This journal is c The Royal Society of Chemistry 2010 Chem. Commun., 2010, 46, 9007–9009 9007

Nanoparticle based enhancement of electrochemical DNA hybridization

signal using nanoporous electrodesw

Alfredo de la Escosura-Munizab and Arben Mekoci*ac

Received 20th July 2010, Accepted 24th September 2010

DOI: 10.1039/c0cc02683b

A novel nanoparticle-based enhanced methodology for the

detection of ssDNA using nanoporous alumina filter membranes,

containing pores of 200 nm in diameter, is reported. The

blockage of the pores due to the hybridization is detected by

measuring the decrease in the differential pulse voltammetric

response of the [Fe(CN)6]4�/3� redox indicator and using screen-

printed carbon electrodes as transducing platform. Furthermore,

20 nm gold nanoparticle (AuNPs) tags are used in order to

increase the sensitivity of the assay. The enhancement mecha-

nism of DNA detection is due to an additional blocking effect

induced by hybridization reaction by bringing AuNPs inside the

pores. The developed methodology can be extended to other

biosensing systems with interest not only for DNA but also for

proteins and cells. The developed nanochannel/nanoparticle

biosensing system would have enormous potential in future

miniaturized designs adapted to mass production technologies

such as screen-printing technology.

Structures from nature have remarkable properties, many of

which have inspired laboratory research. Bioinspired materials

and devices are attracting increasing interest because of their

unique properties, which have paved the way to many signifi-

cant applications.1,2 Ion channels that exist in living organisms

play important roles in maintaining normal physiological

conditions, serve as ‘‘smart’’ gates to ensure selective ion tran-

sport and respond directly to molecules or to physical stimuli.

For example, in these natural ion channels the ionic current

flows through and this current is altered when a molecule

binds to a specific region of the channel.3 The electrostatic

transport through different nanoporous electrodes has been

thoroughly studied in the last years.4–6 These fundamentals are

approached for biosensing purposes, by using biological

(i.e. a-hemolysin protein) or synthetic (i.e. alumina or silicon

nitride membranes) biomimetic nanopores and nanochannels,

simulating this natural behavior.7–9 Nanoporous materials

also show a dramatic increase in surface/volume ratio that

enhances the signals corresponding to interaction between

solutes and surfaces including biomolecule reactions. Based

on this principle nanopore/nanochannel arrays and single

nanopores seem to present promising new features for bio-

sensor development.

Single nanopores have been used to resolve sequences of

individual DNA molecules linked to a degree of partial pore

blockage by the DNA,10–12 measuring changes in conductance

during DNA translocation. Furthermore, the blockage of the

ion current in nanopore arrays by the DNA hybridization has

been approached for the ssDNA detection13,14 performing

voltammetric, conductometric and impedimetric measurements.

In a similar way, the blockage of nanopores by an immuno-

logical reaction has been approached for proteins detection

measuring changes in interferometric responses.15

In spite of these promising perspectives, alternatives are

needed in order to achieve a disposable device for real bio-

sensing applications. In this context we present here a simpler

set-up alternative and the corresponding methodology for the

detection of ssDNA using nanoporous membranes as sensing

platforms. This device, that has been recently reported by our

group for proteins detection,16 combines the properties of the

anodized aluminium oxide (AAO) nanoporousmembranes17 with

the advantages of the screen-printed electrotransducers and the

voltammetric detection mode. Furthermore AuNPs tags are used

for the first time as blocking agents that affect the diffusion of

[Fe(CN)6]4�/3�, used as electroactive species, through the electro-

transducer surface, improving the sensitivity of the assay.

In a previous work, Smirnov’s group13 detected 21-mer

ssDNA in probe ssDNAmodified AAO nanoporous membranes,

thanks to the blocking effect of the hybrid in the diffusion of

electroactive species through 20 nm pore membranes. A three

electrode system that contains a Pt working electrode inserted

inside a Plexiglas cell was used to measure the decrease of the

cyclic voltammetric peaks of the [Fe(CN)6]4�/[Fe(CN)6]

3�

system used as analytical signal for ssDNA detection.

Based on these fundamentals, in the present work the use

of AuNPs tags has been investigated in order to enhance the

blockage in the pores and consequently, to achieve the detec-

tion of lower levels of target ssDNA. Furthermore, screen-

printed carbon electrodes (SPCEs) have been selected as

disposable electrotransducers and 200 nm pore membranes

have been chosen in order to use 20 nm AuNPs tags,

whose synthesis and biofunctionalization has been extensively

studied by our group. In addition to this, differential pulse

voltammetry (DPV) as a more sensitive and reproducible

technique than cyclic voltammetry (data not shown) is selected

for the electrochemical measurements. The value of the peak

current corresponding to the oxidation of [Fe(CN)6]4� to

[Fe(CN)6]3� was chosen as analytical signal.

The functionalization of the pores (internal walls of

the nanochannels) is achieved in three steps:14 (i) generation

aNanobioelectronics & Biosensors Group, Institut Catala deNanotecnologia, CIN2 (ICN-CSIC), Barcelona, Spain.E-mail: arben.merkoci.icn@uab.es; Fax: +34 935868020;Tel: +34 935868014

b Instituto de Nanociencia de Aragon, Universidad de Zaragoza,Zaragoza, Spain. E-mail: alfredo.escosura.icn@uab.es;Fax: +34 976762776; Tel: +34 976762777

c ICREA, Barcelona, Spain. Fax: +34 932687700;Tel: +34 932687700w Electronic supplementary information (ESI) available: Materials,methods, optimization of parameters affecting the analytical signaland pictures of the electrotransducers and the electrochemical cellset-up. See DOI: 10.1039/c0cc02683b

COMMUNICATION www.rsc.org/chemcomm | ChemComm

Publ

ishe

d on

18

Oct

ober

201

0. D

ownl

oade

d by

Uni

vers

ity o

f W

inds

or o

n 27

/10/

2014

15:

54:0

9.

View Article Online / Journal Homepage / Table of Contents for this issue

9008 Chem. Commun., 2010, 46, 9007–9009 This journal is c The Royal Society of Chemistry 2010

of amino groups by a silanization with 3-amino-propyl-

trimethoxysilane (APS); (ii) generation of carboxyl groups

by reaction with glutaraldehyde and (iii) immobilization of

amino-modified probe ssDNA through the peptide bond. A

scheme of this procedure is shown in Fig. 1A. Plan and cross-

sectional SEM views of the functionalized AAO nanoporous

membranes used are shown in Fig. 1B.

The sensing principle for the detection of target ssDNA is

schematized in Fig. 2 (see details of the electrochemical cell

set-up in the ESIw). Considering the size of a 21-mer ssDNA

(approximately diameter of 1.84 nm and length of 0.38 nm)18

the hybridization reaction inside the porous membranes

is possible allowing the formation of hybrids. When high

concentrations of target ssDNA are tested (5 mg mL�1), the

formed hybrids are present to a sufficient extent to produce a

partial blockage and consequently a change in the electro-

active species diffusion along the nanochannel can be observed

as a decrease in the voltammetric signal of [Fe(CN)6]4�

oxidation to [Fe(CN)6]3� (Fig. 2b; Fe2+/Fe3+ are listed in

the scheme instead of [Fe(CN)6]4�/[Fe(CN)6]

3� in order to

simplify the cartoon), compared with that obtained for the

same concentration of a non-specific target ssDNA (blank

assay). In this case, it did not generate any blockage inside the

channel, so a higher voltammetric signal is observed (Fig. 2a).

The main parameters affecting the analytical signal have

been optimized: probe ssDNA concentration, probe ssDNA

immobilization time and hybridization reaction time, for a

5 mg mL�1 concentration of target ssDNA (see Fig. S1 in the

ESIw). A saturation of binding sites for 5 mg mL�1 of probe

ssDNA after an overnight immobilization has been observed.

Regarding the hybridization reaction, 120 min for a 5 mg mL�1

target ssDNA solution was found enough to achieve the

maximum blockage inside the nanochannels.

When the hybridization reaction is carried out for a

5 mg mL�1 solution of a target ssDNA labeled with AuNPs,

under the optimized conditions, a high decrease in the voltam-

metric peak current is observed compared with that obtained

for the unlabeled target, as can be seen in Fig. 2c. This

behavior evidences the blocking effect of the AuNPs that

can be approached for the detection of smaller quantities of

target ssDNA. From Fig. 2 it can also be noticed that the

voltammetric peak is shifted to less negative potentials when

the pores are partially blocked (DE E 50 mV and 120 mV for

the unlabeled and labeled based assays respectively). This can

be probably due to the effect of a mixed phenomena occurring

during detection: first, the blockage in the diffusion of the

electroactive species through the carbon surface and second,

the behavior of both the electrode and the nanochannel

platform as an ‘integrated’ unique conductor platform.

The selectivity of the sensor and the absence of non-specific

adsorptions inside the pores were tested by doing different

reference assays. The voltammetric peak currents obtained for

each assay are summarized in Fig. 3. It can be observed that

there is a decrease in the current registered for a SPCE

modified with a functionalized membrane compared with that

obtained for the bare SPCE, due to the blocking effect on the

Fig. 1 (A) Scheme of the biofunctionalization procedure of the AAO

nanoporous membranes: (i) silanization in APS; (ii) generation of

carboxyl groups by reaction with glutaraldehyde; (iii) immobilization

of the amino-modified probe ssDNA by the peptide bond. (B) SEM

images of a plan (left) and a cross-sectional view (right) of the 200 nm

pore AAO nanoporous membranes.

Fig. 2 (Top) Scheme of the sensing principle for a non-specific assay (left)

and for a specific assay with unlabeled (middle) and 20 nm AuNPs labeled

(right) target ssDNA. (Bottom) The corresponding differential pulse

voltammograms (DPVs) for 5 mg mL�1 of the non-specific target ssDNA

(a) and for the same concentration of unlabeled (b) and 20 nm AuNPs

labeled specific target ssDNA (c). DPVs measurements are recorded

in 1 mM K3[Fe(CN)6]/0.1 M NaNO3 solution using 200 nm pore

AAO nanoporous membranes. Pre-concentration potential: �0.55 V;

pre-concentration time: 30 s; step potential: 10 mV; modulation amplitude:

50 mV; scan rate: 33.5 mV s�1.

Fig. 3 Summary of the voltammetric peak currents (analytical

signals) obtained for bare SPCEs and for SPCEs modified with the

AAO nanoporous membranes after performing different assays inside

the channels. The experimental conditions are the optimized in the

study of Fig. S1 (ESIw). Target ssDNA concentration: 5 mg mL�1.

Publ

ishe

d on

18

Oct

ober

201

0. D

ownl

oade

d by

Uni

vers

ity o

f W

inds

or o

n 27

/10/

2014

15:

54:0

9.

View Article Online

This journal is c The Royal Society of Chemistry 2010 Chem. Commun., 2010, 46, 9007–9009 9009

ions diffusion that exerts the membrane itself. When a ssDNA

non-modified with amino-groups is added (non-specific probe)

no differences in the analytical signal are observed. However,

when the amino-modified probe ssDNA is added to the

functionalized membrane a significant decrease in the signal

is observed, evidencing that the probe is attached to the

membrane through the peptide bond, exerting a certain

blocking effect. When the non-specific target ssDNA is added

after the probe immobilization, no changes are observed in the

signal, demonstrating the selectivity of the assay. A similar

situation is observed when a non-specific target ssDNA

labeled with AuNPs is added, also demonstrating that there

are no non-specific adsorptions of AuNPs inside the pores.

However, when the reaction is carried out with the specific

target ssDNA, a lower current peak intensity is measured due

to the formation of the hybridization duplex. If the same assay

is performed using the specific target ssDNA labeled with

AuNPs, this decrease is observed to a higher extent, due to the

blocking effect of the AuNPs.

Finally, the effect of the concentration of target ssDNA

labeled with AuNPs on the DPV peak current used as analytical

signal was evaluated (Fig. 4), obtaining a linear correlation in

the range 50–250 ng mL�1, adjusted to the following equation:

peak current (mA) = �0.0029 [target ssDNA (ng mL�1)]

+ 0.927; r = 0.998.

The limit of detection (calculated as the concentration of

target ssDNA corresponding to three times the standard

deviation of the estimate) was 42 ng mL�1. The reproducibilityof the method shows an RSD of 9% for three repetitive assays

performed for a 100 ng mL�1 solution of target ssDNA

labelled with AuNPs, using different SPCEs and different

AAO nanoporous membranes.

In summary a novel nanoparticle-based enhancement of the

voltammetric DNA hybridization detection using a nanoporous

based platform and a methodology for the ssDNA detection

have been developed. The experimental set-up and the voltam-

metric detection based on the blockage of the diffusion of

electroactive species through the nanoporous based platforms

is performed in a simple, rapid and selective way, allowing the

detection of 21-mer ssDNA in a novel and very efficient mode,

with an improved sensitivity, thanks to the blocking effect of

the AuNPs used as labels. The proposed methodology could

be extended to biosystems which possess strong bioaffinity

interactions and has enormous potential in future miniaturized

designs. It could also be adapted to mass production techno-

logies such as screen-printing technology.

Currently, protein voltammetric detection in both label-free

and AuNPs amplified format assays is being investigated in

our group. This methodology has enormous potential applica-

tions, for example for the analysis of real samples, where the

membranes can act at the same time as filter, minimizing

matrix effects, and as a simple sensing platform. Furthermore,

the integration of AAO nanoporous membranes with trans-

ducing surfaces such as ITOs17 or other conducting surfaces

may open the way to important technological developments in

electrochemical biosensing field. The use of less-porous

membrane and larger AuNP labels may additionally affect

the nanochannel performance bringing advantages for even

lower detection limits. The nanochannel response tuning by

pore size control and AuNP labels size is now under develop-

ment at our laboratories.

We acknowledge funding from the MEC (Madrid) for

the projects MAT2008-03079/NAN, CSD2006-00012

‘‘NANOBIOMED’’ (Consolider-Ingenio 2010) and the Juan

de la Cierva scholarship (A. de la Escosura-Muniz).

Notes and references

1 E. Munch, M. E. Launey, D. H. Alsem, E. Saiz, A. P. Tomsia andR. O. Ritchie, Science, 2008, 322, 1516–1520.

2 H. Lee, B. P. Lee and P. B. Messersmith, Nature, 2007, 448,338–341.

3 X. Hou and L. Jiang, ACS Nano, 2009, 3, 3339–3342.4 G. Wang, B. Zhang, J. R. Wayment, J. M. Harris and H. S. White,J. Am. Chem. Soc., 2006, 128, 7679–7686.

5 G. Wang, A. K. Bohaty, I. Zharov and H. S. White, J. Am. Chem.Soc., 2006, 128, 13553–13558.

6 B. Yameen, M. Ali, R. Neumann, W. Ensinger, W. Knolld andO. Azzaroni, Chem. Commun., 2010, 46, 1908–1910.

7 H. Bayley and P. S. Cremer, Nature, 2001, 413, 226–230.8 C. R. Martin and Z. S. Siwy, Science, 2007, 317, 231–332.9 Y. Tian, X. Hou, L. Wen, W. Guo, Y. Song, H. Sun, Y. Wang,L. Jiang and D. Zhua, Chem. Commun., 2010, 46, 1682–1684.

10 P. Chen, T. Mitsui, D. B. Farmer, J. Golovchenko, R. G. Gordonand D. Branton, Nano Lett., 2004, 4, 1333–1337.

11 A. Singer, M. Wanunu, W. Morrison, H. Kuhn, M. Frank-Kamenetskii and A. Meller, Nano Lett., 2010, 10, 738–742.

12 M. Wanunu, W. Morrison, Y. Rabin, A. Y. Grosberg andA. Meller, Nat. Nanotechnol., 2010, 5, 160–165.

13 I. Vlassiouk, P. Takmakov and S. Smirnov, Langmuir, 2005, 21,4776–4778.

14 I. Vlassiouk, A. Krasnoslobodtsev, S. Smirnov and M. Germann,Langmuir, 2004, 20, 9913–9915.

15 S. D. Alvarez, C. P. Li, C. E. Chiang, I. K. Schuller andM. J. Sailor, ACS Nano, 2009, 3, 3301–3307.

16 A. de la Escosura-Muniz and A. Merkoci, Electrochem. Commun.,2010, 12, 859–863.

17 T. R. B. Foong, A. Sellinger and X. Hu, ACS Nano, 2008, 2,2250–2256.

18 R. E. Dickerson, H. R. Drew, B. N. Conner, M. L. Kopka andP. E. Pjura, Cold Spring Harbor Symp. Quant. Biol., 1982, 47, 13.

Fig. 4 (A) Differential pulse voltammograms (DPVs) obtained for

different concentrations of target ssDNA labeled with 20 nm AuNPs:

(a) 50, (b) 100, (c) 150, (d) 200, (e) 250 and (f) 300 ng mL�1. Theexperimental conditions are the same as those used for obtaining the

DPVs shown in Fig. 2. (B) Effect of the concentration of the target

ssDNA labeled with 20 nm AuNPs on the analytical signal.

Publ

ishe

d on

18

Oct

ober

201

0. D

ownl

oade

d by

Uni

vers

ity o

f W

inds

or o

n 27

/10/

2014

15:

54:0

9.

View Article Online