Supporting information
Silicon-based highly reproducible and active
surface-enhanced Raman scattering substrates
for sensitive, specific, and multiplex DNA
detection
Ziyun Jiang,1 Xiangxu Jiang,1 Shao Su,1 Xinpan Wei,1 Shuit-Tong Lee,*2 and Yao
He*1
1
Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Institute of
Functional Nano & Soft Materials (FUNSOM), Soochow University, Suzhou 215123, China
2
Center of Super-Diamond and Advanced Films (COSDAF), City University of Hong Kong,
Hong Kong SAR, China and Department of Physics and Materials Science, City University of
Hong Kong, Hong Kong SAR, China
E-mail: yaohe@suda.edu.cn (Y. H), apannale@cityu.edu.hk (S. T. L)
1. Experimental methods
Silicon wafer (p type, 10-20 Ω sensitivity) was purchased from the city university of
Hong Kong (Silicon (100) wafer (phosphate-doped (p-type), 0.01-0.02 Ω sensitivity)
was purchased from Hefei Kejing Materials Technology Co. Ltd (China).
Hydrofluoric acid (5%), hydrogen peroxide (30%), acetone and sulphuric acid (98%)
were purchased from Sinopharm Chemical Reagent Co., Ltd (China). Silver nitrate
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was purchased from Nanjing Chemical Reagent Co. Ltd (China). DNA
oligonucleotides were purchased from invitrogen Inc. (Shanghai, China). The
sequences are listed in Table 1. All solutions were prepared using Nanopure water
from a Millipore Milli-Q system. The DNA hybridization buffer was 10 mM Tris-HCl
and 1.0 M NaCl.
The silver nanoparticles decorated-silicon wafer (AgNPs@Si) was characterized
by Scanning electronic microscopy (SEM) and atomic force microscopy (AFM). The
SEM overview images were recorded using scanning electron microscopy (FEI
Quanta 200F) equipped with energy-dispersive X-ray (EDX) spectroscopy. The AFM
overview images were recorded using MultiMode V AFM (Vecco Corporation, USA).
The SERS analysis was performed using on a HR800 Raman microscope instrument
produced by HORIBA Jobin Yvon (France). The instrument is equipped with a
standard 633 nm HeNe 20mW laser, and other two external lasers (514 Ar ion laser
and 785 Laser diode). Four kinds of objectives (10 × NA 0.25, 50 × NA 0.7, 100 × NA
0.9, and long work distance) are available in the instrument. In the range between 450
nm and 850 nm, the wavenumber accuracy is ± 1 cm-1. Under normal conditions of
temperature stability (± 1 oC), the repeatability is 1 pixel with a standard CCD.
LabSpec 5 software is used for Raman data acquisition and data analysis.
Enhancement factor (EF) calculation is similar to the established methods described
elsewhere.1-4 In details, the EF value was calculated using the following equation,
EF = (Isers × Nbulk) / (Ibulk × Nsers) (1)
where Isers and Ibulk represent the intensities of the same band of the SERS spectra and
the Raman spectra (non-SERS) respectively; Nsers and Nbulk represent the number of
molecules on the substrates within the laser spot. Herein for SERS examination, a
certain volume Vsers R6G aqueous solution was dispersed to an area of Ssers at a
concentration of Csers on the AgNPs@Si substrate. For non-SERS Raman spectra, a
certain volume Vbulk R6G solution was dispersed to an area of Sbulk at a concentration
of Cbulk on a clean Si wafer. Both the substrates were dried in the air. The foregoing
equation thus becomes,
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EF = (Isers / Ibulk) × (Ssers × Vbulk × Cbulk) / (Sbulk × Vsers × Csers)
(2)
Raman measurements were conducted under identical experimental conditions (laser
wavelength, laser power, microscope objective/lenses, spectrometer, etc.). In our
experiment, 10 μL of 10−8 M R6G aqueous solution (the detection limit of R6G) was
dispersed to the AgNPs@Si substrate and 10 μL 1 × 10−2 M R6G solution was
dispersed to the clean Si wafer. The intensities for the C-C stretching mode at 1517
cm−1 are 9850 for the AgNPs@Si substrate, and 760 for the clean Si substrate,
respectively.5,6 We calculated the average EF from the SERS substrate point of view
under plausible assumption that all the probe molecules within the laser spot are
illuminated and contribute to the SERS spectra and the Raman spectra. Therefore, the
average EFs are calculated to be 8.79 × 106 for the AgNPs@Si.
A Si wafer was cleaned with acetone by ultrasonic treatment for 10 min, and washed
with Milli-Q water (Millipore) for 3 times. Afterwards, the Si wafer was immersed in
an oxidant solution containing H2SO4 (98%) and H2O2 (30%,) with a volume ration of
3:1 for 20 min, and rinsed with Milli-Q water for three times to remove organics. The
cleaned silicon wafer was further immersed in hydrogen fluoride (HF, 5%) aqueous
solution for 30 min to achieve fresh H-terminated silicon wafer covered by Si-H
bonds. The H-terminated silicon wafer was then immediately placed into a freshly
prepared reduction solution containing silver nitrate (AgNO3) with slowly stirring for
90 s. AgNPs were readily coated on the surface of Si wafer through the reduction
reaction, producing the AgNPs@Si.6-11 The resultant AgNPs@Si was dried with a
gentle flow of nitrogen and collected as SERS-active substrates in following
experiment.
The DNA self-assembly procedure is similar to the protocol described in our previous
reports.10 As shown in Fig. 1, the prepared AgNPs@Si was first immersed into 100 μL
capture DNA solution (10 nM), which was then kept in a sealed box overnight. The
modified AgNPs@Si was next treated with 0.1 M NaCl phosphate-buffered solution
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for 12-h incubation to guarantee adequate self-assembly between SH-DNA and
AgNPs@Si. Afterward, the prepared capture DNA-modified AgNPs@Si was
hybridized with the capture and probe DNA (100 μL, 10 nM) in a sealed box
overnight, respectively. The resultant AgNPs@Si was rinsed with Milli-Q water to
remove excess DNA and then dried with a gentle flow of nitrogen for SERS detection.
The manipulations were repeated for detection of DNA with different concentrations
in the range of 1 pM-10 nM.
As a control, when free AgNPs were used instead of the AgNPs@Si, all the
manipulations were identical to those described above.
Target DNA with serial concentrations (1 pM-10 nM) was assembled with the
AgNPs@Si via the same manipulations mentioned in Section 1.3, producing the
silicon-based SERS sensors for quantitative detection of DNA. SERS Raman spectra
were acquired using a ULWD 100× objective (NA: 0.7) on a HR800 Raman
microscope instrument equipped with a 633 nm HeNe 20 mW laser (polarized 500 :
1). The diameter of the laser spot was 1 μm. The Raman microscope system has a
motorized stage with a precision XYZ stage controller. Typically, Raman spectra were
acquired from a 1 μm × 1 μm on the substrate.
Single base mutations target DNA (T mutated to either A, G, or C at the 14 site, 1pM)
was used instead of the full complementary target DNA. Besides, all manipulations
were identical.
The whole procedure of DNA multiplexed detection was schematically shown in Fig.
4a. In brief, the prepared AgNPs@Si was first immersed into 100 μL solution
containing with two kinds of capture DNA (10 nM), which was then kept in a sealed
box overnight. The modified AgNPs@Si was next treated with 0.1 M NaCl
phosphate-buffered solution for 12-h incubation to guarantee adequate self-assembly
between capture DNA and AgNPs@Si. Afterward, the prepared capture
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DNA-modified AgNPs@Si was simultaneously hybridized with two kinds of target
DNA (1 pM) and reporter DNA (10 nM) in a sealed box overnight, respectively. The
resultant AgNPs@Si was rinsed with Milli-Q water to remove excess DNA and then
dried with a gentle flow of nitrogen for multiplexed DNA detection.
2.Additional data
Table Ⅰ shows sequence of oligonucleotides employed in the experiment.
Figure S1 shows the size distribution of the AgNPs coated on surface of silicon
wafers.
Figure S2 shows corresponding Raman intensity of SERS spectra recorded from 25
random points on the substrates assembled with 10 pM target DNA.
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Table Ⅰ Sequence of oligonucleotides employed in the experiment
Name
Sequence
Capture DNA 1 (Pa)
5’-TGA GTG GAC GTC AAC GAG CAA-SH-3’
Capture DNA 2 (Pb)
5’-ATT TGA GGA GAA AGA-SH-3’
Target DNA 1 (Ta)
5’-TTG CTC GTT GAC GTC CAC TCA GTG TGA
TTC CAC CTT CTC CAA GAA CTA TAT TGT CTT
TCA-3’
Target DNA 2 (Tb)
5’-TTA GAG TTG CAT GGA ACA GTG TTA ACT
CCT CTT TCT-3’
Reporter DNA 1 (Ra)
5’-(R6G)-TGA AAG ACA ATA TAG TTC TTG GAG
AAG GTG GAA TCA CAC-3’
Reporter DNA 2 (Rb)
5’-CY3-TCC ATG CAA CTC TAA-3’
One-base mismatch DNA
5’-TTG CTC GTT GAC GXC CAC TCA GTG TGA
TTC CAC CTT CTC CAA GAA CTA TAT TGT CTT
TCA-3’ (“X” stands for A, G, or C )
Non-cognate DNA
5’-GAA CAT ACA TAG GGT TTC TCT TGG TTT
CTT TGA TTA TCA TTC ATA CGG CAT GTC ACC
GTC-3’
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Figure S1. The size distribution of the AgNPs coated on surface of silicon wafers,
which is calculated by measuring more than 500 particles.
Figure S2. Corresponding Raman intensity of SERS spectra recorded from 25 random
points on the substrates assembled with 10 pM target DNA.
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