfactant adsorption c) ndohedral method[17]
Scheme15
Scheme16 , Functionalization of SWNTs with PU & PS [ 19 ]
Carbon Nanotubes Decorated with Magnetic Nanoparticles
Hybrid systems based on iron oxides/carbon nanotubes have many potential applications in
electric device, magnetic data storage, and heterogeneous catalysis. The removal of azo dyes
is an important issue. In fact, most dyes used in the manufacturing industries contain
aromatic rings that are generally toxic or potentially carcinogenic/mutagenic agents . With
the aim to remove azo dyes (i.e., methyl orange) dissolved in aqueous solution, magnetic
CNTs have been also prepared by a straightforward Fenton’s reagent method (Figure 17)
[43]. This method consists in the slow addition of H2O2 to a solution of FeSO4, in which
CNTs have been suspended. The resulting solution is the so-called Fenton’s reagent.
Fig.17
The oxidant solution not only allows the conversion of Fe(II) to Fe(III) but also the
generation of reactive functional groups on the CNT’s surface. Further precipitation of
Fe(OH)3 followed by heat treatment under a nitrogen/hydrogen flow, produced Fe2O3
nanoparticles that uniformly dispersed on the surface of CNTs with high loading (>50%).
The advantage of this method consists in the preparation of magnetic CNTs without the use
of strong acids or exploiting reactions for the formation of covalent bonds. Moreover, this
system is able to remove the azo dye methyl orange from aqueous solution by adsorption, to
be separated by an external magnetic field and easily regenerated by UV photocatalysis.
Decoration of CNTs by spinel ferrites nanoparticles with the chemical formula MFe2O4 (M
= Mn, Co, Ni, Mg, or Zn) has been reported to improve optical, magnetic and
electrochemical properties of pristine CNTs. In a recent work, a special electrode has been
designed with the aim to determine analytically the concentration of the antibiotic cefixime
with voltammetric techniques .
Pristine CNTs (diameter of 10–30 nm, length of 5–15 μm) have been treated with HNO3 to
introduce reactive functional groups then they have been dropped onto the surface of a
glassy carbon disk electrode. To obtain magnetic metal functionalized CNTs the authors
developed an in situ chemical citrate gel method. This procedure consists in the treatment of
functionalized CNTs into 1M citric acid followed by addition of a (1:2) solution of Ni/Fe
nitrate. The pH was increased to 9 with ammonia and stirred at 30 °C for 48 h to complete
the reaction. The substance was finally calcinated at 620 °C for 2 h in argon atmosphere to
obtain a powder of NiFe2O4-MWCNTs. This powder has been supported on a carbon disk
electrode and employed in the analytical determination of cefixime (in tablet, blood
22
plasma, and urine samples) by exploiting the presence of its NH2 groups that can be oxidized
by anodic reactions .
A recent paper dealing with the decoration of CNTs with magnetic iron oxide nanoparticles
and exploiting two different reactions (ligand exchange and chemo-selective ligation or
“click chemistry”) has been reported (Figures 7 and 8) ]. The authors suggested that these
systems can be employed in applications for cell labeling, MRI cell tracking and magnetic
manipulations. First, iron oxide superparamagnetic nanoparticles were synthesized by
thermal decomposition of iron stearate in octyl ether and oleic acid. Then, the ligand
exchange reaction took place by adding oxidized CNTs (bearing COOH groups) and stirring
the suspension in THF for 24 h (Figure 18).
Fig.18
The click ligation has been performed in mild conditions exploiting the Cu(I)-catalyzed
azide–alkyne Huisgen 1,3-dipolar cycloaddition reaction in an efficient and highly selective
way.
CNTs have been functionalized with alkyne moieties, whereas iron oxide nanoparticles have
been coated with a dendrimer bearing a terminal azide group. Both derivatized CNTs and
nanoparticles have been reacted with sodium ascorbate and Cu(II) sulphate in a THF/H2O
(3:1) solution (Figure 19).
The degree of functionalization is different for the two reactions owing to the different
degree of active groups (COOH and alkyne groups) on the surface of the CNTs. Oxidized
CNTs have a greater percentage of substitution but alkyne derivatives favor a more
homogeneous derivatization. Moreover, aggregation is reduced when the click chemistry
approach is followed. These compounds enter easily into cells, are moderately toxic and
display good magnetic properties. These properties can be exploited to monitor these
systems by MRI techniques and to manipulate them with magnetic devices.
24
The control of CNTs by external magnetic fields and the monitoring with non-invasive
techniques opens new perspectives for targeted therapy or tissue engineering.
Application:
Amino-carbon nanotubes (amino-CNTs) can conjugate with the DNA by electrostatic
interactions and shuttle the DNA to the cell cytoplasm or even the nucleus.[20] The
functionalized-amino-CNTs are very soluble in water and can be conjugated with nucleic
acids and transfer them into the mammalian cells to produce biological effects (Pantarotto et
al. 2004[20]; Singh et al. 2005[21]).
Huang et al 2013 demonstrated simultaneous attainment of high sensitivity and selectivity in
thin-film field effect transistors (TFTs) based on outer-wall selectively functionalized
double-walled carbon nanotubes (DWCNTs). With carboxylic acid functionalized DWCNT
TFTs, , the author and co-workers obtained excellent gate modulation (on/off ratio as high
as 4000) with relatively high ON currents at a CNT areal density as low as 35 ng/cm2. The
devices displayed an NH3 sensitivity of 60 nM (or 1 ppb), which is comparable to small
molecule aqueous solution detection using state-of-the-art SWCNT TFT sensors while
concomitantly achieving 6000 times higher chemical selectivity toward a variety of amine-
containing analyte molecules over that of other small molecules. These results highlight the
potential of using covalently functionalized double-walled carbon nanotubes for
simultaneous ultrahigh selective and sensitive detection of chemicals and illustrate some of
the structural advantages of this double-wall materials strategy to nanoelectronics. [22]
biosensor for of detecting glucose:
It is reported that a novel amperometric glassy carbon biosensing electrode for glucose
which is based on the immobilization of a highly sensitive glucose oxidase (GOx) by
affinity interaction on carbon nanotubes (CNTs) functionalized with iminodiacetic acid and
metal chelates. The new technique for immobilization is exploiting the affinity of Co(II)
ions to the histidine and cysteine moieties on the surface of GOx. The direct
electrochemistry of immobilized GOx revealed that the functionalized CNTs greatly
improve the direct electron transfer between GOx and the surface of the electrode to give a
pair of well-defined and almost reversible redox peaks and undergoes fast heterogeneous
electron transfer with a rate constant (ks) of 0.59 s−1. The GOx immobilized in this way
fully retained its activity for the oxidation of glucose. The resulting biosensor is capable of
detecting glucose at levels as low as 0.01 mM, and has excellent operational stability (with
no decrease in the activity of enzyme over a 10 days period). The method of immobilizing
GOx is easy and also provides a model technique for potential use with other redox enzymes
26
and proteins (Scheme1).(Tu et al 2012)[23
28
Project Proposal for:
Modification of Carbone Nanotube (CNTs) with metal
nanoparticles for electrochemical immunoassay of alpha-
fetoprotein
Abstract
A new flow-through electrochemical immunosensor is suggesting to be designed for
sensitive detection of alphafetoprotein (AFP) in human serum by using nanogold-
functionalized magnetic Carbon Nanotube (CNTs) as immunosensing probes. Initially,
CNTs will be modified with Carboxylic acid group then amino functionalized magnetic
beads will be covalently immobilized on the surface of CNTs (MCNTs), then nanogold
particles will be adsorbed on the amino groups of the MCNTs to construct GoldMag
functionalized Carbone nanotube (GMCNTs), and then horseradish peroxidase-anti-AFP
conjugates (HRP-anti-AFP) will be assembled onto the surface of nanogold particles
(bio-GMCNTs). With the aid of an external magnet, the formed bio-GMCNTs will be
attached onto the base electrode in the flow system. Sample containing AFP antigens can
be tested by capability of the producing transparent immunoaffinity reaction with the
immobilized HRP-anti-AFP on the bio-GMCNTs.
Introduction
An immunosensor is a kind of biosensor that provides concentration-dependent signals by
using antibodies (Ab) or antigens (Ag) as the specific sensing element [24, 25]. Recently,
electrochemical immunosensors have incited the interest of scholars because of their
sensitivity, highly selectivity, convenience and inexpensiveness, and they have been
successfully applied in environmental analysis [26], the food industry [27,28 ], and clinical
chemistry [29,30].
Sensitive determination of disease-related proteins based on the immunoassay has gained
increasing attention in early disease diagnostic and highly reliable predictions (Choi et al.,
2011)[31]. Among these immunoassays, the homogeneous immunoassays usually involve in
the immobilization of the biomolecules on the nano-/microbeads, and take place in the
solution, thus allowing the integration of multiple liquid handling processes (Song et al.,
2011)[32]. Especially combining with microfluidic device, the homogeneous immunoassay
can be used for the detection of complex samples, such as urine or blood, without the large
sample consumption and sample pretreatment, resulting in a relatively inexpensive and easy
performance (Kang and Li, 2009; Lin et al., 2010)[33,34]. Microfluidic lab-on-a-chip
technology has the advantages of portability, portability, inte- gration, and automation
(Wong and Ho, 2009)[35].
30
multi-walled carbon nanotubes ( MWCNTs)
As is well known, semiconductor multi-walled carbon nanotubes ( MWCNTs), have unique
electrical and mechanical properties, high surface area, and are proven to promote electron
transfer between electrochemically active compounds and electrodes [36,38]. Cao et al. have
developed an electrochemical immunosensor using poly(L-arginine)/multi-walled carbon
nanotubes composite film with functionalized gold nanoparticles for the sensitive detection
of casein [39 ].
The review papers by Masotti et al 2013 emphasized the role of magnetic carbon
nanotubes (Mag-CNTs) as novel and promising drug delivery vectors for applications in
biomedical and biotechnological applications. [40]
fluidMAG-nanoparticles
fluidMAG-nanoparticles (such as fluidMAG-Amine )are ferrofluids consisting of an
aqueous dispersion of magnetic iron oxides with diameters of 50 nm, 100 nm and 200 nm.
The particles are covered with hydrophilic polymers which protect them against aggregation
by foreign ions. Terminal functional groups such as ion-exchange groups or reactive groups
for covalent immobilization can be used for binding to biomolecules. Ferrofluids can further
be used for MRI-diagnostics and magnetic drug targeting applications. fluidMAG-Amine is
used in Covalent coupling of biomolecules application.[64]
Alpha-fetoprotein (AFP):
Alpha-fetoprotein (AFP) is a major plasma protein produced by the yolk sac and the liver.
The AFP expression is often associated with diseases of hepatocellular, testicular
nonseminomatous origin, and occasionally of other entodermal origin and has been widely
accepted as a tumor marker for monitoring the therapeutic effectiveness of hepatocellular
cancer and nonseminomatous testicular cancer (Tomasi, 1977).[41]
Alpha-fetoprotein (AFP) is a glycoprotein that is produced in early fetal life by the liver and
by a variety of tumors including hepatocellular carcinoma, hepatoblastoma, and
nonseminomatous germ cell tumors of the ovary and testis (eg, yolk sac and embryonal
carcinoma). Most studies report elevated AFP concentrations in approximately 70% of
patients with hepatocelllular carcinoma. Elevated AFP concentrations are found in 50% to
70% of patients with nonseminomatous testicular tumors[42]
AFP is elevated during pregnancy. Persistence of AFP in the mother following birth is a rare
hereditary condition.[43] Neonates have markedly elevated AFP levels (>100,000 ng/mL) that
rapidly fall to below 100 ng/mL by 150 days and gradually return to normal over their first
year.[43] Concentrations of AFP above the reference range also have been found in serum of
32
patients with benign liver disease (eg, viral hepatitis, cirrhosis), gastrointestinal tract tumors
and, along with carcinoembryonic antigen, in ataxia telangiectasia.
The biological half-life of AFP is approximately 5 days. <6.0 ng/mL Reference values are
for nonpregnant subjects only; fetal production of AFP elevates values in pregnant women.
Range for newborns is not available, but concentrations over 100,000 ng/mL have been
reported in normal newborns, and the values rapidly decline in the first 6 months of life.
Serum markers are not specific for malignancy, and values may vary by method. Alpha-
fetoprotein (AFP) levels may be elevated in association with a variety of malignancies or
benign diseases. Failure of the AFP value to return to normal by approximately 1 month
after surgery suggests the presence of residual tumor. Elevation of AFP after remission
suggests tumor recurrence; however, tumors originally producing AFP may recur without an
increase in AFP.[ 45,42,46]
Alpha-fetoprotein (AFP) is an oncofetal protein found in high concentration in fetal and
maternal blood and in patient with certain neoplastic and non-neoplastic disorders[47]. AFP
was first identified in 1956, in 2 separete laboratories during electrophoretic experiments on
plasma proteins of neonates, as a protein which migrated between albumin and alpha-
globulin[48] . clinical interist in AFP developed when it was discovered that transplantable
hepatocellular carcinoma of the mouse synthesized and secreted AFP into the blood[49]. High
serum levels of AFP were subsequently detected in patients with hepatoma,germ cell
tumors[47,50,51] . in addition to mammals , birds and even several species of sharks have been
found to synthesize a fetal specific plasma alpha-globulin analogous to mammalian AFP[51].
Ding etal 2009, reported preparing a novel and effective electrochemical immunosensor for
the rapid determination of alpha-fetoprotein (AFP) based on carbon paste electrode (CPE)
consisting of room temperature ionic liquid (RTIL) N-butylpyridinium hexafluorophosphate
(BPPF(6)) and graphite. The surface of the CPE was modified with gold nanoparticles for
the immobilization of the alpha-fetoprotein antibody (anti-AFP). By sandwiching the
antigen between anti-AFP on the CPE modified with gold nanoparticles and the secondary
antibody, polyclonal anti-human-AFP labeled with horseradish peroxidase (HRP-labeled
anti-AFP), the immunoassay was established. The concentration of AFP was determined
based on differential pulse voltammetry (DPV) signal, which was generated in the reaction
between O-aminophenol (OAP) and H(2)O(2) catalyzed by HRP labeled on the sandwich
immunosensor. The immunosensor exhibited high sensitivity and good stability, and would
be valuable for clinical assay of AFP. [52]
Alpha-fetoprotein (AFP) detection by using a localized surface plasmon coupled
fluorescence (LSPCF) fiber-optic biosensor is setup and experimentally demonstrated by
Chang et al 2008. It is based on gold nanoparticle (GNP) and coupled with localized surface
plasmon wave on the surface of GNP. In this experiment, the fluorophores are labeled on
anti-AFP which are bound to protein A conjugated GNP. Experimentally, the ability of real
time measurement in the range of AFP concentration from 0.1ng/ml to 100ng/ml was
detected. To compare with conventional methods such as enzyme-linked immunosorbent
assay (ELISA) or radioimmunoassay (RIA), the LSPCF fiber-optic biosensor performs
higher or comparable detection sensitivity, respectively. [53]
Hsu et al 2011 reported a new highly sensitive biosensor approach which has the ability to
determine 5~100 ng/ml alpha-fetoprotein (AFP) quantitatively using gold nanoparticle
arrays slightly embedded in glass substrates which can be quickly prepared by one-step
microwave-plasma dewetting process [54].
34
Huang et al 2011reported investigation of high sensitive and quantitative detection of alpha-
fetoprotein (AFP) by biosensor based on imaging ellipsometry (BIE) through biological
amplification.. AFP firstly reacted with the rat monoclonal antibody (rat-mAb) initially
immobilized on glutaraldehyde modified silicon surface, then rabbit anti-human AFP
polyclonal antibodies (Rabbit-pAb) and goat anti-rabbit IgG (goat-IgG) were sequentially
applied to amplify signal. Results revealed that signal was enhanced approximately six fold.
. The cross-reaction rate was less than 5.2% evaluated by biomarker (carcinoembryonic
antigen, carbohydrate 19-9 and carbohydrate antigen 242) and two common proteins (human
serum albumin, fibrinogen) and their mixture. [55]
Xia et al 2012 reported droplet methods that have been successfully applied in DNA
hybridization analysis and protein-protein interaction. Existing assay methods implemented
in droplet platforms are severely limited by expensive and high-maintenance equipment. As
a convenient detection method, colorimetry provides a new path for microscale assay since it
can enhance assay efficiency and simplify the detection procedure. a microscale
immunoassay for α-fetoprotein (AFP) was developed for the first time by the incorporation
of colorimetry and droplet platform. Ru(bpy)2(mcbpy-O-Su-ster)(PF6)2 complex (Ru) was
coupled with the monoclonal antibody (Ab) of AFP to form a stable red Ru–Ab complex
both as a quencher for green CdTe quantum dots (QDs) and as a capture probe for AFP. In
the absence of AFP, the mixed droplet showed a red color. With the increase of AFP
concentration, the color change of the droplet was from red to green as a result of the
competition of AFP with QDs for Ru–Ab. The biosensor exhibited not only good sensitivity
and specificity for AFP with a detection limit of 0.06 ng ml−1, but also satisfactory
performance in diluted human sera with a detection limit of 0.4 ng ml−1. [56]
Objective:
In this research proposal , we propose to design a new Gold- Mag-modified CNTs
(SWCNTs, DWCNTs and MWCNT) as biosensor for electrochemical detection of Alpha-
fetoprotein (AFP) in a flow system (human-blood).
The aim of this work is to explore a new electrochemical immunoassay method using
(Gold- Mag-modified CNTs) for the determination of cancer marker in human serum .
Hopefully, the methodology can be applied for the determination of real samples with high
sensitivity and feasibility in clinical screening of cancer markers and diagnostics.
Methodology
Modification Carbon Nanotube with Carboxylic Acid group :
The multi-wall CNTs will be pretreated according to the procedure with minor
modifications According to the method used by (Tang et al 2009, Yu et al 1998, [57,58]. The
multi-wall carbon nanotubes (95%) with outer diameters of ca 10–20 nm and lengths of 5–
10 mm will be oxidized by refluxing in concentrated HNO3 and H2SO4 (volume ratio of 1:3)
for 12 h. The resultant mixture then will be diluted with pure water to about three times of
the original volume and stirred for 24 h. The mixture will be subjected to high-speed
centrifugal sedimentation.
36
Fig. modification CNTs with –COOH group
Carbon Nanotubes modified with Magnetic Nanoparticles
After modifying CNTs with Carboxylic group, Magnetic CNTS can be synthesized
according to the literatures with some modification (Koo et al., 2011; He and Gao, 2010 and
Zhang etal 2011). Amount of CNTs can be initially dispersed into distilled water, and then
sonicated for certin time at room temperature . Following that, amount ( mg) of NHS (N
-ydroxysulfosuccinimide) and amount (mg) of EDC (N-(3- dimethylaminopropyl)-N
-ethylcarbodiimidhydrochlorid) will be added into the mixture, and incubated for certain
time at 4 ℃ to activate the –COOH groups on the CNTS. Afterwards, amount ( mg) of
amino functionalized magnetic nanoparticles (fluidMAG-Amine) will be added into the
mixture, and further stirred for 12 h at 4 ℃. The unconjugated magnetic nanoparticles were
removed by filtration for 10 times with the distilled water. The obtained CNTs then will be
dried in a vacuum oven at 60 ◦C.[59,60,61 ]
Synthesis and bioconjugation of GoldMag -functionalized CNTs
According to the literatures by ( Zhang etal 2011) with miner modification ,
Bioconjugation of GoldMag -functionalized CNTs, can be Synthesized as following,
certain amount (mg) of Magnetic CNTS will be dispersed into amount ( mL) of distilled
water, and the mixture will be then sonicated for 30 min at RT. Following that, amount
(mL) of gold colloids (C[Au] ≈ 24 mM) will be added into the mixture, and incubated for 6
h at 4 ◦C with slight stirring to make nanogold particles assemble on the Magnetic CNTS
surface. Afterwards, the GoldMag -functionalized Magnetic CNTS will be separated and
purified by using an external magnetic field. The obtained GoldMag -functionalized
Magnetic CNTS ( it can be called as GMCNTs) will be then dispersed into amount of
distilled water, and used for the conjugation of HRP-anti-AFP. Amount of HRP-anti-AFP
will be injected into the GMCNTs suspension, and incubated for 6 h at 4 ◦C with slight
stirring. Afterwards, HRP-anti-AFP-labeled GMCNTs (designed as bio- GMCNTs) will be
collected through an external magnet. The obtained pellet will be re-suspended into 2.5 wt%
BSA(bovine serum albumin ) for 60 min at RT to eliminate non-specific binding effect and
block the remaining active groups. Finally, the bio- GMCNTs will be e suspended into
certine amount of pH 6.0 PBS(phosphate-buffer saline (PBS)) containing sodium azide,
and then can be stored at 4 ◦C .[61] Noticeably , this method was applied for grapheme
oxide , and it can be apply for CNTs as a result of the similarity in properties between the
two.
38
Scheme 1. Schematic representation of the process of the bio-GMGPs
Principle the magneto-controlled electrochemical immunoassay
In this research We intend to follow the similar explanation of the principle and
charactrisitic of the magneto-electrochemical immunoassay experiment which was reported
by Zhang et al 2011 and others.
Since AFP and anti-AFP are biomacromolecules with weak conductivity, the formed
immunocomplex can act as an inert layer and partly shield the active center of the HRP
enzyme, which weaken the catalytic efficiency of the HRP toward the reduction of H2O2 in
the solution. As a result, the current response is decreased. The decrease in the current
depends on the concentration of AFP in the sample. The assay principle can be summarized
as follows:
ImmunoreactionHRP-anti-AFP +AFP→ HRP-anti-AFP-AFP (1)Electrochemical measurementH2O2 +HRP(red)→ H2O +HRP(ox) (2)HRP(ox) +2[Fe(CN)6]3−→ HRP(red) +2[Fe(CN)6]4− (3)[Fe(CN)6]4−→ [Fe(CN)]3− + e− (4)
Thus, the access of the active center of HRP catalyzing the oxidation reaction of
[Fe(CN)6]4−/3− by H2O2 was partly inhibited by AFP, which connected on the surface of the
immunosensor by immunoreaction. In addition, the assay principle was also described in
detail in these literatures (Zhao et al., 2007; Liang and Mu, 2006)[62,63]
Caractrization of bio- GMCNTs
To verify the successful synthesis of the bio- GMCNTs, we intend to use some techniques
such as (1)transmission electron microscopy (TEM) to characterize the synthesized
GMCNTs. Because of Nanometer-sized particles could be formed on the surface of CNTS.
(2) the UV–vis absorption spectroscopy of variously functionalized CNTs ( such as (a)
CNTs, (b) MCNTS, (c) GMCNTs and (d) bio-GMCNTS Especially, when HRP-anti-AFP
molecules are assembled on the GMCNTs. On the basis of the results of these methods , we
might make a conclusion that the bio-GMCN Ts could be synthesized by using this new
developed method.
40
Electrochemical characteristics of of bio- GMCNTs
According to the method which explained by Zhang et al 2011 and others, we will use
AFP as an example to investigate the feasibility of the electrochemical immunoassay. In
the experiment, we will investigated the cyclic voltammograms of the bio- GMCNTs-
modified electrode in pH 6.0 PBS-[Fe(CN)6]4−/3− solution at the absence and presence of
H2O2.[61]
Electrochemical characteristics of the magneto-controlled immunosensor
Fig. 2. (A) Cyclic voltammograms of (a) BSA/Au, (b) MGP/BSA/Au, (c) GMGP/BSA/Au and (d) bio-
GMGP/BSA/Au in 0.1 M pH 6.0 PBS-[Fe(CN)6]4−/3− solution; (B) cyclic voltammograms of the bio-
GMGP/BSA/Au at various scan rates (20, 30, 40, 50, 60, 70, 80, 90, and 100 mV s−1 from inner to
outer) in 0.1 M pH 6.0 PBS-[Fe(CN)6]4−/3− solution (Inset: dependence of peak current on square
root of potential sweep rate); and (C and D) cyclic voltammograms of the bio-GMGP/BSA/Au at the
absence (solid dots) and presence (hollow dots) of 1.5 mM H2O2 in 0.1 M pH 6.0 PBS-
[Fe(CN)6]4−/3− solution (Note: before (C) and after (D) incubation with 10 ng mL−1 AFP).
42
Zhang et al 2011 explained the Electrochemical properties of the magneto-controlled
immunosensor using GoldMag -functionalized graphene saying that.
Fig. 2A displays cyclic voltammograms of various nanostructures-modified gold electrodes
in 0.1 M PBS (pH 6.0) containing 5 mM [Fe(CN)6]4−/3− at 50 mV s−1.
During the measurement, 100 µL of MGPs, GMGPs and bio-GMGPs were injected
in the detection cell, respectively (C[GP] ≈ 1.5 mg mL−1). A couple of well-defined redox
peak was observed at the BSA-modified gold electrode (curve ‘a’ in Fig. 2A).
The difference between the cathodic- and anodic-peak potential (_Ep) was 70 mV,
indicating the ferricyanide was a good electron mediator. When the MGPs were
immobilized on the BSA-modified gold electrode, the currents were decreased (curve ‘b’ in
Fig. 2A), suggesting that the amino functionalized magnetic nanoparticles hindered the
electron transfer. Moreover, the peak currents were decreased again when nanogold particles
were assembled onto the MGPs (curve ‘c’ in Fig. 2A). The reason may be the fact that gold
nanoparticles are a highly negatively charged species as a result of the adsorption of citrate
in the fabrication process, and they repulse the negatively charged ferricyanide (Tang et al.,
2011c). The decrease in the current was obviously attained when HRP-anti-AFP was
conjugated to the GMGPs (curve ‘d’ in Fig. 2A). These results adequately revealed that
HRP-anti-AFP could be immobilized on the GMGPs. Typical cyclic voltammograms of the
bio-GMGP-modified gold electrode at different scan rates of 20–100 mV s−1 were studied
(Fig. 2B). As shown the inset of Fig. 2B, the peak currents were linearly proportional to the
square root of the relevant scan rate. These phenomena can be explained by the Randles
Sevcik equation (Bard and Faulkner, 1980), applicable to the case of a semi-infinite volume
of a diffusing reactant in contact with the electrode.
References:
[1] Ai Zhong XU, Ming Shu YANG, "Flow Field Induced Steady Alignment of Oxidized Multi-walled Carbon Nanotubes", Chinese Chemical Letters, Vol. 16, No. 6, 2005, pp 849-852.[2] Lee, S., Oda, T., Shin, P. & Lee, B. 2009, "Chemical modification of carbon nanotube for improvement of field emission property", Microelectronic Engineering, vol. 86, no. 10, pp. 2110-2113.
[3]Wan, Q., Yang, P., Cai, H., Song, H. & Yang, N. 2013, "Voltammetry of nanomolar leveled environmental hazards on the polymer/CNT coated electrodes", Journal of Electroanalytical Chemistry, vol. 689, no. 0, pp. 252-256.
[4]Zhou, H., Wang, T. & Duan, Y.Y. 2013, "A simple method for amino-functionalization of carbon nanotubes and electrodeposition to modify neural microelectrodes", Journal of Electroanalytical Chemistry, vol. 688, no. 0, pp. 69-75.
[5]Malingappa Pandurangappa and Gunigollahalli Kempegowda Raghu (2011)." Chemically Modified Carbon Nanotubes: Derivatization and Their Applications", Carbon Nanotubes Applications on Electron Devices.
[6] Hwa-Jeong Lee, Sang-Wook Han, Young-Do Kwon, Loon-Seng Tan, Jong-Beom Baeka," Functionalization of multi-walled carbon nanotubes with various 4-substituted benzoic acids in mild polyphosphoric acid/phosphorous pentoxide" C A R B ON 4 6 ( 2 0 0 8 ) 1 8 5 0 –1 8 5 9
[7] Gebhardt,B 2012 "ype Selective Functionalization of Single-Walled Carbon Nanotubes" Friedrich-Alexander-Universität Erlangen-Nürnberg
[8] Pierre R. Marcoux, Philippe Hapiot, Patrick Batail and Jean Pinsonc "Electrochemical functionalization of nanotube films: growth of aryl chains on single-walled carbon nanotubes" New. J. Chem. , 2004, 28, 302–307.
[9] Hirsch, A · Vostrowsky,O 2005, "Functionalization of Carbon Nanotubes", Top Curr Chem (2005) 245: 193–237
[10] Wu.H.C, Chang.X, Liu.L, Zhao.F and Zhao.Y2010 "Chemistry of carbon nanotubes in biomedical applications", J. Mater. Chem. , 2010, 20, 1036-1052
[11]. Chen J, Hamon MA, Hu H, Chen Y, Rao AM, Eklund PC, Haddon RC (1998)" Solution properties of single-walled carbon nanotubes " Science 282:95
44
[12]Chen Y, Haddon RC, Fang S, Rao AM, Eklund PC, Lee WH, Dickey EC, Grulke EC,Pendergrass JC, Chavan A,Haley BE, Smalley RE (1998) " Chemical attachment of organic functional groups to single-walled carbon nanotube material" J Mater Res 13:2423
[13]Jiang, Y., Jin, C., Yang, F., Yu, X., Wang, G., Cheng, S., Di, Y., Li, J., Fu, D. & Ni, Q. 2011, "A new approach to produce amino-carbon nanotubes as plasmid transfection vector by [2 + 1] cycloaddition of nitrenes",Journal of Nanoparticle Research, vol. 13, no. 1, pp. 33-38.
[14] Hirsch A and Vostrowsky.O, "C-60 hexakisadducts with an octahedral addition pattern - A new structure motif in organic chemistry", EUR J ORG C, (5), 2001, pp. 829-848
[15] McCarthy B, Coleman JN, Czerw R, Dalton AB, Carroll DL, Blau WJ. Microscopy studies of nanotube-conjugated polymer interactions. Synth Met 2001;121:1225–6.
[16] Hill DE, Lin Y, Rao AM, Allard LF, Sun YP. Functionalization of carbon nanotubes with polystyrene. Macromolecules 2002;35:9466–71.
[17]Ma, P., Siddiqui, N.A., Marom, G. & Kim, J. 2010, "Dispersion and functionalization of carbon nanotubes for polymer-based nanocomposites: A review", Composites Part A: Applied Science and Manufacturing, vol. 41, no. 10, pp. 1345-1367.
[18] Elena Bekyarova, Bin Zhao, Rahul Sen, Mikhail E. Itkis, Robert C. addon 2004"APPLICATIONS OF FUNCTIONALIZED SINGLEWALLED CARBON NANOTUBES" Prepr. Pap.-Am. Chem. Soc., Div. Fuel Chem. 2004, 49(2), 936
[19]Sen,R; Zhao,B;Perea,D;Itkis,M;Hu,H;Love,J; Bekyarova,E;Haddon,R.C, preparation of Single-Walled Crbon Nanotube Reinforced polystyrene and polyurethane Nanofibers and Membranes by Electrospining. Nano letters 2004,4,(3) ,459-464.
[20]Pantarotto D, Singh R, McCarthy D, Erhardt M, Briand J-P, Prato M, Kostarelos K, Bianco A (2004) Functionalized carbon nanotubes for plasmid DNA gene delivery. Angew Chem Int Ed Engl 43:5242–5246
[21] Singh R, Pantarotto D, McCarthy D, Chaloin O, Hoebeke J, Partidos CD, Briand J-P, Prato M, Bianco A, Kostarelos K (2005) Binding and condensation of plasmid DNA onto functionalized carbon nanotubes: toward the construction of nanotube-based gene delivery vectors. J Am Chem Soc 127:4388–4396
[22]Jia Huang, Allen L. Ng, Yanmei Piao, Chien-Fu Chen, Alexander A. Green, Chuan-Fu Sun, Mark C. Hersam, Cheng S. Lee, and YuHuang Wang 2013 "Covalently Functionalized Double-Walled Carbon Nanotubes Combine High Sensitivity and Selectivity in the
Electrical Detection of Small Molecules"Journal of the American Chemical Society 2013 135 (6), 2306-2312
[23]Tu, X., Zhao, Y., Luo, S., Luo, X. & Feng, L. 2012, "Direct electrochemical sensing of glucose using glucose oxidase immobilized on functionalized carbon nanotubes via a novel metal chelate-based affinity method",Microchimica Acta, vol. 177, no. 1, pp. 159-166.
[24]Jiang, X.S.; Li, D.Y.; Xu, X.; Ying, Y.B.; Li, Y.B.; Ye, Z.Z.; Wang, J.P. Immunosensors for detection of pesticide residues. Biosens. Bioelectron. 2008, 23, 1577–1587.
[25]. Li, X.L.; Yuan, R.; Chai, Y.Q.; Zhang, L.Y.; Zhuo, Y.; Zhang, Y. Amperometric immunosensor based on toluidine blue/nano-Au through electrostatic interaction for determination of carcinoembryonic antigen. J. Biotechnol. 2006, 123, 356–366.
[26] Campanella, L.; Eremin, S.; Lelo, D.; Martini, E.; Tomassetti, M. Reliable new immunosensor for atrazine pesticide analysis. Sens. Actuators B Chem. 2011, 156, 50–62.
[27] Mello, L.D.; Kubota, L.T. Review of the use of biosensors as analytical tools in the food and drink industries. Food Chem. 2002, 77, 237–256.
[28] Li, Y.; Cheng, P.; Gong, J.H.; Fang, L.C.; Deng, J.; Liang, W.B.; Zheng, J.S. Amperometric immunosensor for the detection of Escherichia coli O157:H7 in food specimens. Anal. Biochem.2012, 421, 227–233.
[29] Pupim, F.A.A.; Colli, W.; Inácio da Costa, P.; Yamanaka, H. Immunosensor for the diagnosis of Chagas’ disease. Biosens. Bioelectron. 2005, 21, 175–181.
[30]. Luppa, P.B.; Sokoll, L.J.; Chan, D.W. Immunosensors-principles and applications to clinical chemistry. Clin. Chim. Acta 2001, 314, 1–26.
[31]Choi, S., Goryll, M., Sin, L., Wong, P., Chae, J., 2011." Microfluidic-based biosensors toward point-of-care detection of nucleic acids and proteins " Microfluid. Nanofluid. 10, 231–247.
[32] Song,Y H. Zhang, C. Chon, X Pan and D. Li, "Nanoparticle Detection by Microfluidic Resistive Pulse Sensor with a submicron sensing gate and dual detecting channels-two stage differential amplifier", Sensors and Actuators, B, 155 (2011) 930-936.
[33] Kang, Y., Li, D., 2009." Electrokinetic motion of particles in microchannels”" Microfluid. Nanofluid. 6, 431–460.,
[34] Lin, F., Gao, Y., Li, D., Sherman, P., 2010 “Development of microfluidic-based heterogeneous immunoassays" . Front. Biosci. S2, 73–84.
46
[35] Wong, T.-S. and Ho, C.-M., “Dependence of Macroscopic Wetting on Nanoscopic Surface Textures”, Langmuir, vol. 25, pp. 12851 – 12854, 2009.
[36] Trocino, S.; Donato, A.; Latino, M.; Donato, N.; Leonardi, S.G.; Neri, G. Pt-TiO2/MWCNTs hybrid composites for monitoring low hydrogen concentrations in air. Sensors 2012, 12, 12361–12373.
[37] Zhao, G.Y.; Zhan, X.J.; Dou, W.C. A disposable immunosensor for Shigella flexneri based on multiwalled carbon nanotube/sodium alginate composite electrode. Anal. Biochem. 2011, 408, 53–58.
[38] Sui, K.Y.; Li, Y.J.; Liu, R.Z.; Zhang, Y.; Zhao, X.; Liang, H.C.; Xia, Y.Z. Biocomposite fiber of calcium alginate/multi-walled carbon nanotubes with enhanced adsorption properties for ionic dyes. Carbohydr. Polym. 2012, 90, 399–406.
[39] Cao, Q.; Zhao, H.; Yang, Y.M.; He, Y.J.; Ding, N.; Wang, J.; Wu, Z.J.; Xiang, K.X.; Wang, G.W. Electrochemical immunosensor for casein based on gold nanoparticles and poly(l-Arginine)/ multi-walled carbon nanotubes composite film functionalized interface. Biosens. Bioelectron.2011, 26, 3469–3474.20. Sun, A.L.; Chen, G
[40] Andrea Masotti 1,* and Andrea Caporali 2 2013 " Preparation of Magnetic Carbon Nanotubes (Mag-CNTs) for Biomedical and Biotechnological Applications" Int. J. Mol. Sci. 2013, 14, 24619-24642; doi:10.3390/ijms141224619
[41] Tomasi TB Jr: Structure and function of alpha –fetoprotein. Ann Rev Med 28:453-465,1977.
[42]Yachnin.S the clinical significant of human alpha-fetoprotein Ann Clin Lab Sci8:84-90,1978.
[43]Bergstrand CG, Czar B: Demonstration of anew protein fraction in serum from the human fetus. Scand J Clin Lab Invest 8:174-4,1956
[44] Abelev GI, perova SD,Khramkova NI, Postnikova ZA, Irlin IS: Production of embryonal alpha-globulin by transplantable mouse hepatoma. Transpl Bull 1:174-180, 1963.
[45]Alpert E: Human alpha-fetoprotein (AFP) ; Developmental biology and clinical significance . prog Liv Dis 5:337-349,1976
[46] Lester EP, Miller JB, Yachnin S, Human alpha-fetoprotein as a modulator of human lymphocyte transformation :Correlation of biological potency with electrophoretic variants . Proc Natl Acad Sci USA 73:4645-4648,1976
[47]Yachnin.S the clinical significant of human alpha-fetoprotein Ann Clin Lab Sci8:84-90,1978.
[48] Bergstrand CG, Czar B: Demonstration of anew protein fraction in serum from the human fetus. Scand J Clin Lab Invest 8:174-4,1956
[49] Abelev GI, perova SD,Khramkova NI, Postnikova ZA, Irlin IS: Production of embryonal alpha-globulin by transplantable mouse hepatoma. Transpl Bull 1:174-180, 1963.
[50] Tomasi TB Jr: Structure and function of alpha –fetoprotein. Ann Rev Med 28:453-465,1977.
[51]Alpert E: Human alpha-fetoprotein (AFP) ; Developmental biology and clinical significance . prog Liv Dis 5:337-349,1976
[52]Ding C, Zhao F, Ren R, Lin JM. An electrochemical biosensor for alpha-fetoprotein based on carbon paste electrode constructed of room temperature ionic liquid and gold nanoparticles. Talanta. 2009 May 15;78(3):1148-54.
[53] ChangY.F ; ChenR.C ; Li.Y.C ; YuC.J ;HsiehB.Y, et al. 2008"Alpha-fetoprotein detection by using a localized surface plasmon coupled fluorescence fiber-optic biosensor", Proc. SPIE 6826, Optics in Health Care and Biomedical Optics III, 68261B (January 08, 2008); doi:10.1117/12.754617;
[54] Hsu C.Y, Huang N.W and KLin.K.J,2011 "High sensitivity and selectivity of human antibody attachment at the interstices between substrate-bound gold nanoparticles"Chem. Commun., 47 (2011) 872-874.
[55] Huang.C, Chen.Y, Wang.C, Zhu.W, Ma.H,Gang Jin 2011"Detection of alpha-fetoprotein through biological signal amplification by biosensor based on imaging ellipsometry" ,Thin Solid Films (Impact Factor: 1.6). 01/2011; 519(9):2763-2767. DOI:10.1016/j.tsf.2010.11.064
[56] Xia Xiang, Lu Chen, Cuiling Zhang, Ming Luo, Xinghu Ji and Zhike He 2012" A fluorescence-based colorimetric droplet platform for biosensor application to the detection of α-fetoprotein " Analyst, 2012,137, 5586-5591
[57] Tang.C.R,Tian.G, WangY.J, Su.Z.H, LiC.X, Lin.B.G, Huang.H.W, Yu.X.Y, Li.X.F, Long.Y.F and Zeng.Y.L, 2009 " selective pesponse of dopamine in the presence of ascorbic acid and uric acid at gold nanoparticles and MWCNTS grafted with tetraacitic acid modified electrode" Bull. Chem. Soc. Ethiop. 2009, 23(3), 317-326.
[58] Yu RQ, Chen LW, Liu QP, Lin JY, Tan KL, Xu GQ, et al. Platinum deposition on carbon nanotubes via chemical modification. Chem Mater 1998;10(3):718–22.27.
48
[59] Koo.H.Y, Lee.H.J, Go.H.A, Lee.Y.B, Bae.T.S , Kim.J.K and Choi .W.S 2011 " Graphene-Based Multifunctional Iron Oxide Nanosheets with Tunable Properties" Chem. Eur. J. 2011, 17, 1214 – 1219
[60] He, H., Gao, C., 2010. "3210Supraparamagnetic, Conductive, and Processable Multifunctional Graphene Nanosheets Coated with High-Density Fe3O4 Nanoparticles " ACS Appl. Mater. Interface 2, 3201–3210
[61]Zhang, B., Tang, D., Liu, B., Chen, H., Cui, Y. & Chen, G. 2011, "GoldMag nanocomposite-functionalized graphene sensing platform for one-step electrochemical immunoassay of alpha-fetoprotein", Biosensors and Bioelectronics, vol. 28, no. 1, pp. 174-180.
[62] Liang, K., Mu, W., 2006.". flow-injection immune-bioassay for interleukin-6 in human based on gold nanoparticales modified screen-printed graphite electrod" Anal. Chim. Acta 580, 128–135.
[63] Zhao, G., Xing, F., Deng, S., 2007"A disposable amperometric enzyme immunosensor for rapid detection of Vibrio parahaemolyticus in food based on agarose/Nano-Au membrane and screen-printed electrode ." . Electrochem. Commun. 12, 1263–1268.[64] http://www.chemicell.com/products/ferrofluid/ferrofluids.html