Supporting Information

© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2008

© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2008

Supporting Information

for

Ultrasensitive Detection of DNA by PNA and Nanoparticle-Enhanced

Surface Plasmon Resonance Imaging

Roberta D’Agata, Roberto Corradini, Giuseppe Grasso,

Rosangela Marchelli, and Giuseppe Spoto*

Materials and reagents: Reagents were obtained from commercial suppliers and

used without further purification. Wild-type streptavidin (WT-SA) was purchased from

Invitrogen (Italy). Nitrocellulose membrane filters was purchased from Whatman

(U.K.). Trisodium citrate dihydrate, tetrachloroauric(III) acid (HAuCl4·3H2O), triethan-

olamine (TEA), ethanol, dimethyl sulfoxide (DMSO), hexane, sodium hydroxide solu-

tions (10 M in water), dithiobis(N)succinimidylpropionate (Lomant's Reagent or DTSP)

were purchased from Sigma-Aldrich (Italy). Methoxy-polyethyleneglycol amine

(mPEG-NH2, MW = 5000) was purchased from Nektar Therapeutics (USA). Phos-

phate-buffered saline (PBS) solutions at pH 7.4, (NaCl 137 mM, 2.7 mM KCl, phos-

phate-buffered 10 mM) was obtained from Amresco (Italy). SPRI gold chips were pur-

chased from GWC Technologies (USA). Ultra-pure water (Milli-Q Element, Millipore)

was used for all the experiments.

Microfluidic devices fabrication: Two different microfluidic devices with parallel and

Y-shaped microchannels respectively were used for the study. Both of them were

fabricated in poly(dimethylsiloxane) (PDMS) polymer through the well established

replica molding technique elsewhere described.[1] Briefly, PDMS channels were creat-

ed by replication from masters in polyvinyl chloride (PVC) with patterns of six (80 µm

depth, 1.4 cm length, 400 µm width) parallel channels and two (80 µm depth, 1.0 cm

length, 500 µm width) Y-shaped channels respectively, bearing circular reservoirs

(diameter = 400 µm) at the ends of each channel. Replicas were formed from a 1:10

mixture of PDMS curing agent and prepolymer (Sylgard 184, Dow Corning, USA).

The mixture was degassed under vacuum and then poured onto the master in order

to create a layer with a thickness of about 3-4 mm. The PDMS was then cured for at

least 2 hours at 60 °C before it was removed from the masters. The PDMS mold

bearing the negative pattern of the masters was peeled off and washed repeatedly

with ethanol, hexane, ultra-pure water and dried before use. PEEK tubes (UpChurch

Scientific) were inserted in such reservoirs in order to connect the PDMS microfluidic

cell to an Ismatec IP-N (Ismatec SA, Switzerland) peristaltic pump. The microfluidic

device was assembled by fixing the PDMS mold on the SPRI gold chip surface. A

refractive index matching liquid was used to obtain the optical contact between the

flow cell and the prism.

PNA 1 synthesis and surface immobilization: The PNA sequence was designed

as described previously[2] and first checked to minimize any secondary structure,

avoiding loops and hairpins which would result in a loss of hybridization efficiency,

and the corresponding target DNA was analyzed using the online available program

Mfold (version 3.1),[3] though literature data suggest probe and target secondary

structure do not affect surface hybridization rate.[4,5] Two 2-(2-aminoethoxy)ethoxy-

acetic acid (AEEA) linkers were added at the N terminus in order to increase acces-

sibility of the target DNA, according to our previous studies using microarray tech-

nology.[2]

The PNA was synthesized using automatic solid phase synthesis and purified by

HPLC (Figure S1) as described previously. [2] The purified product was then identified

by mass spectrometry (Micromass ZMD). PNA 1 H-AEEA-AEEA-AAACCCTTAATC-

CCA-NH2 calcd m/z 1067.3 (MH44+), 854.0 (MH5

5+), 711.9 (MH6 6+), 610.3 (MH77+),

534.2 (MH88+); found 1067.3, 854.0, 711.8, 610.3, 534.2.

An optimal surface probe density is required in order to obtain reproducible and con-

sistent SPRI results, since an overly dense surface decreases the measurement sen-

sitivity in detecting oligonucleotide hybridization.[6-8]

Gold chips previously functionalized with the DTPS reagent were used to immobilize

the PNA 15-merRR probe. The procedure adopted for the gold chip functionaliza-

tion[9] is the following: the bare gold chip substrates were immersed in a DTSP sol-

ution (4 mM in DMSO) for 48 h. The modified chips were then thoroughly rinsed with

DMSO, ethanol and ultra-pure water, respectively.

The PNA 1 probes were immobilized on the DTPS-modified gold chips through the

amine-coupling reaction between the N-hydroxysuccinimidyl (NHS) ester ends of

DTPS and the N-terminal group present at the 5’-position of the two 2-(2-aminoeth-

oxy)ethoxyacetic acid (AEEA) linkers[10] inserted in the PNA probe sequence. The

probe immobilization was obtained by injecting the probe solution (0.1 µM in PBS,

flow rate 5 µL min-1; Figure S2) into microchannels in contact with DTPS-modified

gold surface. The AEEA double spacers were used in order to minimize surface ef-

fects caused by the steric hindrance of the immobilized systems.[11,12] An average ad-

layer thickness of d=1.6 nm was calculated.[13,14] An average PNA 1 surface coverage

of 3 x 1012 molecules cm-2 was obtained by multiplying the average adlayer thickness

value by the bulk number density of the adsorbate (molecules nm-3). The latter pa-

rameter was estimated from Park et al.[15] Similar surface coverage values are report-

ed to minimize both steric interactions between adjacent probes as well as electro-

static interactions between target molecules.[16,17] The unreacted NHS ester groups

were deactivated by reaction with mPEG-NH2 (4 mM in TEA 0.1 M).

Time5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00

%

0

100

rr15 1: Scan ES+ TIC

9.05e815.99

14.652.20

RR15 renew

m/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400

%

0

100

rr15 309 (15.993) 1: Scan ES+ 3.87e6610.27

152.25534.20

515.41279.32153.15

711.82

854.04 1067.29

Figure S1. Upper Panel: HPLC-MS profile of the PNA 1 after purification: ESI-MS detector in

TIC mode. Lower panel: multicharge ESI-MS spectrum for the peak at 15.99 min. Conditions:

column Phenomenex C18 (300?, 250 x 4.6 mm, 5 µm); eluent A 0.2% formic acid in water;

eluent B 0.2 % formic acid in water/acetonitrile = 60:40; gradient elution from 100%A to

100% B in 40 min.

Figure S2: The changes in percent reflectivity over time obtained for the immobilization of

the PNA 1 probe at three different concentrations (0.1 µM, 0.5 µM and 1 µM in PBS). The

lower PNA 1 concentration (0.1 µM) was used for the hybridization experiments.

SPRI measurements: All the SPRI experiments were carried out by using an SPR

imager apparatus (GWC Technologies, USA) elsewhere described in detail.[18]

SPR images were analyzed by using the V++ software (version 4.0, Digital Optics

Limited, New Zealand) and the software package Image J 1.32j (National Institutes of

Health, USA). SPRI provides data as pixel intensity units (0-255 scale). Data were

converted in percentage of reflectivity (%R), or ?%R in the case of difference imag-

es, by using the formula:

%R = 100 * (0.85 Ip / Is)

where Ip and Is refer to the reflected light intensity detected using p- and s-polarized

light, respectively. The experiments were carried out by sequentially acquiring 15

frames averaged SPR images with 5 seconds time delay between them. Kinetic data

were obtained by plotting the difference in percent reflectivity (∆%R) from selected

regions of interest (ROIs) of the SPR images as a function of time. All the SPRI ex-

periments were carried out at room temperature.

Single-stranded DNA modified AuNps: Gold nanoparticles were synthesized by ci-

trate reduction of HAuCl4·3H2O according to methods elsewhere described.[19] Briefly,

20 mL of trisodium citrate (38.8 mM) were quickly added with vigorous stirring to 200

mL of a boiling solution of HAuCl4 3H2O (1mM). The color of the solution changed

from pale yellow to deep red in a few seconds. A complete reduction of trisodium

citrate was obtained after 6-8 min under boiling. The solution was cooled to the room

temperature and filtered through a 0.45 µm membrane filter. The AuNps solution was

stored at 4 °C in a clean brown glass bottle. Similar conditions assured the nanoparti-

cles stability for several months.

The AuNps were characterized by UV-vis spectroscopy (Agilent 8453 spectrometer)

and by transmission electron microscopy (TEM) (Jeol JEM-2000 FX II, operating at

200 kV). The average diameter of AuNps was 20 ± 5 nm (Figure S3).

AuNps were conjugated to the biotinylated DNA 12-mer sequence (Tm=36.0 °C) ac-

cording to procedures elsewhere described.[20] 1 mL of a solution obtained by diluting

(1:1) the AuNps with H2O was added to 40 µL of a 1 mg mL-1 streptavidin solution

after adjusting the pH to 8.5 with NaOH (0.1 M). The solution was incubated in ice for

1 h and centrifuged (13500 g, 30 min). After the separation of the liquid phase, 200

µL of the biotinylated oligonucleotide (10 µM in H2O) (DNA 12-mer) was incubated for

30 min with the AuNps conjugate. After the centrifugation and the removal of the su-

pernatant solution the DNA 12-mer-AuNps conjugate was suspended in PBS buffer

by achieving a final concentration of about 10 nM. The DNA 12mer-AuNps conjugate

concentration was obtained from UV-vis spectroscopy (ε524=2x108 M-1cm-1).[21] The

amount of immobilized DNA 12mer was determined by quantifying the unreacted

DNA 12-mer in the supernatant solution with UV-vis spectroscopy.[22]

Figure S3: A) Representative UV-vis spectrum of the citrate-stabilized AuNps in water;

B) representative TEM image of the AuNps (diameter =20±5 nm)

Detection of target or mismatched DNA: The detection of target or mismatched

DNA was obtained by using a sandwich hybridization strategy. The target or mis-

matched DNA solutions (in PBS. Seven different concentrations were investigated: 1

µM, 10 nM, 1 nM, 10 pM, 1 pM, 500 fM, 250 fM, 50 fM, 1 fM) were injected for 30 min

into microchannels in contact with the surface immobilized PNA 1 probe (flow rate 5

µL min-1; sample volume 150 µL). Hybridization occurred between the portion of the

DNA-FM complementary to the PNA 1 and the surface immobilized probe. No detect-

able variations in the SPRI signal were revealed at this stage by using target or mis-

matched DNA concentrations lower than 1 µM. The achieved hybridization was then

revealed by injecting a solution of DNA 12-mer-AuNps (10 nM in PBS, flow rate 10 µL

min-1). The DNA 12-mer sequence was complementary to the portion of the DNA-FM

non involved in the PNA 1 probe hybridization. The specificity of the target adsorption

was checked by comparing the SPRI response with those obtained when the mis-

matched sequences (DNA-MIS-T, DNA-MIS -A, DNA-MIS-C) and a control target se-

quence (DNA-CTR) were allowed to interact with the PNA 1 probe. The control target

sequence differs from the target sequence only in the sequence portion involved to

the probe hybridization. Aspecific interactions were also evaluated by allowing the

sequential adsorption of the relevant DNA-FM and DNA 12-mer-AuNps solutions on

mPEG-functionalized gold surfaces. mPEG surfaces were obtained after the mPEG-

NH2 (4 mM in TEA 0.1 M) immobilization on DTPS-modified gold chips. Figures S4

and S5 show the time-dependent SPRI curves obtained when nanoparticle -enhanced

SPRI detection experiments were carried out with 10 pM and 50 fM target, mismatch

and control sequence concentrations.

0 900 1800 2700

0

3

6

PEGPEG

C

B

∆%R

t / sec

A

Figure S4: Time-dependent SPRI curves obtained after the adsorption of the DNA 12-mer-

AuNps on A) DNA-FM hybridized to the surface immobilized PNA 1 probe, B) DNA-CTR

adsorbed on the surface immobilized PNA 1 probe and C) DNA-FM adsorbed on a mPEG

modified gold surface. The concentration of DNA-FM and DNA-CTR used for this experiment

was 10 pM.

Figure S5: Time-dependent SPRI curves showing the nanoparticle-enhanced SPRI detec-

tion of the A) DNA-FM (50 fM) hybridized to the PNA 1. The SPRI response is clearly differ-

ent from those generated by the nanoparticle-based detection of the adsorption of the single

mismatch carrying sequences (C) DNA-MIS-C, D) DNA-MIS-A, E) DNA-MIS-T. Concentra-

tion 50 fM) on PNA 1 probe. Mismatch sequences cause a nanoparticle-enhanced SPRI re-

sponses similar to that obtained from a 50 fM control sequence solution B) DNA-CTR) and to

that generated when the 50 fM DNA-FM solution was adsorbed on to a mPEG surface F).

Data validation: Five replicate control experiments in absence of DNA provided the

average SPRI response ∆%R=0.46 with a standard deviation SD= 0.03. Such values

allow to compute the 99% confidence interval on the mean (µ=0.46 ±0.06). The aver-

age SPRI response obtained in the absence of DNA is different (t-test, confidence le-

vel P=0.99) from the average SPRI response obtained by the nanoparticle -enhanced

SPRI detection of the 1 fM DNA–FM hybridization (average SPRI response ∆%R=

2.14, SD=0.2, n=5, ..

calct 0050 = 18.57, ν=4.26). The limit of detection (LOD) calculated by

assuming the SPRI response in absence of DNA as the blank value is LOD= 0.46+

3x0.03= 0.55, while the limit of quantification (LOQ) is LOQ= 0.46+ 10x0.03= 0.76.

The SPRI response obtained by the nanoparticle-enhanced SPRI detection of the 1

fM DNA–FM hybridization is well beyond the above mentioned limits.

Similar conclusion can be drawn by considering the average SPRI response obtained

with the adsorption of single mismatch carrying sequences (1 fM). In fact, even

though the average SPRI response (average ∆%R=0.29, SD=0.04, n=5) is different

from the average response obtained in the absence of DNA (t-test, P=0.99; ..

calct 0050 =

6.869), it is also different from the average SPRI response obtained by the nanopar-

ticle-enhanced SPRI detection of the 1 fM DNA–FM hybridization (P=0.99; ..

calct 0050 =

20.28, ν=6.47).

In conclusion, we can affirm that all the selected control measurements indicate that

the SPRI signal obtained after the nanoparticle-enhanced SPRI detection of the 1 fM

DNA–FM hybridization is clearly different from those obtained by the adsorption of

single mismatch carrying sequences.

Regeneration: Regeneration of the SPRI device surfaces was performed by pulses

of 10 mM NaOH followed by the injection of the DNA-FM and the DNA 12-mer-AuNps,

respectively. Two successive regeneration cycles allowed to obtain a 70 % regenera-

tion of the surface (Figure S6).

Figure S6: Regeneration of a surface after the nanoparticle-enhanced SPRI detection of the

DNA-FM (50 fM) hybridized to the PNA 1. The NaOH 10mM spike generated an SPRI

response visible at t=3550 s.

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