Highly sensitive DNA sensor based on polypyrrole nanowire

Hai, Highly sensitive DNA sensor based on polypyrrole nanowire, Applied Surface Science 2014, http://dx.doi.org/10.1016/j.apsusc.2014.05.032 This is a PDF file of an unedited manuscript

Accepted Manuscript

Title: Highly sensitive DNA sensor based on polypyrrole

nanowire

Author: Mai Anh Tuan Pham Duc Thanh Chu Thi Xuan

Nguyen Minh Hieu Nguyen Hoang Hai

DOI: http://dx.doi.org/doi:10.1016/j.apsusc.2014.05.032

Reference: APSUSC 27850

To appear in: APSUSC

Received date: 11-6-2013

Revised date: 6-5-2014

Accepted date: 6-5-2014

Please cite this article as: M.A Tuan, P.D Thanh, C.T Xuan, N.M Hieu, N.H Hai,

Highly sensitive DNA sensor based on polypyrrole nanowire, Applied Surface Science

(2014), http://dx.doi.org/10.1016/j.apsusc.2014.05.032

This is a PDF file of an unedited manuscript that has been accepted for publication

As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain

Accepted Manuscript

Highly sensitive DNA sensor based on polypyrrole nanowire

Mai Anh Tuan1, Pham Duc Thanh1, Chu Thi Xuan1, Nguyen Minh Hieu2, Nguyen Hoang Hai2,*

1International Training Institute for Material Science, Hanoi University of Science and

Technology, No 1, Dai Co Viet Road, Hanoi, Vietnam

2Nano and Energy Center, Vietnam National University (Hanoi)

Corresponding authors

* Nguyen Hoang Hai

Nano and Energy Center, Vietnam National University (Hanoi) Phone:

E-mail:

Post address:

84 983500726 nhhai@vnu.edu.vn

334 Nguyen Trai Road, Hanoi, Viet Nam

* Mai Anh Tuan

International Training Institute for Materials Science, Hanoi University of Science and Technology

Phone:

Fax:

E-mail:

Post address:

84 4 38680787

84 4 38692963 mtuan@itims.edu.vn / tuan.maianh@hust.edu.vn No.1 Dai Co Viet, Hanoi, Vietnam

Accepted Manuscript

Abstract

This paper describes the development of a DNA sensor based on polypyrrole nanowire By using

potentiostatic technique, in the presence of gelatin as the soft mold, the polypyrrole nanowires

were synthesized on the surface of the micro-sensor The surface enhanced Raman spectroscopy

shows that the N-H ends of the polypyrrole nanowires orientate upward from the surface

facilitating the DNA probe immobilization through the simple linkage with the phosphate groups

of the probe DNA The label-free signal readout was carried out by lock-in amplifier technique

The response time of the DNA sensor is 10 seconds and the measurement time was 5 minutes

The lowest detectable concentration of E.coli DNA was 0.1 nM

Keywords: DNA sensor, E.coli, electrochemical synthesis, gelatin, polypyrrole nanowire

1 Introduction

Since the birthday of the electrical conducting polymer (CP) in 1960s In particular, after Alan

MacDiarmid, Hideki Shirakawa, and Alan Heeger reported a 10 million-fold increase in the

conductivity of poly-acetylene doped with iodine in 1977 [1,2], dozens of CPs have been

synthesized and characterized However, not many of them have been found to be well suited for

biomedical applications due to the requirement of biocompatibility, conductivity, and stability in

biological systems

In biosensor, the specific matching between the complementary DNA sequences or the key-lock

interaction between the enzyme-substrate is a well-studied But the transduction and signal

amplification of the biological recognition has challenged the scientists and engineers [3,4]

For biomedical application, PPy is still one of the most studied polymers Especially, in

electrochemical approach, the researchers have made many efforts to modify the interface where

the change in electrochemical properties occurred in order to enhance the characteristics of the

CPs based biosensors [5-8]

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In our recent paper, we reported the development of DNA sensor using the DNA attached PPy

membrane The PPy were also electrochemically synthesized but created in cauliflower form

which was supposed not to facilitate the electron transfer between the electrolyte where the

reaction occurred and the surface of the transducer As the results, the lowest detectable

concentration of DNA was 2 nM [9] In this work, PPy membrane was improved in form of the

nanowire And, it’s expected that the modified surface would improve the sensitivity of the DNA

sensor

2 Experiment

2.1 Chemicals

Pyrrole monomer was purchased from Merck H2SO4 solution (0.5M), LiClO4 buffer (0.1M),

phosphate buffer solution (pH=7) were prepared with double distilled water All chemicals were

at analytical grade

The DNA sequences used in this work, table 1, were supplied by Invitrogen Life Technologies

Company through the National Institute of Hygiene and Epidemiology of Vietnam (NIHE) The

PCR amplified E.Coli DNA sequence was provided by NIHE The initial concentration of DNA

probe was 0.05μM

2.2 Instrumentations and sensor

The electrochemical biosensor based on interdigitated microelectrodes was designed and

fabricated at the Hanoi University of Science and Technology The dual electrode was fabricated

using a conventional photolithographic method with a finger-width of 10 µm and a gap size of

10 µm The fingers of inter-digitated electrodes were fabricated by sputtering 10 nm Ti and 200

nm Pt on a layer of silicon dioxide (SiO2) with thickness of approximately 100 nm thermally

grown on top of a silicon wafer [10]

The controlled-potential experiments (for PPy synthesis) were performed with the Autolab

PGSTAT 302 (from Metrohm, the Netherland) The three-electrode system consisted of a

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platinum working electrode, a counter electrode (of the above mentioned micro-sensor) an

Ag/AgCl reference electrode (in saturated KCl)

The measurement of the hybridization of the DNA probe and target was conducted by using SR

830 DPS Lock-in Amplifier from Stanford Research, figure 1 Detail of the measurement set-up

is described in 2.5

2.3 Polymerization of the PPy

The active area of the working electrode was, first of all, cleaned with double distilled water,

then acetone (95%) to remove the physically adsorbed molecules The surface was then

electrochemically activated by sweeping voltage (from -0.4 to +1.8 VDC; scan rate of 0.1Vs-1

and cycle number of 20-50) in H2SO4 buffer (0.5M) till stable CV curve recorded

A typical electrolyte was prepared in a 50-ml volume of phosphate buffer solution (PBS pH=7),

0.1M LiClO4 containing gelatin which required slightly heating to achieve dissolution and 0.5 ml

yrrole Then, the test solution were force-pumped continuously at least 15 min with N2 to

eliminate interfering oxygen The sensor was immersed in the electrolyte solution, and within the

sweeping range The suitable oxidized voltage was found and recorded for the next usage The

detail synthesis of PPy was discussed in [11] The scanning range was between -1.0 V and

+1.0V; the scan rate was at 250 mV/s, and 0.75V was applied as the potential polymerization of

pyrrole in potentiostatic mode The polypyrrole was 0.5 mL in the presence of 0.08% gelatin,

and 400 s used throughout the whole polymerization

2.4 Immobilization of the DNA probe

The DNA was immobilized to the nanowire PPy through simple the linkage between the NH-

groups (upward from one end of the PPy nanowires) and the phosphate groups of the probe

DNA The sensor was soaked into the solution containing 0.05 µM of probe DNA for 02 hours at

room temperature followed by 5-time DI water rinse to wash away the unsaturated and

non-specific binding sites followed by air-dried

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2.5 Label-free measurement

The measurement was described in figure 1 A referential signal of alternative current density,

has frequency of 10 kHz and amplitude of 100 mV, was taken out from the sine generator of the

Lock-in Amplifier SR830, and was set on the two identical micro-electrodes of which one serves

as working electrode (DNA immobilized) and the rest works as the reference electrode (without

the immobilized DNA) This differential technique is helpful to minimize the environmental

noise in the measuring solution

When the DNA strands are hybridized, the concentration of charges in conductive membrane is

changed The output signal was gathered up on two 1 kΩ resistances by the A and B channels of

the Lock-in Amplifier interfaced with a computer The principle of the measurement is adopted

from Manual book of Lock-in Amplifier-model SR830 of the Stanford Research System Each

measurement was realized 4 times for data acquisition All measurements were performed at

room temperature

3 Results and discussion

3.1 Electrochemical synthesis of PPy nanowire

The polypyrrole was synthesized by adding different quantity of pyrrole in 50 mL electrolyte

solution containing Li+ 0.1M, PBS (pH=7) and given quantity of gelatin When the

electrochemical synthesis was without gelatin, the polypyrrole were grown randomly in

orientation and size as shown in figure 2 a, and no wire was observed At low concentration

(C<0.08), gelatin was not close enough to form the mold for polymer to grow, figure 2 b At

specific concentration (0.08%), the gelatin molecules bonded together to create the soft molds on

which the PPy nanowires grown As can be seen in figure 2c, the nanowire were around 50 nm

in diameter, and very consistent on the surface of the electrode When the gelatin was denser

(greater than 0.08%), a cross-link among the gelatin molecules occur to form the islands on

which polypyrrole synthesized, figure 2d

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3.2 Surface Enhanced Raman Spectroscope (SERS)

In previous report, the FTIR spectrum analysis confirmed the synthesized polypyrrole on the

surface of working electrode [11] In addition to that, in this paper, SERS was again used to

investigate the functional groups on the active surface of the sensor which functionalized by PPy

and E.coli The data given in the figure 3 was in good matching with previous reports [12-16]

In figure 3, the most exciting peak at 1244 cm-1 (of the SERS) is assigned to the vibration of

β(CH)/δNH [17] Although, the peak of N-H exists but the position shifts to a lower wave

number compared with the peak at 1300 cm-1 of N-H in previous study [18] Through the weak

enhanced peak, it demonstrates that there are not many N-H groups which are close to the Pt

surface due to In other expression, N-H groups are generally orientated upward and far from the

surface of the membrane which facilitates the DNA immobilization later on Furthermore, this

may be ascribed to the change in chemical structure of PPy due to the presence of gelatin as a

‘soft template’ in potentiostatic polymerization

3.3 Determination of DNA hybridization

To read out the hybridization signal, the probe DNA-attached sensor is soaked into a measuring

cell filled with 250 μL of double distilled water Sample’s concentration was controlled by

adding a repeated volume of 5μL target DNA, respectively

Figure 4 demonstrates the response time and reaction time of the DNA sensor A drop containing

5 nM target DNA was injected into measuring cell The target hybridizes very rapidly with the

immobilized probe leading to the change of the readout The response time of the DNA sensor

was just a few seconds After the first jump step of output signal, the conductivity observed was

constant after 2÷3 minutes To avoid fluctuation of output signal, the reaction time chosen for the

other measurements was 5 minutes

In order to evaluate the response of the DNA sensor, similar experiments were repeated with

increasing concentration of target DNA In figure 5, when adding different non-matching DNA

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sequence, no response was detected In contrast, the signal was changed rapidly when full

matching target was injected into the measuring cell The lowest concentration of the target DNA

that the DNA sensor can detect was 0.1 nM

3.4 Reproducibility of the DNA sensor

The reproducibility of the DNA sensor was evaluated by first of all heating up the DNA sensor

to 800C which is higher the melting point of the given sequence, in double distilled water for 5

minutes then then quickly frozen in an ice bath for 2 minutes to obtain the single DNA strand

Afterwards, the DNA sensor was immersed into the cell containing the sample to detect the

DNA target as the second time The different output signal between the matching and

re-matching was recorded and illustrated in figure 6

The output signal of the DNA sensor changes slightly compared with that before denaturation

(Δδ= 5.3%) Several measurements were carried out by using different sensors (prepared at the

same condition) with the same DNA target concentration variation and DNA probe (0.05μM)

immobilized on the sensor surface The calculated results indicated that the relative standard

deviation of the output signal of these sensors was less than 9% These results recommended that

the DNA sensor is reproducible and can be proposed to reusable in some specific circumstances

4 Conclusion

We report, in this paper, the synthesis of PPy nanowires obtained by using potentiostatic

technique in the presence of gelatin as the soft mold The obtained PPy nanowires

orientate the N-H bond upward which takes advantage for the DNA probe

immobilization through the simple linkage with the phosphate groups of probe DNA The

response time of the DNA sensor is 10 seconds and the measurement time was 5 minutes

The sensor can detect as low as 0.1 nM of E.coli DNA (20 bases) which is much better

than that of our previous result The initial experiment showed that, the sensor has good

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reproducibility Further study will be realized to evaluate the selectivity when

mismatching DNA is also present in the sample; to investigate the influence of the pH,

temperature and also the initial probe concentration

Acknowledgement

This work was financially supported by the Vietnamese National Foundation for Science and

Technology Development (NAFOSTED) for a basic research project, code 103.01-2011.59

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