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Title

Surface‑Enhanced Raman Spectroscopy for Facile
DNA Detection using Gold Nanoparticle Aggregates
Formed via Photoligation

Author(s)Nguyen, T. B. Thuy; Yokogawa, Ryoko; Yoshimura,
Yoshinaga; Fujimoto, Kenzo; Koyano, Mikio;
Maenosono, Shinya

Citation

Analyst, 135: 595‑602

Issue Date

2010‑01‑19

Type

Journal Article

Text version

author

URL

http://hdl.handle.net/10119/10307

RightsCopyright (C) 2010 Royal Society of Chemistry.
Nguyen T. B. Thuy, Ryoko Yokogawa, Yoshinaga
Yoshimura, Kenzo Fujimoto, Mikio Koyano andShinya Maenosono, Analyst, 135, 2010, 595‑602.
http://dx.doi.org/10.1039/B919969A ‑ Reproducedby permission of The Royal Society of Chemistry

Description

Japan Advanced Institute of Science and Technology

CREATED USING THE RSC ARTICLE TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILSARTICLE TYPE

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Surface-Enhanced Raman Spectroscopy for Facile DNA Detection using
Gold Nanoparticle Aggregates Formed via Photoligation
Nguyen T. B. Thuy,a Ryoko Yokogawa,a Yoshinaga Yoshimura,b Kenzo Fujimoto,a,b Mikio Koyanoa andShinya Maenosono*a5Received (in XXX, XXX) Xth XXXXXXXXX 200X, Accepted Xth XXXXXXXXX 200X
First published on the web Xth XXXXXXXXX 200X
DOI: 10.1039/b000000x

We present a new type of nanoparticle-based DNA sensor using surface-enhanced Raman
scattering (SERS) on gold nanoparticle (Au NP) aggregates formed by DNA photoligation. The
10 DNA sensor exploits the photoligation reaction between oligodeoxynucleotides (ODNs) attached tothe surfaces of Au NPs in the presence of target DNA (T-DNA). When hybridization takes place
between the ODNs and T-DNA, Au NPs are covalently crosslinked to form aggregates via
photoligation. Once the NP aggregates are formed, the interspace between Au NPs in the aggregate
act as a stable “hot spot”, and a SERS signal from the Raman-active molecules (sodium
15 cacodylate) present in the hot spot is easily and sensitively detected. In contrast, a SERS signal is
not detected if the hybridization is unsuccessful, because the stable hot spot does not form. ThisDNA sensor does not require an enzymatic reaction, fluorescent dye, precise temperature control,or complicated operating procedures.

Introduction
The development of highly sensitive, selective andinexpensive DNA detection methods is of great importance inthe fields of medical research, clinical diagnosis,1 forensic
investigations and gene therapy because of the very low
concentration of DNA in cells.2-4 Up to now, many techniques
25 have been proposed to improve the sensitivity, selectivity andreduce the cost of genetic diagnostics.5-8 Conventional DNA
detection methods involve the implementation of mass
spectrometry 9 or gel electrophoresis.10 However, thesemethods have some limitations because of relatively high cost
30 and time-consuming operations. Other disadvantages such as
photobleaching and overlapping peaks limit DNA detection
systems based on optical methods using fluorescent dyes.11 To
overcome these problems, many researchers have proposed
various other approaches for colorimetric detection of DNA
35 hybridization based on the unique optical properties of gold
nanoparticle (Au NP) aggregates.12-17 For example, Mirkin
and coworkers have developed a method for detecting
polynucleotides that uses the distance-dependent opticalproperties of Au NPs modified with oligonucleotides. 2,12 Li
40 and Rothberg showed that after the adsorption of singlestranded DNA on Au NPs, the NPs were stabilized against
salt-induced aggregation. 18 This observation formed the basis
for a colorimetric assay to identify specific DNA sequences.
Recently, Storhoff et al. have developed a “spot-and-read”
45 colorimetric detection method for identifying DNA which isalso based on the distance-dependent optical properties of Au
NPs with extraordinary sensitivity.19 Unfortunately, these
newly emerging techniques require carefully trained andskillful personnel as well as a relatively long analysis time.
50 This is because their principles are based on the difference inthe melting temperatures of the duplex formed between the
20

This journal is © The Royal Society of Chemistry [year]

allele-specific probe and the target DNA so they require
stringent temperature control. Moreover, these techniques
might have difficulty in distinguishing single-base
55 imperfections that display a major difference in melting
temperature. The limitations of these existing techniques
would be inconvenient for practical clinical applications.16
Recently, various metal NP-based biosensors using
localized surface plasmon resonance (LSPR) have been
20-24
60 proposed,and development of applications of LSPR has
been a topical research subject.25 The Au NP-based DNA
sensors mentioned above are some noteworthy cases of LSPR
biosensors. In particular, one of the most attractive extensionsof LSPR sensors is their biosensing applications using
26-32
65 surface-enhanced Raman scattering (SERS).
SERS is asurface sensitive phenomenon which results in theenhancement of Raman scattering by molecules absorbed onrough metal surfaces or metal colloid aggregates. The
significant amplification of Raman scattering intensity is
70 because of the electric field enhancement that occurs in thevicinity of small, interacting metal NPs that are illuminatedwith light resonance or near resonance with the LSPR
frequency. SERS has attracted great interest in recent years as
a powerful analytical tool that yields enormous structural
about the analyte, as well as quantitative
75 information
information.33 SERS-based sensors are highly sensitive, andthus, can be exploited in biosensing applications and havebeen successfully applied to analyze biologically relevantmolecules, such as DNA,34-38 proteins39 and cells,40 with high80 sensitivity and selectivity. However, the development ofrobust and reproducible SERS-enhancing substrates has long
been a central task in SERS research.
To that end, we propose a unique and easy-to-operate NPbased SERS sensor for the detection of DNA sequences. The
41,42
85 proposed DNA sensor exploits the photoligation reaction

Journal Name, [year], [vol], 00–00 | 1

synthesis, thiol-modifier C6 S-S, and 3’-thiol-modifier C3 S-S
CGP, the reagents for the DNA synthesizer such as A, G, C,
T-β-cyanoethyl phosphoramidite, and CPG support werepurchased from Glen Research, USA. Calf intestine alkaline
phosphatase was purchased from Promega, Japan. Nucleate
45 5’-nucleotidohydrolase (3’-phosphohydrolase) (Nuclease P1)
was purchased from Yamasa Corp., Japan.
40

Equipment
UV-visible spectroscopy
UV-visible absorption spectra of the Au NP dispersions wereobtained using a Beckman Coulter DU800 UV/VIS
spectrometer. The spectra were collected over the range of
200-800 nm.
Dynamic light scattering (DLS) and zeta potential (ZP)
55 measurements
DLS and ZP measurements were performed on diluted Au NP
dispersions at room temperature using a Zetasizer Nano ZS
ZEN 3600 (Malvern, UK).
Raman spectroscopy+
60 Raman spectra were obtained with a Kr ion laser (wavelength
647.1 nm, power 100 mW), using a Horiba-Jobin Yvon
Ramanor T64000 triple monochromator equipped with a CCD
detector. The non-polarized Raman scattering measurements
were set under a microscope sample holder using a 180°
65 backscattering geometry at room temperature. The laser spot
diameter was 1 µm. As an acquisition time was 30 sec per
spectrum, we averaged three spectra taken from same area.
Transmission electron microscopy (TEM)
TEM measurements were performed to determine the average
70 diameter and the morphology of Au NPs and their aggregatesusing a Hitachi 7100 transmission electron microscope
operated at 100 kV. TEM samples were prepared by dropping
5 µL of the Au NP dispersions onto carbon-coated copper
grids under ambient conditions and allowing them to75 evaporate slowly at room temperature.
UV irradiation for photoligation
Photoligation between the cytosine base at the 3’-end of P1
and 5-carboxylvinyl-2’-deoxyuridine at the 5’-end of P2 was
performed using an UV-LED (Omron, ZUV, wavelength280 366±15 nm, power 1600 mW/cm ) at a distance of 1.5 cm at
0 °C for 15 min.
50

Fig. 1 Illustration of the DNA sensor. (a) Main components of the system.
(b) Procedure to hybridize Au-P1, Au-P2, and T-DNA in cacodylate
buffer. (c) Schematic depiction of the hybridized structure containing Au5 P1, Au-P2, and T-DNA. (d) Chemical structures of the cytosine base at
the 3’-end of P1 and cvU at the 5’-end of P2 before and after photoligation.

between oligodeoxynucleotides (ODNs) attached on thesurfaces of Au NPs in the presence of target DNA (T-DNA).
10 In this technique, two kinds of ODNs, a common probe (P1)
and a specific discriminating probe (P2), are separately
conjugated on the surface of Au NPs. These ODN-conjugated
Au NPs are denoted as Au-P1 and Au-P2, respectively. Whenhybridization takes place between Au-P1 and T-DNA and
15 between Au-P2 and T-DNA, the Au NPs covalently crosslink
to form aggregates via photoligation between P1 and P2 asshown in Figure 1. Once the NP aggregates have formed, theinterspace between Au NPs in the aggregate act as a stable“hot spot”, and a SERS signal from the Raman-active
20 molecules present in the hot spot is easily detected. In contrast,
a SERS signal is not detected if the hybridization is
unsuccessful, because a stable hot spot is not formed. The
DNA sensor proposed here is simple, easy-to-use, rapid androbust, requiring no enzymatic reaction, fluorescent dye,temperature control, or complicated operating
25 precise
conditions.

ODN synthesis

Experimental
Materials
Trisodium citrate hydrate (cat. no 194-13245), 3aminopropyltrimethoxysilane (cat. no 323-74-352), calcium
chloride (CaCl2, purity 95 %), and sodium chloride (NaCl,
purity 99.5 %) were purchased from Wako Pure Chemical
Industries, Ltd., Japan. Gold(III) chloride trihydrate
35 (HAuCl 4 .3H 2 O, cat. no. 1525-26) was purchased from MP
Biomedicals, LLC, France. Methanol, acetone, and sulfuric
acid were purchased from Kanto Chemical Co., Inc., Japan.
Cacodylate buffer solution (0.2 M, cat. no 37237-35) was
purchased from Nacalai Tesque, Inc., Japan. For ODN
30

2 | Journal Name, [year], [vol], 00–00

ODN sequences 5’- HS-ACTCACAGTTTTCAC -3’ (15-mer,5’-SH, P1), 5’-cvUTCAGTGTA-SH -3’ (9-mer, 3’-SH, P2),
and 5’-TACACTGAAGTGAAAACTGTGAGTG -3’ (25-mer,
T-DNA)
weresynthesizedbytheconventional
phosphoramidite method using an Applied Biosystems 3400
DNA synthesizer. Here cvU denotes a 5-carboxylvinyl-2’90 deoxyuridine moiety that acts as a photolinking group. The
coupling efficiency was determined using a trityl monitor. The
coupling efficiency and coupling time of the crude mixture of
the phosphoramidite were 97 % and 999 s, respectively. The
coupled mixtures were deprotected by incubation with 28 %95 ammonia for 8 h at 55 °C and were purified on a Chemcobond
5-ODS-H column (10×150 mm) by reversed-phase high85This journal is © The Royal Society of Chemistry [year]performance liquid chromatography (HPLC); 0.05 M
ammonium formate containing 3-20 % CH3CN was used as
the eluent with a linear gradient (30 min) at a flow rate of 3.0
mL/min. ODNs were fully digested with calf intestine alkaline−1−15 phosphatase (50 U mL ) and Nuclease P1 (50 U mL ) at
37 °C for 4 h. The digested samples were analyzed using
HPLC (Supplementary Information Figures S1-S3). The
concentration of each ODN was determined by comparing thepeak areas with standard solutions that contained dA, dG, dC,
10 and dT at a concentration of 0.1 mM. Preparation of ODNs
was confirmed by MALDI-TOF-MS analysis (Supplementary
Information Figures S4-S6).
Au NP synthesis
Au NPs with average diameter of 15 nm (standard deviation≤15 %) were synthesized by the citrate thermal reduction
method.43 The LSPR extinction peak was located at 520 nm.
The citrate ions were used as a reducing agent, as well as aprotective capping agent to stabilize the Au NPs in the
20 medium. Typically, Au NPs were synthesized in a 100 mL
round-bottom three-neck flask equipped with a thermo-couple.
An aqueous solution of HAuCl4.3H2O (50 mL, 1 mM) was
stirred vigorously and heated to reflux at 100 °C. Then,aqueous trisodium citrate (5 mL, 38.8 mM) was rapidly
25 injected in the flask. After the injection of sodium citrate, thecolor of reaction mixture changed from yellow to burgundy
indicating the formation of Au NPs, the mixture was stirred
for 15 min at 100 °C. The mixture was cooled to room
temperature and then filtered using a 0.45 µm syringe filter
Stedim Biotech GmbH, Germany). The
30 (Sartoriusconcentration of Au NPs was estimated to be 10.4 nM using
the extinction coefficient of 15 nm Au NPs of ε 520 ≅2.40×108
M−1 cm−1.441535Preparation of substrates

Glass substrates were used after surface modification. The
surface modification was conducted according to theprocedure reported by Watts et al.45 Glass slides (Matsunami
micro slide glass, 2.6×7.6 cm2) were cleaned by sonication in
40 acetone for 10 min, followed by 10 min ultrasonic cleaning inmethanol. After drying for 20 min at 100 °C in air, the slides
were immersed in concentrated sulfuric acid for 2 h. The
slides were rinsed thoroughly with distilled water 6 times andthen dried for 20 min at 100 °C in air. Surface modification of
45 the glass substrates was performed by soaking the substrates
in a mixture of 3-aminopropyltrimethoxysilan (APTMS, 3mL) in methanol (60 mL) for 4 h at room temperature. Aftersoaking, the glass substrates were washed thoroughly using a
copious amount of methanol to remove the excess APTMS.
50 The substrates were stored in methanol at room temperaturewhen not in use. The substrates were dried for 20 min at
100 °C in air immediately prior to use. APTMS modificationof the glass slides was confirmed by measuring the watercontact angle.
55

Preparation of Au-P1 and Au-P2
This journal is © The Royal Society of Chemistry [year]

The citrate capped Au NPs were modified with thiolated
ODNs (P1 or P2) through a simple protocol. An aqueous
solution of CaCl2 (50 µL, 10 mM) was added to an aqueous
60 dispersion of the Au NPs (1 mL, 10.4 nM). Subsequently, 50µL of the NP dispersion was divided and mixed with anaqueous solution of ODN (P1 or P2, 50 µL, 100 µM) to
conjugate thiolated ODNs on the surfaces of the Au NPs.
After mixing, the dispersion was incubated at 40 °C for 5 h incv
65 the dark. Note that the ODN containing
U is light sensitive,
and thus, should be protected from exposure to UV radiation.
ODN-modified Au NPs (Au-P1 and Au-P2) were stored at
4 °C in the dark until just prior to use.
70

Hybridization and photoligation

2 µL of each dispersion containing Au-P1 or Au-P2 was takenfrom their respective neat dispersions. Then, both the Au-P1
and Au-P2 dispersions were mixed with 2 µL of T-DNA
solution (100 µM) in a cacodylate (50 mM)/NaCl (100 mM)
75 buffer at room temperature in the dark. The final compositionof the mixture (total volume 10 µL) was as follows: T-DNA
20 µM; sodium cacodylate [(CH3)2AsOONa] 50 µM; NaCl
100 µM. Subsequently, the mixture was cooled down to 0 °C
and irradiated with UV light with a wavelength of 366 ± 1580 nm for 15 min to promote photoligation. The mixture wasthen heated at 90 °C for 3 min using a Thermal Cycler MP
TP-3000 (Takara, Japan) to denature the DNA. Then, 2 µL of
the dispersion was dropped on the APTMS-modified glass
substrate and dried at 90 °C for 5 min.
85

Results and discussion
Preparation of ODN-modified Au NPs

The as-synthesized Au NPs are capped with citrate ions so thesurfaces of the NPs are negatively charged. The ODNs are
90 also negatively charged. Hence, the conjugation reaction of
thiolated ODNs to Au NPs was unsuccessful when one simply
mixed the Au NPs and ODNs because of the electrostatic
repulsion between the Au NPs and ODNs. Therefore, Ca2+
ions were added to the NP dispersion to screen the negative2+
95 charge of both species. Ca
ions were chosen to reduce theelectrostatic repulsion between the Au NPs and ODNs
because CaCl2 solution is routinely used to enhance theefficiency of the genetic transformation of E. coli withplasmid DNA. It is well known that divalent cations, such as
2+
100 Ca , make the cell membrane permeable to plasmid DNA,effectively reducing the electrostatic repulsion between DNA
and cell membrane. However, if the concentration of Ca2+
ions was too high, the Au NPs aggregated before the ODNs
were attached on the NP surfaces. Thus, the concentration of2+
105 Cascreening the charges of NPs and ODNs was carefully
controlled to avoid aggregation of the Au NPs.
Figure 2 shows UV-vis absorption spectra of the Au NP
dispersions taken immediately after adding different volumesof CaCl 2 solution (10 mM). The volume of the Au NP
110 dispersion prior to adding the CaCl 2 solution is 1 mL. Whenthe volume of CaCl2 solution added was larger than 100 µL,

Journal Name, [year], [vol], 00–00 | 3

Fig. 2 Absorption spectra of the Au NP dispersions containing different
volumes of CaCl2 solution. From top to bottom: 0, 50, 100, 200, 300, 400,
and 500 µL of CaCl2 solution added to 1 mL of Au NP dispersion,
5 respectively.

Fig. 3 Time dependence of absorption spectra of the Au NP dispersions
containing (a) 0, (b) 50 and (c) 100 µL of CaCl2 solution. Black, red,
10 orange, green, light blue, blue, and purple lines represent 0, 1, 2, 3, 4, 5,
and 12 h incubation periods, respectively. (d) Time dependence of the ZP
of Au NP dispersions containing 0 (black), 50 (red) and 100 µL (blue) of
CaCl2 solution.

the extended plasmon band (EPB) was clearly seen between
600 and 800 nm, indicating the formation of Au NP
aggregates. A negligible change in the absorption spectrum
was observed when the volume of CaCl2 solution added was
50 µL, signifying that the Au NPs were still well dispersed
the experimental time range. Subsequently, the20 duringaggregation rate (stability) of the Au NPs with the addition of0, 50 and 100 µL of CaCl2 solution was investigated. Because
the ODNs do not conjugate to the Au NPs after aggregation,
the incubation was stopped before significant aggregation
25 took place. Figures 3a, 3b and 3c show the variation of theabsorption spectra of the Au NP dispersions over time withthe addition of 0, 50 and 100 µL of CaCl2 solution,
respectively. As seen in Figure 3a, the Au NP dispersion withno added CaCl2 is remarkably stable over the experimental
30 time range (12 h). In the case of the Au NP dispersion with 50
15

4 | Journal Name, [year], [vol], 00–00µL of CaCl2 solution added (Figure 3b), no sign ofaggregation was observed up to 5 h after the addition of the
CaCl2 solution. However, after 12 h incubation, the EPB was
clearly seen in the absorption spectrum. In the case of the Au
35 NP dispersion containing 100 µL of CaCl 2 solution (Figure3c), the EPB was clearly observed within 1 h of the additionof the CaCl2 solution.
To confirm the screening effect of the Ca2+ ions, the zeta
potential (ZP) of the Au NPs was measured for each2+
40 dispersion containing different Ca
concentrations (Figure3d). The ZP of the as-synthesized Au NPs was constant at
about −39 mV. After addition of the CaCl2 solution, ZP
decreased to about −34 mV regardless of the volume of CaCl2
solution added. However, in the case of the Au NP dispersion
45 containing 100 µL of CaCl 2 solution, ZP quickly returned to
the value of the as-synthesized Au NPs. This can be explainedby the ZP measurement technical mechanism. ZP was
estimated by measuring the mobility of particles using laser
Doppler anemometry under an alternating electric field.
50 Therefore, when the aggregates precipitate in the capillary cell,
their ZP cannot be measured, so only the ZP of the remaining
mobile (non-aggregated) NPs is detected. The non-aggregatedNPs would have almost the same ZP value as the assynthesized Au NPs, so aggregation caused the ZP to return to
55 the value of as-synthesized Au NPs. On the other hand, for the
Au NP dispersion containing 50 µL of CaCl 2 solution, ZP
remained constant at around −34 mV for the first 5 h, and thenreturned to the value of the as-synthesized Au NPs. These
results are consistent with those of the UV-vis spectroscopy.
60 In consequence, the conjugation reaction of thiolated ODNs to
Au NPs was performed for 5 h after the addition of 50 µL of
CaCl2 solution (10 mM). The slight decrease in the ZP isthought to be enough to enhance the conjugation reaction rate,
while preventing aggregation of the NPs.
To confirm the conjugation of thiolated ODNs on the65
surfaces of the Au NPs, Raman measurements were performed.
The ODN sequence used was 5’-ACGCAGGCAC-SH -3’ (10mer, 3’-SH), and the Raman spectrum was obtained
immediately after dropping 2 µL of the dispersion (Au
70 NPs/ODNs/CaCl 2 mixture) on an APTMS-modified glass
substrate. Figure 4a shows the Raman spectra of samples
prepared with different incubation times. Several Ramanpeaks start to appear after a 2 h incubation period. The 610
cm−1 band is assigned to the ring breathing mode of guanine,−1
75 which is sensitive to the conformation, and the 780 cm
band
46
is assigned to the ring breathing mode of cytosine. In Figure
4b, the temporal variations of the 610 cm−1 and the 780 cm−1
band intensities were plotted. The peak intensities increased
with incubation time and were almost saturated after a 3 h
80 incubation period, suggesting that the conjugation reaction of
ODNs on the Au NP surfaces was complete. That is, thenumber of ODN molecules present in the hot spot increases,
because the ODNs were successfully immobilized on the Au
NP surfaces. Considering the results shown in Figs. 3 and 4,
85 the optimum incubation time for the conjugation reaction of
ODNs on the Au NP surfaces was determined to be 5 h.
After conjugation, the ODN-modified Au NPs exhibited
almost identical UV-vis spectra. A negligible shift in theThis journal is © The Royal Society of Chemistry [year]

Fig. 4 (a) Raman spectra of Au NPs incubated with ODNs for different
periods at 40 °C. Black, red, orange, green, light blue, and blue lines
represent 0, 1, 2, 3, 4, and 5 h incubation periods, respectively. (b)−1−1
5 Temporal variations of the 610 cm (blue triangles) and the 780 cm (redsquares) band intensities.

LSPR band from 520 nm to 522 nm was observed between theas-synthesized and ODN-modified Au NPs (data not shown).
2+
10 This suggests that Au NPs were stabilized better against Ca
with ODNs conjugated on their surfaces, because the UV-vis
spectrum of bare Au NPs after the addition of 50 µL of CaCl2
solution (Fig. 3b) was not completely identical to that of assynthesized Au NPs (Fig. 3a). Figure 5a shows the size
15 distributions of as-synthesized and ODN-modified Au NPs
dispersed in water, measured by dynamic light scattering(DLS). Interestingly, the ODN-modified NPs were smaller on
average than the unmodified Au NPs. This result implies thatthe repulsion force between ODN-modified NPs is larger than
20 that of the unmodified, citrate-capped NPs because the higher
electrostatic and steric repulsion potentials of ODNs,
compared with those of the citrate ions, suppress theclustering of Au NPs. Figures 5b and 5c show TEM images of
the as-synthesized and ODN-modified NPs, respectively. The
25 ODN-modified NPs show larger separation from each otherthan the unmodified NPs, which is consistent with the size
distributions of the two types of Au NP.
DNA hybridization detection using SERS spectroscopy
30

Most examples of SERS-based biosensors use a Raman-active
This journal is © The Royal Society of Chemistry [year]

Fig. 5 (a) Size distributions of as-synthesized (blue curve) and ODNmodified (red curve) Au NPs dispersed in water measured by DLS, andTEM images of (b) as-synthesized and (c) ODN-modified Au NPs.
35

dye as a reporter molecule to obtain a strong SERS signal,47
because the SERS signal from the biorelevant molecule itself
is usually weak. In general, the system becomes more
complicated and more difficult to operate as the number of
40 components increases. Therefore, in our DNA detection
system, sodium cacodylate is used as a reporter molecule aswell as a hybridization buffer. The 608 cm−1 vibration of
cacodylate (symmetric AsO2 stretch) was the intensity
standard used by Blazej and Peticolas in their pioneering48
45 studies of Raman excitation profiles of nucleic acid bases.
−1
The Raman scattering cross section of the 608 cm vibrationof cacodylate at 647 nm was determined to be 40±4 µbarns
where a barn is 10−24 cm2.49 Figure 6a shows the Raman
spectra of sodium cacodylate obtained in liquid (solution) and50 solid (crystal) phases. The cacodylate vibrations are visible asa pair of bands centered at 605 cm−1 and 639 cm−1 (AsO2stretch) in the liquid phase sample. In the solid phase, thehigher energy peak shifted toward 600 cm−1, while the lower
energy peak disappeared.
Figure 6b shows the dependence of the UV irradiation time55
on the Raman spectrum of cacodylate in the sample depositedon the APTMS-modified glass substrate. The AsO2 stretchingvibrations are clearly observed as a pair of bands centered at
608 cm−1 and 638 cm−1 in both samples irradiated with UV
60 light for 15 and 30 min. The inset in Figure 6b shows theintensity of the 608 cm−1 peak versus UV irradiation time. The
Raman spectra were taken at ten different positions for eachsample. Consequently, it exemplifies that the intensity of the
Journal Name, [year], [vol], 00–00 | 5

Fig. 6 (a) Raman spectra of sodium cacodylate obtained in liquid (top)and solid (bottom) phases. The inset shows the chemical structure of
sodium cacodylate. (b) Changes in the Raman spectrum of cacodylate in
5 the sample deposited on the APTMS-modified glass substrate plottedversus UV irradiation time (0, 15, and 30 min from bottom to top). The
inset shows the intensity of the peak at 608 cm−1 versus UV irradiationtime.

608 cm−1 band of the samples irradiated by UV is notably
higher than that of the sample not exposed to UV irradiation.
Additionally, the intensity of the 608 cm−1 band did notchange when the UV irradiation time was prolonged from 15
min to 30 min. These results imply that Au-P1 and Au-P2 are
15 successfully linked via photoligation in the presence of TDNA, and thus, the SERS signal of cacodylate present in the
hot spot can be obtained. Moreover, 15-min of UV irradiation
seems to be long enough to allow completion of thephotoligation reaction. Hence, the UV irradiation time was
20 fixed at 15 min.
To confirm that the increased intensity of the 608 cm−1
band was caused by photoligation of Au-P1 and Au-P2, we
carried out control experiments using Raman measurements ofsamples lacking UV exposure, T-DNA, Au-P1 and Au-P2,
25 deposited on the APTMS-modified glass substrate. Figures 7a,7b, 7c, 7d, and 7e show the Raman spectra of the complete(containing Au-P1, Au-P2, T-DNA, and exposed to UV
irradiation), and the samples lacking UV exposure, T-DNA,
Au-P1, and Au-P2, respectively. The Raman spectra taken at
30 eight different positions for each sample were plotted in eachfigure. As seen in Figs. 7b-e, the intensities of the band at 608
cm−1 of the control samples are statistically the same, and are
10

6 | Journal Name, [year], [vol], 00–00

evidently much lower than that of the complete sample (Fig.
7a). This result clearly indicates that the complete set of
35 conditions (Au-P1, Au-P2, T-DNA, and UV irradiation) is
necessary to obtain the SERS signal from cacodylate, that is,
the hybridization detection scheme shown in Figure 1 isrealized in our experiments. It is noteworthy that we were able
to reproduce our experimental results at least three times and
40 always obtained the same results, indicating this system isrobust and highly reproducible. As can be seen in Figure 7a,
however, the intensity of the 608 cm−1 peak varied
considerably within the same sample. This is due to themicroscopic inhomogeneity of the sample, i.e., the probability
45 of finding Au NP aggregates (hot spots) within the laser beamdiffers according to the location in the sample, because theconcentration of Au NPs is quite low. Figure 7f shows thehistogram of the intensity of the 608 cm−1 peak. Amplified
signals with an intensity of greater than 10 counts/s are
50 observed only in the complete sample, and can be attributed to
SERS signals from aggregates formed via photoligation. On
the other hand, all of the samples have weak signals with anintensity of less than 4 counts/s, which are the signals frompositions without a hot spot, and can be regarded as the
55 background. The signals with an intensity ranging from 4 to
10 counts/s observed in the perfect, Au-P1-less and Au-P2less samples may arise from hot spots formed via natural
aggregation during drying. To reduce the variation in theintensity of the SERS and increase the signal-to-noise ratio,
60 the samples must be homogeneous. However, if a monolayer(or multilayer) of Au NPs is formed on the substrate surface,
the background intensity will increase significantly, as thedensity of naturally-formed hot spots will markedly increase
because of decreases in the inter-particle separation distance.
65 The processing of more uniform materials is part of ourongoing work.
To further confirm the formation of aggregates via
crosslinking between Au-P1 and Au-P2 through hybridization
with T-DNA followed by photoligation between P1 and P2,
70 TEM images were taken of all of the samples (Figure 8). In
Fig. 8, TEM images taken at two different arbitrarily selected
positions in the same sample are shown for all of the samples.
Obviously, large aggregates are observed only in the perfect
sample (Fig. 8a), while only single, isolated NPs or small
75 clusters of NPs are seen in the imperfect samples (Fig. 8b-e).
The TEM images shown in Fig. 8b-e are quite similar to Fig.
5c. The mean surface-to-surface separation distance between
the NPs in Fig. 8a is estimated to be 3.2 nm, which is asignificantly larger value than that of citrate-capped Au NPs
80 (1.4 nm), supporting the formation of the Au-P1-P2-Au bond.
However, 3.2 nm is only approximately one third of the total
length of P1 (6.3 nm) and P2 (3.8 nm), and thus, the Au-P1P2-Au bond is thought to be bent in the interspaces between
Au NPs.
85

Conclusions
We present a simple and easy-to-use DNA sensor obtained bymeasuring the surface-enhanced Raman scattering (SERS) of
crosslinked Au NP aggregates. The DNA sensor uses
This journal is © The Royal Society of Chemistry [year]

Fig. 7 Raman spectra of (a) perfect (Au-P1+Au-P2+T-DNA+UV), (b) UV-less (Au-P1+Au-P2+T-DNA), (c) T-DNA-less (Au-P1+Au-P2+UV), (d) AuP1-less (Au-P2+T-DNA+UV), and (e) Au-P2-less (Au-P1+T-DNA+UV) samples. (f) Histogram of the intensity of the 608 cm−1 peak. Red, blue, green,
5 yellow, and light blue bars correspond to the perfect, UV-less, T-DNA-less, Au-P1-less, and Au-P2-less samples, respectively.

Fig. 8 TEM images of the samples: (a) perfect, (b) UV-less, (c) T-DNA-less, (d) Au-P1-less, and (e) Au-P2-less.

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Journal Name, [year], [vol], 00–00 | 7

photoligation to achieve DNA recognition without requiring
an enzymatic reaction, fluorescent dye, precise temperature
control, or complicated operating conditions. The sensor
operates well with a minute amount of target DNA (T-DNA)
5 (~20 pmol), making it extremely sensitive. Au NPs conjugatedwith a common probe (Au-P1) and a specific discriminatingprobe with a photo-linking group at the 5’ end (Au-P2) were
simultaneously hybridized with T-DNA. Then, Au-P1 and AuP2 were covalently linked to form a Au-P1-P2-Au bond by
10 UV irradiation. The SERS signal of cacodylate ions, which act
as reporter molecules, present in the interspaces between Au
NPs was successfully detected. The incomplete set of
conditions, lacking components such as Au-P1, Au-P2, TDNA, or UV exposure, did not clearly produce the SERS
15 signal. The results explicitly indicate that our sensor is
feasible. However, in this study, we only used fully matchedsequences, i.e., there was no mismatch between P1 and TDNA, and between P2 and T-DNA. The match/mismatch
discrimination performance of the DNA sensor is part of our
20 ongoing work on this research topic.
DNA photoligation can be used for the potential application
as a tool for molecular biology research such as the specificdetection of DNA or RNA molecules. We anticipate that thisnew concept of DNA sensor using SERS on Au NP aggregates
25 formed by DNA photoligation will be the starting point for thedevelopment of a practical assay for the quantification of amultitude of DNA and noncoding RNA in living cells.

Acknowledgements
30

The authors would like to thank the Vietnamese Government
for a 322 scholarship. Dr. Ha Minh Hiep (Osaka University) is
thanked for his valuable comments. We thank Dr. Derrick
Mott for his careful reading of, and useful comments about,the manuscript.

35

Notes and referencesaSchool of Materials Science, Japan Advanced Institute of Science andTechnology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan. Fax: +81
761 51 1625; Tel: +81 761 51 1611; E-mail: shinya@jaist.ac.jpb40 Innovation Plaza Ishikawa, Japan Science and Technology Agency, 1-1
Asahidai, Nomi, Ishikawa 923-1292, Japan.
† Electronic Supplementary Information (ESI) available: Results of
HPLC and MALDI-TOF MS analyses of the ODNs. See
DOI: 10.1039/b000000x/

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