Available online at www.sciencedirect.com ScienceDirect Procedia Engineering 168 (2016) 419 – 422 30th Eurosensors Conference, EUROSENSORS 2016 Label-free and Electrochemical Detection of Nucleic Acids Based on Isothermal Amplification in Combination with Solid-state pH Sensor Miyuki Tabataa, Yurika Katayamaa, Fahmida Mannanb, Ayaka Seichic, Koji Suzukic, Tatsuro Godaa, Akira Matsumotoa, Yuji Miyaharaa,* a Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-surugadai, Chiyoda-ku, Tokyo 101-0062, Japan b Faculty of Medicine, Imperial College London, Exhibition Road, London SW7 2AZ, United Kingdum c Department of Applied Chemsitry, Graduate School of Science and Engineering, Keio University, 3-14-1 Hiyoshi, Kohoku, Yokohama, Kanagawa 223-8522, Japan Abstract Label-free quantification methods for nucleic acids are attracting attention, since they provide potential tools in clinical applications such as disease diagnosis or prognosis observation In this research, we developed a simpler and cost-effective electrical monitoring device of nucleic acid amplification, combining an isothermal amplification method and an Iridium/Iridium oxide (Ir/IrOx) electrode without labeling The fabricated Ir/IrOx electrode showed ideal Nernstian response under the various pH buffer solutions, and its potential response was less affected even in the presence of charged proteins Moreover, we successfully real-time monitored primer generation-rolling circle amplification (PG-RCA) at the 0, 10, 100, 1000 pM of target DNA by detection of released proton during amplification reaction at the constant reaction temperature This label-free and portable device might be received considerable attention as a useful platform for a point-of-care testing in clinical use © 2016 2016The TheAuthors Authors Published by Elsevier Ltd.is an open access article under the CC BY-NC-ND license © Published by Elsevier Ltd This (http://creativecommons.org/licenses/by-nc-nd/4.0/) Peer-review under responsibility of the organizing committee of the 30th Eurosensors Conference Peer-review under responsibility of the organizing committee of the 30th Eurosensors Conference Keywords: Biosensor; iridium/iridium oxide; isothermal nucleic acid amplification * Corresponding author Tel.: +80-3-5280-8095; fax: +81-3-5280-8135 E-mail address: miyahara.bsr@tmd.ac.jp 1877-7058 © 2016 The Authors Published by Elsevier Ltd This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Peer-review under responsibility of the organizing committee of the 30th Eurosensors Conference doi:10.1016/j.proeng.2016.11.534 420 Miyuki Tabata et al / Procedia Engineering 168 (2016) 419 – 422 Introduction PCR is widely used as a quantitative nucleic acid detection method Most of the present real-time PCR systems employ fluorescent detection using intercalating reagents (e.g., SYBR Green) or sequence-specific reporter probes to quantify the amplified PCR products Meanwhile, PCR is disadvantageous on miniaturization because it requires precise temperature control using a thermal cycler and an optical detection by fluorescence labeling method Over the last few decades, there have been several emerging infectious diseases including HIV, SARs, H1N1, and Ebola, and they are becoming a threat to the life and the health of the people in the world Recently, miniaturized nucleic acid amplification systems have been intensively studied for point-of-care testing as one of the approaches in this direction To overcome this problem, electrical/electrochemical nucleic acid detection devices were reported because of the compatibility with semiconductor technology and fine processing technology [1, 2] One of the most successful examples of commercialized products is Ion Torrent (Thermo Fisher Scientific) semiconductor chip-based DNA sequencer launched in 2012 [1, 3, 4] However, the electrical properties of ISFET change significantly during a thermal cycle of PCR Currently, combination of isothermal nucleic acid amplification instead of PCR with electrical/electrochemical devices has been studied extensively to overcome this problem [2, 5] In this research, we proposed a new concept on the combination of PG-RCA and Ir/IrOx electrode PG-RCA (Fig 1) is one of the exponential isothermal amplification method and has an advantage in point of not affecting the electrical properties of Ir/IOx during amplification reaction PG-RCA A circular probe was circularized according to the method reported by Murakami et al [6] PG-RCA is an improved method of linear RCA, which amplifies target sequence in an exponential manner by applying nicking reaction to the long ssDNA amplicon followed by initiating another RCA reaction from the fragmented amplicon as a new template (Fig 1) The PG-RCA reaction was performed at 37 oC for h Fig Schematic illustration of primer-generation rolling circle amplification Fabrication and characterization of IrOx wire 3.1 IrOx preparation and pH sensitivity Two types of IrOx fabrication methods were tried To get the Pt/IrOx electrode, Pt wire (φ 0.3 mm) was immersed into [Ir(COO)2(OH)4]2- aqueous solution and then applied 1V for 15min against cathode Pt wire By the anodic Miyuki Tabata et al / Procedia Engineering 168 (2016) 419 – 422 oxidation coating, IrOx was deposited on the Pt surface Thermal oxidation processing of Ir wire (φ 0.3 mm) was carried out at 800oC for 30 under air atmosphere times after immersing Ir wire in M NaOH solution for days These obtained Pt/IrOx and Ir/IrOx electrodes were used as a working electrode a) b) Fig Photographs of IrOx layer a) Pt/IrOx, an b) Ir/IrOx The pH sensitivity and selectivity of IrOx play a major role in the overall performance of our system Two types of IrOx fabrication methods (e.g electrodeposition (Pt/IrOx) and thermal oxidation (Ir/IrOx)) were compared on pH sensitivity and electrical stability A slope of −72.1 mv/pH on Pt/IrOx was found using a three-point extrapolation with standard buffer solutions at pH 4.0, 7.0 and 9.2 On the other hand, Ir/IrOx showed −57.5 mv/pH This value was close to the Nernstian slope (−59.2 mV/pH at 25oC), which demonstrated the excellent proton buffering capacity of fabricated Ir/IrOx 3.2 Electrical behavior of Ir/IrOx on the non-specific adsorption To evaluate electrical stability, potential measurement was carried out in the presence of bovine serum albumin (BSA), which was used as a model protein negatively charged under the physiological condition (Fig 3) Delta potential hardly changed in the electrodes having an oxide layer (Pt/IrOx and Ir/IrOx), although adsorbed BSA to electrode surface was confirmed a) BSA solution Rinse with SDS Buffer c) b) BSA concentration BSA concentration Fig Potential changes of electrodes with or without an oxide layer in presence of BSA presence a) Raw potential data, b) Pt and Pt/IrOx, and c) Ir and Ir/IrOx 421 422 Miyuki Tabata et al / Procedia Engineering 168 (2016) 419 – 422 PG-RCA monitoring PG-RCA fluorescence monitoring using SYBR Green as a conventional method was performed at 37oC for 2hours PG-RCA successfully proceeded, and the reaction speeds increased increasing with a series of different concentrations of target DNA ranged from to 1000 pM Subsequently, electrical detection of PG-RCA was also conducted in which the proton as an indicator The pH calculated from potential measurement using Ir/IrOx electrode was shifted to the acid side depending on the concentration of target Conclusion Conclusively, we have developed a label-free, isothermal electrochemical platform for DNA detection using simply Ir/IrOx electrode This should be a promising tool for the current biosensor scope from the view of point-of-caretensting These devices can be used not only in large hospitals but also in smaller medical facilities or physicians’ offices, or even in a patient’s home In addition, we can take the small instrument out of the laboratory to detect nucleic acids of viruses or microorganisms on site for the purpose of infectious disease testing Acknowledgements This study was supported in part by the Center of Innovation Program from Japan Science and Technology Agency, JST, JSPS KAKENHI Grant-in-Aid for Young Scientists (B) (No 15K16320), and JSPS KAKENHI Grant-in-Aid for JSPS Fellows (No 15J06165) References [1] C Toumazou, L M Shepherd, S C Reed, et al (2013): Simultaneous DNA amplification and detection using a pH-sensing semiconductor system, Nat Methods, 10 (7), 641-646 [2] B Yao, Y Liu, M Tabata, et al (2014): Sensitive detection of microRNA by chronocoulometry and rolling circle amplification on a gold electrode, Chem Commun., 50, 9704-9706 [3] B Merriman, Ion Torrent R&D Team, J.M Rothberg (2012): Progress in Ion Torrent semiconductor chip based sequencing, Electrophoresis, 33, 3397-3417 [4] J Rothberg, W Hinz, T Rearick, J Schultz, W Mileski, M Davey, et al (2011): An integrated semiconductor device enabling non-optical genome sequencing, Nature, 475 (7356), 348-352 [5] A Seichi, N Kozuka, Y.Kashima, M Tabata, T Goda, A Matsumota, et al (2016): Real-time monitoring and detection of primer generationrolling circle amplification of DNA using an ethidium ion-selective electrode, Anal 32 (5), 505-510 [6] T Murakami, J Sumaoka, M Komiyama (2009): Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification, Nucleic Acids Res 37 (3) e19 ... PG-RCA (Fig 1) is one of the exponential isothermal amplification method and has an advantage in point of not affecting the electrical properties of Ir/IOx during amplification reaction PG-RCA A circular... launched in 2012 [1, 3, 4] However, the electrical properties of ISFET change significantly during a thermal cycle of PCR Currently, combination of isothermal nucleic acid amplification instead of. .. amplification and detection using a pH- sensing semiconductor system, Nat Methods, 10 (7), 641-646 [2] B Yao, Y Liu, M Tabata, et al (2014): Sensitive detection of microRNA by chronocoulometry and rolling