Development of a Capillary-driven, Microfluidic, Nucleic Acid Biosensor

Sensors & Transducers Volume 13, Issue 12 December 2011

www.sensorsportal.com

ISSN 1726-5479

Editors-in-Chief: Sergey Y. Yurish, tel.: +34 93 413 7941, e-mail: [email protected] Guest Editors: Elena Gaura, James Brusey, and Ramona Rednic Editors for Western Europe Meijer, Gerard C.M., Delft University of Technology, The Netherlands Ferrari, Vittorio, Universitá di Brescia, Italy Editor for Eastern Europe Sachenko, Anatoly, Ternopil State Economic University, Ukraine

Editor South America Costa-Felix, Rodrigo, Inmetro, Brazil Editor for Africa Maki K.Habib, American University in Cairo, Egypt Editor for Asia Ohyama, Shinji, Tokyo Institute of Technology, Japan

Editors for North America Datskos, Panos G., Oak Ridge National Laboratory, USA Fabien, J. Josse, Marquette University, USA Katz, Evgeny, Clarkson University, USA

Editor for Asia-Pacific Mukhopadhyay, Subhas, Massey University, New Zealand

Editorial Advisory Board Abdul Rahim, Ruzairi, Universiti Teknologi, Malaysia Ahmad, Mohd Noor, Nothern University of Engineering, Malaysia Annamalai, Karthigeyan, National Institute of Advanced Industrial Science and Technology, Japan Arcega, Francisco, University of Zaragoza, Spain Arguel, Philippe, CNRS, France Ahn, Jae-Pyoung, Korea Institute of Science and Technology, Korea Arndt, Michael, Robert Bosch GmbH, Germany Ascoli, Giorgio, George Mason University, USA Atalay, Selcuk, Inonu University, Turkey Atghiaee, Ahmad, University of Tehran, Iran Augutis, Vygantas, Kaunas University of Technology, Lithuania Avachit, Patil Lalchand, North Maharashtra University, India Ayesh, Aladdin, De Montfort University, UK Azamimi, Azian binti Abdullah, Universiti Malaysia Perlis, Malaysia Bahreyni, Behraad, University of Manitoba, Canada Baliga, Shankar, B., General Monitors Transnational, USA Baoxian, Ye, Zhengzhou University, China Barford, Lee, Agilent Laboratories, USA Barlingay, Ravindra, RF Arrays Systems, India Basu, Sukumar, Jadavpur University, India Beck, Stephen, University of Sheffield, UK Ben Bouzid, Sihem, Institut National de Recherche Scientifique, Tunisia Benachaiba, Chellali, Universitaire de Bechar, Algeria Binnie, T. David, Napier University, UK Bischoff, Gerlinde, Inst. Analytical Chemistry, Germany Bodas, Dhananjay, IMTEK, Germany Borges Carval, Nuno, Universidade de Aveiro, Portugal Bousbia-Salah, Mounir, University of Annaba, Algeria Bouvet, Marcel, CNRS – UPMC, France Brudzewski, Kazimierz, Warsaw University of Technology, Poland Cai, Chenxin, Nanjing Normal University, China Cai, Qingyun, Hunan University, China Campanella, Luigi, University La Sapienza, Italy Carvalho, Vitor, Minho University, Portugal Cecelja, Franjo, Brunel University, London, UK Cerda Belmonte, Judith, Imperial College London, UK Chakrabarty, Chandan Kumar, Universiti Tenaga Nasional, Malaysia Chakravorty, Dipankar, Association for the Cultivation of Science, India Changhai, Ru, Harbin Engineering University, China Chaudhari, Gajanan, Shri Shivaji Science College, India Chavali, Murthy, N.I. Center for Higher Education, (N.I. University), India Chen, Jiming, Zhejiang University, China Chen, Rongshun, National Tsing Hua University, Taiwan Cheng, Kuo-Sheng, National Cheng Kung University, Taiwan Chiang, Jeffrey (Cheng-Ta), Industrial Technol. Research Institute, Taiwan Chiriac, Horia, National Institute of Research and Development, Romania Chowdhuri, Arijit, University of Delhi, India Chung, Wen-Yaw, Chung Yuan Christian University, Taiwan Corres, Jesus, Universidad Publica de Navarra, Spain Cortes, Camilo A., Universidad Nacional de Colombia, Colombia Courtois, Christian, Universite de Valenciennes, France Cusano, Andrea, University of Sannio, Italy D'Amico, Arnaldo, Università di Tor Vergata, Italy De Stefano, Luca, Institute for Microelectronics and Microsystem, Italy Deshmukh, Kiran, Shri Shivaji Mahavidyalaya, Barshi, India Dickert, Franz L., Vienna University, Austria Dieguez, Angel, University of Barcelona, Spain Dighavkar, C. G., M.G. Vidyamandir’s L. V.H. College, India Dimitropoulos, Panos, University of Thessaly, Greece Ko, Sang Choon, Electronics. and Telecom. Research Inst., Korea South

Ding, Jianning, Jiangsu Polytechnic University, China Djordjevich, Alexandar, City University of Hong Kong, Hong Kong Donato, Nicola, University of Messina, Italy Donato, Patricio, Universidad de Mar del Plata, Argentina Dong, Feng, Tianjin University, China Drljaca, Predrag, Instersema Sensoric SA, Switzerland Dubey, Venketesh, Bournemouth University, UK Enderle, Stefan, Univ.of Ulm and KTB Mechatronics GmbH, Germany Erdem, Gursan K. Arzum, Ege University, Turkey Erkmen, Aydan M., Middle East Technical University, Turkey Estelle, Patrice, Insa Rennes, France Estrada, Horacio, University of North Carolina, USA Faiz, Adil, INSA Lyon, France Fericean, Sorin, Balluff GmbH, Germany Fernandes, Joana M., University of Porto, Portugal Francioso, Luca, CNR-IMM Institute for Microelectronics and Microsystems, Italy Francis, Laurent, University Catholique de Louvain, Belgium Fu, Weiling, South-Western Hospital, Chongqing, China Gaura, Elena, Coventry University, UK Geng, Yanfeng, China University of Petroleum, China Gole, James, Georgia Institute of Technology, USA Gong, Hao, National University of Singapore, Singapore Gonzalez de la Rosa, Juan Jose, University of Cadiz, Spain Granel, Annette, Goteborg University, Sweden Graff, Mason, The University of Texas at Arlington, USA Guan, Shan, Eastman Kodak, USA Guillet, Bruno, University of Caen, France Guo, Zhen, New Jersey Institute of Technology, USA Gupta, Narendra Kumar, Napier University, UK Hadjiloucas, Sillas, The University of Reading, UK Haider, Mohammad R., Sonoma State University, USA Hashsham, Syed, Michigan State University, USA Hasni, Abdelhafid, Bechar University, Algeria Hernandez, Alvaro, University of Alcala, Spain Hernandez, Wilmar, Universidad Politecnica de Madrid, Spain Homentcovschi, Dorel, SUNY Binghamton, USA Horstman, Tom, U.S. Automation Group, LLC, USA Hsiai, Tzung (John), University of Southern California, USA Huang, Jeng-Sheng, Chung Yuan Christian University, Taiwan Huang, Star, National Tsing Hua University, Taiwan Huang, Wei, PSG Design Center, USA Hui, David, University of New Orleans, USA Jaffrezic-Renault, Nicole, Ecole Centrale de Lyon, France Jaime Calvo-Galleg, Jaime, Universidad de Salamanca, Spain James, Daniel, Griffith University, Australia Janting, Jakob, DELTA Danish Electronics, Denmark Jiang, Liudi, University of Southampton, UK Jiang, Wei, University of Virginia, USA Jiao, Zheng, Shanghai University, China John, Joachim, IMEC, Belgium Kalach, Andrew, Voronezh Institute of Ministry of Interior, Russia Kang, Moonho, Sunmoon University, Korea South Kaniusas, Eugenijus, Vienna University of Technology, Austria Katake, Anup, Texas A&M University, USA Kausel, Wilfried, University of Music, Vienna, Austria Kavasoglu, Nese, Mugla University, Turkey Ke, Cathy, Tyndall National Institute, Ireland Khelfaoui, Rachid, Université de Bechar, Algeria Khan, Asif, Aligarh Muslim University, Aligarh, India Kim, Min Young, Kyungpook National University, Korea South Sandacci, Serghei, Sensor Technology Ltd., UK

Kotulska, Malgorzata, Wroclaw University of Technology, Poland Kockar, Hakan, Balikesir University, Turkey Kong, Ing, RMIT University, Australia Kratz, Henrik, Uppsala University, Sweden Krishnamoorthy, Ganesh, University of Texas at Austin, USA Kumar, Arun, University of South Florida, USA Kumar, Subodh, National Physical Laboratory, India Kung, Chih-Hsien, Chang-Jung Christian University, Taiwan Lacnjevac, Caslav, University of Belgrade, Serbia Lay-Ekuakille, Aime, University of Lecce, Italy Lee, Jang Myung, Pusan National University, Korea South Lee, Jun Su, Amkor Technology, Inc. South Korea Lei, Hua, National Starch and Chemical Company, USA Li, Fengyuan (Thomas), Purdue University, USA Li, Genxi, Nanjing University, China Li, Hui, Shanghai Jiaotong University, China Li, Xian-Fang, Central South University, China Li, Yuefa, Wayne State University, USA Liang, Yuanchang, University of Washington, USA Liawruangrath, Saisunee, Chiang Mai University, Thailand Liew, Kim Meow, City University of Hong Kong, Hong Kong Lin, Hermann, National Kaohsiung University, Taiwan Lin, Paul, Cleveland State University, USA Linderholm, Pontus, EPFL - Microsystems Laboratory, Switzerland Liu, Aihua, University of Oklahoma, USA Liu Changgeng, Louisiana State University, USA Liu, Cheng-Hsien, National Tsing Hua University, Taiwan Liu, Songqin, Southeast University, China Lodeiro, Carlos, University of Vigo, Spain Lorenzo, Maria Encarnacio, Universidad Autonoma de Madrid, Spain Lukaszewicz, Jerzy Pawel, Nicholas Copernicus University, Poland Ma, Zhanfang, Northeast Normal University, China Majstorovic, Vidosav, University of Belgrade, Serbia Malyshev, V.V., National Research Centre ‘Kurchatov Institute’, Russia Marquez, Alfredo, Centro de Investigacion en Materiales Avanzados, Mexico Matay, Ladislav, Slovak Academy of Sciences, Slovakia Mathur, Prafull, National Physical Laboratory, India Maurya, D.K., Institute of Materials Research and Engineering, Singapore Mekid, Samir, University of Manchester, UK Melnyk, Ivan, Photon Control Inc., Canada Mendes, Paulo, University of Minho, Portugal Mennell, Julie, Northumbria University, UK Mi, Bin, Boston Scientific Corporation, USA Minas, Graca, University of Minho, Portugal Moghavvemi, Mahmoud, University of Malaya, Malaysia Mohammadi, Mohammad-Reza, University of Cambridge, UK Molina Flores, Esteban, Benemérita Universidad Autónoma de Puebla, Mexico Moradi, Majid, University of Kerman, Iran Morello, Rosario, University "Mediterranea" of Reggio Calabria, Italy Mounir, Ben Ali, University of Sousse, Tunisia Mrad, Nezih, Defence R&D, Canada Mulla, Imtiaz Sirajuddin, National Chemical Laboratory, Pune, India Nabok, Aleksey, Sheffield Hallam University, UK Neelamegam, Periasamy, Sastra Deemed University, India Neshkova, Milka, Bulgarian Academy of Sciences, Bulgaria Oberhammer, Joachim, Royal Institute of Technology, Sweden Ould Lahoucine, Cherif, University of Guelma, Algeria Pamidighanta, Sayanu, Bharat Electronics Limited (BEL), India Pan, Jisheng, Institute of Materials Research & Engineering, Singapore Park, Joon-Shik, Korea Electronics Technology Institute, Korea South Penza, Michele, ENEA C.R., Italy Pereira, Jose Miguel, Instituto Politecnico de Setebal, Portugal Petsev, Dimiter, University of New Mexico, USA Pogacnik, Lea, University of Ljubljana, Slovenia Post, Michael, National Research Council, Canada Prance, Robert, University of Sussex, UK Prasad, Ambika, Gulbarga University, India Prateepasen, Asa, Kingmoungut's University of Technology, Thailand Pullini, Daniele, Centro Ricerche FIAT, Italy Pumera, Martin, National Institute for Materials Science, Japan Radhakrishnan, S. National Chemical Laboratory, Pune, India Rajanna, K., Indian Institute of Science, India Ramadan, Qasem, Institute of Microelectronics, Singapore Rao, Basuthkar, Tata Inst. of Fundamental Research, India Raoof, Kosai, Joseph Fourier University of Grenoble, France Rastogi Shiva, K. University of Idaho, USA Reig, Candid, University of Valencia, Spain Restivo, Maria Teresa, University of Porto, Portugal Robert, Michel, University Henri Poincare, France Rezazadeh, Ghader, Urmia University, Iran Royo, Santiago, Universitat Politecnica de Catalunya, Spain Rodriguez, Angel, Universidad Politecnica de Cataluna, Spain Rothberg, Steve, Loughborough University, UK Sadana, Ajit, University of Mississippi, USA Sadeghian Marnani, Hamed, TU Delft, The Netherlands Sapozhnikova, Ksenia, D.I.Mendeleyev Institute for Metrology, Russia

Saxena, Vibha, Bhbha Atomic Research Centre, Mumbai, India Schneider, John K., Ultra-Scan Corporation, USA Sengupta, Deepak, Advance Bio-Photonics, India Seif, Selemani, Alabama A & M University, USA Seifter, Achim, Los Alamos National Laboratory, USA Shah, Kriyang, La Trobe University, Australia Sankarraj, Anand, Detector Electronics Corp., USA Silva Girao, Pedro, Technical University of Lisbon, Portugal Singh, V. R., National Physical Laboratory, India Slomovitz, Daniel, UTE, Uruguay Smith, Martin, Open University, UK Soleymanpour, Ahmad, Damghan Basic Science University, Iran Somani, Prakash R., Centre for Materials for Electronics Technol., India Srinivas, Talabattula, Indian Institute of Science, Bangalore, India Srivastava, Arvind K., NanoSonix Inc., USA Stefan-van Staden, Raluca-Ioana, University of Pretoria, South Africa Stefanescu, Dan Mihai, Romanian Measurement Society, Romania Sumriddetchka, Sarun, National Electronics and Computer Technology Center, Thailand Sun, Chengliang, Polytechnic University, Hong-Kong Sun, Dongming, Jilin University, China Sun, Junhua, Beijing University of Aeronautics and Astronautics, China Sun, Zhiqiang, Central South University, China Suri, C. Raman, Institute of Microbial Technology, India Sysoev, Victor, Saratov State Technical University, Russia Szewczyk, Roman, Industrial Research Inst. for Automation and Measurement, Poland Tan, Ooi Kiang, Nanyang Technological University, Singapore, Tang, Dianping, Southwest University, China Tang, Jaw-Luen, National Chung Cheng University, Taiwan Teker, Kasif, Frostburg State University, USA Thirunavukkarasu, I., Manipal University Karnataka, India Thumbavanam Pad, Kartik, Carnegie Mellon University, USA Tian, Gui Yun, University of Newcastle, UK Tsiantos, Vassilios, Technological Educational Institute of Kaval, Greece Tsigara, Anna, National Hellenic Research Foundation, Greece Twomey, Karen, University College Cork, Ireland Valente, Antonio, University, Vila Real, - U.T.A.D., Portugal Vanga, Raghav Rao, Summit Technology Services, Inc., USA Vaseashta, Ashok, Marshall University, USA Vazquez, Carmen, Carlos III University in Madrid, Spain Vieira, Manuela, Instituto Superior de Engenharia de Lisboa, Portugal Vigna, Benedetto, STMicroelectronics, Italy Vrba, Radimir, Brno University of Technology, Czech Republic Wandelt, Barbara, Technical University of Lodz, Poland Wang, Jiangping, Xi'an Shiyou University, China Wang, Kedong, Beihang University, China Wang, Liang, Pacific Northwest National Laboratory, USA Wang, Mi, University of Leeds, UK Wang, Shinn-Fwu, Ching Yun University, Taiwan Wang, Wei-Chih, University of Washington, USA Wang, Wensheng, University of Pennsylvania, USA Watson, Steven, Center for NanoSpace Technologies Inc., USA Weiping, Yan, Dalian University of Technology, China Wells, Stephen, Southern Company Services, USA Wolkenberg, Andrzej, Institute of Electron Technology, Poland Woods, R. Clive, Louisiana State University, USA Wu, DerHo, National Pingtung Univ. of Science and Technology, Taiwan Wu, Zhaoyang, Hunan University, China Xiu Tao, Ge, Chuzhou University, China Xu, Lisheng, The Chinese University of Hong Kong, Hong Kong Xu, Sen, Drexel University, USA Xu, Tao, University of California, Irvine, USA Yang, Dongfang, National Research Council, Canada Yang, Shuang-Hua, Loughborough University, UK Yang, Wuqiang, The University of Manchester, UK Yang, Xiaoling, University of Georgia, Athens, GA, USA Yaping Dan, Harvard University, USA Ymeti, Aurel, University of Twente, Netherland Yong Zhao, Northeastern University, China Yu, Haihu, Wuhan University of Technology, China Yuan, Yong, Massey University, New Zealand Yufera Garcia, Alberto, Seville University, Spain Zakaria, Zulkarnay, University Malaysia Perlis, Malaysia Zagnoni, Michele, University of Southampton, UK Zamani, Cyrus, Universitat de Barcelona, Spain Zeni, Luigi, Second University of Naples, Italy Zhang, Minglong, Shanghai University, China Zhang, Qintao, University of California at Berkeley, USA Zhang, Weiping, Shanghai Jiao Tong University, China Zhang, Wenming, Shanghai Jiao Tong University, China Zhang, Xueji, World Precision Instruments, Inc., USA Zhong, Haoxiang, Henan Normal University, China Zhu, Qing, Fujifilm Dimatix, Inc., USA Zorzano, Luis, Universidad de La Rioja, Spain Zourob, Mohammed, University of Cambridge, UK

Sensors & Transducers Journal (ISSN 1726-5479) is a peer review international journal published monthly online by International Frequency Sensor Association (IFSA). Available in electronic and on CD. Copyright © 2011 by International Frequency Sensor Association. All rights reserved.

Sensors & Transducers Journal

Contents Volume 13 Special Issue December 2011

www.sensorsportal.com

ISSN 1726-5479

Research Articles Foreword Elena Gaura, James P. Brusey, Ramona Rednic ..............................................................................

I

Wireless Sensors for Space Applications William Wilson, Gary Atkinson............................................................................................................

1

Applications of a Navigation Instrument Based on a Micro-Motor Driven by Photons Jorge Valenzuela and Samson Mil’shtein ..........................................................................................

11

Fabrication and Analysis of MEMS Test Structures for Residual Stress Measurement Akshdeep Sharma, Deepak Bansal, Maninder Kaur, Prem Kumar, Dinesh Kumar, Rina Sharma and K. J. Rangra.................................................................................................................................

21

An electro-thermal MEMS Gripper with Large Tip Opening and Holding Force: Design and Characterization Jay J. Khazaai, H. Qu, M. Shillor and L. Smith ..................................................................................

31

Self-alignment of Silicon Chips on Wafers: a Numerical Investigation of the Effect of Spreading and Wetting Jean Berthier, Kenneth Brakke, Sébastien Mermoz, Loïc Sanchez, Christian Fretigny, Léa Di Cioccio ....................................................................................................................................

44

Use of Small-Scale Wind Energy to Power Cellular Communication Equipment B. Plourde, J. Abraham, G. Mowry, W. Minkowycz............................................................................

53

Superhydrophobic Porous Silicon Surfaces Paolo Nenzi, Alberto Giacomello, Guido Bolognesi, Mauro Chinappi, Marco Balucani, and Carlo Massimo Casciola..............................................................................................................

62

Increased Bandwidth of Mechanical Energy Harvester B. Ahmed Seddik, G. Despessse, S. Boisseau, E. Defay..................................................................

73

Simplifying the Design,Analysis, and Layout of a N/MEMS Nanoscale Material Testing Device Richa Bansal, Jason V. Clark .............................................................................................................

87

A Novel Silicon-based Wideband RF Nano Switch Matrix Cell and the Fabrication of RF Nano Switch Structures Yi Xiu Yang, Hamood Ur Rahman and Rodica Ramer ......................................................................

98

Carbon Nanomaterials for Optical Absorber Applications Anupama Kaul, James Coles, Krikor Megerian, Michael Eastwood, Robert Green, Thomas Pagano, Prabhakar Bandaru and Mehmet Dokmeci............................................................

109

Improved Nanoreinforced Composite Material Bonds with Potential Sensing Capabilities David Starikov, Clyde A. Price, Michael S. Fischer, Abdelhak Bensaoula, Farouk Attia, Thomas A. Glenn, Mounir Boukadoum ..............................................................................................

117

Upconverting Phosphor Thermometry for High Temperature Sensing Applications Xiaomei Guo, Hongzhi Zhao, Huihong Song, Elizabeth Zhang, Christopher Combs, Noel Clemens, Xuesheng Chen, Kewen K. Li, Yingyin K. Zou and Hua Jiang...........................................................

124

A Model for Enhanced Chemiluminescence Reactions in Microchambers Combined to an Active Pixel Sensor Jean Berthier, Pierre L. Joly, Florence Rivera, Patrice Caillat ...........................................................

131

Antibody Immobilization on Conductive Polymer Coated Nonwoven Fibers for Biosensors Shannon K. Mcgraw, Michael J. Anderson, Evangelyn C. Alocilja, Patrick J. Marek, Kris J. Senecal, Andre G. Senecal .....................................................................................................

142

Development of a Capillary-driven, Microfluidic, Nucleic Acid Biosensor Fei He, Yuhong Wang, Shenquan Jin and Sam R. Nugen ................................................................

150

Authors are encouraged to submit article in MS Word (doc) and Acrobat (pdf) formats by e-mail: [email protected] Please visit journal’s webpage with preparation instructions: http://www.sensorsportal.com/HTML/DIGEST/Submition.htm International Frequency Sensor Association (IFSA).

Sensors & Transducers Journal, Vol. 13, Special Issue, December 2011, pp. 150-158

Sensors & Transducers ISSN 1726-5479 © 2011 by IFSA http://www.sensorsportal.com

Development of a Capillary-driven, Microfluidic, Nucleic Acid Biosensor Fei HE, Yuhong WANG, Shenquan JIN and Sam R. NUGEN Department of Food Science University of Massachusetts, Amherst, MA, 01003, USA Tel.: +1 (413) 545-1025 E-mail: [email protected]

Received: 29 June 2011 /Accepted: 16 November 2011 /Published: 28 December 2011 Abstract: An ideal point-of-care device would incorporate the simplicity and reliability of a lateral flow assay with a microfluidic device. Our system consists of self-priming microfluidics with sealed conjugate pads of reagent delivery and an absorbent pad for additional fluid draw. Using poly (methyl methacrylate) (PMMA) as a substrate, we have developed a single-step surface modification method which allows strong capillary flow within a sealed microchannel. Conjugate pads within the device held trapped complex consisting of the magnetic beads and nucleic-acid-probe-conjugated horseradish peroxidase (HRP). Magnetic beads were released when sample entered the chamber and hybridized with the complex. The complex was immobilized over a magnet while a luminol co-reactant stream containing H2O2 was merged with the channel. A plate reader was able to quantify the chemiluminescence signal. This new format of biosensor will allow for a smaller and more sensitive biosensor, as well as commercial-scale manufacturing and low materials cost. Copyright © 2011 IFSA. Keywords: Microfluidics, Biosensors, Capillary flow assay, Nucleic acid detection.

1. Introduction Rapid detection technologies with high selectivity and sensitivity for pathogenic bacteria are critical in food safety, environmental monitoring, and clinical diagnosis [1]. In the analysis of complex biological samples, conventional laboratory-scale technologies include culture and colony counting which involve a pre-enrichment or selective enrichment step followed by a biochemical test [2] and [3], immunological techniques, polymerase chain reaction (PCR) or reverse transcriptase PCR (RT-PCR). Although these approaches can allow for low limits of detection, most of them present problems of 150


complex sample handling, lengthy time requirements, high consumption of expensive samples or reagents, and they do not afford the necessary limit of detection and specificity towards the target. Microfluidics-based lab-on-chip systems offer several advantages over existing techniques [4, 5]. The lab-on-chip includes all detection steps in one device allowing assays to become more repeatable by limiting user error. A major benefit of miniaturization is portable devices for the onsite detection of pathogens [7]. A precise fluid control and flow stability are also critical for successful diagnostics in microfluidics-based systems. However, traditional microfluidics relies on external equipment such as syringe pumps, tubing, microscopes or computers for operation. These components are not ideal for resource-limited settings which require portability and durability [8, 9]. Current passive fluid handling techniques like capillary pumps, gravity-induced flow and air-evacuated PDMS pump are gaining some research momentum due to their simplicity and ease of operation [10]. The integration of multiple microfluidic technologies is required for performing an entire point-of-care (POC) assay, and it is increasingly recognized as a central technical challenge in developing functional lab-on-a-chip devices [11]. We have developed a method of rapid prototyping of microfluidic chips at low cost. Polymer substrates are appealing materials for mass production due to the ease of microfabrication techniques such as injection molding, compression molding, extrusion, hot embossing and replication. They are considered as a low-cost alternative to the silicon- or glass- based MEMS technologies, for single-use disposable biosensors [12, 13]. More importantly, electroless Ni-PTFE plating techniques on the copper mold provided fast and integrated separation of the embossed structures from the mold without breakage during hot embossing process. Our UV-bond microfluidic chip, which consists of conjugate pad and absorbent pad, is a surface driven capillary flow device. It allows for the development of disposable and portable diagnostics with the benefits of lateral flow assay, eliminating the need for external pumps and controls. UV irradiation provided long-term hydrophilicity on the PMMA channel surface and enable strong capillary flow. Moreover, we present microfluidic assay format which can successfully be used to produce a functional low-cost diagnostic device to be used in resource-limited areas. Magnetic beads have been used in immunoassays and in various reactions involving enzymes, proteins, and DNA for magnetically controlled transport [14]. Magnetic beads were used as capture probes to improve the limit of detection due to increased capture efficiency. The ability to separate them from a reaction mixture by an external magnetic field, increased surface area and ability to be transported in biological systems and reacting medium is an advantage over conventional support systems.

2. Device Fabrication 2.1. Channel Design Our system consists of self-priming microfluidics with sealed conjugate pads of reagent delivery and an absorbent pad for additional fluid draw. The channel has a total length of 0.18 m and a width of 500 µm. The height of the channel was targeted at approximately 40 µm resulting to a volume of approximately 3.6 µl. Two inlet poles respectively allow for sample and chemiluminescence buffer additions. (Fig. 1 a)

2.2. Copper Stamp Fabrication We fabricated a copper-based stamp with raised structures for microfluidic channels and demonstrated successful replication of the stamp microstructure on poly (methyl methacrylate) (PMMA) substrates 151


[15]. The copper hot embossing masters were fabricated using photolithography and electroplating (Fig. 1 b). A copper plate with a polished mirror finish was cut into 5 cm × 5 cm squares, and cleaned with 1 % Microsoap-90 (Cole Parmer, IL, USA ) at 60 C in ultrasonic cleaner (Branson, CT, USA), rinsed with distilled water 3 times, then dried with nitrogen gas. Next, the copper plate was treated in UV Ozone cleaner (Jelight, CA, USA) for 1h, rinsed with distilled water and again, nitrogen gas dried. A negative photoresist (SU-8 2050, MicroChem, MA) was deposited onto the center of the copper square and spun for 5 seconds at 500 rpm, 100 rpm/s and then 30 s at 1000 rpm, 300 rpm/s to obtain a thickness of approximately 80 µm. The plate was then baked at 95 C on a level surface for 5 min followed by gradual cooling to 37 C. The resist was patterned by exposure to UV light (Karl SUSS MA6 Mask Aligner, SUSS MicroTec) through a 365 nm long pass filter. A post-exposure bake for 6 min at 95 C was followed by developing for 3 min in SU-8 developer with mild agitation, then rinsed with isoproponal alcohol (Fisher Scientific, USA) and dried with nitrogen gas.

Fig. 1. (a) Demonstration of PMMA chip fabrication through hot embossing, laser ablation, and UV-assisted thermal bonding (b) Device fabrication. Copper Master Stamp was fabricated using photolithography, electroplating, electroless Ni-PTFE plating.

Electroplating was performed at room temperature using Acid Copper Plating solution (Caswell, NY, USA), which at a current density of 0.582 mA/mm2. Current was applied to the electroplating bath to produce approximately 80 µm tall copper structures on the exposed copper surface, resulting in the raised microchannel design. After plating, the SU-8 layer was stripped off the copper plate by immersed in 95 C deionized water for 20 min. The copper plate was then rinsed with sulfuric acid to wash off SU-8 residues, and rinsed with distilled water.

2.3. Electroless Ni-PTFE Plating of Copper Stamp A release layer was deposited on the stamp surface by electroless Ni-PTFE (EN-PTFE) plating to increase the performance of copper stamp during de-embossing. Electroless Ni Coating Kit 152


(MacDermid, CT, USA) was used to produce a composite of electroless nickel phosphorus and occlude sub-micro particles of Teflon® (polytetrafluroethylene, PTFE). EN-PTFE bath was made up with 20% by volume nickel particles (ELNIC 101-C5, MacDermid) and PTFE particles (Niklad ICE, MacDermid).

2.4. PMMA Chip Fabrication Hot embossing was used to fabricate microfeatured channel patterns on PMMA substrates using a hydraulic press with heated platens (Carver, NJ, USA). The microfeatures in the copper stamp included microchannel arrays of approximately 80 µm in depth and 400 µm in width. PMMA discs of 1.6 mm thickness were utilized as the molding substrate. Prior to hot embossing, PMMA sheets were cut into 45 mm × 50 mm squares, ultrasonicated in 15 % 2-propanol (Fisher Scientific, USA) for 15 min at 25 C, and rinsed 3 times with distilled water, then placed in 80 C vacuum oven (1k Pa) (Lab-Line, IL) for 12 hours to remove any volatiles that could result in outgassing during embossing process. The PMMA substrate was then sandwiched between the copper master and a glass plate for the duration of the embossing cycle which consisted of 16 kN applied force at 120 C. Then the copper master, PMMA and glass plate were removed and cooled to room temperature. Following the cooling process, the PMMA was removed from the copper master due to the EN-PTFE release layer. The embossed PMMA discs were then visually inspected under microscope. Inlets, outlets and chambers for conjugated pad and absorbent pad were made by laser ablation (EpilogLaser, CO, USA). In this case, the laser was used due to the thickness limitations of the copper master using photolithography. Ideally, the chamber for the conjugate and absorbent pads would have been incorporated onto the master. Vapor bonding [16] was used to bond unstructured PMMA to the embossed PMA resulting in a sealed channel. Prior to bonding, the bonding surfaces of the embossed PMMA and unstructured PMMA were UV treated for 6 min in UV/ozone cleaner. The purpose of this UV treatment was to provide a hydrophilic surface on the PMMA surface. Following UV treatment the PMMA pieces were placed in a closed chamber 3 cm above a chloroform (Fisher Scientific, USA) bath at 25 C for 3 minutes. After the pieces were removed the conjugated and absorbent pads were placed into the laser ablated chambers and the structured and unstructured PMMA pieces were pressed together. Then the combined chip was placed into the hydraulic press (Carver® Lab Press, USA). A force of 500 kN was applied with an embossing temperature of 37 °C for 4 minutes. The final chip dimensions were 4 cm × 2.7 cm × 0.3 cm (l×w×h) (Fig. 2).

3. Nucleic Acid Detection We used a yeast rRNA sequence as the target of the chemiluminescent assay (Table 1). In order to characterize the assay, a synthetic DNA sequence was used which was truncated (Eurofins mwg operon, AL, USA). The HRP-streptavidin (Streptavidin-Horseradish Peroxidase Conjugate, Thermo Scientific, USA) was directly conjugated to a biotin-modified nucleic acid reporter probe. The biotinmodified capture probe was immobilized to streptavidin-coated super paramagnetic beads (Dynabeads® MyOneTM Streptavidin T1, Invitrogen Dynal AS, Oslo, Norway).

153


Fig. 2. (a) Photo of a PMMA chip with conjugated pad and absorbent pad compared with one cent; (b) Photo of empty PMMA chip holder; (c) PMMA chips in a holder and holder cover; (d) PMMA chip in a holder ready for microchip reading. Table 1. Nucleic acid sequence. Probe Capture Reporter Target

Sequence (5’ – 3’) Biotin- GGAGGGCAAGTCTGGT Biotin- AAGACTTGCCCTCC CAGGTCTGTGATGCCCAAAAAAAAAAAGGAGGGCAAGTCTGGT

3.1. Pretreatment of Conjugate Pad Eighty microliters of 1  10-3 g/ml HRP-StAv and 100 µl of 20 µM reporter probe was mixed and incubated at room temperature with agitation for 45 minutes, and then blocked with 18 µl 10 % BSA buffer for 20 min with mild agitation. Then the solution was then mixed with 90 µl of 12 % sucrose solution. The solution was applied to three pieces of 10 mm  16 mm conjugate pad (Conjugate Pad Grade 8975, PALL® Life Sciences, USA) which were then dried for over 24 hours at room temperature. The dried conjugate pads were cut into 2 mm  8 mm pieces for use in chip fabrication. Fifteen microliters of the streptavidin-coated magnetic beads was washed following the manufacturer’s instructions, and resuspended to 30 µl with PBS buffer. The beads were then mixed with 30 µl 2 µM capture probe and incubated at room temperature with agitation for 45 minutes. The magnetic beads were then washed with PBS buffer 3 times and resuspended to 60µl with PBS buffer.

3.2. Chemiluminescence Assay Detection The sample solution consisted of 10 µl of target DNA and 10 µl of the magnetic capture beads. Ideally, this would represent the product of isolation from a lysed yeast sample. Samples containing 0, 25, 100, 300 and 600 fmol of target nucleic acid were tested. The sample solution was prepared by hybridizing 10 µl of target with 10 µl of the magnetic beads for 20 minutes. The sample was added to the 154


microfluidic chip through a sample port and allowed to wick into the conjugate pad. The sample immediately hydrated the conjugate pad and released the reporter probe allowing hybridization to begin. The solution continued to flow through the hydrophilic microchannel towards the absorbent pad at a controlled rate. In order to allow flow to continue, 20 µl of PBS buffer was added into the chip which also served as a washing step. In the conjugate chamber, the hybridization of the probes formed a reporter-target-capture sandwich complex and continued to flow towards the capture zone under the capillary forces of the absorbent pad. With the aid of a magnet in the capture zone, the beads (with or without bound target) were localized onto the surface of microchannels. After the beads were captured and rinsed in the chamber (approximately 10 minutes) a luminol–H2O2 solution (SuperSignal® West Pico Chemiluminescent Substrate, Thermo Scientific, USA) was added to the inlet port and allowed to flow toward the capture zone. An imaging station (Kodak, Rochester, NY) and multimode microplate reader (Biotek® Synergy 2, USA) were used for detection and binding confirmation.

4. Results and Discussion 4.1. Fabrication of PMMA Microfluidic Chip by EN-PTFE Plated Copper Molding Our fabrication method offered a robust and fast way for low-cost thermoplastic microfluidic chip development, including EN-PTFE plating on copper master mold and solvent bonding with increased accuracy of replication process.

4.1.1. Characterization of EN-PTFE Plating The copper mold surfaces with varying concentrations of EN-PTFE were examined by atomic force microscopy (AFM), scanning electron microscopy (SEM), and water contact angle. Atomic force microscopy (AFM) was used to quantify surface roughness, and the results showed that PTFE particles would make the surface rougher (Veeco Corp., Santa Barbara, Calif., U.S.A.). The samples surfaces with PTFE particles were imaged on a JOEL 6320FXV field emission scanning electron microscope (SEM) (Peabody, Mass., U.S.A.) (Fig. 3). The most useful method for characterizing hydrophobicity (that is, wettability) on solid surfaces is contact angle measurement [16-18]. Large contact angles indicate highly hydrophobic surface, which suggest high PTFE concentration on sample surface. Contact Angle θ of pure Ni plating, plain copper, EN-24% PTFE plating, and EN-50 % PTFE plating (3 measurements on each of samples) were measured on a DSA100 (Kruss, Hamburg, Germany) as 33.41°±0.25, 40.77°±0.93, 97.76°±0.24 and 101.64°±0.28 respectively (Values are means of n = 3 determinations ± standard deviations). The contact angles for the water increased from approximately 40° (hydrophilic) to approximately 101° (hydrophobic) in proportion to the plated PTFE concentration. Contact angle result showed that electroless plating with 24 % and 50 % PTFE both increased the hydrophobicity of surfaces compared to plain copper plate.

4.2. Nucleic Acid Detection via Chemiluminescence 4.2.1. Visual Inspection Images from the Kodak imaging station were able to detect the chemiluminescent reaction. The absorbent pad which contained the unbound HRP reporter complex as well as the flowing luminolH2O2 displayed a high degree of chemiluminescence as expected (Fig. 4). In the figure, the reaction in the detection chamber can also be seen. 155


Fig. 3. SEM Examination of copper stamp surfaces (a) pure nickel plating; (b) EN-24 %PTFE (c) EN-48%PTFE.

Fig. 4. Photos of chemiluminescence signals (a) Sample solution contains magnetic beads and PBS; (b) Sample solution contains magnetic beads and target nucleic acid.

4.2.2. Microreader Detection A plate holder with four PMMA chips was placed into the multimode microplate reader for 20 minutes following the luminol/H2O2 addition. Samples containing 0, 25, 100, 300 and 600 fmol of target nucleic acid were tested. The chemiluminescent reaction measured over 20 minutes (Fig. 5a) displayed higher peaks with increasing target concentrations. These signals were integrated and plotted for a dose response curve (Fig. 5b). Using the 0+3SD calculation the limit of detection was estimated to be approximately 1 fmol.

5. Conclusions We have demonstrated a microfluidic platform which requires no pumps and contains many of the necessary reagents within the device. We have also introduced a method to improve the de-embossing issues associated with hot embossing of PMMA. By using a PTFE coated embossing master, we eliminate the need for applied release layer which could result in substrate contamination. Within the PMMA microchannels, fluid transport was achieved through capillarity flow generated by the UV surface modification and the application of an absorbent pad. 156


Fig. 5. Results from chemiluminescent assays. (a) A comparison of the chemiluminescent signals over time between the 0 fmol control (dotted lines) and 25 fmols (solid lines). (b) A dose response curve was constructed by integrating the chemiluminescent signals over time. The data represent a minimum of three replicates with the error bars representing the standard deviation.

The microfluidic platform is a hybrid of traditional microfluidics and lateral flow assays. Using the advantage of both methods will allow the development of an assay for resource-limited areas. Although our assay, required the use of a photomultiplier tube, the transduction method could easily be converted to electrochemical which would allow a more portable device.

Acknowledgments The authors would like to acknowledge USDA special grant #2010-34637-20985 for financial support. This project was supported in part by the UMass Amherst Center for Hierarchical Manufacturing, a nanoscience shared facility funded by the National Science Foundation under NSF Grant #CMMI1025020.

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[10].T. G. Henares, F. Mizutani, H. Hisamoto, Current development in microfluidic immunosensing chip, Analytica Chimica Acta, Vol. 611, 2008, pp. 17-30. [11].R. C. R. Wootton, A. J. de Mello, Microfluidics: Exploiting elephants in the room, Nature, Vol. 464, 2010, pp. 839–840. [12].A. Mathur, S. S. Roy, M. Tweedie, S. Mukhopadhyay, S. K. Mitra, J. A. McLaughlin, Characterization of PMMA microfluidic channels and devices fabricated by hot embossing and sealed by direct bonding, Current Applied Physics, Vol. 9, 2009, pp. 1199–1202. [13].S. R. Nugen, P. J. Asiello, J. T. Connelly, A. J. Baeumner, PMMA biosensor for nucleic acids with integrated mixer and electrochemical detection, Biosens Bioelectron, Vol. 24, No. 8, 2009, pp. 2428-33. [14].C. M. Niemeyer, Nanoparticles, Proteins, and Nucleic Acids: Biotechnology Meets Materials Science, Angew. Chem. Int. Ed., 2001, Vol. 40, pp. 4128−4148. [15].S. R. Nugen, P. J. Asiello, A. J. Beaumner, Design and fabrication of a microfluidic device for near-single cell mRNA isolation using a copper hot embossing master, Microsyst Technol, Vol. 15, 2009, pp. 477-483. [16].I. R. G. Ogilvie, V. J. Sieben, C. F. A. Floquet, R. Zmijan, M. C. Mowlem, H. Morgan, Reduction of surface roughness for optical quality microfluidic devices in PMMA and COC, J. Micromech. Microeng, Vol. 20, Issue 6, 2010, Article Number 065016. [17].L. Gao, T. J. McCarthy, Wetting 101 Degrees, Langmuir, Vol. 25, 2009, pp. 14105–14115. [18].M. Morra, E. Occhiello, F. Garbassi, Knowledge about polymer surfaces from contact-angle measurements, Adv. Colloid Interface Sci., Vol. 32, 1990, pp. 79-116. [19].M. Strobel, C. S. Lyons, An Essay on Contact Angle Measurements, Plasma Processes and Polymers, Vol. 8, 2011, pp. 8–13. ___________________ 2011 Copyright ©, International Frequency Sensor Association (IFSA). All rights reserved. (http://www.sensorsportal.com)

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