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Angewandte
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DOI: 10.1002/anie.201408558
High-Throughput Screening
Direct Surface-Enhanced Raman Scattering Analysis of DNA
Duplexes**
Luca Guerrini,* Željka Krpetić, Danny van Lierop, Ramon A. Alvarez-Puebla, and
Duncan Graham
Abstract: The exploration of the genetic information carried
by DNA has become a major scientific challenge. Routine
DNA analysis, such as PCR, still suffers from important
intrinsic limitations. Surface-enhanced Raman spectroscopy
(SERS) has emerged as an outstanding opportunity for the
development of DNA analysis, but its application to duplexes
(dsDNA) has been largely hampered by reproducibility and/or
sensitivity issues. A simple strategy is presented to perform
ultrasensitive direct label-free analysis of unmodified dsDNA
with the means of SERS by using positively charged silver
colloids. Electrostatic adhesion of DNA promotes nanoparticle
aggregation into stable clusters yielding intense and reproducible SERS spectra at nanogram level. As potential applications, we report the quantitative recognition of hybridization
events as well as the first examples of SERS recognition of
single base mismatches and base methylations (5-methylated
cytosine and N6-methylated Adenine) in duplexes.
DNA is the custodian of the memory of life. Although very
stable, it is subject to modifications that are associated with
evolution but more frequently with genetic diseases or
cancer.[1] Owing to the small content of DNA in cells and its
structural complexity, its analysis require denaturation (heat),
fragmentation (restriction endonucleases), separation (electrophoresis), amplification (polymerase chain reaction, PCR)
and detection (quantitative PCR or microarray techniques).[2]
Besides the cost both in time and money, during these
processes samples are subject to alterations[3] and losses of
epigenetic information.[4] The advent of nanophotonics offers
a unique opportunity for the development of direct, fast, and
ultrasensitive methods for DNA analysis.[5] In the broad field
of plasmonics, surface-enhanced Raman scattering (SERS)
[*] Dr. L. Guerrini, Prof. R. A. Alvarez-Puebla
Departamento de Qumica Fisica e Inorganica
Universitat Rovira i Virgili and Centro de Tecnologia Qumica
Carrer de Marcel l Domingo s/n, 43007 Tarragona (Spain)
and
Medcom Advance SA, Viladecans Bussines Park, Edificio Brasil
C/Bertran i Musitu, 83–85, 08840 Viladecans (Barcelona) (Spain)
E-mail: [email protected]
Dr. Ž. Krpetić
School of Chemistry and Chemical Biology
Centre for BioNano Interactions, University College Dublin
Belfield, Dublin 4 (Ireland)
Dr. Ž. Krpetić, Dr. D. van Lierop, Prof. D. Graham
Centre for Molecular Nanometrology, Pure and Applied Chemistry,
University of Strathclyde (UK)
Prof. R. A. Alvarez-Puebla
ICREA, Passeig Llus Companys 23, 08010 Barcelona (Spain)
1144
spectroscopy has arisen as a powerful analytical tool for
detection and structural characterization of biomolecules.[6]
The large majority of SERS-based DNA detection strategies
rely on the selective recognition between two complementary
strands to identify a specific sequence, and the use of extrinsic
Raman labels for SERS readout.[7] However, labeling of the
DNA strands requires complex and costly chemistry, and does
not provide any chemical-specific information.[8] A second
approach is based on the direct detection of the distinctive
SERS signal from DNA strands directly adsorbed onto the
nanostructured surface. This strategy shows outstanding
potential in terms of sensitivity, selectivity, and chemicalspecific information.[8, 9] To date, however, direct SERS
analysis of unmodified DNA is mainly restricted to singlestranded DNA sequences (ssDNA), as the direct contact of
double-stranded sequences (dsDNA) with nanostructured
metallic surfaces (negative at the physiological pH) is largely
hindered by the negative charge of the phosphate backbone,
unless relatively high DNA concentrations are employed.[9b,c]
As a result, dsDNA SERS spectra traditionally suffer from
inherent poor spectral reproducibility[8, 9b] and/or limited
sensitivity, hampering the extended application of label-free
SERS strategies for the study of unmodified duplexes. Thus,
successful translation of the analytical potential of SERS to
the direct study of dsDNA would be a great leap forward for
the full exploration of the genetic information.
To overcome this major challenge, in this study we use
positively charged silver nanoparticles coated with spermine
molecules (AgNP@Sp). Spermine bound to AgNPs acts as
stabilizer, and upon addition of negatively-charged DNA, it
promotes NP aggregation into stable particle clusters.[10] This
has important consequences for the direct SERS analysis of
[**] This work was funded by the Spanish Ministerio de Economia
y Competitividad (CTQ2011-23167), the European Research Council
(CrossSERS, FP7/2013 329131, PrioSERS FP7/2014 623527), and
Medcom Advance SA. D.G. acknowledges support from the Royal
Society through a Wolfson Research Merit Award. We are grateful to
Dr. Matteo Masetti (Department of Pharmacy and Biotechnology,
Universit di Bologna, Bologna, Italy) for the dsDNA drawings.
Supporting information for this article (experimental, AgNP@Sp
characterization (Section S1), DNA-driven aggregation of
AgNP@Sp and spectral reproducibility (Section S2), vibrational
assignment of dsDNA SERS spectra (Section S3), SERS of ss and ds
sequences on silver hydroxylamine colloids (Section S4), ds1 SERS
spectrum and the pondered sum of the individual spectra of ss1 and
ssc (Section S5), and SERS spectra of ds2, ds3 and ds4 (Section S6)) is available on the WWW under http://dx.doi.org/10.1002/
anie.201408558.
2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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dsDNA. Firstly, the DNA adsorption occurs by non-specific
electrostatic interaction of the phosphate groups rather than
by base-specific nucleoside–metal binding. Secondly, DNA
promotes the nanoparticle aggregation removing the need of
external aggregating agents. On the one hand, this significantly simplifies the sensing scheme, reducing the number of
variables to be controlled (type and concentration of the
aggregating agent, time and order of addition, and so on). On
the other hand, DNA sequences are selectively sandwiched
between interparticle junctions (that is, hot-spots[11]), thus
maximizing the intensification of the Raman signal (Figure 1 a). Thirdly, the nanoparticle aggregation occurs fast and
independently of the base composition, generating long-term
stable clusters in suspension, as demonstrated by the extinc-
Figure 1. a) Diagram of dsDNA sandwiched between two positively
charged AgNP@Sp. b) Extinction spectra of pure AgNP@Sp colloids
and in the presence of ds1 (final conc. 630 ng mL1) acquired 2 h and
24 h upon DNA addition (the AgNP@Sp + ds1 mixture was sonicated
for few seconds before the spectral acquisition). c) SERS spectra of
ss1 and ssc (3.15 mg mL1), and ds1 (630 ng mL1) on AgNP@Sp.
SERS spectra were normalized to the PO2 stretching at 1090 cm1.
The ds1 SERS spectrum in the 2700–3050 cm1 region was divided by
two. d) Detail of the 680–890 cm1 spectral region for the SERS spectra
of ssc + ds1 mixtures at different molar ratios
Rhybr = [ds1]/([ds1] + [ssc]) (from top to bottom: 0.011, 0.024, 0.041,
0.063, 0.091, 0.130, 0.189, 0.286, 0.474, and 1). The ds1 concentration
was progressively increased from 0 to 630 ng mL1 while the ssc
concentration was simultaneously decreased from 3.15 mg mL1 ng to
zero. The dotted line is the difference spectrum ssc-ds1 obtained by
digitally subtracting the SERS spectra shown in (c). e) Ratiometric
peak intensities I724/I738 vs. Rhybr = [ds1]/([ds1] + [ssc]) molar ratio.
Angew. Chem. Int. Ed. 2015, 54, 1144 –1148
tion spectra illustrated in Figure 1 b. This also allows us to
perform the SERS measurements after several hours upon
the analyte addition (that is, when the process of DNA
adhesion has reached its equilibrium). Finally, SERS spectra
are acquired in colloidal suspensions with a long working
distance objective. In this experimental set-up, the recorded
SERS spectra are the statistically-averaged result of a large
ensemble of nanoparticle/DNA clusters in continuous Brownian motions within the scattering volume. Such averaged
bulk SERS regime yields good-quality spectra with welldefined average band centers, bandwidths, and relative
intensities.[12] As a result, dsDNA electrostatic adhesion
onto the AgNP@Sp nanoparticles allows for the ultrasensitive
analysis of duplexes and their modifications at the nanogram
regime with high batch-to-batch reproducibility, yielding
SERS spectra that are truly spectral representations of the
nucleoside composition and organization within the helix. To
confirm this, we show an ample set of examples of direct labelfree SERS detection of unmodified duplexes, including
quantification of hybridization events as well as recognition
of single-base mismatches and base methylations (5-methylated cytosine and N6-methylated adenine), which to date
were restricted to single-stranded sequences.[9a,d]
The average SERS spectra of two complementary singlestranded sequences (ss1 and ssc) and their corresponding
double-helix structure (ds1) on AgNP@Sp colloids are shown
in Figure 1 c. The overall dsDNA concentration was kept
constant throughout the entire study at ca. 630 ng mL1
(corresponding to less than 3.0 ng in the probed volume; see
the Supporting Information, Section S2) and appropriately
selected to yield long-term stable clusters in suspension with
reproducible and unvaried SERS spectral profiles (Section S2). Vibrational assignment of dsDNA was based on
literature references and comparative spectral analysis with
homo- and bi-polymeric sequences (Section S3). In contrast
with previous results on negatively charged colloids (Section S4),[13] when the individual ss units hybridize into the ds
a large reshaping of their SERS spectra is observed (Figure 1 c), influencing peak position, bandwidths, and relative
intensity. Notably, if ss1 and ssc spectra show small changes in
relative intensity that are associated with the base composition, the stacking and pairing of the nucleobases into the
hybridized native structure is revealed by several characteristic features of the Raman melting profiles.[14] Among others,
we highlight the marked intensity decrease and peak-shifting
of the ring breathing modes (700–800 cm1) and the carbonyl
stretching modes (1653 cm1), the latter being extremely
sensitive to the disruption of Watson–Crick hydrogen bonding.[14] We also observe a general narrowing of the Raman
features in the 1150–1600 cm1 spectral region, containing
many overlapping bands mainly arising from in-plane vibrations of base residues.[15] Such spectral modification can be
ascribed to the adoption of a more rigid and well-defined
conformation by the two individual ss when they self-organize
into the rigid duplex geometry. The investigation of a set of
solutions containing different ds1 and ssc molar ratios, Rhybr =
[ds1]/([ds1] + [ssc]) (Figure 1 d) shows a progressive shift of
the ring breathing bands of adenine (from 734 cm1 to
730 cm1) when the DNA population is enriched with ds1.
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The spectral subtraction of the SERS spectra allows us to fully
disclose the spectral alterations associated with changes in the
DNA structure. In particular, the difference spectrum ds1-ssc
(dotted line, Figure 1 d) reveals a minimum at 724 cm1 and
a maximum at 738 cm1. The corresponding ratiometric peak
intensities I724/I738 were then selected to monitor the relative
ds1/ssc populations in the samples, and plotted against Rhybr
(Figure 1 e). Data shows that ds sequences can be clearly
identified within the mixture even when present at values
< 1 %, whereas SERS spectra are fully dominated by ds1
contributions for duplex populations 20 %. Such result is
consistent with the higher affinity of dsDNA to AgNP@Sp
owing to the larger availability of negatively charged phosphate groups as compared to ssDNA. Linear correlation (r2 >
0.99) is observed in the 0.01–0.19 Rhybr range.
To test the possibility of detecting single-base mismatches
in DNA duplexes, SERS spectra of the full-complementary
ds1 and the heteroduplexes ds2, ds3, and ds4 were acquired
and compared (Figure 2). These heteroduplexes contain one
from a marked change in the relative intensity. These results,
together with the lack of significant changes in SERS intensity
of the pyrimidine bases, clearly indicate that the spectral
alterations are ascribed to the A!G base-mismatch in the
ds2. On the other hand, ds3ds1 and ds4ds1 difference
spectra show very similar spectral patterns where, in addition
to the positive A (730 and 1507 cm1) and the negative C
(1250 and 1528 cm1) contributions, a consistent intensity
increase of the purine bands (1325, 1487, and 1577 cm1) is
observed. The pyrimidine ring breathing (787 cm1, C + T)
also undergoes a drastic intensity decrease whereas thymine
marker bands do not reveal significant alterations. These
spectral changes can therefore be associated with the A!C
base-mismatch in ds3 and ds4. Minor differences between ds3
and ds4 difference spectra (Figure 2) can be ascribed to the
different position of the base mismatch within the
sequence.[15]
The potential application of AgNP@Sp in SERS analysis
of 5-methylated cytosine bases (mC) and N6-methylated
adenine bases (mA) within ds structures was examined by
acquiring the SERS spectra of dsmC and dsmA and comparing
to their non-methylated analogous ds1. In dsmC and dsmA, the
C or A nucleobases of one of the strands were all replaced by
their respective 5-methylated and N6-methylated counterparts (see sequence structures in Figure 3 and Figure 4). All
Figure 2. SERS spectra of ds1 and ds2; and digitally subtracted SERS
spectra ds2ds1, ds3ds1, and ds4ds1. For the sake of clarity, the
digitally subtracted spectra were multiplied by 3 (for ds2ds1) and 4
(for ds3ds1 and ds4ds1). Frequency positions of characteristic
nucleotide bands are highlighted in: blue (adenine), yellow (cytosine),
orange (guanine), and green (thymine). The overall ds concentration
was kept constant at 630 ng mL1.
Figure 3. a) Molecular structure of 5-methyl cytosine (mC). SERS spectra of ds1 and dsmC, and the difference spectrum dsmC-ds1. For the
sake of clarity, the digitally subtracted spectrum was multiplied by
a factor of two. b) Detail of the 700–840 cm1 region for the SERS
spectra of ds1 + dsmC at different cytosine base ratios RmC = [mC]/
([C] + [mC]) (from the top to the bottom, RmC = 0, 0.045, 0.091, 0.182,
0.273, 0.364, and 0.455). The overall ds concentration was kept
constant at 630 ng mL1. c) Ratiometric peak intensities I732/I788 vs. the
molar ratio RmC = [mC]/([C] + [mC]).
adenine base, A, in place of: (ds2) one guanine, G, (ds3) one
cytosine, C, (terminal position), and (ds4) one cytosine
(internal position). Subtraction of the SERS spectra of ds1
from the other samples generates difference spectra containing vibrational signatures associated with the additional
(positive features) and removed nucleobase (negative features). Positive A bands emerge in all difference spectra at
730 and 1507 cm1, whereas negative G features are observed
at ca. 620, 675, and 1354 cm1 in the ds2ds1. In this case,
negative bands also appear at 1487 and 1577 cm1, ascribed to
purine modes (mainly G contribution) while the other purine
feature at 1325 cm1 (mainly A contribution) does not suffer
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the spectral changes are in good agreement with the normal
Raman studies of the substitution of the methyl group into the
5C and N6 atom in cytosine[16] and adenosine[17] nucleosides,
respectively. The major spectral markers of the cytosine
methylation can be easily recognized through the whole
difference spectrum (Figure 3 a). Among others, we observe
a red-shift and relative intensity decrease of the pyrimidine
ring breathing band at 787 cm1, together with the appearance
of a new weak feature at ca. 755 cm1. In the 1200–1450 cm1
2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 4. a) Molecular structure of N6-methyladenine (mA). SERS spectra of ds1 and dsmA, and the difference spectrum dsmAds1. For the
sake of clarity, the digitally subtracted spectrum was multiplied by two.
b) Detail of the 700–760 cm1 region for the SERS spectra of
ds1 + dsmA at different adenine base ratios RmA = [mA]/([A] + [mA])
(from the top to the bottom RmA = 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6).
The overall ds concentration was kept constant at 630 ng mL1.
c) Ratiometric peak intensities I743/I730 vs. molar ratio RmA.
spectral region, the normal Raman of unmodified cytidine
aqueous solution contains two main bands at 1242 and
1292 cm1, whereas the 5-methyl substituted shows a very
different spectral signature with three discernible bands at
1224, 1258, and 1302 cm1 and a new very intense feature at
1362 cm1.[16] Accordingly, the difference spectrum in Figure 3 a reveals two negative bands at ca. 1244 and 1287 cm1;
three positive contributions broadly centered at 1218, 1268,
and 1315 cm1, and a very strong and sharp band at 1362 cm1.
Moreover, the C ring band at about 1510 cm1 also undergoes
a marked red-shift and intensity decrease while the broad
carbonyl stretching band centered at 1653 cm1 shows a large
red-shift (well highlighted in the difference spectrum by the
negative feature at ca. 1622 cm1 and the positive one at ca.
1662 cm1).
The spectral modifications of the SERS spectral profile of
the normal DNA upon methylation of six of their eleven the
A bases are also striking (Figure 4 a, difference spectrum).
The A ring breathing band at 731 cm1 undergoes a drastic
11 nm up-shift together with a notable intensity decrease,
whereas the pyrimidine stretching of the adenine at 1509 cm1
almost disappears from the spectrum. Furthermore, we
observe up-shifts of the phosphate stretching mode at
1090 cm1 (together with a marked intensity increase); the
v(CN) imidazole feature at 1487 cm1 and the 1577 cm1 band
ascribed to base ring modes (mainly G + A).
Quantification of relative methylated base populations
within the sample was investigated by monitoring the
ratiometric peak intensities I732/I788 for mC and I743/I730 for
m
A, at different molar ratios (RmC = [mC]/([C] + [mC]) and
RmA = [mA]/([A] + [mA])). Details of the ring breathing spectral regions are illustrated in Figure 3 b and Figure 4 b. In the
case of dsmC (Figure 3 b), we highlight the progressive
intensity decrease of the C + T ring breathing band at
787 cm1 upon increasing of the relative mC populations.
Differently, for dsmA (Figure 4 b), the A ring breathing band
at 730 cm1 suffers a dramatic intensity decrease and large
Angew. Chem. Int. Ed. 2015, 54, 1144 –1148
peak red-shift as the RmA value becomes larger. Linear
correlations were obtained in both cases (r2 > 0.99 for mA and
> 0.94 for mC) with detection limits of one mC per 22 total
cytosine bases and one mA per 10 total adenine bases in the
sample (Figure 3 c and Figure 4 c, respectively).
In summary, we have demonstrated the potential application of SERS for the development of a fast and affordable
high-throughput screening method for gaining detailed
genomic information on DNA duplexes. Importantly, owing
to the low amount of DNA required (comparable to only 10–
100 cells), the analysis can be performed without the necessity
of amplification, thus providing realistic direct information of
the DNA in its native state. Owing to the direct nature of this
sensitive and selective assay, we anticipate these findings of
key importance in a number of different fields including
diagnosis, genetic engineering, biotechnology, drug discovery
(antibiogram in microbiology), agriculture, and forensic
science.[1, 18]
Received: August 26, 2014
Revised: September 30, 2014
Published online: November 24, 2014
.
Keywords: DNA · hybridization · methylated bases ·
single-base mismatch · surface-enhanced Raman spectroscopy
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Supporting Information
Wiley-VCH 2015
69451 Weinheim, Germany
Direct Surface-Enhanced Raman Scattering Analysis of DNA
Duplexes**
Luca Guerrini,* Željka Krpetić, Danny van Lierop, Ramon A. Alvarez-Puebla, and
Duncan Graham
anie_201408558_sm_miscellaneous_information.pdf
Abbreviations
ssDNA – single stranded DNA
dsDNA – double stranded DNA
AgNP – silver nanoparticles
AgNP@Sp – Spermine-coated silver nanoparticles
AgHX – hydroxylamine-reduced silver nanoparticles
A – adenine
C – cytosine
G – guanine
T – thymine
C – 5-methylated cytosine
m
A – N6-methylated adenine
m
Experimental Section
Materials. All materials were of highest purity available and obtained from Sigma Aldrich, unless
stated otherwise. DNA oligonucleotides were purchased from Eurofins MWG Operon. The
oligonucleotide base sequences are listed in Table 1. Stock solutions of each oligonucleotide were
prepared in Milli-Q water (final concentration ca. 4x10-4 M). Annealing was conducted by heating to
95°C for 10 minutes equimolar solutions of oligonucleotides ss1, ss2, ss3, ss4, ss5, ssmC and ssmA;
and their complementary strand ssc in PBS (0.3 M). This yielded the corresponding double-stranded
DNA solutions ds1, ds2, ds3, ds4, ds5, dsmC and dsmA (final concentration 10-5 M) which were
stored at -20 °C until required. As reference samples, we selected 21 base homopolymeric sequences
(pA, pC, pT and pG) as well as 22 base self-complementary oligonucleotides (ssCG and ssAT). The
reference oligonucleotide base sequences are listed in Table 2. Annealing of ssCG and ssAT was
conducted as indicated before and the resulting dsCG and dsAT samples (final concentration 10 -5 M)
were stored at -20 °C.
Table 1. Single-stranded DNA sequences (ssDNA) used in the study.
Single-strand
Sequences
ss1
CAT CGC AGG TAC CTG TAA GAG
ss2
CAT CGC AGG TAC CTG TAA GAA
ss3
AAT CGC AGG TAC CTG TAA GAG
ss4
CAT CGC AGG TAA CTG TAA GAG
m
ss C
m
CAT mCGmC AGG TAmC mCTG TAA GAG
ssmA
CmAT CGC mAGG TmAC CTG TmAmA GmAG
ssc
GTA GCG TCC ATG GAC ATT CTC
m
C and mA indicate 5-methyl Cytosine and N6-methyl Adenine residues, respectively (see Fig. S5a for
nucleobase structures).
Table 2. Reference single-stranded DNA sequences (ssDNA) used in the study.
Single-strand
Sequences
pA
AAA AAA AAA AAA AAA AAA AAA
pC
CCC CCC CCC CCC CCC CCC CCC
pT
TTT TTT TTT TTT TTT TTT TTT
pG
GGG GGG GGG GGG GGG GGG GGG
ssCG
CCG CGC CGC GCG CGC GGC GCGG
ssAT
AAT ATA ATA TAT ATA TTA TATT
2
Synthesis of Silver Colloids. Synthesis of Positively-Charged Silver Nanoparticles (AgNP@Sp).
Spermine coated-silver nanoparticles (AgNP@Sp) were prepared as previously reported.[1] Briefly,
20 μL of a 0.5 M AgNO3 solution were added to 10 mL of Milli-Q water, followed by addition of 7
μL of a 0.1 M spermine tetrahydrochloride. Subsequently, under vigorous stirring, 250 μL of a
freshly prepared NaBH4 (0.01 M aqueous solution) were added drop wise to the mixture under
stirring. Finally, the solution was gently stirred for 20 min. Glass vials were previously coated with
polyethylene immine (PEI, average mw ca. 25,000 by LS) by an overnight immersion into an
aqueous 0.2% v/v PEI solution, followed by extensive rinsing with Milli-Q water and N2 dried. See
section S1 for AgNP@Sp characterization.
Synthesis of Negatively-Charged Silver Hydroxylamine Nanoparticles (AgHX). Silver nanoparticles
obtained by chemical reduction of Ag+ using hydroxylamine hydrochloride (AgHX) were
synthesized as described by Leopold et al.[2] Briefly, 300 μL of a NaOH 0.1 M solution were added
to 90 mL of a hydroxylamine hydrochloride 1.66 mM solution. Subsequently, 10 mL of a AgNO3
9.06 mM solution were added drop by drop to the mixture under vigorous stirring.
SERS Experiments. For SERS studies, 1 μL of PBS (0.3 M) solutions of dsDNA (10 -5 M) or 1 μL
of PBS (0.3 M) ssDNA solutions (10-4 M) were mixed with 200 μL of AgNP@Sp. The final analyte
concentration in the sample was 5x10-8 M for dsDNAs and 5x10-7 M for ssDNAs, which corresponds
to approximately 630 ng/mL and 3.15 μg/mL, respectively. After the addition of the ds/ss DNA, the
colloids were left to equilibrate for 2 h and redispersed by quick sonication before running the SERS
measurements.
DNA Hybridization Study. Mixed ss and ds DNA samples were prepared by combining different
volumes of 10-5 M ssc and ds1 solutions (the final ssc+ds1 concentration in the mixtures was kept
constant to 10-5 M) corresponding to the following molar ratios, R= [ds1]/([ds1]+[ss5]) = 0.011,
0.024, 0.041, 0.063, 0.091, 0.130, 0.189, 0.286, 0.474 and 1.
Detection of methylated bases in dsDNA. Mixed ds samples were prepared by combining different
volumes of 10-5 M solutions of ds1 and dsm (either mC or mA). The final ds concentration in the
mixtures was kept constant to 10-5 M. Since the DNA spectra depend only on base composition, and
the interaction of DNA with the metal surface takes place via the external phosphate groups, we
could expect that having unmodified and methylated Adenine on different sequences should be
similar that having them on the same ds structure.[3]
Equipment and Instrumental Settings. CPS disc centrifuge DC24000 (CPS Instruments Inc.) was
used to measure the particle size distribution. A gradient fluid, 8-24 wt % sucrose solution in Milli-Q
3
water, was freshly prepared and filled successively in nine steps into the disc, rotating at a speed of
24 000 rpm starting with the solution of highest density. Calibration was performed using
poly(vinylchloride) particles (0.476 μm, Analytik Ltd.) as calibration standard before each
measurement. For calculations, particle density of 10.49 g/cm3 was used and a density of 1.385
g/cm3 of the calibration standard.
FEI Tecnai G2 20 Twin TEM operating at accelerating voltage of 200 kV was used for imaging.
UV-vis spectra were recorded using a Thermo Scientific Evolution 201 UV-visible
spectrophotometer.
The nanoparticle hydrodynamic diameter distribution was measured on a Malvern Nanosizer
(ZSeries) using a low-volume cuvette, averaging 11 runs at 25ºC.
The ζ (Zeta) potential measurements were carried out using a Malvern 2000 Zetasizer, using the
default method protocol and a minimum sample volume of 3 ml. Before each measurement a
standard solution of -68.0 ± 6.8 mV was measured.
SERS experiments were conducted using a Renishaw InVia Reflex confocal microscope equipped
with a high-resolution grating consisting of 1800 grooves/cm for visible wavelengths, additional
band-pass filter optics, and a CCD camera. A 532 nm laser was focused onto the sample by a longworking distance objective (0.17 NA, working distance 30 mm) and the spectra were typically
acquired with an exposure time of 5x10 s. All SERS spectra reported in the manuscript were
obtained by averaging the SERS response of 4 different sample replications (N=4) obtained by
combining the same colloids (prepared the day before the measurement and left aging overnight)
with the specific DNA buffered solution. Baseline correction was applied to all spectra, unless stated
otherwise. Importantly, difference SERS spectra were calculated from the original no baselinecorrected spectra to avoid any generation of spectral artefacts.
4
Section S1. AgNP@Sp characterization
Differential Centrifugal Sedimentation (DCS) measurements of spermine coated-silver nanoparticles
(AgNP@Sp) yielded a centered value at 31.5 nm (weight distribution, Fig. S1a). Additionally,
AgNP@Sp colloids show a hydrodynamic diameter of 48.0 nm, with a ζ-potential of +35.3 ± 7.6
mV. The final bulk pH is 6.1.
Nanoparticle concentration in the AgNP@Sp colloids (ca. 0.3 nM) was calculated by Beers law
using the extinction coefficient for silver nanoparticles of 1.85x1010 M-1 cm-1, derived from
literature.[4]
Background SERS spectrum of AgNP@Sp was obtained by aggregating the colloids with MgSO4 0.1
M. MgSO4 is a “passive” electrolyte that induces nanoparticle aggregation by simply increasing the
ionic concentration without firmly adsorbing onto the silver surface (as it occurs when halide salts
such as NaCl are used to aggregate the nanoparticles)[5]. AgNP@Sp colloids are characterized by a
low/null SERS background signal (Fig. S1c) which, in contrast to what occurs for citrate-reduced
colloids, does not obligate to the digital subtraction of intense spurious bands, thus removing an
additional factor that may affect the spectra-to-spectra reproducibility.
Figure S1. (a) Characterization of spermine coated-silver nanoparticles (AgNP@Sp) by Differential Centrifugal
Sedimentation (DCS). (b) Representative TEM micrograph of AgNP@Sp colloids prepared by evaporating 10 μL of
diluted colloidal dispersion onto formvar-coated copper grids. (c) Background SERS spectrum of AgNP@Sp aggregated
with MgSO4 0.1 M.
5
Section 2. DNA-driven aggregation of AgNP@Sp into stable clusters in
suspension and spectral reproducibility.
Spectral reproducibility is a key factor in determining the successful application of label-free SERS
in the DNA analysis. Aggregated nanoparticle systems are known to generate clusters with high
SERS activity but with poor control over the final geometry, size and distribution of the aggregates.
This lack of control may severely affect the reproducibility of the overall SERS intensity but, when
analyzed in the averaged bulk SERS regime, “the good-quality signal from the statistical average
SERS of an ensemble of scatterers normally produces stable and reproducible spectra with welldefined average band centers, FWHMs and relative intensities”.[6] In this study, we are not
interested in the overall SERS intensity but we do analyze changes in the spectral profile (i.e.
average band centers, FWHMs and relative intensities). For this reason, SERS spectra are acquired in
colloidal suspensions with a long working distance objective adopting an experimental set-up where
a large ensemble of nanoparticle/DNA clusters, in continuous Brownian motions within the
scattering volume of the objective, are simultaneously investigated by the laser. The averaging effect
of these numerous contributions is a critical factor for obtaining ensemble signals which are stable
and reproducible.
Another key feature that contributes to the high spectral reproducibility relies on the fact that DNA
both acts as an aggregating agent of individual nanoparticles as well as a very effective stabilizing
agent of the so-formed clusters. This is particularly important since it allows us to measure the
sample after several hours upon the DNA addition and not during the aggregation process when the
conformational equilibrium of the DNA onto the metal is likely not achieved yet.
The DNA concentration employed throughout the whole study was carefully selected to yield longterm stable clusters in suspension and unvaried SERS spectra. Fig. S2 illustrates the extinction and
SERS spectra of AgNP@Sp (nanoparticle concentration ca. 0.3 nM) in the presence of different
concentration of ds1, acquired 2 hours after the addition of the analyte. The spectra are stacked for
visual comparison. As can be seen in Fig. S2a, the LSPR of the colloids at 391 nm undergoes a
progressively weakening with the simultaneous appearance of a broad contribution at longer
wavelength when the ds1 concentration is raised from 0 to ca. 0.126 μg/mL. The samples, however,
do not show long-term stability and the Eppendorf walls, where the samples are stored, are
progressively coated with a layer of silver nanoparticles, indicating that the DNA coverage onto the
metal surface is not sufficient to “quench” the spermine positively charges and provide protection
against the wall-adhesion. Differently, when the ds1 concentration is further increased, we clearly
observe a change in the aggregation pattern with the progressive blue-shift of the broad plasmon
6
Figure S2. (a) Extinction spectra and (b) SERS spectra of AgNP@Sp colloids in the presence of ds1 at different
concentration, 2 hours after the initial DNA addition (the samples were quickly sonicated prior to the measurements).
Top: representative TEM images corresponding to AgNP@Sp and AgNP@Sp+ds1 (0.63 μg/mL). These images provide
a qualitative visualization of the DNA-promoted aggregation. In this case, in the attempt to minimize drying-induced
aggregation effects, 30 μL of each colloidal sample were deposited onto formvar-coated copper grids in a humidity
chamber and left to settle for 20 min before being removed. The substrate was finally dried by N 2 flow.
contribution associated with the cluster formation, from ca. 600 nm to ca. 500 nm. In this second
aggregation regime, the silver nanoparticles are fully stabilized by the DNA adsorption and the
extent of the aggregation decreases as the ds1 concentration increases (i.e. as much as the
nanoparticle surfaces are saturated with the analyte). The samples corresponding to ds1
7
concentration above 0.315 μg/mL have shown long-term stability with null/minimal changes in the
extinction spectra even after several days.
The original non-baselined SERS spectra of the same samples are illustrated in Fig. S2b.
First of all, the results show that the Raman features of the DNA can be detected down to 63 ng/mL,
which corresponds to 12.6 ng in the 200 μL of colloids employed in the sample preparation.
However, the actual volume investigated by the laser is much smaller. The illuminated volume (V),
when using a macrosampling objective, is defined by the focal length, f, and the lens diameter, D. [7]
In our case, f= 30 mm and D= 10 mm. Therefore, for a 532 nm laser, V is approximately 40 μm3.
However, several experimental factors, often impossible to evaluate, contribute to make the
scattering volume much larger than the theoretical calculations do. For instance, it is known that
resolution criteria that are perfectly adequate for imaging are not sufficient for Raman spectroscopy
and the observed signal originates not only within the focal volume but tails off with distance (and
the extension of this spatially remote contribution is very case specific and difficult to estimate).[8] In
addition, the continuous diffusion of the NP-DNA clusters in-and-out the scattering volume increases
the actual number of investigated sequences and particles, and is strictly dependent of the cluster size
distribution, laser exposure time, number of accumulations etc.[9] To safely include all these
experimental factors and avoid unreliable claims on the levels of sensitivity, we conservatively
assume the laser investigated volume as a cylinder of 1 mm diameter and the depth of the sample (in
this case, 6 mm well). This corresponds to a volume of 4.7 μL and, therefore, the limit of detection
for ds1 analysis can be re-estimated as less than 0.3 ng in the probed volume.
However, more importantly than the limit of detection itself, the analysis of the SERS
spectral profiles indicates that the Raman features of the DNA remain unaltered when the ds1
concentration lies within the second aggregation regime (> 0.315 μg/mL) whereas, for lower analyte
amount, the SERS bands significantly broaden, weaken and, in some cases, undergo spectral shifts.
Based on these results, we selected 0.63 μg/mL as the optimum dsDNA concentration to perform our
studies (corresponding to less than 3.0 ng in the scattering volume), since it provides long-term
stable clusters in suspension and a SERS signal which is not affected by fluctuations of the
[DNA]/[NP] ratio within a large ds1 interval. It is important to note that potential minor fluctuations
of the SERS spectra that may arise from colloidal batch-to-batch dissimilarities (such as NP
concentration and size distribution) are “washed away” by the direct comparison of SERS spectra of
the “original” dsDNA and the “modified” one (both acquired by using the same colloidal batch).
8
A further example of the spectral reproducibility achieved under this experimental conditions is
illustrated in Fig. S3a, where 8 SERS spectra of 8 different repetitions of ds1 (0.63 μg/mL) onto
AgNP@Sp ([NP]~0.3 nM) are reported as acquired. The repetitions were performed as follows: 200
μL of AgNP@Sp from the same colloidal batch were placed in 8 different vials, then mixed with the
same volume of a ds1 buffered solution and finally investigated by SERS. As can be seen in Fig.
S3b, only minimal fluctuations of the background are observed whereas the spectral profile remains
unperturbed, showing a consistent sample-to-sample reproducibility.
Figure S3. Original no-baselined SERS spectra of 8 different repetitions of AgNP@Sp + ds1 (0.63 μg/mL) visualized as
(a) stacked and (b) overlapped. (c) Corresponding average SERS spectrum.
9
Section S3. Vibrational Assignment of the Main Bands of the dsDNA SERS Spectra
Vibrational assignment of ds1 sequence was based on both the data reported in the literature[10] and
by measuring the SERS spectra of 21-mer homopolymeric sequences of the four bases (pA, pC, pT
and pG) and 22 base self-complementary dsCG and dsAT double-stranded sequences (Fig. S5b).
The spectral interval 600-800 cm-1 contains Raman features associated with vibrations involving
concerted ring stretching motions (ring breathing) of purine or pyrimidine residues, often in
combination with stretching of the glycosidic bond and possibly also stretching of bonds within the
linked deoxyribose ring.[10e] Thus, these features are very informative about the helical geometry. In
this region, we identify bands mainly ascribable to the guanine residues at 502, 621, and 665/677 cm1
, whereas strong bands at 730 and 787 cm-1 are mainly due to ring breathing vibrations of adenine
and the combination of cytosine + thymine residues, respectively. Analysis of the purine breathing
modes coupled to the deoxyribose in the SERS spectrum of ds1 reveals a large predominance of the
Z tertiary structure (bands at 621 and 665 cm-1) whereas the B-form (band at 686 cm-1) was the
dominant geometry in the SERS spectra of single-stranded oligonucleotides[11]. Interestingly, in
contrast to what observed for negatively charged colloids,[5, 12] (Fig. S6) the characteristic phosphate
band at 1090 cm-1, originating from the localized symmetric stretching vibration of the
phosphodioxy moiety, is rather intense in the SERS spectra of both single- and double-stranded
DNA structures on AgNP@Sp. This data clearly indicates that the phosphate groups are in close
proximity to the metal surface, providing a further evidence of the electrostatic-driven adsorption of
the DNA sequences onto the spermine-coated silver nanoparticles. The spectral region 1150–1600
cm-1 contains many overlapping bands, mainly arising from in-plane vibrations of base residues[10e].
Based on a spectral comparison with the SERS spectra of the experimental controls and on the
analysis of the vibrational studies reported in the literature, we tentatively assign the broad multiple
peak bands centered at 1246 cm-1 mainly to cytosine residues (with guanine contribution); the sharp
intense feature at 1325 cm-1 mainly to adenine residues (with guanine and thymine contributions);
the weaker features at 1354 and 1376 cm-1 to guanine and thymine residues, respectively; the 1421
cm-1 band to the DNA backbone vibrations; the base ring modes at 1487 cm -1 mainly to guanine
residues with some adenine contribution; the two weaker features at 1507 and 1528 cm-1, to adenine
and cytosine ring modes, respectively; and the intense band at 1577 cm-1 to ring modes mainly
ascribable to similar contributions of guanine and adenine residues. Finally, in the 1600-1800 cm-1
region overlap carbonyl group stretching vibrations of thymine (C2=O and C4=O), guanine (C6=O),
and cytosine (C2=O)[10e]. Thymine residues are known to generate intense Raman features around
1652 and 1672 cm-1, whereas the guanine mode at ca. 1720 cm-1 is only moderately intense.
10
Conversely, the cytosine C2=O mode near 1680 cm-1 is usually extremely weak[10e]. It is worth
noticing, however, that Raman signature associated with each DNA base sequence cannot be
approximated by a simple weighted sum of the Raman spectra of alternating AT/TA or GC/CG
repeats[10e] since base sequences play a key role in determining the final vibrational signature.
11
Figure S5. (a) Molecular structures of the 4 DNA nucleobases and the two methylated cytosine and adenine derivatives
(at the position C5 and N6, respectively). (b) Normalized SERS spectra of homopolymeric single stranded sequences
(pA, pG, pC and pT), and dsCG and dsAT, and ds1 duplexes. ssDNA concentrations was kept fixed at 5x10-7 M and
dsDNA at 5x10-8 M, which correspond to a final amount of analyte in the probed volume equals to 3.15 μg/mL (pA),
2.94 μg/mL (pC), 3.36 μg/mL (pG), 3.01 μg/mL (pT) and 630 ng/mL (dsCG, dsAT and ds1). Table (c) lists the main
Raman shifts and the tentative vibrational assignment of the main SERS bands of ds1 on AgNP@Sp colloids.
12
Section S4. SERS of single and double-stranded DNA sequences on negatively
charged silver colloids.
Based on the excellent results recently reported by Bell and co-workers[5,
12a]
in the direct SERS
investigations of ssDNA in solution, we selected hydroxylamine-reduced silver nanoparticles
(AgHX) aggregated with MgSO4 as a reference colloidal substrate. As it can be seen from Fig. S6,
no significant band shifts can be observed when ss spectra are compared to ds spectra, whereas we
do observe a certain degree of sample-to-sample irreproducibility in terms of relative intensity in the
case of the duplex sample (see for instance the nucleobase ring breathing band in the 700-800 cm-1
spectral region and the bands above 1500 cm-1, highlighted in yellow). The lack of differentiation
between the SERS spectra of ss vs. ds (unmodified sequences) using negatively-charged colloids is
consistent with the previous findings reported by Marotta et al.[13] and Papadopoulou et al.[12a] Thus,
it may be speculated that the observed SERS signal for ds1 could be due either to the partial
denaturation of the dsDNA or to the small fraction of non-hybridised sequences in the ds1 solution
resulting from uncontrollable experimental errors in the preparation of equimolar mixtures of the two
complementary strands.
13
Figure S6. Representative SERS spectra of four different repetitions of hydroxylamine-reduced silver nanoparticles
(AgHX) aggregated with MgSO4 in the presence of a constant concentration of (a) ss1 and (b) ds1 (3.15 and 0.63 μg/mL
of analyte, respectively).
14
Section S5. Reshaping of the SERS spectrum of the single-stranded
complementary sequences upon hybridization.
The calculation of the pondered sum of the individual SERS spectra of ss1 and ssc relies on the
experimental evidence that the peak intensity of the symmetric phosphodioxy stretching vibration
band at 1090 cm-1 is largely independent to the DNA hybridisation
[14]
, i.e. the peak height at 1090
cm-1 is approximately proportional to the number of phosphate groups in the sequence, either single
or double-stranded. Thus, ss1+ssc can be approximated as the combined spectral result of the
hypothetical equimolar mixture of non hybridised ss1 and ssc sequences.
Figure S7. SERS spectrum of ds1 (630 ng/mL) and digitally calculated SERS spectrum of ss1+ssc, both normalized to
the phosphate band at 1090 cm-1. The ss1ssc pondered spectrum was obtained by summing the two separated SERS
spectra of ss1 and ssc (3.15 ng/mL), both normalized to the 1090 cm-1 band.
15
Section S6. Single-base Mismatch
DNA damages or biosynthetic errors of DNA polymerases during the DNA replication can result in
heteroduplex sequences containing base-base mismatches. Deficiency in the cell mismatch repair
machinery leads to the accumulation of such mutations which, in turn, results in a high level of
microsatellite instability, a characteristic feature of a number of cancers, including gastric,
endometrial, ovarian, lung, and colorectal cancer.[15] Nowadays, the application of SERS
spectroscopy to the identification of single-nucleotide polymorphisms in unmodified sequences is
limited to single-stranded structures.[5]
Figure S8. SERS spectra of ds1, ds2, ds3 and ds4 (each 630 ng/mL).
16
Section S7. 5C and N6 methylation of cytosine and adenine.
In mammals, cytosine methylation at position C5 is the most abundant epigenetic modification and
mainly occurs at the CpG regions of DNA, where the rate of methylation usually lays in between the
70% and 80% of all cytosine bases[16]. The chemical instability of 5-methylcytosine (mC) renders the
CpG islands mutational hot spots as demonstrated by the evidence that a large number of human
genetic disease are found to be strictly associated with such transitions within CpG sequences[17]. If
the 5-methylcytosine is considered as the 5th DNA base, recent data point to the biological
importance of N6-methyladenine (mA) as the “other” methylated base[18].
m
A is mainly found in the
genomes of bacteria, but accumulating evidence indicate its presence in some archaea and eukaryotic
cells, where its role remain largely unknown[18-19]. Indirect evidence seems to suggest that mA might
be present also in mammalian DNA[18]. In bacterial DNA, the function of mA has been associated
with genome defense, DNA replication and repair, nucleoid segregation, regulation of gene
expression, control of transposition, and host–pathogen interactions[18-19]. In particular, DNA-adenine
methyltransferases has been recognised as a key factor in determining bacterial viability and
virulence[19]. To date, the application of SERS to the identification of methylated bases in
unmodified sequences is limited to qualitative studies on single-stranded structures[3].
17
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19