Directly immobilized DNA sensor for label-free detection of herpes virus

doi:10.4236/jbise.2009.26054

Paper Menu >>

Journal Menu >>

Vol.2, No.6, 374-379 (2009)

doi:10.4236/jbise.2009.26054

JBiSE

Directly immobilized DNA sensor for l abel-free

detection of herpes virus

Phuong Dinh Tam1,2, Mai Anh Tuan 2, Nguyen Duc Chien3

1Hanoi Advanced School of Science and Technology, Hanoi University of Technology, Hanoi, Vietnam; 2International Training Insti-

tute for Materials Science, Hanoi University of Technology, Hanoi, Vietnam; 3Institute of Engineering Physics, Hanoi University of

Technology, Hanoi, Vietnam.

Email: [email protected]

Received 24 March 2008; revised 12 May 2009; accepted 29 June 2009.

ABSTRACT

This paper reports the direct immobilization of

deoxyribonucleic acid (DNA) sequences of Her-

pes simplex virus (5’–AT CAC CGA CCC GGA

GAG GGA C–3’) on the surface of DNA sensor

by using the cyclic voltammetric method with

the presence of pyrrole. The potential was

scanned from –0.7 volt to + 0.6 volt, the scan-

ning rate was at 100mV/s. This kind of DNA

sensor was developed to detect Herpes virus

DNA in real samples. The FTIR was applied to

verify specific binding of DNA sequence and

conducting polymer, the morphology of con-

ducting polymer doped with DNA strands was

investigated by using a field emission scanning

electron microscope (FE-SEM). The results sh ow-

ed that output signal given by co-immobilized

DNA/PPy membrane sensor was better than that

given by APTS immobilized membrane sensors.

The sensor can detect as low as 2 nM of DNA

t arget in real samples.

Keywords: DNA Sensor; Hybridization; APTS

1. INTRODUCTION

The detection of specific DNA/RNA sequences is of

great importance in numerous applications of modern

life science, including identification of medical research

and clinical diagnosis [1,2], controlling the food quality

[3,4], environmental analysis [5,6]. Many methods have

been used for this purpose such as polymerase chain

reaction (PCR) [7,8,9], quartz crystal micro-balance

(QCM) [10,11], fluorescence [12], surface plasmon

resonance [13], microfluidic system [14], cell culture

and real-time PCR, etc. These methods are precise, and

allow a wide, dynamic range of detection. However, they

are complex, costly and time consuming. In addition, it

is impossible to carry the on-site/in-field tests. Thus,

development of a cheap, reliable device allowing rapid

detection is always the challenge for scientists and engi-

neers. In this context, DNA sensor based on electro-

chemical detection is one of the feasible and promising

tools.

We reported, in this paper, the direct co-immobiliza-

tion of DNA sequence of Herpes simplex virus and

polypyrrole onto the surface of a sensor by cyclic volt-

ammetry to determine the herpes DNA target sequence

in the sample. The herpes simplex virus (HSV) is an

enveloped double-stranded DNA virus. There are two

distinct forms of HSV, serotype 1 and serotype 2 (HSV-

1 and HSV-2). HSV-2 is the most common cause of

genital herpes, whereas HSV-1 is the most common

cause of facial herpes or cold scores. HSV-1 is transmit-

ted through contact with oral secretions. Diseases caused

by Herpes virus are commonly found in patients in Viet-

nam.

2. EXPERIMENTS

2.1. Chemical Reagents

DNA sequences used in this work (Ta ble 1 ) were sup-

plied by Invitrogen Life Technologies Company through

National Institute of Hygiene and Epidemiology of Viet-

nam. Pyrrole was purchased from Merck. Other chemi-

cals are of analytical grade.

2.2. Sensor Fabrication

The microelectrode based DNA sensor was designed and

fabricated at clean room of ITIMS. The sensor consists

of pairs of microelectrodes on the surface of silicon sub-

strate, one of which acts as working sensor and the other

as a reference electrode. The dimension of the inter-

electrodes was 20 µm x 20 µm (Figure 1). The detailed

fabrication process was discussed in [15].

2.3. Cyclic Volttametry Electropolymerization

Electropolymerization was carried out by using IM6EX

P. D. Tam et al. / J. Biomedical Science and Engineerin g 2 (2009) 374-379

375

Table 1. DNA sequences used in this work.

Types DNA sequences

Probe 5’–AT CAC CGA CCC GGA GAG GGA C–3’

Non-complementary 5’–AT CAC CGA CCC GGA GAG GGA C–3’

Target 3’-TA GTG GCT GGG CCT CTC CCT G-5’

Mis/1 3’-TA GTG GGT GGG CCT CTC CCT G-5’

Mis/2 3’-TA GTG GGT GGG AAT CTC CCT G-5’

Figure 1. 20 µm x 20 µm microelectrode sensor was fabricated at ITIMS.

(Germany) impedance analyzer at room temperature in

which the micro-sensor acted as working electrode while

auxiliary electrode was a platinum wire. Reference elec-

trode is Ag/AgCl in saturated KCl.

The sensor was first surface cleaned by KCr2O7 in

H2SO4 98% followed by cyclic voltammograms (swept

potential from –1.5V to +2.1V, scan rate: 25mV/s) in

0.5M H2SO4 to activate the surface of the sensors. Fi-

nally, the potential was swept on the working electrode

from –0.7 volt to 0.6 volt versus standard counter elec-

trode (SCE). The scanning rate was 100mV/s.

2.4. Measurement

Differential measurements were realized to determine

the changes in conductance of DNA membrane. An AC

reference signals (10 KHz, 100mV sine wave), generated

by the generator of Lock-in Amplifier SR830, and was

applied on two identical micro-electrodes of DNA sensor.

The output signal was acquired by measuring the voltage

drop on two 1 KΩ resistances by the A and B channels

of the Lock-in Amplifier and processed by a PC through

RS 232 interface. All measurements were performed at

room temperature. In this experiment, five DNA sensors

were used to test the hybridization of DNA sequences.

3. RESULTS AND DIS CUS SION

3.1. The Polymerization of PPy/DNA

Normally, pyrrole is polymerized with the presence of an

anionic dopant which contributes to film conductivity.

Variety of anions can be used as dopant for polypyrrole

(Ppy) polymerization such as Cl-, NO3

-. In this work

ClO4

- and DNA sequence were used.

According to Wang et al [16], DNA can be considered

as sole counter anion in the electropolymerization proc-

ess at the working electrode. This allowed maximum

possible incorporation of DNA in the conductive poly-

mer throughout the film thickness and full contribution

of oligonucleotides charged phosphates to the polymer

conductivity.

The cyclic voltammograms of synthesized Ppy and

Ppy/DNA film is shown in Figure 2 where the oxidation

of pyrrole monomer leads to the formation of radical

cation, subsequent oxidation of the dimer and coupling

will result in the formation of an insoluble polymer,

positively charged on the surface. This electrochemical

procedure allowed the formation of a copolymer which

Figure 2. Cyclic voltammograms of 0.5mM Ppy doped

0.05µM DNA probe sequence in LiClO4 solution.

Swept potential from –0.7 V to 0.6V, scanning rate is at

100mV/s.

P. D. Tam et al. / J. Biomedical Science and Engineerin g 2 (2009) 374-379

376

is a mixture of polypyrrole and an oligonucleotides that

shows an increasing current along with conducting film

growth which corresponds to incorporation of oligo into

the Ppy film. The film was rinsed and used for detection

of DNA hybridization.

3.2. FTIR Spectrum of Ppy and DNA/Ppy

In this work, the FTIR spectroscopy was used to verify

the existence of polypyrrole and DNA sequence on the

microelectrode surface after the polymerization process.

The infrared spectrum of the DNA/Ppy complexes and

pure Ppy were performed on Niconet 6700 FT-IR ma-

chine with the effective range from 400 cm-1 to 4000

cm-1 at room temperature. As shown in Figure 3, the

absorption band at 1889 cm-1–1629 cm-1 vibration plane

implied G-C and A-T base pairs while the backbone

phosphate group at 1095 cm-1 was perturbed upon Ppy

interaction [17,18].

The absorption band at 1254 cm-1 was assigned to the

biopolaronic species formed in the over oxidation pro-

cess of Ppy [19]. The C-H and N-H bonds were also

observed at 735 cm-1 (for DNA/Ppy film); 734 cm-1 for

Ppy film; 894 cm-1 for DNA/Ppy and 897 cm-1 for Ppy

membrane, respectively. These results show very good

agree- ment with earlier reported work [20].

3.3. Morphology of Conducting Polymer Film

The morphology of sensor surface coated with Ppy film

was studied by FE-SEM. Figure 4 indicated micro-

graphs of polypyrrole doped with LiClO4 (4a) and with

both 0.1 M LiClO4 and 0.05 M DNA sequence (4b)

membrane given by direct electropolymerization method.

In Figure 4(a), the pure PPy doped with LiClO4 was

cauli-flower structure matching other works [21]. This

structure is related to the dopant intercalation in the

polymeric chain. As in Figure 4(b) the DNA strands was

observed as white dots in host polymer membrane. Good

distribution of DNA in PPy membrane makes it advan-

tage for hybridization process of the probe in target solu-

tion.

Figure 3. The FTIR spectra of Ppy/DNA and Ppy (upper curve:

Ppy/dopant, lower curve: Ppy/dopant/DNA).

(a) (b)

Figure 4. The FE-SEM of Ppy and Ppy–DNA coated onto microelectrode surface. a) Ppy doped Li-

ClO4; b) © Ppy doped LiClO4 and DNA.

P. D. Tam et al. / J. Biomedical Science and Engineerin g 2 (2009) 374-379

377

3.4. The Hybridization of DNA Sensor

As above-mentioned, the probe-attached sensor is com-

monly soaked into solution containing target DNA. A

DNA helix sequence is formed on the surface of the

sensor when target/immobilized DNA matching oc-

curred.

Such hybridization is detected by changes in the con-

ductance of the conductive membrane on the surface of

sensors leading to the change in output signal of the sys-

tem. In Figure 5, the hybridization illustrated by linear

curve that described the relation between the target DNA

concentration and output signal of the DNA sensor. For

both APTS and Ppy/DNA attachment method, the sensor

can detect as low as 2 nM of target DNA. However, the

intensity of the output signal found to be better when

direct immobilization was used than that given by APTS.

This is explained by the contribution of Ppy and dopant

which improve the conductivity of the membrane

namely enhancing the electric charge transfer within the

film.

Figure 5. DNA sequence hybridization curve at

room temperature, 0.05M probe DNA.

Figure 6. The detection mismatch DNA sequence at

room temperature, 0.05M probe DNA.

3.5. Detection of Mismatches DNA

Sequence Using the DNA Sensor

Mismatch detection is our first trial step for sensor selec-

tivity investigation. In this work, we used two different

mismatched sequences of Herpes virus (Table 1), and

then compared their hybridization signals with those of

fully complementary targets to investigate the effects of

the base pair mismatches. As presented in Figure 6, a

much stronger signal of DNA hybridization containing

fully matched DNA compares to system containing

mismatched DNA was clearly found. This study is still

in progress for statistic conclusion.

The influence of the mismatch positions was investi-

gated by change of the DNA single base pair mismatch

(mis/1) possessed a C (Cytosine) instead of a G (Gua-

nine) at the 7th oligo and the three base pair mismatch

sequence (mis/2) additionally contained CC instead of

AA at 12th and 13th position close to the 5’ end of the

DNA molecule. Figure 6 presents the evident decrease

of signal was observed when probe DNA was hybridized

with two mismatches. It also can be seen that, the signal

reduction of mis/2 was stronger than mis/1. From these

results, it can be deduced that the electrochemical DNA

sensor has enough high sensitivity to detect a single base

pair mismatch DNA at some of positions within the se-

quence.

3.6. Hybridization with PCR Amplified

Sample

The DNA sequence of herpes virus in real sample was

used in our work. Firstly, the DNA sequences of herpes

virus were amplified by PCR method (20µg/l) and then

divided into 2 parts: 10g/µl DNA sequence for gel

electrophoresis; 10g/µl DNA sequence for DNA hy-

bridization detection using the DNA sensor. The Figure

7 showed results of 92 base pairs fragment–amplified

by PCR method in which lance M indicates the Marker,

Figure 7. Agarose gel electrophoresis of the

PCR products amplified in the thermal cycler.

P. D. Tam et al. / J. Biomedical Science and Engineerin g 2 (2009) 374-379

378

Figure 8. PCR amplified DNA se-

quence detected by DNA sensor.

lane 1: PCR performed in H2O Enppendorf tubes, lanes

27: the DNA template with various DNA concentra-

tions, and lanes 8, 9 are for the Herpes virus PCR prod-

ucts with 92 base pairs.

Note that, the PCR amplified sample was double

strands, thus, before the performance of DNA sequence

detection, the sample was thermally denatured at 98oC

for 5 minutes and then was quickly decreased to 50oC to

obtain the single DNA strand. Afterwards, our DNA

sensors were immerged into the cell containing the sam-

ple to detect the target. Figure 8 described the relation

between target DNA concentration and output signal of

DNA sensor at room temperature, phosphate buffer solu-

tion with 0.5 µM probe DNA. It can be seen that, output

signal of DNA sensor is linear with target DNA concen-

tration. This result went well that given by PCR method

and matched our purpose to develop the DNA sensor as

pre-diagnostic device.

4. CONCLUSIONS

This paper described the direct immobilization of DNA

strand on the surface of sensor by electrochemical

method. The DNA sensor was used to determine the

Herpes simplex virus DNA in the sample. The results

showed that, the DNA sensor can detect as small as 2

nM of herpes virus DNA concentration at room tem-

perature and the intensity of the output signal is better

than by using APTS attachment method. The influence

of mismatch DNA was determined with decrease of 50%

signal (mis/1) and 75% signal (mis/2) compares to full

matching at 12 nM target DNA concentration. We still

keep doing further research to verify selectivity and sen-

sitivity of the sensors at different measuring conditions.

5. ACKNOWLEDGEMENTS

The work has been supported by Ministry of Education and Training

under research project code B2008-01-175

REFERENCES

[1] K. Jalava, S. Nikkari, J. Jalava, E. Eerola, M. Skurnik, O.

Meurman, O. Ruuskanen, A. Alanen, E. Kotilainen, P.

Toivanen, and P. Kotilainen, (2000) Direct amplification

of rRNA genes in diagnosis of bacterial infections, J. of

Clinical Microbiology, 38, 32–39.

[2] J. Wang, G. Rivas, C. Parrado, X. Cai, and M. N. Flair,

(1997) Electrochemical biosensor for detecting DNA se-

quences from the pathogenic protozoan Cryptosporidium

parvum, Talanta, 44, 2003–2010.

[3] M. Passamano and M. Pighini, (2006) QCM DNA- sen-

sor for GMOs detection, Sensors and Actuators B, 118,

177–181.

[4] A. Rang, B. Linke, and B. Jansen, (2005) Detection of

RNA variants transcribed from the transgene in Roundup

ready soybean, Eur. Food Res. Tech., 220, 438–443.

[5] J. Wang, G. Rivas, X. Cai, E. Palecek, P. Nielsen, H.

Shiraishi, N. Dontha, D. Luo, C. Parrado, M. Chicharro,

P. A. M. Farias, F. S. Valera, D. H. Grant, M. Ozsoz, and

M. N. Flair, (1997) DNA electrochemical biosensors for

environmental monitoring, Analytical Chemical Act.,

347, 1–8.

[6] Y. Lu, J. Liu, J. Li, P. J. Bruesehoff, C. M. B. Pavot, and

A. K. Brown, (2003) New highly sensitive and selective

catalytic DNA biosensors for metal Ions, Biosensors and

Bioelectronics, 18, 529–540.

[7] P. Rossmanith, M. Krassnig, M. Wagner, and I. Hein,

(2006) Detection of Listeria monocytogenes in food us-

ing a combined enrichment/real-time PCR method tar-

geting the prfA gene, Research in Microbiology, 157,

763–771.

[8] K. E. Yoder and R. Fishel, (2006) PCR-based detection

is unable to consistently distinguish HIV 1LTR circles,

Journal of Virological Methods, 138, 201–206.

[9] B. D. Rio, A. G. Binetti, M. C. Martín, M. Fernández, A.

H. Magadán, and M. A. Alvarez, (2007) Multiplex PCR

for the detection and identification of dairy bacterio-

phages in milk, Food Microbiology, 24, 75–81.

[10] I. Mannelli, M. Minunni, S. Tombelli, and M. Mascini,

(2003) Quartz crystal microbalance (QCM) affinity bio-

sensor for genetically modified organisms (GMOs) de-

tection, Biosensor and Bioelectronics, 18, 129–140.

[11] X. D. Zhou, L. J. Liu, M. Hu, L. L. Wang, and J. M. Hu,

(2002) Detection of hepatitis B virus by piezoelectric

biosensor, J. Pharm. and Biomed. Anal, 27, 341–345.

[12] R. Epstein, I. Biran, and D. R. Walt, (2002) Fluores-

cence- based nucleic acid detection and micro arrays,

Anal. Chimica Acta., 469, 3–36.

[13] T. Jiang, M. Minunni, P. Wilson, J. Zhang, A. P. F.

Turner, and M. Mascini, (2005) Detection of TP53 muta-

tion using a portable surface plasmon resonance DNA-

based biosensor, Biosensors and Bioelectronics, 20,

1939–1945.

[14] K. Yamashita, Y. Yamaguchi, M. Miyazaki, H. Naka-

mura, H. Shimizu, and H. Maeda, (2004) Microfluidic

system for DNA sequence detection, Chem. Eng. Journal,

101, 157–161.

[15] P. D. Thanh, M. A. Tuan, N. D. Chien, and C. Jean-Marc,

(2004) Investigation on interferences of conductometric

biosensor using tyrosinase enzyme, in Proc. 7th Viet-

namese-German Seminar on Physics and Engineering,

Vietnam, 158–161.

[16] J. Wang, M. Jiang, A. Fortes, and B. Mukherjee, (1999)

New label-free DNA recognition based on doping nu-

P. D. Tam et al. / J. Biomedical Science and Engineerin g 2 (2009) 374-379

379

cleic-acid probes within conducting polymer films, Anal.

Chim. Acta, 402, 7–12.

[17] H. A. Tajmir-Riahi, (2006) An overview of protein-

DNA and protein-RNA interactions, J. of the Iranian

Chem. Soc., 3, 297–304.

[18] Y. Zhou and Y. Li, (2004) Studies of interaction between

poly (allylamine hydrochloride) and double helix DNA

by spectral methods, Biophysical Chemistry, 107,

273–281.

[19] L. S. Andréa and O. A. S. Maria, (2007) Electrodeposi-

tion of polypyrrole films on aluminum from tartrate

aqueous solution, J. Braz. Chem. Soc., 18, 143–152.

[20] M. Omastová, J. Pionteck, and S. Koina, (1996) Prepara-

tion and characterization of electrically conductive poly-

propylene/polypyrrole composites, Eur. Polym. J., 32,

681– 689.

[21] H. N. Cong, K. E. Abbassi, J. L. Gautier, and P. Chartier,

(2005) Oxygen reduction on oxide/polypyrrole compos-

ite electrodes: effect of doping anions, Electrochimica

Acta., 50, 136–1376.