Highly sensitive DNA sensor based on polypyrrole nanowire

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 PII: DOI: Reference: S0169-4332(14)01049-6 http://dx.doi.org/doi:10.1016/j.apsusc.2014.05.032 APSUSC 27850 To appear in: APSUSC Received date: Revised date: Accepted date: 11-6-2013 6-5-2014 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 Highly sensitive DNA sensor based on polypyrrole nanowire Mai Anh Tuan1, Pham Duc Thanh1, Chu Thi Xuan1, Nguyen Minh Hieu2, Nguyen Hoang Hai2,* International Training Institute for Material Science, Hanoi University of Science and ip t Technology, No 1, Dai Co Viet Road, Hanoi, Vietnam Nano and Energy Center, Vietnam National University (Hanoi) cr Corresponding authors * Nguyen Hoang Hai us Nano and Energy Center, Vietnam National University (Hanoi) 84 983500726 E-mail: nhhai@vnu.edu.vn Post address: 334 Nguyen Trai Road, Hanoi, Viet Nam M an Phone: * Mai Anh Tuan d International Training Institute for Materials Science, Hanoi University of Phone: Ac ce p Fax: te Science and Technology 84 38680787 84 38692963 E-mail: mtuan@itims.edu.vn / tuan.maianh@hust.edu.vn Post address: No.1 Dai Co Viet, Hanoi, Vietnam Page of 16 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 ip t 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 cr 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 us The response time of the DNA sensor is 10 seconds and the measurement time was minutes an The lowest detectable concentration of E.coli DNA was 0.1 nM Keywords: DNA sensor, E.coli, electrochemical synthesis, gelatin, polypyrrole nanowire M Introduction Since the birthday of the electrical conducting polymer (CP) in 1960s In particular, after Alan d MacDiarmid, Hideki Shirakawa, and Alan Heeger reported a 10 million-fold increase in the te 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 Ac ce p 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] Page of 16 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 ip t reaction occurred and the surface of the transducer As the results, the lowest detectable concentration of DNA was nM [9] In this work, PPy membrane was improved in form of the cr nanowire And, it’s expected that the modified surface would improve the sensitivity of the DNA sensor us Experiment an 2.1 Chemicals Pyrrole monomer was purchased from Merck H2SO4 solution (0.5M), LiClO4 buffer (0.1M), M phosphate buffer solution (pH=7) were prepared with double distilled water All chemicals were at analytical grade d The DNA sequences used in this work, table 1, were supplied by Invitrogen Life Technologies te 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 Ac ce p 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 Page of 16 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 ip t 830 DPS Lock-in Amplifier from Stanford Research, figure Detail of the measurement set-up is described in 2.5 cr 2.3 Polymerization of the PPy The active area of the working electrode was, first of all, cleaned with double distilled water, us then acetone (95%) to remove the physically adsorbed molecules The surface was then an 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 M 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 d yrrole Then, the test solution were force-pumped continuously at least 15 with N2 to te 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 Ac ce p 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 NHgroups (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 nonspecific binding sites followed by air-dried Page of 16 2.5 Label-free measurement The measurement was described in figure 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 ip t 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 cr the immobilized DNA) This differential technique is helpful to minimize the environmental noise in the measuring solution us When the DNA strands are hybridized, the concentration of charges in conductive membrane is an changed The output signal was gathered up on two kΩ resistances by the A and B channels of the Lock-in Amplifier interfaced with a computer The principle of the measurement is adopted M from Manual book of Lock-in Amplifier-model SR830 of the Stanford Research System Each measurement was realized times for data acquisition All measurements were performed at te Results and discussion d room temperature 3.1 Electrochemical synthesis of PPy nanowire Ac ce p 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 a, and no wire was observed At low concentration (C