Plasmonics (2010) 5:189–198
DOI 10.1007/s11468-010-9133-z
Highly Sensitive Resonance Scattering Detection of DNA
Hybridization Using Aptamer-Modified Gold Nanopaticle
as Catalyst
Huilin Tao & Lili Wei & Aihui Liang & Jianfu Li &
Zhiliang Jiang & Hesheng Jiang
Received: 18 March 2009 / Accepted: 12 February 2010 / Published online: 26 February 2010
# Springer Science+Business Media, LLC 2010
Abstract Gold nanoparticle particles in size of 10 nm were
used to label the thiol-modified single-stranded DNA
aptamer (SH-ssDNA) to obtain an aptamer-modified gold
nanoparticle probe (AussDNA) for target DNA (tDNA). In
pH 7.4 NaH2PO4–Na2HPO4 buffer solution, the hybridiza-
tion reaction between AussDNA and tDNA took place to
form larger aptamer-modified gold nanoparticle cluster
complex. The excess aptamer-modified gold nanoparticle
probe in the supernatant solutions was obtained by
centrifuging and can be used as nanocatalyst for the
0.276 mmol/L CuSO4-65.4 mmol/L potassium-sodium
tartrate-0.37 mmol/L glucose system at 70 °C. The cubic
Cu2O particles generated by the nanocatalytic reducing
exhibit a strong resonance scattering (RS) peak at 620 nm.
In the selected conditions, the RS intensity at 620 nm
decreased with addition of tDNA, and the decreased
intensity ΔI620 nm is proportional to tDNA concentration
(CtDNA) from 0.12 to 72 pM, with regress equation of
ΔI620 nm = 1.29CtDNA + 4.05, correlation coefficient of
0.9917, and detection limit of 0.084 pM tDNA.
H. Tao : L. Wei : A. Liang : J. Li : Z. Jiang (*)
Key Laboratory of Ecology of Rare and Endangered Species
and Environmental Conservation of Education Ministry,
Guangxi Normal University,
Guilin 541004, China
e-mail: zljiang@mailbox.gxnu.edu.cn
H. Jiang
Animal Science and Technology Collage, Guangxi University,
Nanning 531004, China
Keywords Aptamer-modified gold nanoparticle probe .
DNA hybridization . Nanocatalysis . Fehling reagent .
Cubic Cu2O particle . Resonance scattering spectral assay
Introduction
DNA is known as inherited substance in life body. With
the increase of knowledge about the mutations of genes
being responsible for numerous inherited human disorders,
the analysis of sequence-specific DNA and DNA mutant
detection play fundamental roles in diagnostics of genetic,
bacterial, and viral diseases and environmental monitoring
of micro organisms [1, 2]. Currently, a variety of
techniques have been developed for DNA hybridization
detection, including fluorescence imaging [3, 4], electro-
chemical [5–8], micro-gravimetrical [9], chemilumines-
cence [10–12], and electrogenerated chemiluminescence
techniques [13, 14]. For all that, to satisfy the needs of
different research, basing on the combination modern
analytical technique with DNA hybridization, the explora-
tion for a simple, rapid, and low-cost method to determine
DNA with high sensitivity, and selectivity is one of the
challenging frontiers in analytical chemistry.
Gold nanoparticle has high electron density, good
biocompatibility, high catalytic activity, and novel optical
properties, and plays an important role in biochemical
nanoanalysis. Because of the high-density electron, and
easy combining with -COOH, -NH3 and -SH, the gold
nanoparticle particles were labeled easily with biological
macromolecules such as protein, DNA, RNA, and glyco-
190
protein, and the macromolecular activity keeps invariant
after labeling. Thus, it has become the fourth immune
label following radioactive elements, enzyme and fluores-
cein. In 1997, Mirkin and co-workers developed an
entirely new colorimetric detection scheme for DNA
hybridization in a homogeneous solution based on
aggregation of oligonucleotide-functionalized gold nano-
particles and the optical properties of gold nanoparticles,
with detection limit of 10 fmol. In addition, they also
reported another new colorimetry for DNA detection,
using Agcore–Aushell nanoparticles [15]. Recently, to
improve the sensitivity, some sensitive detection techni-
ques, such as surface plasmon resonance [16], laser
diffraction [17], surface-enhanced Raman spectroscopy
[18], and array-based electrical detection [19], linear light-
scattering technique [20], have been introduced into DNA
detection using gold nanoparticle, with satisfactory results.
On the other hand, based on the high catalytic activity of
gold nanoparticle [21], and gold nanoparticle-gold/silver
enhancement DNA detection technique with high sensi-
tivity has been established [22, 23]. Xu et al. [24] reported
an immunogold nanoparticle-silver enhancement light-
scattering technique to assay nucleic acid, with a detection
limit of 10 fmol/L. Gold nanoparticle exhibited catalytic
effect on NADH–HAuCl4 particle reaction, and was
utilized to assay NADP with an optical sensor [25]. Pan
et al. [26] reported an electrochemical approach for
sequence-specific DNA-binding transcription factor detec-
tion by gold nanoparticle-catalyzed silver enhancement at
interface between electrodes and electrolyte solutions. It
was found that this method for the detection of sequence-
specific DNA-binding protein showed pronounced speci-
ficity, and that the detection limit was as low as 0.1 pM.
Zhao et al. [27] established a simple gold nanoparticle-based
method to detect microamount of polynucleotide molecules
with silver staining enhancement. This sandwich colorimetric
assay can detect as low as 0.1 fmol/L single-strand target
polynucleotides and 10 fmol/L double-strand target polynu-
cleotides. However, the gold nanoparticle-catalyzed silver
enhancement technique can be influenced by natural light and
Cl−, and the gold nanoparticle-catalytic gold enhancement
reaction was stopped difficultly at room temperature, the
operation was not convenience and precious reagent of
HAuCl4 was used. Thus, it was necessary to explore a new
gold nanoparticle-catalytic reaction technique for the deter-
mination of DNA with high sensitivity, good reproducibility,
simplicity, and low cost.
Resonance scattering technique, using common spectro-
fluorimeter, was simple, sensitive and inexpensive, and has
been widely applied in analysis of nucleic acids, proteins,
and small molecular drug [28–36], with satisfactory results.
Our studies indicate that the use of catalytic amplification
can obtain high sensitivity and selectivity. Combining RS
Plasmonics (2010) 5:189–198
technique with inorganic catalytic and enzyme catalytic
amplification reactions, a highly sensitive, selective, and
simple catalytic-RS method has developed to determine
trace inorganic ion and organic compound, with satisfactory
results [37, 38]. On the other hand, based on the
immunogold nanoparticle-catalyzed gold/silver enhanced
particle reactions, the RS technique also applied to protein
and nucleic acid analysis [24, 39, 40]. However, those gold
nanoparticle-catalyzed gold/silver enhancement techniques
have some shortages as mentioned above. Recently, our
research group has developed a highly sensitive, selective,
and simple immunogold nanoparticle-catalyzed Cu2O par-
ticle RS assay for Malb [41], which overcome the
disadvantages in the immunogold nanoparticle-catalyzed
gold/silver enhancement technique. Up to now, there is no
report about aptamer-modified gold nanoparticle-catalytic
Cu2O particle RS assay for DNA hybridization by using
gold nanoparticles as the labels of SH-ssDNA aptamer.
Herein, a convenient and low cost, and highly sensitive
aptamer-modified gold nanoparticle-catalytic Cu2O particle
RS assay was developed for tDNA, coupling the gold
nanoparticle-labeled DNA aptamer hybridization reaction
and RS effect of Cu2O particles.
Experimental section
Reagents and apparatus
Single-stranded DNA used in this paper was purchased from
Sanbo Yuanzhi Biotechnology Limited Company, Beijing,
China. The sequences are as follows: 5′-(SH-C6)-(T)10-TTG
TGC CTG TCC TGG-3′(SH-ssDNA1); 5′-TTG TGC CTG
TCC TGG-(T)10-(C3-SH)-3′(SH-ssDNA2), 5′-GAG AGA
CCG GCG CAC-(T)10-(C3-SH)-3′ (SH-ssDNA3), 5′-(SH-
C6)-(T)10-GAG AGA CCG GCG CAC-3′ (SH-ssDNA4);
target DNA strand (tDNA for short): 5′-GTG CGC CGG
TCT CTC CCA GGA CAG GCA CAA-3′; a HAuCl4
solution (National Pharmaceutical Group Chemical Reagents
Company, China) and a sodium citrate solution were used to
prepare nanogold particles. Fehling solution including
0.28 mol/L CuSO4 and 1.23 mol/L potassium-sodium
tartrate (KNaT) containing 6.25 mol/L NaOH, 1.0 mg/mL
glucose, 2 mol/L NaCl, pH 6.0–8.0 NaH2PO4-Na2HPO4. All
reagents were of analytical grade, and water was doubly
distilled.
A Model Cary Eclipse fluorescence spectrophotometer
(Varian Company, Palo Alto, CA) was used to record the RS
spectra by means of synchronous scanning excited wave-
length lex and emission wavelength lem (lex−lem =Δl=0)
and the RS intensity. A model 79-1 magnetic heat agitator
(Zhongda Instrumental Plant, Jiangsu, China), model
SK8200LH ultrasonic reactor (Kedao Ultrasonic Instrument
Plasmonics (2010) 5:189–198
Limited Company, Shanghai, China), model Sigma 3K3D
high-speed refrigeration centrifuge (Sigma Company, Harz,
Germany), model HH-S thermostated water bath (Dadi
Automatic Instrument Plant, Jiangsu, China), model FEI
Quanta 200 scan electron microscope (Philips Limited
Company, Holland), and model H-600 transmission electron
microscope (Electronic Stock Limited Company, Japan)
were used.
Preparation of nanogold
Gold nanoparticles in size of 10 nm were prepared by citrate
reduction of HAuCl4 according to the procedure described in
Frens [42] with a slight modification. All glassware used in
the following procedures were thoroughly cleaned with
freshly prepared HNO3–HCl (HNO3/HCl=1:3, by volume),
washed with double-distilled water. In brief, a 100 mL of
0.01% HAuCl4 solution was heated to boiling. After 2 min,
3.5 mL of 1% sodium citrate solution was added quickly
with vigorous stirring. About 8 min later, the heating was
stopped. The solutions were stirred continuously until it
cooled to room temperature. Finally, the cooled solution was
diluted with water to 100 mL and stored in the refrigerator at
4 °C. With this method, we obtained gold colloid with a
consistent particle size of 10 nm.
Optimization of ssDNA amounts
We added 0, 0.2, 0.4, 0.6, 0.8, 1.0 nmol of SH-ssDNA to
500 μL 57.69 μg/mL nanogold solution, and incubated at
room temperature for 24 h. Then, we added 500 μL pH 7.0
Na2HPO4–NaH2PO4 (20 mmol/L PO43−), and mixed well.
After 8 h, 50 μL 2 mol/L NaCl solution was added in twice
and aged at room temperature for 48 h. Then the solution
was diluted to 2.0 mL with water and mixed well. We
measured the RS intensities at 548 nm in each tube, with
PMT detector voltage of 500 V, the excitation and emission
slit of 2.5 nm. From Table 1, we concluded the minimal
ssDNA that stabilized 500 μL nanogold approximately was
0.8 nmol. In the experiment, a 0.8 nmol ssDNA was chosen
for labeling 500 μL nanogold solutions.
Fabrication of AussDNA probe
The nanoparticles were functionalized with SH-ssDNA by
following the procedure described by previous literature
with a minor modification [20, 43]. Briefly, 0.8 nmol of
SH-ssDNA was incubated with 500 μL of nanogold
Table 1 Effect of SH-ssDNA amount on nanogold labeling
nSH-ssDNA (nmol) 0 0.2 0.4 0.6 0.8 1.0
I548 nm 68.5 42.1 31.8 28.7 24.9 24.2
191
solution at room temperature for 24 h. Upon addition of
500 μL, 20 mmol/L Na2HPO4–NaH2PO4, and was kept for
8 h. Then, 25 μL 2 mol/L NaCl solution was added and
placed again. After 10 h, another 25 μL 2 mol/L NaCl
solution was added and aged at room temperature for 30 h.
To remove uncoated SH-ssDNA, the solution was centri-
fuged at 14,000 rpm for 30 min at 0 °C and the supernatant
was discarded. The red oily precipitate was dispersed by
2 mL of 10 mM phosphate buffer (pH 7.0) containing
0.1 M NaCl. After another centrifugation under the same
conditions, the precipitate was re-dispersed with doubly
distilled water to make a stock solution, which was used for
the following experiments. The concentration of AussDNA
is 14.5 μg/mL calculated as Au, and 0.40 μmol/L
calculated as ssDNA.
Procedure
Into a graduated test tube, 150 μL pH 7.4 NaH2PO4–
Na 2 HPO 4 buffer solutions, 120 μL 14.5 μg/mL
AussDNA2, and 120 μL 14.5 μg/mL AussDNA4, a certain
amount of tDNA, and 500 μL 2 mol/L NaCl solutions were
added successively, and then diluted to 1.5 mL with water,
mixed well and incubated in an ultrasonic reactor for
15 min. The solution was centrifuged at 8,000 rpm for
30 min at 0 °C to obtain the supernatant, and the
supernatant was taken out for use.
A 15 μL 0.276 mol/L CuSO4 solution, 80 μL 1.23 mol/L
KNaT, 75 μL of the supernatant, and 100 μL 1.0 mg/mL
glucose was added successively to a 5-mL graduated test
tube, diluted to 1.5 mL mark, mixed well, and placed them in
a bath at 70 °C for 10 min. Stop the reaction by tap-water
cooling. A part of the solutions was transferred into a quartz
cell. The settings were as follows, volt=500 V, excited slit=
emission slit=2.5 nm. The RS spectrum, the RS intensity at
620 nm (I620 nm), and the (I620nm)b of blank solutions without
tDNA were recorded. The values for ΔI620 nm =(I620 nm)b −
I620 nm was calculated.
Results and discussion
The gold nanoparticle-labeled DNA aptamer could hybrid-
ize with the tDNA sequence according to the strict principle
of DNA bases match by electrostatic interaction and
hydrogen bonds, and the tDNA specifically bind with gold
nanoparticle-labeled ssDNA in a form of double stranded
under the suitable circumstances. Based on this phenome-
non, we can realize the assay of DNA sequences using
AussDNA aptamer-modified gold nanoparticle probe. To
detect tDNA by optical and electrical technique, the ssDNA
probe must be modified, using an appropriate coupling
mode such as –SH, and it did not affect the biological
192
Fig. 1 Hybridization fashion between AussDNA and tDNA. a Tail-
to-tail, b head-to-tail, c head-to-head
recognized functions such as hybridization. By functional-
izing the gold nanoparticle with either 3′ or 5′ termini of
the SH-ssDNA, the gold nanoparticle would combine with
the SH-ssDNA based on strong Au–S covalent bond on the
surface. In this experiment, the gold nanoparticle-labeled
DNA aptamer which modified with either 3′ or 5′ termini
was stable because the gold nanoparticle coated by the SH-
ssDNA, and a certain concentration of salt could not make
them aggregation. However, when tDNA hybridize with
AussDNA in form of double-strand mode, ssDNA-
functionalized gold nanoparticles would be released and
aggregated to form larger aptanaogold clusters. As shown
in Fig. 1, the tDNA is a 30-base gene fragment, and the two
SH-ssDNA aptamers containing 15 bases are complemen-
tary to part of the tDNA. By functionalizing the gold
nanoparticles with either 3′or 5′ termini of the SH-ssDNA,
Fig. 2 Principle of the aptamer-
modified gold nanoparticle-
catalytic reaction
Plasmonics (2010) 5:189–198
the gold nanoparticles would align in a tail(AussDNA4)-to-tail
(AussDNA2) (Fig. 1a), head(AussDNA3)-to-tail(AussDNA2)
(Fig. 1b), or head(AussDNA3)-to-head(AussDNA1) (Fig. 1c)
fashion onto the tDNA after the hybridization. Owing to
many ssDNA immobilized on the surface of one nano-
particle, the hybridization in each alignment fashion can
result in the formation of extended polymeric network
aggregates with different degrees. The tail-to-tail form was
most sensitive and was chosen to detect tDNA.
After the hybridization between AussDNA and tDNA,
the aggregated gold nanoparticle particles and the excess
aptamer-modified gold nanoparticle probe AussDNA can
be separated by centrifugation. Similar to the immunogold
nanoparticle catalysis [41], both exhibit catalytic effect on
the Fehling reagent-glucose reaction. However, the opera-
tion with the excess aptamer-modified gold nanoparticle is
simple, and was chosen for use. Aptamer-modified gold
nanoparticle, like a common catalyst, catalyze the Cu(II)/
Cu(I) reduction in solutions to produce Cu2O particles
(Fig. 2). Thus, the RS intensity at 620 nm enhanced greatly.
With addition of tDNA, the aptamer-modified gold nano-
particle complex increased, the excess AussDNA in the
supernatant reduced, and the RS intensity at 620 nm
decreased. The decreased RS intensity at 620 nm was
linear to the tDNA concentration. Therefore, an aptamer-
modified gold nanoparticle-catalytic Cu2O particle RS
method can be proposed for the determination of tDNA.
Electron microscopy
Gold nanoparticle was characterized by transmission
electron microscopy, and the results showed that the
10 nm gold nanoparticle was spherical and uniformity
[39]. The gold nanoparticle functionalized by HSssDNA
had the same size and good dispersion. After the hybrid-
ization between AussDNA and tDNA, the labeled gold
nanoparticle were liberated and aggregated to form large
Plasmonics (2010) 5:189–198 193

Fig. 3 SEM of Cu2O particles.

Left magnified ×10,000; right
magnified 100,000 times

aptamer-modified gold nanoparticle clusters. In the medium
of NaKT–NaOH at 70 °C, Cu(II) was slowly reduced to
Cu2O particles by weak reducer glucose. Addition of gold
nanoparticle, large amounts of Cu2O particles in yellow
were observed. In the different conditions, Cu2O particles
produced are in the different shape such as sphere, cube,
octahedron, and so on [41–44]. The Cu2O particles were
prepared according to the procedure and were centrifuged
at 10,000 rpm for 5 min to remove the mass salt, then re-
dispersed with doubly distilled water in the ultrasonic
apparatus; 50.0 μL Cu2O sample was dropped in the
conductive adhesive, and then dried by airing. The
scanning electron microscopy (SEM) of the Cu2O particles
was shown in Fig. 3. The SEM indicated that Cu2O
particles are cubic, with the average length × width × high
of 400×400×400 nm. This work provided a simple and
rapid gold nanoparticle-catalytic synthetic procedure for
cubic Cu2O particles.
Resonance scattering spectra
Aptamer-modified gold nanoparticle has weak RS signal at
548 nm. Upon addition of tDNA, the RS peak at 548 nm
The scattering signal is very weak at less than 300 nm since
the molecules absorption in the system is strong. Because
of the weak absorption of Cu(II) in the wavelength of 400–
700 nm, the effect of its absorption on the scattering signals
can be ignored. We known that glucose was a weak
reductant and was difficult to reduction of Cu(II) to Cu(0)
without the supernatant as catalyzer and the scattering
signal of the system is very weak. When the supernatant
was added to the Fehling reagent-glucose solution, the
catalytic system exhibited a strongest RS peak at 620 nm
(Fig. 4). Thus, the peak at 620 nm is the RS peak that
produced by Cu2O particles, and the synchronous scattering
spectrum is RS spectrum. When tDNA concentration
a
b
c
d
RS

enhanced, owing to the hybridization reaction of tDNA-

AussDNA, that can be also used to detect 5–120 pM tDNA,
with detection limit of 2 pM tDNA, regress equation of
ΔI548 nm =0.27CtDNA +2.41 and relative coefficient of e
0.9931. The Fehling reagent-glucose-tDNA-AussDNA sys-
tem has two synchronous scattering peaks at 465 and
620 nm that are different to the aptamer-modified gold
nanoparticle RS peak and are belonging to Cu2O particles.
The synchronous scattering spectrum of liquid inorganic
nanoparticles shows that the light source, free molecules
absorption, and resonance scattering effect of nanoparticles Wavelength/nm
are the three major factors which cause the synchronous
Fig. 4 Resonance scattering spectra of tDNA–AussDNA–glucose–Cu
scattering peak. The strongest emission peak of the light
(II) particle reaction. a 0.276 mmol/L CuSO4-65.4 mmol/L KNaT-
source is located at 465 nm, so there is a synchronous 0.37 mmol/L glucose–58 ng/mL AussDNA-0.0 pM tDNA; b a-6.0 pM
scattering peak at 465 nm originating from light source. tDNA; c a-24 pM tDNA; d a-48 pM tDNA; e a-72 pM tDNA
194
Table 2 Relationship between
gold nanoparticle concentration Sugar Regression equation
(C) and the ΔI
Glucose ΔI=11.1 C+4.29
Fructose ΔI=9.46 C+0.54
Xylose ΔI=7.87 C+7.88
increased, the AussDNA in the supernatant reduced, and
the RS intensity at 620 nm decreased linearly. Thus,
wavelength of 620 nm was chosen for use.
Sugar-Fehling reagent-gold nanoparticle-catalytic reaction
Fehling reagent has advantages including easy obtained
reagents and low cost. The Fehling reaction for reducing
Fehling reagents with glucose to form Cu2O precipitate was
widely used in the analytical determination of reducing
saccharides. Qi's research group found that Pd nanoparticle
has catalytic effect on the reaction between Fehling reagent
and glucose that was utilized to prepare octahedral Cu2O
nanocages [44]. According to the literature [41], the
catalytic effect of the gold nanoparticle in size of 10 nm
is the most effective. Here, we studied the catalytic effect of
10 nm gold nanoparticle on the Fehling reagent-different
reducing sugar system. With increasing the nanocatalyst
concentration the RS intensity at 620 nm (ΔI) increased.
From the Table 2, we could see that there were differences
in the catalytic effect in glucose, fructose and xylose
systems. In which, the Fehling reagent-glucose system has
the strongest catalytic effect, highest sensitivity, widest
linear range. Therefore, a Fehling reagent-glucose-gold
nanoparticle system was selected for the following study.
In the gold–silver staining procedure and silver/gold/
copper enhancement assays based on gold nanoparticle
Fig. 5 Principle of gold
nanoparticle-catalytic Cu2O
amplification
Plasmonics (2010) 5:189–198
Linear range (nM) Correlation coefficient Detection limit (nM)
0.098–15.68 0.9929 0.081
0.196–11.76 0.9965 0.17
0.392–19.60 0.9877 0.33
modified electrode, Ag+, Au3+, and Cu2+ can be reduced to
produce Ag(0), Au(0), and Cu (0) under certain conditions,
in succession they were coated on the gold nanoparticle
surface to form relatively large Au core composite particles.
In the non-fixed gold nanoparticle catalyst solutions, gold
nanoparticle also catalyze the reduction to produce new
small particles, and the number of the new particles is much
higher than the Au core composite particles [45]. This
implies that the products of the gold nanoparticle-catalytic
reaction are mostly new particles which enlarge the
scattering signals. Likewise, the reaction between the
Fehling reagent and glucose using gold nanoparticle as
the catalyst mostly generated new small Cu2O nanoparticles
that also have strong catalytic effect on the Fehling reaction
to form larger cubic-structured Cu2O particles, which is
autocatalytic action (Fig. 5) [46]. Consequently, the Cu2O
particles generated by reducing cause an increase in the RS
intensity at 620 nm. In addition, the organic compounds are
helpful to stabilizing and protecting Cu2O particles by
intermolecular forces.
Optimization of the hybridization conditions
Effect of pH 6.0–8.0 NaH2PO4–Na2HPO4 buffer solution
on the ΔI620 nm was tested. Results showed that when pH
was 7.4, and the volume was 150 μL, the system had the
maximal ΔI620 nm. Thus, 150 μL of pH 7.4 NaH2PO4–
Plasmonics (2010) 5:189–198 195

60 70

50 60

50
40

I620nm
I620 nm

40
30
30
20 20

10 10

0
0 0 0.1 0.2 0.3 0.4 0.5 0.6
0 20 40 60 80 100
c(ng/mL) c(mmol/L)

Fig. 6 Effect of AussDNA concentration. pH 7.4 NaH2PO4- Fig. 8 Effect of glucose concentration on ΔI620 nm; 0.276 mmol/L
Na2HPO4-48 pM tDNA-33.5 mmol/L NaCl-0.276 mmol/L CuSO4- CuSO4-65.4 mmol/L KNaT-58 ng/mL AussDNA-48 pM tDNA
65.4 mmol/L KNaT-0.37 mmol/L glucose

tration is too low. But the high-concentration salt has

negative effect on diffusion motion of AussDNA in the
Na2HPO4 buffer solutions was selected for use. AussDNA
solution. Therefore, a 33.5 mmol/L NaCl was selected.
is not only the probe for tDNA, but also the nanocatalyst,
Results showed that ultrasonic irradiation can accelerate the
and its concentration has effect on both hybridization
hybridization reaction, and the ΔI620 nm was maximal and
reaction and catalytic reaction. The influence of AussDNA
stable after 15 min. Thus, ultrasonic irradiation time of
concentration on ΔI620 nm was studied. The ΔI620 nm
15 min at room temperature was chosen.
increased along with the concentration of AussDNA
including AussDNA2 and AussDNA4. When the concen-
Effect of centrifugal speed, time, and the supernatant
tration was 58 ng/mL, the ΔI620 nm was maximal (Fig. 6).
volume
A 58 ng/mL AussDNA was chosen. Electrolyte can destroy
hydrated layer around the gold nanoparticle particles and
Effect of centrifugal speed and time on the ΔI620 nm was
beak the stable colloid state, which lead to the aggregation
examined, respectively. When centrifugal speed was in the
of dispersed gold nanoparticle particles. Thus, NaCl
range of 6,000–8,000 rpm, the ΔI620 nm increased. When it
concentration has great influence on the ΔI620 nm. We
was higher than 8,000 rpm, the ΔI620 nm decreased (Fig. 7).
studied the effect of NaCl concentration and the results
The ΔI620 nm increased when centrifugal time was within
showed that the ΔI620 nm decreased when the NaCl
30 min. Prolonged the time, the ΔI620 nm decreased. Thus,
concentration is too high or too low. It cannot be provided
8000 rpm for 30 min was chosen. Influence of the
the necessary salinity for the hybridization process and the
supernatant volume (V) on ΔI620 nm value showed that the
aggregation of gold nanoparticle when the NaCl concen-

60 70

60
50
50
I 620 nm

40
40
I620nm

30
30
20 20

10 10

0 0
6000 8000 10000 12000 14000 30 40 50 60 70 80 90
rpm T(Celsius)

Fig. 7 Effect of centrifugation speed on ΔI620 nm; 0.276 mmol/L Fig. 9 Effect of reaction temperature on ΔI620 nm; 0.276 mmol/L
CuSO 4 -65.4 mmol/L KNaT-0.37 mmol/L glucose-58 ng/mL CuSO4-6.54×10-2 mol/L KNaT-0.37 mmol/L glucose-58 ng/mL
AussDNA-48 pM tDNA AussDNA-48 pM tDNA
196 Plasmonics (2010) 5:189–198

Table 3 Analytical features for the different hybridization fashion

Hybridization fashion Regression equation Linear range (pM) Correlation coefficient Detection limit (pM)

Tail-to-tail ΔI620 nm =1.29CtDNA +4.05 0.12–72 0.9917 0.084

Tail-to-head ΔI620 nm =0.99CtDNA +2.60 0.24–72 0.9905 0.22
Head-to-head ΔI620 nm =0.65CtDNA +2.02 0.6–48 0.9924 0.51

volume in the range of 0-75 μL was linear to ΔI620 nm. The
linear regression equation was ΔI620 nm =0.5878 V+0.64,
with correlation coefficient of 0.9988. When the V was
more than 75 μL, the ΔI620 nm was stable. A 75 μL of the
supernatant was used.
Selection of the AussDNA-glucose–Cu(II) catalytic
reaction conditions
KNaT was a good complex for Cu(II) to avoid formation
of Cu(OH)2 precipitation to hold active concentration of
copper ions [47]. Effect of KNaT concentration on the
ΔI620 nm was considered. A 65.4 mmol/L KNaT, giving
biggest ΔI620 nm value, was chosen for use. Effect of
CuSO4 and glucose concentration on ΔI620 nm was
examined, respectively. Results showed that when CuSO4
concentration was 0.276 mmol/L, the ΔI620 nm was
biggest, and was chosen for use. Glucose was a weak
reducer and a good stable reagent for Cu2O particles, and
can be used to control reaction rate to obtain good
dispersion Cu2O particles [48]. As in Fig. 8, when the
glucose concentration was 0.37 mmol/L, the ΔI620 nm was
maximal. So a 0.37 mmol/L glucose and was chosen for
use.
Effect of reaction temperature in the range of 30–90 °C
on the ΔI620 nm was considered. Owing to the nanocatalytic
reaction being endothermic reaction [49, 50], the reaction
do not take place below 40 °C, the ΔI620 nm was very small.
When the temperature was higher than 40 °C, the ΔI620 nm
increased greatly (Fig. 9). When the temperature was higher
than 80 °C, the uncatalytic reaction rate of Cu(II)–glucose
increased also, and the ΔI620 nm decreased quickly. To
obtain high sensitivity and low blank value, a reaction
temperature of 70 °C was chosen for use. The ΔI620 nm
value increased linearly with reaction time within 10 min at
70 °C. After 10 min, the ΔI620 nm was biggest and stable. A
reaction time of 10 min was selected. After stopping the
catalytic reaction by tap-water cooling, the influence of
the measurement time on ΔI620 nm was examined. The
ΔI620 nm was stable within 40 min, longer than 40 min the
ΔI620 nm decrease slowly. If the solution was mixed again,
the ΔI620 nm remains original value. According to this
operations, the ΔI620 nm remains within 2 h. The reason
was that the O atoms in the Cu2O particle can interact with
those water-soluble organic molecules such as glucose by
H-bond and molecular interaction forces that protected and
suspended the bigger Cu2O particle in the basic medium.
Therefore, although Cu2O particles is in comparatively big
size, the gold nanoparticle–glucose–Cu(II) system still has
good stability.
Linear relationship
According to the procedure, the ΔI620 nm for different
concentration of tDNA was recorded and the working
curves were drawn according the relationship between
CtDNA and their corresponding ΔI620 nm. As shown in
Table 3, the tail-to-tail fashion had the highest sensitivity
(0.084 pM) and the widest linear range (0.12–72 pM), the
tail-to-head fashion took second place, and the head-to-

Table 4 Effect of coexistence substances (48 pM tDNA)

Coexistent substance Tolerance Relative error Coexistent substance Tolerance Relative error
(nM) (%) ( nM) (%)

L-tyrosine 110 +7.0 Urea 15,300 −6.6

L-lysine 5,470 −-5.6 Glycine 33,300 −4.7
Vitamin B6 8,100 −6.4 Tryptophan 330 −5.0
HSA 150 +7.4 Fe2+ 1260 −5.6
Terabutyl ammonium chloride 6,700 −5.9 Cetyltrimethyl ammonium bromide 13,300 −6.9
ctDNA 23 −6.8 hsDNA 18 −6.6
ssDNA3 0.4 +4.5 ssDNA1 2 +5.6
Zn2+ 670 +5.9 Mg2+ 330 +5.8
Plasmonics (2010) 5:189–198
head fashion was the worst. Owing to the gold nanoparticle
particles functionalized by the ssDNA which align in the
terminal of DNA molecule was not enwrapped easily after
the hybridization. It is much easy to aggregate to form
bigger clusters for the exposed gold nanoparticle particles
in the high-concentration NaCl solution. The decreased
intensity ΔI620 nm had good linear relationship with the
tDNA concentration in the range of 0.12–72 pM. Compared
with nucleic acid assays using HAuCl4–NH2OH–nogold
nanoparticle, Ag(I)-hydroquinone-nogold nanoparticle, Cu
(II)-ascorbic acid-nogold nanoparticle reaction reported [24,
27], this catalytic reaction can be stopped by tap-water
cooling, and overcame the shortcoming of bad stability in
all above methods. Moreover, the reagents are easily
available, low cost and stable.
Detection specificity
According to the procedure, the influence of coexistent
substances on the determination of 48 pM tDNA was
examined, with a relative error of ±8%. The tolerated
amount for coexistent substances was listed in Table 4. The
results showed that the coexistent substances did not
interfere with the tDNA determination, which indicated
that this method had good selectivity. Although the base
sequence of ssDNA1 and ssDNA3 is similar to the ssDNA2
and ssDNA4, respectively, 0.4 nmol/L ssDNA1 and
2.0 nmol/L ssDNA3 did not interfer with the detection of
tDNA. One is AussDNA2 and AussDNA4 existed in higher
concentration of 1.6 nmom/L, other is the –SH position in
the ssDNA molecule.
Conclusion
A simple and highly sensitive aptamer-modified gold
nanoparticle-catalytic Cu2O-particle RS technique for
tDNA was developed, coupling the hybridization reaction
between aptamer-modified gold nanoparticle probes and
target DNA, nanocatalytic reaction of the Fehling reaction,
RS effect of Cu2O particles, and centrifugal technique.
Comparing with the silver, gold and copper particle
reaction techniques reported, the Cu2O particle reaction
technique has high sensitivity and selectivity, simplicity
and low cost. In addition, a simple and rapid gold
nanoparticle-catalytic synthetic procedure for cubic Cu2O
particles in length × width × high of 400×400 ×400 nm
was proposed.
Acknowledgements This work was supported by the National
Natural Science Foundation of China (Nos. 20667001, 20365001),
Natural Science Foundation of Guangxi (No.0728213, 0991021Z) and
the Foundation of New Century Ten-Hundred-Thousand Talents of
Guangxi.
197
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