Analytica Chimica Acta 545 (2005) 21 26
Electrochemical DNA biosensors based on platinum nanoparticles
combined carbon nanotubes
a b b," b,"
Ningning Zhu , Zhu Chang , Pingang He , Yuzhi Fang
a
Department of Chemistry, Shanghai Normal University, 100 Guilin Road, Shanghai 200234, China
b
Department of Chemistry, East China Normal University, Shanghai 200062, China
Received 12 January 2005; received in revised form 5 April 2005; accepted 6 April 2005
Available online 24 May 2005
Abstract
Platinum nanoparticles were used in combination with multi-walled carbon nanotubes (MWCNTs) for fabricating sensitivity-enhanced
electrochemical DNA biosensor. Multi-walled carbon nanotubes and platinum nanoparticles were dispersed in Nafion, which were used to
fabricate the modification of the glassy carbon electrode (GCE) surface. Oligonucleotides with amino groups at the 5 end were covalently
linked onto carboxylic groups of MWCNTs on the electrode. The hybridization events were monitored by differential pulse voltammetry
(DPV) measurement of the intercalated daunomycin. Due to the ability of carbon nanotubes to promote electron-transfer reactions, the
high catalytic activities of platinum nanoparticles for chemical reactions, the sensitivity of presented electrochemical DNA biosensors was
remarkably improved. The detection limit of the method for target DNA was 1.0 × 10-11 mol l-1.
© 2005 Elsevier B.V. All rights reserved.
Keywords: Carbon nanotubes; Platinum nanoparticles; Electrochemical DNA biosensor
1. Introduction and power requirements of DNA diagnostics [4,5]. The use
of nanomaterials in electrical detection is relatively new and
The demands for innovative analytical device capable of offers unique opportunities for electrochemical transduction
delivering the genetic information in a faster, simpler, and of DNA sensing events.
cheaper manner at the sample source are becoming increas- The emergence of nanotechnology is opening new hori-
ingly important. DNA biosensors and high-density DNA ar- zons for the application of nanoparticles in analytical
rays can fit these demands [1 3]. Hybridization of nucleic chemistry. The unique physical and chemical properties of
acids to their complementary sequences is the essence of nanoparticles offer excellent prospects for chemical and bi-
DNA biosensor and DNA chip technology. Detection of hy- ological sensing [6,7]. One of the attractive applications for
bridization on an electrode surface was commonly based on bioanalytical is the catalytic property of noble metal nanopar-
detecting the signal changes from labeled-target DNA hy- ticles. Platinum nanoparticles have been an intensive research
bridized with surface-bound DNA probes. Fluorescent de- subject for the design of electrodes [8]. Platinum films mod-
tection is successfully used, but bulky and expensive control ified microelectrodes were shown to be excellent ampero-
instrumentation hampers its wide application, which has also metric sensors for H2O2 in a wide range of concentrations
encouraged the development of lower-cost detection tech- [9]. Carbon nanotubes (CNTs) are of special interest due to
niques. Electrochemical devices offer promising routes for their unique electronic, metallic, and structural characteristic
interfacing the DNA recognition and signal transduction el- [10]. Among the diversified applications of CNTs, there has
ements, and are uniquely qualified for meeting the size, cost, been growing interest to use CNTs in biological devices ow-
ing to the ability of CNTs to promote electron-transfer reac-
" tions with biomolecules [11]. CNTs have been suspended in
Corresponding authors. Fax: [phone].
Nafion, which were used to modify the electrode for the devel-
E-mail address: [e-mail] (Y. Fang).
0003-2670/$  see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2005.04.015
22 N. Zhu et al. / Analytica Chimica Acta 545 (2005) 21 26
opment of an amperometric biosensor for glucose [12]. CNTs exchange resin (5 wt.%), sodium dodecyl sulfate (SDS) and
have also been used as carrier platforms for CdS nanoparticle EDAC were purchased from Sigma (US). H2PtCl6·6H2O,
trace for enhanced electrical detection of DNA hybridization 0.3 mol l-1, PBS solution (0.3 mol l-1 NaCl + 0.1 mol l-1
[13]. Fang and co-workers have also used MWCNTs-COOH phosphate buffer, pH 7.3) and 0.1 mol l-1 PBS solution
for DNA immobilization and electrochemical detection [14]. (0.1 mol l-1 NaCl + 0.1 mol l-1 phosphate buffer, pH 7.3)
Platinum nanoparticles and single-walled carbon nanotubes were used. Other reagents were commercially available and
(SWCNTs) were combined to modify a glassy carbon elec- were all of analytical reagent grade.
trode to improve their electro-activity for H2O2. The results 24-Base synthetic oligonucleotides were purchased
showed that the Pt nanoparticles/CNTs modified electrode from Shenggong Bioengineering Ltd. Company (Shanghai,
responds more sensitively to glucose than those modified by China):
platinum nanoparticles or CNTs alone [15].
" 24-base probe sequence: 5 -NH2-GAGCGGCGCAACAT-
In this paper, we use platinum nanoparticles combined
TTCAGGTCGA-3
MWCNTs for the modification of electrode and DNA hy-
" its fully complementary sequence: 5 -TCGACCTGAAA-
bridization detection. MWCNTs were suspended in Nafion,
TGTTGCGCCGCTC-3
a perfluorosulfonated polymer, which also interacted with
" a non-complementary sequence: 5 -GAGCGGCGCAA-
platinum nanoparticles to form a network, which connected
CATTTCAGGTCGA-3
platinum nanoparticles to the electrodes surface. Oligonu-
cleotides probe with an amino group at 5 end were cova-
2.3. Preparation of platinum nanoparticles and
lently linked onto the carboxyl groups of the MWCNTs in the
modification electrode
presence of the water-soluble coupling reagent l-ethyl-3-(3-
dimethylaminopropy-l)-carbodiimide (EDAC). Hybridiza-
Platinum nanoparticles (Ptnano) were prepared according
tion was conducted by immersing the electrode immobi-
to the literature [16]. Briefly, H2PtCl6·6H2O (4 ml 5% aque-
lized with DNA probe into the buffer solution containing its
ous solution) was added to distilled water (340 ml) and heated
complementary sequence. Then difference pulse voltamme-
ć%
to 80 C with stirring in a 500 ml flask. After adding 60 ml of
try (DPV) measurement was performed using electro-active
sodium citrate (1% aqueous solution), the resulting solution
daunomycin as an indicator. The results showed that the DNA
ć%
was maintained at 80 Ä… 0.5 C for about 4 h. The course of
biosensors using MWCNTs combined platinum nanoparti-
the reduction was followed by absorption spectroscopy, and
cles respond more sensitively to target DNA than those based
the end of the reaction was marked by the disappearance of
on platinum nanoparticles or MWCNTs alone. The perfor-
the absorption bands of PtCl62-.
mance of the DNA biosensors with respond to selectivity,
Glassy carbon electrode was carefully polished with pol-
linear range, and sensitivity was discussed.
ishing paper and subsequently with alumina until a mirror
finish was obtained. After 5 min of sonication to remove the
alumina residues, the electrode was immersed in concen-
2. Experimental
trated H2SO4 for 3 min followed by thorough rinsing with
water and ethanol. The electrode was then transferred to the
2.1. Apparatus
electrochemical cell for cleaning by cyclic voltammetry be-
tween -0.5 and +1.2 V (versus Ag/AgCl) in 50 mM phos-
The cyclic voltammetry (CV) and DPV measurements
phate buffer, pH 7.2, until a stable CV profile was obtained.
were performed with a CHI 630 Electrochemical Analyzer
The prepared electrode was dried and use immediately for
(CHI Instruments Inc., USA). The three-electrode system
modification.
consisted of a glassy carbon working electrode (effective area
An amount of 2.0 mg of MWCNTs were dissolved in a
7.07 mm2), an Ag/AgCl reference electrode (saturated KCl)
mixture of 100 l of Nafion and 900 l of Ptnano solution
and a counter electrode made of platinum. All electrochem-
[15], as the stock solution. After about 40 min of sonication,
ical measurements were conducted in a 10 ml cell. A trans-
uniformly dispersed MWCNTs and Ptnano were formed. GCE
mission electron microscope (TEM) (Hitachi, Japan), a JB-1
was then modified by a 5 l drop of MWCNTs/Ptnano, and
stirring machine and a TDL-16B centrifuge were used.
dried in air. After the electrode was thoroughly rinsed with
water, the MWCNTs/Ptnano-modified electrodes were pre-
2.2. Chemicals
pared.
All stock solutions were prepared with ultrapure wa-
ter from an Aquapro system. MWCNTs (with a diameter 2.4. Immobilization of ssDNA on
of about 40 60 nm and length of around 1 10 m) with MWCNTs/Ptnano-modified GCE
carboxylic groups were obtained from Shenzhen Nanotech
Co. Ltd. (Shenzhen, China). Daunomycin hydrochloride The immobilization of oligonucleotides probe on the
was obtained from Shanghai Institute for Drug Control and MWCNTs/Ptnano-modified GCE was carried out as the fol-
used without further purification. Nafion-perfluorinated ion- lowing:
N. Zhu et al. / Analytica Chimica Acta 545 (2005) 21 26 23
Fig. 1. Schematic representation of the electrochemical detection of DNA hybridization based on platinum nanoparticles combined MWCNTs.
The MWCNTs/Ptnano-modified GCE was immersed in 3. Results and discussion
a 2.25 × 10-5 mol l-1 oligonucleotide solution containing
0.1 mol l-1 ED AC and 10 mM acetate buffer (pH 5.2) for The electrochemical DNA biosensor based on platinum
10 h with stirring at room temperature. Then the electrode nanoparticles and MWCNTs for DNA hybridization de-
was washed with a 0.2% SDS phosphate buffer (pH 7.3) tection using daunomycin as indicator was illustrated as
for 5 min to remove the unbound DNA probes and prevent Fig. 1.
the nonspecific binding. Thus the oligonucleotide probe
was immobilized through the formation of amide bonds 3.1. Electrochemical characteristics of
between the  COOH on the MWCNTs and  NH2 of the MWCNTs/Ptnanoparticles/GCE
oligonucleotides at 5 end.
CNTs are very hydrophobic, and most metals could not
easily adhere to CNTs. Pt nanoparticles can be deposited
2.5. Hybridization and electrochemical detection
on Nafion-modified CNTs due to charged interactions [15].
TEM micrograph of CNTs in the presence of Pt nanoparti-
Hybridization reaction was carried out by immersing
cles is shown in Fig. 2. As can be seen, platinum nanoclus-
the ssDNA probe captured MWCNTs/Ptnano/GCE into
ters agglomerate to some extent and disperse on the surface
a stirred hybridization solution (0.3 mol l-1 PBS buffer)
of CNTs, indicating that Pt nanoparticles are deposited on
containing different concentration of DNA target for 30 min
ć%
Nafion-modified CNTs.
at 37 C. After that, the electrode was rinsed three times
with a 0.2% SDS phosphate buffer (pH 7.3) to remove
the non-hybridized target DNA. The hybridized electrode
was placed in the stirred daunomycin (1.0 × 10-5 mol l-1)
solution containing 0.1 mol l-1 phosphate buffer for 5 min.
After that, the electrode was washed with 0.1 mol l-1 phos-
phate buffer for 5 min to remove the physically adsorbed
molecules.
The electrochemical detection of hybridization was
performed in a 10 ml of electrochemical cell with hy-
bridized working electrode, an Ag/AgCl electrode and a
platinum wire counter electrode. The DPV measurements
were conducted from +0.70 to 0.0 V (versus Ag/AgCl)
in a 0.1 mol l-1 phosphate buffer (pH 7.3). The peak
current related to the reduction of daunomycin at about
+0.44 V was taken as the electrochemical measurement
Fig. 2. TEM micrograph of platinum nanoparticles combined MWCNTs.
signal.
24 N. Zhu et al. / Analytica Chimica Acta 545 (2005) 21 26
Fig. 3. Cyclic voltammograms of Ptnano/GCE (A), MWNTs/GCE (B), and
Fig. 4. Cyclic voltammograms of (A) Ptnano/GCE, (B) MWNTs/GCE, and
Ptnano/MWNTs/GCE (C) recorded in 0.3 mol l-1 PBS blank solution (pH
(C) Ptnano/MWNTs/GCE recorded in 0.1 mol l-1 PBS blank solution (pH
7.2), scan rate: 0.1 V/s. Scan range: -0.2 to 0.8 V (vs. Ag/AgCl).
7.2) containing 1 × 10-5 mol l-1 daunomycin, scan rate: 0.10 V/s. Scan
range: 0.0 0.7 V (vs. Ag/AgCl).
Electrochemical characteristics of three types of elec-
trodes: GCE modified by MWCNTs, GCE modified by
surface area, the quantities of ssDNA immobilized on the
Ptnano, and GCE modified by MWCNTs/Ptnano were con-
electrode surface can be greatly enhanced, therefore the de-
ducted in PBS buffer and electro-active daunomycin so-
tection limits of sequence-specific DNA in solution should
lution by CV scans. As shown in Fig. 3, curve A
be greatly lowered. Such results show that Pt nanoparticles
is the CV response of Ptnano/GCE in 0.3 mol l-1 PBS,
were in contact with MWCNTs, which were also in electri-
curve B is that of MWCNTs/GCE and curve C is that
cal contact with the GCE. enabling the use of the resulting
of MWCNTs/Ptnano/GCE. Compared with the Ptnano/GCE
composite structure as electrode materials.
(Fig. 3 A) and MWCNTs/GCE (Fig. 3B), the back current of
the MWCNTs/Ptnano/GCE (Fig. 3C) is the largest. This shows
that the effective electrode surface of MWCNTs/Ptnano/GCE 3.2. Optimum of DNA assay conditions
is larger than that of Ptnano-modified GCE or MWCNTs-
modified GCE alone. After MWCNTs or MWCNTs/Ptnano Pt nanoparticle solutions were used in combination with
is deposited on the GCE, a pair of stable redox wave ap- Nafion for the solubilization of MWCNTs and then for
pears, which is corresponding to the redox of carboxylic the modification of GCE. To optimize the concentration
group of MWCNTs [17]. So MWCNTs also provide an ac- of MWCNTs in a mixture of Pt nanoparticles and Nafion,
tive binding group for oligonucleotides conjugation. Fig. 4 the amount was varied from 0.5 to 2.0 g 1-1. The optimum
is the CV response of three types of electrodes in dauno- concentration for the detection of DNA hybridization was
mycin solution respectively. The GCE modified by MWC- 2.0 g1-1. The amount of MWCNTs/Ptnano-modified on the
NTs+Ptnano (Fig. 4C) exhibits the highest electro-active sur- GCE was also optimized for DNA immobilization and hy-
face area according to the Randles Sevcik equation [19]. bridization detection. The experimental results showed that
Ip = 2.69 × 105ADl/2n3/2Å‚1/2C, where n is the number of elec- the hybridization peak current increased with the increase
trons participating in the redox reaction, A the area of the elec- of the amount of MWCNTs/Ptnano, the optimum amount of
trode (cm), D the diffusion coefficient of the molecule in solu- MWCNTs/Ptnano was 5.0 l. So, 5.0 l of MWCNTs/Ptnano
tion (cm2 s-1), C the concentration of the probe molecule in was used for the modification of GCE. The effect of the hy-
the bulk solution (mol cm-3), and y the scan rate of the poten- bridization time was investigated (Fig. 5). As the hybridiza-
tial perturbation (V s-1). For the MWCNTs-modified GCE, tion time prolonged, the electrochemical signal rose gradu-
the peak current is still pronounced (Fig. 4B), but it exhibits a ally, and reached a constant value after 30 min, so 30 min was
decrease compared with that of MWCNTs/Ptnano/GCE, likely used as hybridization time.
due to the lack of Pt nanoparticles that were in contact with To prevent the nonspecific binding, ssDNA-modified
MWCNTs. For the Ptnano/GCE, the current value (Fig. 4A) MWCNTs/Ptnano/GCE are pretreated with 0.2% SDS be-
was much smaller, which illustrated a decrease in the electro- fore the intercalator binding process. It has been reported
active surface area of the electrode [15], mainly due to the lack that the long aliphatic chains of SDS molecules can inter-
of CNTs that act as nanoconnectors between Pt nanoparticles act attractively with the bases of ssDNA [18], when dauno-
and the electrode. Nevertheless, the resultant current value mycin molecules approach the SDS-treated single-stranded,
was significantly higher than that of the bare GCE (figure some of the SDS molecules on the ssDNA form micelles
not shown). With MWCNTs + Ptnano having a much larger that can be washed out in the subsequent rinsing step. The
N. Zhu et al. / Analytica Chimica Acta 545 (2005) 21 26 25
Fig. 6. The DPV responses of daunomycin intercalating in the dsDNA
Fig. 5. Effect of the hybridization time on daunomycin DPV peak current.
recorded in 0.1 mol l-1 PBS. (A) Ptnano/MWNTs/GCE for probe caption and
complementary DNA detection (2.25 × 10-7 mol l-1). (B) MWNTs/GCE
experimental results show that after the ssDNA-modified
for probe caption and the same concentration of the complementary DNA
detection. Amplitude: 50 mV; pulse period: 0.2 s; pulse width: 60 ms.
MWCNTs/Ptnano/GCE has been pretreated with 0.2% SDS,
the redox current of daunomycin decreases remarkably. That
bridization and daunomycin intercalates, DPV measurements
is to say, this process can avoid the strong nonspecific in-
were conducted from +0.7 to 0.0 V. As shown in Fig. 7, a pro-
teractions between daunomycin and ssDNA, and the detec-
nounced increase in the current value of the daunomycin lo-
tion limit of DNA hybridization detection can be improved
cated at +0.44 V is observed when hybridized with its comple-
greatly. After DNA hybridization, a DNA double helix forms.
mentary sequence; and negligible electrochemical response
The sugar-phosphate backbone lies on the outside and the
is obtained when incubating with non-complementary se-
bases on the inside. When treated with SDS, the hydropho-
quence, which is similar to that of the blank measurement.
bic chains of SDS molecules hardly interact with the in-
The results show that the MWCNTs/Ptnano-based hybridiza-
ternally stacked bases. Therefore, daunomycin is relatively
tion assay has high selectivity.
freely intercalated into a double helix. With the increase of
The analytical performance of the DNA sensor based on
the target DNA concentration, the peak current of intercalated
MWCNTs/Ptnano was also explored by using the immobi-
daunomycin increases. It is observed that SDS treatment does
lized probe to hybridize with the different concentrations
not significantly change the electrochemical character of the
of the complementary sequence according to the described
duplex-modified electrodes.
procedure. The peak current in DPV response of inter-
3.3. Electrochemical DNA hybridization detection based
on MWCNTs+Ptnano and daunomycin
Fig. 6 illustrates the difference in DPV responses be-
tween the oligonucleotides covalently immobilized on the
MWCNTs/Ptngno/GCE (Fig. 6A) and that on the MWC-
NTs/GCE in the absence of Pt nanoparticles (Fig. 6B) for
2.25 × 107 mol l-1 of target-complementary sequence assay.
It shows that the use of MWCNTs/Ptnanno/GCE for the fabri-
cation of the DNA hybridization assays, the electrochemical
signals of daunomycin are two times larger than that in the
MWCNTs/GCE-based biosensor without Pt nanoparticles.
Such a result reconfirmed that Pt nanoparticles were in elec-
trical contact, through the MWCNTs with the glassy carbon
electrode, enabling the composite structure to be used as an
electrode for DNA hybridization detection.
Fig. 7. The DPV response of the daunomycin recorded for the
The selectivity of the DNA hybridization assay was in-
oligonucleotides/Ptnano + MWNTs/GCE probe without hybridization (1),
vestigated with the probe/MWCNTs/Ptnano/GCE hybridized
hybridized with non-complementary oligonucleotides sequence (2), and hy-
with its complete complementary sequence and non-
bridized with complementary oligonucleotides sequence (3). Amplitude:
complementary sequence in certain conditions. After hy- 50 mV; pulse period: 0.2 s; pulse width: 60 ms.
26 N. Zhu et al. / Analytica Chimica Acta 545 (2005) 21 26
modification. CNTs have the ability to promote electron-
transfer reactions and large surface area; platinum nanopar-
ticles possess the high catalytic activities for chemical
reactions, so the sensing signal for DNA hybridization
is greatly amplified. By minimizing the background sig-
nal, the sensitivity of the DNA biosensors is remarkably
improved.
Acknowledgements
Financial support from the National Nature Science Foun-
dation of China (NSFC, grants no. 29875008) and Shanghai
Municipal Education Commission (no. 04DB27) is gratefully
acknowledged.
Fig. 8. (A) Differential pulse voltammograms for different target con-
centrations: (a) 2.25 × 105 pM; (b) 2.25 × 104 pM; (c) 2.25 × 103 pM; (d)
2.25 × 102 pM; (e) 2.25 × 101 pM; (f) 0.0 pM. (B) The resulting logarithmic
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