![There, too, the gentle, great black ox will stand:
Folk say he knelt at night in strawy stall;
Perchance he knows these kings from Eastern land,
For now he lifts his head with lowing call!
[1] Weihnachten—Christmas
[2] In many parts of Southern Germany it is a custom to place on
the outer door the initials of the three kings—C. M. B.
[3] Esel—German for “donkey,”](https://image.slidesharecdn.com/2159032-250610162101-f2b5af9a/85/Microfluidic-Diagnostics-Methods-And-Protocols-1st-Edition-Lawrence-Kulinsky-56-320.jpg)
Microfluidic Diagnostics Methods And Protocols 1st Edition Lawrence Kulinsky
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- 6. ME T H O D S I N MO L E C U L A R BI O L O G Y ™ Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651
- 8. Microfluidic Diagnostics Methods and Protocols Edited by Gareth Jenkins KeyLaboratoryforOrganicElectronicsandInformationDisplays(KLOEID),NanjingUniversity ofTechnology,InstituteofAdvancedMaterials(IAM),Jiangsu,China Colin D. Mansfield Scientific and Medical Communications Consultant, Lyon, France
- 9. Editors Gareth Jenkins Institute of Advanced Materials (IAM) Key Laboratory for Organic Electronics and Information Displays (KLOEID) Nanjing University of Technology Jiangsu, China Colin D. Mansfield Scientific and Medical Communications Consultant Lyon, France ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-62703-133-2 ISBN 978-1-62703-134-9 (eBook) DOI 10.1007/978-1-62703-134-9 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2012951427 © Springer Science+Business Media, LLC 2013 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is a brand of Springer Springer is part of Springer Science+Business Media (www.springer.com)
- 10. v Preface Microfluidic techniques are becoming widely incorporated into medical diagnostic systems due to the inherent advantages of miniaturization. In particular, the application of microfluidics to point-of-care testing (POCT) devices and high-throughput screening is predicted to become increasingly important, and consequently, the interest in microfluidic diagnostics is rapidly growing. The inherent advantages of scaling down include increased speed, efficiency, a reduction in the demand for sample and reagents, and the potential for multiplexing and parallelization. Other often cited advantages, yet by no means universally achievable, include increased portability, lower device costs (through mass production), and more highly integrated and automated systems leading to powerful yet easy to use devices. Such potential has led to widespread predictions that such technologies will help revolutionize health care provision at a particularly timely moment. When faced with the multiple challenges of increased costs, an aging population, bringing health care to devel- oping countries, and the need to shift the business models of pharmaceutical companies away from palliative care to more responsive and personalized therapeutics, it is easy to see that microfluidic diagnostics is well placed to take a centrally important role. The sheer number of different methods and applications available has, however, led to a diffuse and fragmented field with little standardization. From a practical and commercial perspective, microfluidic diagnostics have not yet had as much of an impact in “real-world” applications as had been widely predicted although steady progress has been made. This may be partially attributed to the difficulty in translating academic research into practical solutions. In particular, the highly interdisciplinary nature of the field can be daunting to new researchers, especially those coming from more established and well-defined disciplines who seek to apply the benefits of microfluidics to their own work. Many challenges are faced in order to convert promising concepts from the lab bench through to practical and commercially viable devices. As well as technological challenges, regulatory hurdles and issues relating to intellectual property (IP) and other commercial concerns further compli- cate the routes for technology transfer. This book seeks to partly address some of these problems by providing a set of proto- cols necessary for the development of a variety of microfluidic diagnostic technologies. It pulls together a range of methods from leading researchers in the field, covering subjects such as microfluidic device fabrication, on-chip sample preparation, diagnostic applications and detection methodologies. The protocols described range from cutting-edge develop- ments to established techniques and basic demonstrations suitable for education and train- ing; from basic fabrication methods to commercializing research. What you need to know and how to do it: each protocol offers step-by-step instruc- tions, including an introductory overview of the technique, a list of materials and reagents required, as well as helpful tips and troubleshooting advice. Insightful reviews along with advice on how to successfully develop and commercialize microfluidic diagnostic technolo- gies makes this volume indispensable reading for scientists entering the field as well as pro- viding a reference text for those already established. Due to the multidisciplinary nature of
- 11. vi the field, little background knowledge is assumed, providing an accessible text for scientists from a range of disciplines including biomedical researchers, engineers, biochemists, and clinicians. This book is organized into three parts: “Microfluidic Diagnostics: From the Classroom to the Boardroom” contains a number of protocols suitable for the educational demonstra- tion of microfluidic techniques, as well as chapters relating to commercialization issues, such as the microfluidic device market, patent filing, and regulatory affairs. In addition, the opening chapter provides an overview of present technology and future trends in point-of- care microfluidic diagnostics. “Fabrication and Manipulation Protocols” contains a number of protocol and review chapters detailing various microfluidic fabrication methods for the manipulation of fluidic samples on the microscale. “Application Protocols” contains proto- cols and reviews for various applications of microfluidic diagnostics and a range of detection methodologies. In preparing this book we would first and foremost like to express our gratitude to all the authors whose hard work and excellent contributions will, we hope, form a useful and informative text for many other researchers in the field. We appreciate their time and espe- cially their patience during a long and arduous review process. Dr Jenkins would like to express his thanks to his friends and colleagues at Imperial College London, in Nanjing, and at Xiamen University and also to the members of the European Consortium of Microfluidics (hosted by the Centre for Business Innovation) for many useful discussions during the preparation of this work. He would also like to acknowledge financial support from the UK Department for Business Innovation and Skills and from the State Key Laboratory of Physical Chemistry of Solid Surfaces at Xiamen University, China for the support of his UK-China Fellowship. Special thanks are due to his wife and newborn son whose support has been unending and indispensable throughout. Dr Mansfield would like to thank the series editor, John Walker, for inviting him to participate in this project, as well as for his guidance during preparation of the book. He would especially like to express gratitude to his wife, Fidji, and sons, James and Ryan, for their support and patience while he spent countless weekends and evenings away from them working on this book. Jiangsu, China Gareth Jenkins Lyon, France Colin D. Mansfield Preface
- 12. vii Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi SECTION I MICROFLUIDIC DIAGNOSTICS: FROM THE CLASSROOM TO THE BOARDROOM 1 Present Technology and Future Trends in Point-of-Care Microfluidic Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Lawrence Kulinsky, Zahra Noroozi, and Marc Madou 2 Teaching Microfluidic Diagnostics Using Jell-O® Chips . . . . . . . . . . . . . . . . . . . . . 25 Cheng Wei T. Yang and Eric T. Lagally 3 Fundamentals of Microfluidics for High School Students with No Prior Knowledge of Fluid Mechanics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Vishal Tandon and Walter Peck 4 Measuring Microchannel Electroosmotic Mobility and Zeta Potential by the Current Monitoring Method. . . . . . . . . . . . . . . . . . . . . . 55 Chenren Shao and Don L. DeVoe 5 Overview of the Microfluidic Diagnostics Commercial Landscape. . . . . . . . . . . . . . 65 Lily Kim 6 Practical Aspects of the Preparation and Filing of Patent Applications . . . . . . . . . . . 85 Fiona Bessoth 7 Introduction to In Vitro Diagnostic Device Regulatory Requirements . . . . . . . . . . 103 Jonathan Day SECTION II MICROFLUIDIC DIAGNOSTICS: FABRICATION AND MANIPULATION PROTOCOLS 8 Microfluidic Device Fabrication by Thermoplastic Hot-Embossing . . . . . . . . . . . . . 115 Shuang Yang and Don L. DeVoe 9 Introduction to Glass Microstructuring Techniques . . . . . . . . . . . . . . . . . . . . . . . . 125 Radoslaw Mazurczyk and Colin D. Mansfield 10 Glass Microstructure Capping and Bonding Techniques . . . . . . . . . . . . . . . . . . . . . 141 Radoslaw Mazurczyk, Colin D. Mansfield, and Marcin Lygan 11 Rapid Prototyping of PDMS Devices Using SU-8 Lithography. . . . . . . . . . . . . . . . 153 Gareth Jenkins 12 Microfluidic Interface Technology Based on Stereolithography for Glass-Based Lab-on-a-Chips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 Song-I Han and Ki-Ho Han 13 Three-Dimensional, Paper-Based Microfluidic Devices Containing Internal Timers for Running Time-Based Diagnostic Assays . . . . . . . . . 185 Scott T. Phillips and Nicole K. Thom
- 13. viii Contents 14 Thread Based Devices for Low-Cost Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . 197 Meital Reches 15 Droplet-Based Microfluidics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 Sanjiv Sharma, Monpichar Srisa-Art, Steven Scott, Amit Asthana, and Anthony Cass 16 Droplet-Based Microfluidics for Binding Assays and Kinetics Based on FRET . . . . . 231 Monpichar Srisa-Art and Sanjiv Sharma 17 Surface Treatments for Microfluidic Biocompatibility . . . . . . . . . . . . . . . . . . . . . . . 241 N.J. Shirtcliffe, R. Toon, and P. Roach 18 Superhydrophobicity for Antifouling Microfluidic Surfaces . . . . . . . . . . . . . . . . . . . 269 N.J. Shirtcliffe and P. Roach SECTION III MICROFLUIDIC DIAGNOSTICS: APPLICATION PROTOCOLS 19 The Application of Microfluidic Devices for Viral Diagnosis in Developing Countries. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 Samantha M. Hattersley, John Greenman, and Stephen J. Haswell 20 Applications of Microfluidics for Molecular Diagnostics . . . . . . . . . . . . . . . . . . . . . 305 Harikrishnan Jayamohan, Himanshu J. Sant, and Bruce K. Gale 21 Quantitative Heterogeneous Immunoassays in Protein Modified Polydimethylsiloxane Microfluidic Channels for Rapid Detection of Disease Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335 Peng Li 22 Breast Cancer Diagnostics Using Microfluidic Multiplexed Immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349 Minseok S. Kim, Seyong Kwon, and Je-Kyun Park 23 Charged-Coupled Device (CCD) Detectors for Lab-on-a Chip (LOC) Optical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365 Avraham Rasooly, Yordan Kostov, and Hugh A. Bruck 24 Multilayer Microfluidic Poly(Ethylene Glycol) Diacrylate Hydrogels. . . . . . . . . . . . 387 Michael P. Cuchiara and Jennifer L. West 25 Purification of DNA/RNA in a Microfluidic Device . . . . . . . . . . . . . . . . . . . . . . . . 403 Andy Fan, Samantha Byrnes, and Catherine Klapperich 26 Agarose Droplet Microfluidics for Highly Parallel and Efficient Single Molecule Emulsion PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413 Xuefei Leng and Chaoyong James Yang 27 Integrated Fluidic Circuits (IFCs) for Digital PCR . . . . . . . . . . . . . . . . . . . . . . . . . 423 Ramesh Ramakrishnan, Jian Qin, Robert C. Jones, and L. Suzanne Weaver 28 microFIND® Approach to Fluorescent in Situ Hybridization (FISH) . . . . . . . . . . . 433 Andrea Zanardi, Emanuele Barborini, and Roberta Carbone 29 An ELISA Lab-on-a-Chip (ELISA-LOC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451 Avraham Rasooly, Hugh A. Bruck, and Yordan Kostov 30 Multiplexed Surface Plasmon Resonance Imaging for Protein Biomarker Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473 Eric Ouellet, Louise Lund, and Eric T. Lagally
- 14. ix Contents 31 Surface Acoustic Wave (SAW) Biosensors: Coupling of Sensing Layers and Measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491 Kerstin Länge, Friederike J. Gruhl, and Michael Rapp 32 Microchip UV Absorbance Detection Applied to Isoelectric Focusing of Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 507 Junjie Ou and Carolyn L. Ren Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 523
- 16. xi Contributors AMIT ASTHANA • Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Hyderabad, Andhra Pradesh, India EMANUELE BARBORINI • Tethis SPA, Milan, Italy FIONA BESSOTH • Patent Attorneys Ter Meer Steinmeister & Partner, Munich, Germany HUGH A. BRUCK • Department of Mechanical Engineering, University of Maryland, College Park, MD, USA SAMANTHA BYRNES • Department of Biomedical Engineering, Boston University, Boston, MA, USA ROBERTA CARBONE • Tethis SPA, Milan, Italy ANTHONY CASS • Institute of Biomedical Engineering & Department of Chemistry, Imperial College, London, UK MICHAEL P. CUCHIARA • Department of Bioengineering, MS-142, BRC, Rice University, Houston, TX, USA JONATHAN DAY • DNA Electronics Ltd, Institute of Biomedical Engineering, Imperial College, London, UK DON L. DEVOE • Department of Mechanical Engineering, University of Maryland, College Park, MD, USA ANDY FAN • Department of Biomedical Engineering, Boston University, Boston, MA, USA BRUCE K. GALE • Department of Mechanical Engineering, State of Utah Center of Excellence for Biomedical Microfluidics, University of Utah, Salt Lake City, UT, USA JOHN GREENMAN • Postgraduate Medical Institute, University of Hull, Hull, UK FRIEDERIKE J. GRUHL • Karlsruhe Institute of Technology (KIT), Institute for Microstructure Technology (IMT), Eggenstein-Leopoldshafen, Germany SONG-I HAN • School of Nano Engineering, Inje University, Gimhae, Republic of Korea KI-HO HAN • School of Nano Engineering, Inje University, Gimhae, Republic of Korea STEPHEN J. HASWELL • Department of Chemistry, University of Hull, Hull, UK SAMANTHA M. HATTERSLEY • Postgraduate Medical Institute, University of Hull, Hull, UK HARIKRISHNAN JAYAMOHAN • Department of Mechanical Engineering, State of Utah Center of Excellence for Biomedical Microfluidics, University of Utah, Salt Lake City, UT, USA GARETH JENKINS • Institute of Advanced Materials (IAM), Key Laboratory for Organic Electronics and Information Displays, Nanjing University of Posts and Telecommunication, China; Institute of Advanced Materials, Nanjing University of Technology, China; Institute of Biomedical Engineering, Imperial College, London, UK ROBERT C. JONES • Fluidigm Corporation, South San Francisco, CA, USA LILY KIM • Wyss Institute of Biologically Inspired Engineering at Harvard University, Brookline, MA, USA MINSEOK S. KIM • Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology (KAIST), Yuseong-gu, Daejeon, Republic of Korea CATHERINE KLAPPERICH • Department of Biomedical Engineering, Boston University, Boston, MA, USA
- 17. xii Contributors YORDAN KOSTOV • Steven Sun Division of Biology Office of Science and Engineering, FDA Center for Devices and Radiological Health (CDRH), Silver Spring, MD, USA; University of Maryland Baltimore County, Baltimore County, MD, USA LAWRENCE KULINSKY • Department of Mechanical and Aerospace Engineering, University of California, Irvine, CA, USA SEYONG KWON • Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology (KAIST), Yuseong-gu, Daejeon, Republic of Korea ERIC T. LAGALLY • Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada; Department of Chemical and Biological Engineering, University of British Columbia, Vancouver, BC, Canada KERSTIN LÄNGE • Karlsruhe Institute of Technology (KIT), Institute for Microstructure Technology (IMT), Eggenstein-Leopoldshafen, Germany XUEFEI LENG • Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, People’s Republic of China PENG LI • Department of Mechanical, Industrial and Systems Engineering, University of Rhode Island, Kingston, RI, USA LOUISE LUND • Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada; Center for High Throughput Biology, University of British Columbia, Vancouver, BC, Canada MARCIN LYGAN • Institut des Nanotechnologies de Lyon (INL), Ecully Cedex, France MARC MADOU • Department of Mechanical and Aerospace Engineering, University of California, Irvine, CA, USA; Department of Biomedical Engineering, University of California, Irvine, CA, USA; Ulsan National Institute of Science and Technology (UNIST), Ulsan, South Korea COLIN D. MANSFIELD • Institut des Nanotechnologies de Lyon (INL), UMR CNRS, Ecully Cedex, France RADOSLAW MAZURCZYK • Institut des Nanotechnologies de Lyon (INL), UMR CNRS 5270, Ecully Cedex, France ZAHRA NOROOZI • Department of Mechanical and Aerospace Engineering, University of California, Irvine, CA, USA JUNJIE OU • Department of Mechanical and Mechatronics Engineering, University of Waterloo, Waterloo, ON, Canada ERIC OUELLET • Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada; Department of Chemical and Biological Engineering, University of British Columbia, Vancouver, BC, Canada; Biomedical Engineering Program, University of British Columbia, Vancouver, BC, Canada JE-KYUN PARK • Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology (KAIST), Yuseong-gu, Daejeon, Republic of Korea; KAIST Institute for the NanoCentury, Yuseong-gu, Daejeon, Republic of Korea WALTER PECK • Whitney Point High School, Whitney Point, NY, USA SCOTT T. PHILLIPS • The Pennsylvania State University, University Park, PA, USA JIAN QIN • Fluidigm Corporation, South San Francisco, CA, USA RAMESH RAMAKRISHNAN • Fluidigm Corporation, South San Francisco, CA, USA MICHAEL RAPP • Karlsruhe Institute of Technology (KIT), Institute for Microstructure Technology (IMT), Eggenstein-Leopoldshafen, Germany AVRAHAM RASOOLY • Division of Biology, Office of Science and Engineering, FDA Center for Devices and Radiological Health (CDRH), Silver Spring, MD, USA; National Cancer Institute, Rockville, MD, USA
- 18. xiii Contributors MEITAL RECHES • Institute of Chemistry and Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem, Israel CAROLYN L. REN • Department of Mechanical and Mechatronics Engineering, University of Waterloo, Waterloo, ON, Canada P. ROACH • Institute for Science and Technology in Medicine, Keele University, Staffordshire, UK HIMANSHU J. SANT • Department of Mechanical Engineering, State of Utah Center of Excellence for Biomedical Microfluidics, University of Utah, Salt Lake City, UT, USA STEVEN SCOTT • Institute of Biomedical Engineering & Department of Chemistry, Imperial College, London, UK CHENREN SHAO • Department of Mechanical Engineering, University of Maryland, College Park, MD, USA SANJIV SHARMA • Institute of Biomedical Engineering & Department of Chemistry, Imperial College, London, UK N.J. SHIRTCLIFFE • Biomimetic Materials, Hochschule Rhein-Waal, Rhine-Waal University of Applied Sciences, Kleve, Germany MONPICHAR SRISA-ART • Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand VISHAL TANDON • Department of Biomedical Engineering, Cornell University, Ithaca, NY, USA NICOLE K. THOM • The Pennsylvania State University, University Park, PA, USA R. TOON • Nemaura Pharma Limited, Loughborough, Leicestershire, UK L. SUZANNE WEAVER • Fluidigm Corporation, South San Francisco, CA, USA CHENG WEI T. YANG • Michael Smith Laboratories & Department of Chemical and Biological Engineering, University of British Columbia, Vancouver, BC, Canada JENNIFER L. WEST • Department of Bioengineering, MS-142, BRC, Rice University, Houston, TX, USA SHUANG YANG • Department of Mechanical Engineering, University of Maryland, College Park, MD, USA CHAOYONG JAMES YANG • Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, People’s Republic of China ANDREA ZANARDI • Tethis SPA, Milan, Italy
- 20. Section I Microfluidic Diagnostics: From the Classroom to the Boardroom
- 22. 3 Gareth Jenkins and Colin D. Mansfield (eds.), Microfluidic Diagnostics: Methods and Protocols, Methods in Molecular Biology, vol. 949, DOI 10.1007/978-1-62703-134-9_1, © Springer Science+Business Media, LLC 2013 Chapter 1 Present Technology and Future Trends in Point-of-Care Microfluidic Diagnostics Lawrence Kulinsky, Zahra Noroozi, and Marc Madou Abstract This work reviews present technologies and developing trends in Point-of-Care (POC) microfluidic diagnostics platforms. First, various fluidics technologies such as pressure-driven flows, capillary flows, electromagneti- cally driven flows, centrifugal fluidics, acoustically driven flows, and droplet fluidics are categorized. Then three broad categories of POC microfluidic testing devices are considered: lateral flow devices, desktop and handheld POC diagnostic platforms, and emergent molecular diagnostic POC systems. Such evolving trends as miniaturization, multiplexing, networking, new more sensitive detection schemes, and the impor- tance of sample processing are discussed. It is concluded that POC microfluidic diagnostics has a potential to improve patient treatment outcome and bring substantial savings in overall healthcare costs. Key words: Point of care, POC, POCT, Microfluidics, Diagnostics, Lateral flow, Molecular diagnostics, Immunoassay Microfluidic diagnostics had an explosive growth in the last 20 years spurred by the convergence of clinical diagnostic techniques (such as blood gas analysis, immunoassays, and molecular biology testing) and mature microfabrication technology (1) that allowed production of submillimeter-size fluidic channels and reservoirs in a variety of material systems (for example: silicon, polydimethylsi- loxane (PDMS), poly(methyl methacrylate) (PMMA), etc.). Miniaturization of a chemical lab has apparent immediate benefits: dramatically smaller amount of sample and reagents needed for the analysis; lower test costs; exponential reduction in test times due to the fact that diffusion distances in microfluidic systems are very small compared to the macroscopic lab tests; multiplexing— performing multiple types of tests from the same sample; possibil- ity of integration and automation of all process steps on the same 1. Introduction
- 23. 4 L. Kulinsky et al. platform; and development of a wide variety of Point-of-Care (POC) testing devices. It is our belief that POC diagnostic systems will revolutionize the practice of medicine and have a strong potential to dramatically reduce healthcare costs. In some cases where tests should be per- formed immediately POC testing, often referred to as POCT, is the only option—for example in critical care and operating rooms or threatening infectious diseases. In other cases, when all the required tests can be done in a physician’s office (as opposed to the patient’s sample being sent to a central laboratory for processing and then the patient visiting the doctor’s office again when test results are ready) POC testing offers the following benefits: (a) substantial savings in overall healthcare costs (as a result of reduced number of patient visits to medical offices); (b) an improvement in patient quality of life (avoidance of psychological stress caused by the uncertainty of his/her possible state of health or prognosis while waiting for results); (c) a possibility to start treatment earlier, which in some cases can affect the outcome of the treatment; and (d) a reduction of errors related to mixing up the results of various patients as compared to large test laboratories. It seems to us that one of the possible future trends will be an emerging support for POC testing by health insurance companies and government insur- ance programs (such as Medicare), which will benefit from the lowering of medical costs. Eventually some of these cost savings will be passed on to consumers in the form of lower health insur- ance premiums. Before we commence our review of the state of microfluidic POC development it is necessary to clarify the terminology that we use. We consider that POC test platforms are self-contained diag- nostic Lab-on-a-Chip (LOC) platforms (2), micro Total Analysis Systems (3), and fluidic cartridges or Lateral Flow (LF) strips (4)— all either with integrated or dedicated readout systems with foot- prints ranging from the small chip to a desktop system that can be placed in doctors’ offices, hospitals, or mobile first responders’ vehicles and designed to be operated by minimally trained person- nel. In other words, POC diagnostic devices should require mini- mal manual operations (other than sample collection) and thus should contain all the necessary reagents, and it is highly desirable that all process steps (including sample preparation and pretreat- ment) are automated and integrated within the system. It is under- standable that due to the complex processing steps not every clinical test can be performed outside of the laboratory, but we believe that whenever possible, there is a great benefit to be derived from the development of a wider array of POC tests. As the size of a system shrinks into the micrometer range, the surface area-to-volume ratio increases, and surface forces (rather than body forces such as gravitational forces that dominate physics at the everyday “macro”scale) become the most significant influence
- 24. 5 1 Future Trends in POC Microfluidic Diagnostics for operation of microdevices (5, 6). For example, capillary and electrostatic forces play dominant roles at microscopic dimensions, and there are also challenges to mix fluids in reservoirs and microfluidic channels where convection is limited and diffusion becomes a key transport mode. We will first briefly review various types of microfluidic devices according to the predominant propul- sion forces in each system (i.e., capillary-driven devices, pressure- driven devices, etc.). The details of fluidic functions (including metering, gating, separation, etc.) that control the microfluidics of each system type are not however elaborated on in this work as they are discussed in several comprehensive reviews (2, 7, 8). We can categorize microfluidic diagnostic devices into three subsets of POC testing platforms: (a) LF test devices; (b) handheld or benchtop POC testing systems for blood gas, electrolytes, blood chemistry, and detection of certain protein markers—these systems typically use cartridges, tubes, and other non-LF platforms; and (c) molecularbiology-basedPOCtesting(suchasnucleicacidtesting)— the newest and fastest-growing sector of the POC testing market. In each case we will consider the examples of POC testing devices that are available commercially and will observe some emerging technology and trends for each class of diagnostic platforms. The newly released TriMark Publications’ report (9) indicates that the global POC testing market reached $7.7 billion in 2010 and is growing at 7% per year. During 2010, 950 million POC tests were carried in US hospitals and the annual POC test number will grow to 1.5 billion by 2012. Presently, the vast majority of commercial POC testing is focused on blood glucose testing and other LF POC testing platforms. During the same period, academic research has been very active in developing other (non-capillary) fluidic plat- forms leading to a wide arsenal of fluidic handling techniques for non-LF platforms, including molecular diagnostic techniques. We will conclude our review with a summary of the observed present and evolving trends in the development and commercialization of POC microfluidic diagnostic devices. Microfluidic diagnostics uses microfluidic technologies to accomplish a predetermined set of operations (i.e., to bring the sample and reagents together, to add buffer, to implement wash, to facilitate the readout, etc.) required by the specific biochemistry of the tests and detection techniques. This section lists an arsenal of available microfluidic techniques and the next section describes commercial and developing POC diagnostic platforms followed by analyses on how microfluidic technologies are used in these tests. We catego- rize most of the microfluidic techniques according to the force 2. Microfluidic Technologies
- 25. 6 L. Kulinsky et al. employed for fluid propulsion (such as pressure-driven flow, electromagnetically driven flow, etc.). It should be noted that besides the main force used for fluid propulsion, other forces might also be utilized for specific fluidic operations on the same platform (gating, separation, etc.). For instance, while centrifugal fluidic platforms rely on a centrifugal pseudo-force as the principle means for fluid propulsion, other forces are employed as well, for exam- ple, capillary forces are used in valving, and electromagnetic forces are employed in the cell lysis process on the same platform. In addition to the classification of fluidic devices based on the pre- dominant fluid propulsion force, it is also possible to classify fluidic devices according to the type of flow employed on the platform— i.e., whether there is a continuous flow or the so-called segmented flow (where fluid is advanced in discrete packets or droplets). We will see that segmented flows (also called droplet microfluidics), an extremely important emerging technology, can be achieved on a variety of platforms, for example, centrifugal platforms, pressure- driven platforms, electromagnetically driven flows, etc. Thus, we will consider “droplet microfluidic devices” as a distinct category of microfluidic devices. When the size of the fluidic channels is reduced to hundreds of microns and below, surface forces (rather than body forces such as gravitational forces) start to dominate the behavior of fluid sys- tems. For example, aqueous solutions whisk along hydrophilic cap- illary walls (such as a piece of fleece) with the fluid advancing through the hollow capillary (or along the interfiber spaces) with- out any applied pressure. This technology is very appealing as it does not require external pumps. Capillary flow devices such as LF immunoassays or blood glucose test strips are the most successful commercial microfluidic diagnostic platforms existing today. While capillary diagnostic test devices tend to be inexpensive and widely accepted as POC testing platforms, complex tests where multiple steps such as mixing, dilutions, washing, etc. are required are difficult to adapt to them. However, as we will see later, there are some efforts under way to introduce multiplexing on capillary plat- forms. It is expected that there will be a strong continued push to increase the variety of POC tests performed using capillary fluidic diagnostic platforms. In this type of pressure-driven fluidic device, external pumps (or various ingenious built-in micro-pumps) (10, 11) are used to drive fluids (samples, reagents) through the system. This type of plat- form is very flexible as many fluidic operations such as mixing, valving, metering, separation, etc. have been developed over the years (12). Flow of liquids in microchannels is a low-Reynolds number process, with a consequence that flow is laminar and mixing between two liquid streams coming together happens by diffusion 2.1. Capillary Flow Devices 2.2. Platforms with Applied Lateral Pressure
- 26. 7 1 Future Trends in POC Microfluidic Diagnostics across the liquid/liquid interface. This process is relatively slow and many types of mixers (both active and passive) have been developed to promote more effective mixing (13, 14), including mixers designed to promote chaotic advection to create “folds” in the fluid in order to decrease the effective diffusion distances (15–17). Other fluidic operations utilize a laminar flow profile to separate and sort cells in microchannels (18, 19). The advantage of this type of platform is that a wide variety of fluidic operations are available and this platform is used extensively in academic research. Disadvantages include the presence of pumps, the need for fluidic connections to the pump, and relatively large dead volumes. This type of platform uses transverse pressure, to propel, or to stop the fluid flow. The microchannels and reservoirs are made of soft plastic that can be squeezed or pinched off by the external pressure caused by fluid flowing in the adjacent channels (see Fig. 1) or by other transverse forces. The material choice for this platform is lim- ited to soft elastomers (20). This platform makes it possible to cre- ate very large fluidic networks (21) and thus might be an attractive choice for drug screening and other high-throughput applications. On the centrifuge-based fluidics platforms (often manufactured in the shape of a disk and called Lab-on-a-CD (22)), the centrifugal pseudo-force directs the flow of the fluid from the reservoirs placed close to the center of the disk to the reservoirs located near the edge of the disk. A motor is required for the disk rotation, but there is no need for external pumps, thus no need for a fluidic con- nection between the pump and the fluidic platform. The valving on the CD platform is accomplished either based on the so-called passive valving, relying on the interplay between centrifugal forces (dependent on the rotation speed) and the capillary forces (depen- dent on channels’ material and geometry), or based on active valv- ing, where some form of external actuation is utilized (for example, the use of an infrared focused light source to melt wax plugs (23)). A wide variety of fluidic functions are available (including valving, mixing, aliquoting, blood fractionation, and cell lysis), making CD devices a very appealing sample-to-answer fluidic platform for diagnostic applications (24, 25). Electrically and magnetically driven flows are governed by such processes as electrophoresis, electro-osmosis, dielectrophoresis, electrowetting, and ferrohydrodynamics. An electro-osmotic flow is produced as a result of fixed charges present on the surface of microfluidic channel walls. These surface charges cause a charge separation within the solution near the channel walls and the for- mation of an electrical double layer (EDL). When an electric field is then applied along the length of the microchannel, the mobile charges within the EDL will be swept towards the oppositely 2.3. Platforms with Applied Transverse Pressure 2.4. Centrifugal Fluidics Platforms 2.5. Electrically and Magnetically Driven Flows
- 27. 8 L. Kulinsky et al. charged electrode, moving fluid with them. Electro-osmosis is most pronounced in channels of several hundred microns in diam- eter and smaller, since in these geometries the EDL occupies a considerable part of the channel’s cross section (26). Electrophoresis is the motion of charged molecules and particles under the influence of a spatially uniform electric field—it is used extensively to separate and purify charged macromolecules such as proteins and nucleic acids (27). Usually electrophoretic separation is accompanied by electro-osmosis, and these two processes are sum- marized under the heading of electrokinetic flow (28). One of the advantages of the electrokinetic flow is the plug-like (non-para- bolic) velocity profile that helps to avoid dispersion of analytes or reagents within the fluid. Dielectrophoresis can be used to trans- port, trap, separate, and sort different types of particles and cells based upon their polarizability in nonuniform electric fields (29). Electrowetting is a modification of the wetting properties (contact angle) of dielectric surfaces caused by an applied electric field— i.e., a hydrophobic surface can become hydrophilic and vice versa, with the effect being reversible with a change of the applied volt- age. A coordinated change in the hydrophobicity of several adja- cent pads can cause a droplet to move from one pad to another and thus, it is possible to program the movement of a fluid in a complex pattern by applying potential to (and thus changing the Fig. 1.A two-layer polydimethylsiloxane (PDMS) push-down microfluidic valve.An elastomeric membrane is formed where the flow channel is positioned orthogonal to the control chan- nel directly above. Adapted from ref. 20.
- 28. 9 1 Future Trends in POC Microfluidic Diagnostics contact angle of) individually addressable pads (30). Some of the newest fluidic devices employ ferrofluids—a suspension of mag- netic particles that is moved around by switching on and off elec- tromagnets underneath different regions of the fluidic platform. Magnetic particles drag fluid with them and thus, similarly to the electrowetting application, droplets can be moved, merged, and separated with ease (31). Surface acoustic waves (SAWs) that travel over the surface of a substrate and propel a liquid droplet in the direction of the wave propagation have been used in microfluidic applications to gener- ate, propel, mix, and break up liquid droplets (32). An attractive feature of acoustically driven flow is that in the MHz frequency range, sessile drops on a piezoelectric surface can be propelled at velocities as high as 1–10 cm/s. Enhanced mixing and micro- centrifugation can be achieved within individual droplets to sepa- rate or mix different phases on a microscale (33). However, open architecture of SAWs might not work well for molecular diagnostic POC applications as the heating step during the polymerase chain reaction (PCR) step will cause evaporation and therefore, addi- tional measures should be implemented to prevent evaporation, further complicating the setup. In chemical and biochemical processes it can be advantageous to have a fluid travel in separate packets or droplets, either as single droplets traveling on a surface or between two planes (as for example, in the electrowetting processes described above) or as droplets of one phase separated and carried by another fluid (of different phase) that can be generated on centrifugal or pressure- driven platforms. These droplets can carry samples and they can be combined with other droplets, carrying buffers, washes, reagents, etc. to implement very complex multistep chemical or biochemical processes (such as molecular diagnostics) on a com- pact platform. This is also a powerful way to multiplex the analysis as each droplet (out of virtually millions) can carry a different type of reagent—thus, for example, it would be a very flexible platform for drug screening or process optimization. The dispersion of the sample in the microchannel can be minimized if the sample is moved within a droplet. Droplets can also serve as protective vehi- cles for drug delivery (34), as microreactors where reaction speeds will be very fast due to the short diffusion distances (35), and in many other applications (36). The toolset for creating droplets, transporting droplets, combining droplets, splitting droplets, put- ting cells within droplets, or droplets within droplets has been developed on a wide variety of platforms (including pressure driven, centrifugal, electrokinetic, etc.) (37, 38). 2.6. Acoustically Driven Flows 2.7. Segmented Flow (Droplet Microfluidic1 ) Devices 1 The term “digital microfluidics” is also used.
- 29. 10 L. Kulinsky et al. Microfluidic diagnostic technologies can be separated into lab-based testing technologies/devices and POC testing platforms. Both types of platforms utilize similar microfluidic handling processes as out- lined above, but there are also significant differences. First, lab- based testing does not need to be completely integrated (i.e., sam- ple preparation steps can be performed on separate platforms and then pipetting robots can transfer metered sample between differ- ent stations) and second, there is no need to limit the overall weight/size of the testing platform and readout equipment. In contrast, POC testing platforms require a significant integration, where sample preparation steps as well as compact detection tech- nologies can present additional problems for the creation of com- pact, inexpensive, portable (or desktop) diagnostic systems. There are a number of different tests that can be conducted on POC diag- nostic platforms: blood glucose monitoring, blood gas and electro- lytes, rapid coagulation tests, drugs of abuse screening, pregnancy and fertility tests, fecal occult blood testing, hemoglobin testing, cancer marker testing, cholesterol testing, infectious disease testing, etc. We will consider these tests from the microfluidic (not biologi- cal or biochemical) perspective. Depending on the complexity of the biochemical steps, some of these tests are performed without dilution and washing steps and are adapted directly to LF plat- forms—the most widespread and commercially successful type of POC testing to date. Another type of platform for POC testing is the so-called “handheld” and “desktop” POC test stations that can perform multiple assays from the same sample (multiplex) or do quantitative automated multiple tests (such as blood gases and blood chemistry in addition to immunoassays). The final type of the POC diagnostic platform is molecular biology-based testing, which represents the fastest-growing POC testing segment, and even though there are currently very few molecular diagnostic POC tests available commercially it is a subject of very active academic interest and we should expect new products on the market in the near future. This review is not exhaustive and it does not cover some POC testing devices such as in vivo diagnostic sensors. Strip-based tests that use capillary forces are among the most ubiquitous and commercially successful POC tests. These tests can roughly be separated into two categories—qualitative tests that identify the presence or absence of a particular analyte in the sample and quantitative (or semiquantitative) tests that typically also use readout devices. An example of a qualitative LF test is the pregnancy test. A typical over-the-counter pregnancy test is an immunoassay where the presence or absence of a specific protein (such as human chorionic 3. Types of Point- of-Care Testing Platforms 3.1. Strip-Based POC Testing Platforms: Lateral Flow Tests 3.1.1. Lateral Flow POC Diagnostic Technologies
- 30. 11 1 Future Trends in POC Microfluidic Diagnostics gonadotrophin (hCG)) is detected based on the interaction of antibodies immobilized on a substrate with antigens in the sample. Figure 2 (2) presents a schematic design of one type of an LF immunoassay test that utilizes immunochromatography for the detection of a specific protein. While the sample passes over the con- jugate pad, the antigens in the analyte form complexes with mono- clonal antibodies conjugated to latex or gold nanoparticles or to an enzyme. The enzyme by reacting with multiple substrate molecules provides for optical signal amplification (absorption or fluorescence) by activating multiple dye molecules in a substrate. Capillary action continues to carry antibody–antigen pairs (with the attached tags) over the detection pad where a test line contains an array of immobi- lized polyclonal antibodies specific to the same antigen. The antigen (that is already paired with the tagged antibody) attaches itself to the immobilized polyclonal antibody, producing an antibody–antigen– antibody sandwich that is anchored on the test line and thus change of the test line can be observed directly or with fluorescent micro- scope (depending on the type of tag). The control line contains anchored immunoglobulin G (IgG) that binds to a nonspecific Fig. 2. Schematic design of a lateral flow test: (a) sample pad (sample inlet and filtering), conjugate pad (reactive agents and detection molecules), incubation and detection zone with test and control lines (analyte detection and functionality test), and final absorbent pad (liquid actuation); (b) start of assay by adding liquid sample; (c) antibodies conjugated to colored nanoparticles bind the antigen; (d) particles with antigens bind to test line (posi- tive result),particles without antigens bind to the control line (proof of validity).(Reproduced from ref. 2 with permission from RCS Publishing).
- 31. 12 L. Kulinsky et al. region of the antibodies and therefore, tagged antibodies will also be captured on the control line in a properly functioning LF immu- noassay test unit. The most popular quantitative LF test is utilized in POC sys- tems for home blood glucose level measurements. Typically, a drop of blood is placed on a test strip where capillary action carries the sample to a region of the strip where an enzyme (such as glucose oxidase, GOx) is embedded. The reaction of glucose with oxygen in the presence of GOx can be detected electrochemically in a handheld reader, e.g., by amperometric detection of the peroxide reaction product, where the current is proportional to the patient’s blood sugar level. Quantification in other LF tests is accomplished with auto- mated detection using reflectometry. For example, Metrika, Inc. (now with Bayer HealthCare) has introduced such a portable device for measurement of HbA1c that was approved by the FDA for home use (39). Similarly, the RAMP™ System (from Response Biomedical Corp.) provides a quantitative measurement of cardiac markers to assist in the diagnosis of a heart attack (40). In the latter LF device immunochromatography is used with fluorescently labeled latex particles for detection as well as for internal calibra- tion to measure the levels of Troponin I and CK-MB in a whole blood sample in less than 15 min. Evolving trends in LF testing include the use of more sensitive and selective recognition elements. Examples include: nucleic acid hybridization-based LF devices (41) or a combination of antibody– antigen recognition with nucleic acid hybridization in nucleic acid LF immunoassays (42); and utilization of advanced labels such as resonance-enhanced absorption (43), chemiluminescence (44), up-converting phosphors (45), silver-enhanced gold nanoparticle labels (46), etc. Other trends involve developing a larger number of immunoassays (47), advancing quantification of the detection (48), and quality control in manufacturing to increase reproduc- ibility of tests (49). In order to further increase the test sensitivity for LF assays some manufacturers have introduced microfabricated posts and grooves in the fluidic microchannels to better control fluid flow through the system (see Fig. 3) (50, 51). Some of the immunoassays incorporate wash steps and thus cannot be in the format of LF (immuno) assays. Implementation of more sensitive detection techniques leads to the creation of more expensive and complex automated POC desktop platforms that we consider below. In addition to the goal of developing more sensitive tests, there is another pressing need—that of developing inexpensive, easy-to- use, disposable POC diagnostic tests for the resource-limited set- tings of developing countries (52, 53). The latest area of intense research activity is in the simplification of LF devices to their bare 3.1.2. Evolving Trends in Lateral Flow Testing Platforms
- 32. 13 1 Future Trends in POC Microfluidic Diagnostics ingredients—patterned filter paper impregnated with reagents (the so-called bioactive paper (54, 55)) forms a basis of simple microfluidic paper-based analytical devices (μPADs) (56–58). This extremely simple test platform was used in proof-of-concept dem- onstration for urinalysis, for quantitative colorimetric detection, for multiplexing, and even in constructing a simple filter paper and adhesive tape-based, multilayer three-dimensional fluidic network (see Fig. 4) (59). In addition, fast prototyping and production techniques for paper-based diagnostic devices have been proposed (60). So far we have considered some fairly simple one-step assays (mostly LF immunoassays and glucose test strips) that are either qualitative and do not require a readout device or semiquantitative and only need a simple readout device (e.g., a reflectometer). Now we will discuss multistep POC tests that are somewhat more complex and require a desktop station (or, in a few cases, a handheld device) that will automate sample preprocessing, assay steps, and detection. While LF tests mostly use capillary forces for fluid transport, the handheld/desktop POC platforms employ a full range of microfluidic technologies—from centrifugal fluidics to electrokinetic flows. Many desktop POC platforms have a set of microfluidic car- tridges for various types of tests (e.g., blood gases and electrolytes, protein markers, etc.). For example, one of the most versatile plat- forms, namely, the i-STAT Analyzer (Abbott Point-of-Care) (61, 62), is a handheld device that has separate fluidic cartridges for blood gases and blood electrolytes, lactate, coagulation, hematology, and cardiac markers. Several drops of whole blood are dispensed 3.2. Automated Handheld and Desktop POC Platforms 3.2.1. Handheld and Desktop POC Diagnostic Technologies Fig. 3. An array of microfabricated features such as 50 μm posts and micromachined grooves controls fluid flow on the LF immunoassay platform. (Reproduced from ref. 50 with permission from American Association for Clinical Chemistry, Inc.).
- 33. 14 L. Kulinsky et al. into the cartridge, which is then sealed and inserted into the ana- lyzer. The self-testing and calibration routine is initiated when a barb punctures a sealed reservoir containing the calibration solu- tion, which washes over the sensor array. The fluidic control is based on an applied transverse pressure, when the analyzer device presses the internal air pouch that, in turn, displaces the calibrant into the waste reservoir and sends the blood sample into the sens- ing chamber. The diagnostic tests of the i-STAT Analyzer are per- formed via amperometric or potentiometric detection on the thin-film biosensor array. There are many other commercial hand- held and desktop analyzers on the market, but in contrast with the i-STAT device, the majority of manufacturers produce stand-alone blood gas analyzers, stand-alone immunoassay testing platforms, or two (or more) separate instruments for various types of tests. For example, the Biosite Triage® MeterPlus specializes in immuno- assay test panels for cardiac markers (63) (see Fig. 5), while Roche has a dedicated Cobas b221 POC blood analyzer and a separate handheld CoaguCheck coagulation monitor (64); Siemens pro- duces POC RapidPoint blood gas analyzers, another POC test sta- tion Stratus® CS Acute Care™ Diagnostic System for cardio markers, and yet another CLINITEK platform for urinalysis (65). Centrifugal fluidics serves as a basis for several POC diagnos- tics applications: Abaxis’ Piccolo xPress provides whole blood anal- ysis on a disk (66), Gyros’ Bioaffy CD are designed for POC diagnostics and development of immunoassays (67, 68); Samsung has developed an immunoassay from whole blood on a disk with active valving executed by laser-irradiated ferrowax microvalves (69); IMTEK’s researchers have designed an integrated disk with built-in Fig.4.Three-dimensional paper-tape stack demonstrating possibility for fluidic networks in paper-based analytical devices. (Reproduced from ref. 59 with permission from National Academy of Sciences, USA).
- 34. 15 1 Future Trends in POC Microfluidic Diagnostics total internal reflection surfaces to optimize rapid determination of alcohol level in whole blood (70). One of the attractive features of the centrifugal platform for POC testing is that the sample prepa- ration steps can be integrated seamlessly within the device—for example, whole blood fractionation can be achieved by centrifuga- tion (for example, just the plasma fraction can be selected for sub- sequent processing) (71); also efficient mechanical cell lysis has been developed on CD platform (72, 73). The field of micro Total Analysis Systems (μTAS) or LOC is quite mature with new detection techniques being developed constantly (74, 75). We envision incorporation and adaption of μTAS and LOC technologies to accelerate detection rates and sensitivities in POC diagnostic platforms, including a range of tools and processes such as employment of electrokinetic techniques (27, 76, 77), use of magnetic beads (78, 79), etc. A new generation of biosensors will be developed (80, 81) (especially those relying on electro- chemical detection, as evidenced by the success of i-STAT platform discussed above) and novel detection technologies (such as optofluidics (82, 83) and label-free detection (84)) will be imple- mented on POC testing platforms. Presently “LOC” strongly resembles “Chip on a Lab” where all of the processing steps are conducted on a small chip with tiny amounts of sample and reagent, but to achieve a good sensitivity, powerful and bulky detection devices are used—thus the overall size of the POC desktop plat- form is often dominated by the detection device. The miniaturiza- tion trend will continue, especially for detection systems, in order to decrease the footprint of the POC diagnostic platforms. The POC testing fluidics propulsion systems will become even less 3.2.2. Evolving Trends in Handheld and Desktop POC Diagnostic Platforms Fig. 5. Biosite’s POCT Desktop Platform Triage® MeterPlus with a test cartridge. (Reproduced with permission from Alere San Diego, Inc.).
- 35. 16 L. Kulinsky et al. power-demanding and multiplexing technology (such as barcodes) will become more widespread. This last set of trends is demon- strated by the recent development of an LF chip for multiplexed protein detection (85). The information collection, sharing, com- puterization of patient files, and integrity of test data are receiving an increased attention as an area for potential cost savings and improvement of health care. In order to store patient data to facili- tate POC test data sharing with health providers, and to avoid pos- sible mix-ups of the test results, almost all of the newest POC testing handheld and desktop platforms have built-in data storage, printers, and/or special barcode labels (with patient information and test data) and they have dedicated communication ports for Ethernet or wireless data transmission to Laboratory Information System (LIS) and/or Hospital Information System (HIS) (86). There are two primary reasons why molecular diagnostic detection is growing in demand despite the fact that there is an extensive list of available immunoassays. The first reason is the so-called serocon- version—a period that is needed for our body to accumulate enough of the antibodies against a specific antigen to be detectable via immunoassays. This delay (for immunoassay detection) in the case of many infectious diseases has serious consequences such as perma- nent damage or even death and the possibility that an undiagnosed virus carrier will continue to infect other people. The second reason is amplification that increases test sensitivity—nucleic acids can be amplified via PCR technology, but proteins cannot be amplified. The field of academic research in the area of molecular diag- nostics systems is too extensive to be discussed here, even superficially, and we invite interested readers to look at the recent reviews on the subject (87–90). Instead of a survey of academic research on POC molecular diagnostic systems, we will describe several molecular diagnostic platforms that are commercially avail- able or under development. These technologies use disposable units (cartridges or pouches) with pre-packed reagents for sample preparation as well as a non-disposable portable closed system for housing permanent components including electronics, pumps, and controls for processing assays. Cepheid’s GeneXpert® (91) (represented in Fig. 6) is a real-time PCR-based DNA analysis system incorporating sample preparation (DNA extraction), real-time DNA amplification, and detection. This modular system has multiple bays allowing it to simultaneously analyze multiple samples, with the degree of multiplexing limited to its five fluorescent channels. The system provides “sample-to- answer” results from an unprocessed sample in about 1 h. The dis- posable cartridge incorporates the body for housing preloaded reagents, a valve that moves the sample and reagents to the active area located in the base of the cartridge by rotating to different 3.3. Molecular Diagnostic POC Platforms 3.3.1. Molecular Diagnostic Technologies for POC Applications
- 36. 17 1 Future Trends in POC Microfluidic Diagnostics positions, and a reaction tube for performing real-time PCR (RT-PCR). The cell lysis is carried out by ultrasonic energy gener- ated by a sonic horn that acts upon the base of the cartridge where the active area is located. The extracted DNA solution is subse- quently washed, purified, and concentrated by moving through dif- ferent chambers, and then advanced to the reaction chamber where RT-PCR takes place. The platform utilizes an integratedfluorescence detection system. The final results are processed using dedicated software. A barcode system is used to store related information about each test. Other advantages of the GeneXpert® system are its small footprint and low power consumption. Iquum’s Liat (Lab-in-a-tube) System (92), composed of an ana- lyzer and a disposable tube, processes a single sample with a com- plete “sample-to-answer” time (that includes a sample preparation step) of 30–60 min. Raw sample, such as whole blood, plasma, urine, or a swab, is placed within a tube that is inserted into the analyzer where the RT-PCR process takes place. The fluidic trans- port during assay processing is directed by actuators applying a transverse pressure to collapsible compartments of the tube to dis- place liquid in the adjoining reservoir. Magnetic beads (controlled by a built-in magnet) are used to bind and transport nucleic acid of the sample. One notable advantage of the Liat tube system is that all assay steps are performed inside the tube that contains all the reagents and a sample. After the test is complete, the tube contain- ing the biohazardous waste is safely disposed of. Fig. 6. GeneXpert® platform and disposable cartridge with components such as (1) processing chambers that contain reagents, filters, and capture technologies necessary to extract, purify, and amplify target nucleic acids; (2) optical window that enables real- time optical detection; (3) reaction tube for rapid thermal cycling; and (4) valve that enables fluid transfer from chamber to chamber and may contain nucleic acid lysis and filtration components. (Reproduced with permission from Cepheid).
- 37. 18 L. Kulinsky et al. Enigma ML (93) or “minilab” is a modular multiplexed RT-PCR system for analyzing both DNA and RNA targets from different body liquids or swabs. The results are obtained in 30–45 min. The Idaho Technology’s FilmArray (94) is also a single sample fluorescence-based system for simultaneous detection of 18 viral and three bacterial pathogens. All reagents required to perform the assay are preloaded and lyophilized in a disposable pouch. No pre- cise sample metering prior to testing is required; the disposable pouch with a built-in vacuum system draws in the required volume upon injection of a sample. Cell lysis is performed using a bead beating process. The released nucleic acids are then bound to mag- netic beads and are transferred to the purification chamber where a wash buffer removes all the debris while the nucleic acids remain bound to the beads. An elution buffer separates the nucleic acids from the magnetic beads and flows them into the PCR chamber where they are amplified through a two-stage PCR process: during the first stage of PCR, the FilmArray performs a single, large-volume, multiplexed reaction; in the second stage PCR is performed in small wells, each of which contains a primer designed to detect one specific target. Image processing software is used for analysis of the fluorescence intensity in each well. Results from a raw sample are obtained in 1 h. In all four systems described here, barcode scanning is used for recording of information about the test and the pouch ID. Table 1 presents a quick comparison between the features of the four dis- cussed molecular diagnostic POC platforms. There are a growing number of companies developing POC Molecular Diagnostic Platforms with both PCR as well as RT-PCR technologies. The main difference between these two nucleic acid amplification technologies is that, even though implementation of RT-PCR is somewhat easier, at the moment there is a limit to the multiplexing capacity of solution-based RT-PCR because of the 3.3.2. Evolving Trends in POC Molecular Diagnostic Platforms Table 1 Comparison between four POC molecular diagnostic platforms Company System Hands-on time (min) Run time (min) Multiplex capacity Throughput Status Cepheid GeneXpert 2 45 5 Modular (48 samples) FDA cleared— available commercially Iquum Liat 5 30 6 One sample In development Enigma Enigma ML 1 30 6–12 Modular (six samples) In development Idaho Technology FilmArray 5 60 20+ One sample In development
- 38. 19 1 Future Trends in POC Microfluidic Diagnostics limited number of distinct types of fluorophores (five or six), while for PCR with microarrays there is almost unlimited multiplex capacity. Future developments will emphasize the adaptation of nucleic acid amplification and detection technologies demonstrated on LOC platforms (for example, digital fluidics (95), electropho- resis (96), and other technologies (97)) to POC molecular diag- nosticdevices.Weanticipatemoredevelopmentandimplementation of the isothermal amplification strategies (98) and of novel detec- tion techniques such as bioluminescence (99). It is also expected that, as molecular diagnostic technologies develop higher sensitiv- ity, much less intrusive (but also less concentrated) types of samples such as saliva, sweat, or tears will be used more often. Microfluidic diagnostics for POC testing has the potential to reduce health care costs and improve patents’ outcomes. It is likely that government as well as health insurance providers will help in the wider adaptation of POC tests, through more favorable coverage and reimbursement policies and the encouragement of widespread use in doctors’ offices. We can summarize evolving general trends in POC diagnostics as miniaturization (especially needed for detection systems), multiplexing, and networking (collection, storage, and transfer of data). Present demands are in the areas of developing POC testing platforms for resource-limited environments (53); in automation, simplification, and integration of sample preparation and preprocessing steps (100–102); and also in transfer of the wide range of tests performed in the academic setting based upon LOC formats into commercial POC diagnostic platforms. It was observed that the field of microfluidics, which initially showed a great promise to be used in a large number of unique and useful applications, has never quite found a commercially success- ful, ubiquitous “killer” application (103, 104). It is feasible that POC diagnostics might just be that “killer” application (or range of applications) for the microfluidic toolbox. Acknowledgments This work was supported by the National Science Foundation grants ECCS-0801792 and NIRT-0709085, National Institute of Health grant 1 R01 AIO89541-01, and UC Lab Fees Award 09-LR-09-117362 and sponsored by World Class University (WCU) program (R32-2008-000-20054-0) through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology. 4. Conclusions
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- 44. 25 Gareth Jenkins and Colin D. Mansfield (eds.), Microfluidic Diagnostics: Methods and Protocols, Methods in Molecular Biology, vol. 949, DOI 10.1007/978-1-62703-134-9_2, © Springer Science+Business Media, LLC 2013 Chapter 2 Teaching Microfluidic Diagnostics Using Jell-O® Chips Cheng Wei T. Yang and Eric T. Lagally Abstract Microfluidics has emerged as a versatile technology that has found many applications, including DNA chips, fuel cells, and diagnostics. As the field of microfluidic diagnostics grows, it is important to introduce the principles of this technology to young students and the general public. The objective of this project was to create a simple and effective method that could be used to teach key microfluidics concepts using easily accessible materials. Similar to the poly(dimethylsiloxane) soft lithography technique, a Jell-O® “chip” is produced by pouring a mixture of Jell-O® and gelatine solution into a mold, which is constructed using foam plate, coffee stirrers, and double-sided tape. The plate is transferred to a 4°C refrigerator for curing, and then the Jell-O® chip is peeled off for experimental demonstrations. Three types of chips have been fabricated with different molds: a JELLO mold, a Y-channel mold, and a pH-sensor mold. Using these devices, the basics of microfluidic diagnostics can be demonstrated in one or two class periods. The method described in this chapter provides teachers with a fast and inexpensive way to introduce this tech- nology, and students with a fun and hands-on way to understand the basics of microfluidic diagnostics. Key words: Microfluidics, Microfluidic diagnostics, Lab-on-a-chip, Microfluidics education, Teaching methods, Jell-O microfluidics Microfluidics is a multidisciplinary field that utilizes fundamentals of physics, biology, chemistry, and engineering to create miniatur- ized and integrated devices for various applications, including DNA chips, biological assays, and chemical synthesis (1). Because it uses small volumes of fluid samples, microfluidics has the potential to revolutionize modern biology and medicine by significantly reduc- ing costs and reaction times associated with an analysis (2). Many types of materials have been explored for creating microfluidic channels and chips. Because it is inexpensive, optically transparent, and biocompatible, poly(dimethylsiloxane) (PDMS) elastomer has been extensively used in microfluidics (3). Soft lithography is the common technique for fabricating PDMS microfluidic chips (4). 1. Introduction
- 45. 26 C.W.T. Yang and E.T. Lagally In our laboratory, PDMS soft lithography is being used to create microfluidic chips for affinity reagent isolation (Fig. 1a) and bacte- rial pathogen detection (Fig. 1b). A general workflow of the soft lithography fabrication process is presented in Fig. 2. Other extensions of microfluidics are being explored in other materials as well. For example, much effort is currently focused on producing low-cost microfluidic diagnostics for addressing the issue of global public health using both paper- and thread-based microfluidic devices (5–7). As the field of microfluidic diagnostics continues to grow and becomes an integral part of our daily lives, it is important to transfer the current research efforts and applications of this technology to young students and the general public. Recently, we have devised a set of demonstrations to illustrate the use of Jell-O® and other inexpensive materials for teaching microfluidics (8). Using these experiments, people can easily and effectively learn concepts such as microfluidic chip fabrication, lami- nar flow, dimensionless numbers, pH sensing, and diagnostics. These demonstrations can also serve as a bridge between nonscien- tists and scientists by creating a platform for discussing current microfluidics research. Moreover, these educational endeavors can help to inspire the next generation of young scientists into the field of microfluidics. This chapter describes the use of Jell-O® chips for teaching microfluidics and microfluidic diagnostics to young stu- dents and the general public. 1. Six 6 in. foam plates, round (see Note 1). 2. Several flat wooden coffee stirrers. 3. Single- and double-sided tape. 2. Materials 2.1. General Mold Construction Fig. 1. (a) Photograph of a PDMS chip for affinity reagent isolation. (b) Photograph of a PDMS chip platform for bacterial pathogen detection.
- 46. 27 2 Teaching Microfluidic Diagnostics Using Jell-O® Chips 4. Scissors. 5. Personal protective equipment (gloves, lab coat, and safety goggles). 1. Two 85 g boxes of lemon-flavored Jell-O® jelly powder (see Note 2). 2. Two 7 g pouches of unflavored (the Original) Knox® Gelatine (see Note 3). 3. Two beakers of 120 mL of purified water for dissolving Jell-O® and Knox Gelatine. 4. One metal stirrer. 5. Hot plate. 6. Six premade molds with specific patterns. 7. PAM® Original no-stick cooking spray. 8. Some tissue paper. 9. Refrigerator with temperature of 4°C. 10. Flat 5 in. aluminum pans. 11. Personal protective equipment (gloves, lab coat, and safety goggles). 2.2. General Jell-O® Chip Fabrication Fig. 2. Scheme for producing Jell-O® chips using soft lithography. (a) A negative mold is made with desired features. (b) Liquid chip material is poured onto the mold. (c) Mold with liquid material is cured. (d) Solidified chip is peeled off and (e) placed on a rigid substrate for experiments. (Reproduced from ref. 8 with permission from American Chemical Society).
- 47. 28 C.W.T. Yang and E.T. Lagally 1. Jell-O® microfluidic chips, each with a continuous channel depicting the letters “JELLO.” 2. Round drinking straws. 3. One disposable transfer pipet per Jell-O® chip. 4. Food-grade color dye, green. 5. Small vials of water with a few drops of green food coloring dye (~30 mL each). 6. Personal protective equipment (gloves, lab coat, and safety goggles). 1. Jell-O® microfluidic chips, each with a Y-shaped channel. 2. Round drinking straws. 3. Two disposable transfer pipets per Jell-O® chip. 4. Food-grade color dye, blue. 5. Small vials of clear water (~30 mL each). 6. Small vials of water with a few drops of blue food coloring dye (~30 mL each). 7. Personal protective equipment (gloves, lab coat, and safety goggles). 1. Jell-O® microfluidic chips, each with two straight channels. 2. Round drinking straws. 3. Two disposable transfer pipets per Jell-O® chip. 4. Two small pieces of acid-sensing pH paper and two small pieces of base-sensing pH paper. 5. Double-sided tape. 6. Small vial of 1 M hydrochloric acid (or cooking vinegar). 7. Small vial of 1 M sodium hydroxide (or dissolved antacid solution). 8. Personal protective equipment (gloves, lab coat, and safety goggles). In general, the molds are created using foam plates, wooden coffee stirrers, and double- and single-sided tape. The coffee stirrers are first cut into various shapes and sizes, depending on the purpose of the mold, using a pair of scissors. These pieces of coffee stirrers are taped onto a foam plate using double-sided tape to create the desired mold pattern. Single-sided tape is then adhered to the junctions of the wooden sticks to reduce the gap. Three types of 2.3. Module 1: “JELLO Chip” Demonstration 2.4. Module 2: “Y-Channel Chip” Demonstration 2.5. Module 3: “pH-Sensor Chip” Demonstration 3. Methods
- 48. 29 2 Teaching Microfluidic Diagnostics Using Jell-O® Chips molds have been constructed to illustrate the diverse concepts that can be taught using this teaching method: a “JELLO” mold, a Y-channel mold, and a pH-sensor mold. In general, the Jell-O® chips are made by pouring a liquid mixture of both Jell-O® and gelatine into the molds. These plates are left to cure in a 4°C refrig- erator for about 2 days. When ready, the Jell-O® chips are removed from the refrigerator, peeled from the molds, and placed on alumi- num dishes for demonstrations. The high sugar content from the Jell-O® and gelatine mixture provides a natural seal on the alumi- num dishes, and the strength of the seal is suitable for the low- pressure applications presented here. A general workflow for fabricating these Jell-O® chips is shown in Fig. 3. Instructors should allocate two 1-h class periods to conduct the demonstration(s). The first class period is dedicated to introducing microfluidics and soft-lithography, constructing the molds, preparing the Jell-O® and gelatine mixture, pouring the mixture into the molds, and moving the plates to the refrigerator (see Note 4). The second class period is focused on conducting the hands-on experiments, observing the microfluidics phenomena, elucidating the accompany- ing theory, and discussing some current and relevant applications. For more mature audiences (high-school, university, general public), Fig. 3. General workflow for producing Jell-O® chips using soft lithography approach. (a) Foam plate and wooden coffee stirrers are starting materials for making the mold. (b) A negative mold is made with desired features using double-sided tape. (c) Jell-O® and gelatine liquid mixture is poured onto the mold. (d) The molds with liquid material are left to cure in a 4°C refrigerator. Solidified chips are peeled off and placed on aluminum pans for experiments at room temperature (repro- duced from ref. 8 with permission from American Chemical Society).
- 49. 30 C.W.T. Yang and E.T. Lagally the learners should conduct both chip fabrication and experimentation. For younger audiences (grade-school and middle-school), the mold and the chips should be made in advance by instructors; these students would participate by manipulating the chips to form a seal on an aluminum pan, and conducting the experiments. The main learning outcomes are summarized in Table 1 below. 1. “JELLO” Chip: Cut the coffee stirrers into rectangular shapes of various lengths, according to the letters “JELLO.” Using double-sided tape, attach these small pieces of wooden sticks onto a foam plate to form a continuous channel depicting the letters “JELLO.” Use small pieces of single-sided tape to cover 3.1. General Mold Construction Table 1 A summary of learning outcomes for Module I: “JELLO Chip” demonstration, Module II: “Y-Channel Chip” demonstration, and Module III: “pH-Sensor Chip” demonstration (reproduced from ref. 8 with permission from American Chemical Society) Parameter Module I Module II Module III Target learners Grade-school science students High school science students High school science students Mold fabrication difficulty Medium Medium Easy Experimental difficulty Easy Medium Medium Learning objectives Basics of microfluidic fabrication Visualization of laminar flow Differences between acids and bases Soft lithography Differences between laminar flow and turbulent flow Fundamentals of pH sensing Concept of pressure-driven flow Significance of dimen- sionless parameters Concept of parallelization Diversity, complexity, and flexibility of designs Current microfluidic applications of laminar flow Current microfluidic parallelization applications Questions to be answered What is microfluidics and how are microfluidic chips made? Why do the two solutions not mix in this Jell-O chip? What are acids and bases? How are channels formed in microfluidic chips? What is the difference between turbulent flow and laminar flow? How can we determine the pH of solutions using pH papers? How do liquids flow in microfluidic chips? What are dimensionless numbers? What is parallelization? Can fluid be passed through the chip with only one inlet and no outlet? How can dimensionless numbers help us to build our devices? What are current microfluidic applica- tions of parallelization?
- 50. 31 2 Teaching Microfluidic Diagnostics Using Jell-O® Chips the junctions of coffee sticks to ensure a smooth overall mold surface (see Note 5). 2. Y-Channel Chip: Two pieces of coffee stirrers are needed for forming one Y-channel mold. Cut the first coffee stirrer at both ends using a pair of scissors to obtain a long rectangular-shaped wooden stick of ~3 in. long. One end of this stick should be flat (outlet) and the other end should be further cut into a dag- ger shape. Cut the second coffee stirrer to obtain two smaller rectangular-shaped sticks of the same length (~1 in. long) (see Note 6). Using double-sided tape, tape the longer stick near the bottom half of a foam plate. Similarly, tape the two smaller sticks at the dagger-shaped end of the longer stick to form a mold with the letter “Y” (see Note 5). Use small pieces of single-sided tape to cover the junctions of coffee sticks to ensure a smooth overall mold surface. 3. pH-Sensor Chip: Two pieces of coffee stirrers are required for forming the pH-sensor mold. Cut both of the coffee stirrers to obtain two long rectangular-shaped wooden sticks of the same length (~3 in. long). Using double-sided tape, attach these sticks to the middle of the foam plate at ~1 in. apart. 1. After constructing the mold plates (see Note 7), mix two pouches of Jell-O® jelly powder in 120 mL of purified water in one beaker (using a metal stirrer). Mix two pouches of Knox Gelatine powder in another beaker with the same amount of water (see Note 8). 2. Place the first beaker (containing partially dissolved Jell-O® ) on a hot plate and heat the solution to a boil (see Note 9). Remove beaker from the heat and pour the content of the sec- ond beaker (containing partially dissolved gelatine) into the first beaker. Reheat the mixture of Jell-O® and gelatine solu- tion to a boil on the hot plate, and finally remove this beaker from the heat. 3. Apply a small amount of cooking spray onto the inside rim of the foam plate (with tissue paper) to facilitate the peeling of the Jell-O® chips after curing. Pour the mixture of Jell-O® and gelatine solution into the molds (an amount that can ade- quately cover the wooden sticks). Approximately six mold plates can be filled with the amount of solution prepared. 4. Transfer the molds with liquid mixture to a 4°C refrigerator for curing (see Note 10) and cure the chips for about 2 days to obtain more robust Jell-O® chips. 5. When ready for demonstration, carefully peel the cured Jell-O® chips off of the mold. Bending the foam plate at the rim may help with the peeling process. Be careful when peeling the Jell-O® chip near the wooden sticks to prevent any tears, which can result in leakage in the chip. 3.2. General Jell-O® Chip Fabrication
- 51. 32 C.W.T. Yang and E.T. Lagally 6. Determine the side of the chip with hollow channel(s). Place this side against an aluminum pan to create a natural and revers- ible seal, and to form an enclosed channel. Make sure to elimi- nate all visible air bubbles between the chip and aluminum pan (see Note 11). 1. These instructions assume that chips with a continuous channel depicting the letters “JELLO” are cured and ready to be used for demonstration. 2. To motivate this demonstration, questions including “What is microfluidics?,” “How are microfluidic chips made?,” and “How are channels formed in microfluidic chips?” can be pre- sented to the class. It may be helpful to use Figs. 1 and 2 to facilitate the discussion. 3. After class discussion, peel the “JELLO” chips from their foam plates and place them on aluminum pans as described in Subheading 3.2, then proceed with experimental demonstra- tion. These instructions explain the procedure for working with one chip (see Note 7). 4. Using a round drinking straw, puncture an inlet hole at the tip of letter “J” and an outlet hole at the end of letter “O” with a gentle twisting motion. 5. Add a few drops of green food coloring dye into a small vial of water, and load the green water into a disposable transfer pipet. 6. By gently squeezing the pipet bulb, inject the green water into the channel via the inlet hole. The resulting fluid flow can be directly visualized without the use of any imaging apparatus. An example of the results produced is shown in Fig. 4. 7. Learning Objectives including “Basics of microfluidic fabrica- tion” and “Soft lithography” can be easily taught using the “JELLO” demonstration presented above. After conducting the hands-on experiment, the similarities and differences between 3.3. “JELLO Chip” Demonstration Fig. 4. A chip made into the letters “JELLO” and filled with green colored water demon- strates the ease of making complex patterns with the Jell-O® technique (reproduced from ref. 8 with permission from American Chemical Society).
- 52. 33 2 Teaching Microfluidic Diagnostics Using Jell-O® Chips Jell-O® chip and PDMS chip fabrication can be highlighted. For example, negative molds for PDMS chip fabrication are produced using photolithography; the PDMS pre-polymer is cured in an oven at 60°C; and the feature sizes of PDMS microfluidic chips are usually a few micrometers in width (4). However, the general fabrication concepts and PDMS soft lithography can be easily explained using this demonstration. 8. The concept of pressure-driven flow can also be explained using this demonstration. Questions including “How do liquids flow in microfluidic chips?” and “Can fluid be passed through the chip with only one inlet and no outlet?” can be used to moti- vate the class discussion. When pressure is applied to the pipet bulb, a pressure is applied to the colored water in the transfer pipet, and a fluid flow is observed. The fluid flow stops as soon as the pressure from the pipet bulb is released. In contrast, if there is only one inlet and no outlet, then the fluid cannot flow through the channel. This phenomenon occurs because the air present in the channel has no place to escape. Furthermore, if a large enough pressure is exerted on the fluid in this inlet-only system, then the reversible seal between chip and aluminum pan would break. The outlet provides a path for the air inside the channel to escape, therefore allowing the fluid to flow. 9. Finally, this demonstration can be used to illustrate the level of creativity that can occur in designing microfluidic chips. Depending on our specific needs, we can fabricate molds and chips with varying flexibility, diversity, and complexity. Currently, microfluidic chips are being designed to address specific problems in microfluidic diagnostics. 1. These instructions assume that chips with a continuous chan- nel depicting the letter “Y” are cured and ready to be used for demonstration. 2. To motivate this demonstration, pose the question “What would happen when you pour clear water and blue water in a cup?” to the class. Evidently, the students would expect mixing of the two solutions. Subsequently, pose the question “What would happen when you inject clear water and blue water in the ‘Y-channel’ chip?.” The majority of the students would most likely answer that mixing of the fluids would occur. Without revealing the answer, obtain the aluminum pans with “Y-channel” chips and proceed with experimental demonstra- tion. These instructions explain the procedure for working with one chip (see Note 7). 3. Using a round drinking straw, puncture two inlet holes at the top of letter “Y” and an outlet hole at the bottom of letter “Y” with a gentle twisting motion. 3.4. “Y-Channel Chip” Demonstration
- 53. 34 C.W.T. Yang and E.T. Lagally 4. Prepare two small vials of purified water, and add a few drops of blue food coloring dye to one of the vials. Obtain two dis- posable transfer pipets: load one pipet with blue water and the other one with clear water. 5. Simultaneously inject clear water and blue water into left chan- nel and right channel, respectively (see Note 12). The resulting laminar fluid profile can be directly visualized without the use of a microscope (see Note 13). An example of the results pro- duced is shown in Fig. 5. 6. Furthermore, the flow rate of one solution can be changed (by changing the pressure applied to the pipet bulb), and the shift- ing of the interface between clear and colored water can be observed. It is counterintuitive to see that the two fluids do not mix in the Y-channel chip, so this demonstration provides a convenient starting point for discussing the differences between turbulent flow and laminar flow. 7. Students who are more mathematically advanced can be intro- duced to dimensionless parameters or numbers for a more comprehensive understanding (see Note 14). For examples, dimensionless parameters including the Reynolds number (Re) (see Note 15) and Péclet number (Pe) (see Note 16) can be discussed and calculated in class. To gain a better understand- ing of Pe numbers, diffusion and diffusion coefficient (D) may also need to be discussed (see Note 17). 8. The significance of dimensionless numbers can also be high- lighted, because learning how to use dimensionless numbers is an important skill for scientists and engineers. For example, dominant forces in the fabricated microfluidic devices can be calculated using dimensionless numbers. Reynolds number can be used to determine whether laminar or turbulent flow dominates; Péclet number can be used to determine whether Fig. 5. (a) A Jell-O® Y-channel chip with a Reynolds number of 30.The injection of colored water to one inlet and clear water to the second results in the classic laminar flow profile, in which both streams remain separate and mix solely by diffusion along the length of the channel. (b) Diagram of laminar flow diffusive mixing occurring at the interface between two different fluids along the channel length. This phenomenon is governed by the Péclet number (reproduced from ref. 8 with permission from American Chemical Society).
- 54. 35 2 Teaching Microfluidic Diagnostics Using Jell-O® Chips convective mass transfer or diffusion dominates. Conversely, the value of a parameter can also be changed to switch between the analytical regimes, and dimensionless numbers can facili- tate in the designing of microfluidic chips. 9. In these Jell-O® Y-channel chips, reliable separation of analytes based solely on diffusion or molecular size cannot be easily achieved (Fig. 5a); however, this result can be achieved in smaller microfluidic systems (Fig. 5b). For example, diffusive mixing has been used in microfluidic T-sensors for chemical concentrations measurements (9) and for rapid determination of diffusion coefficients for molecules of varying sizes (10). 10. After discussing the theory behind the Y-channel chip, some current applications of this device can be highlighted. In addi- tion to the two T-sensor examples discussed above, laminar flow can also be used to separate the anode and cathode streams in Y-shaped fuel cells, without the use of a polymer electrolyte membrane (11–13). 1. These instructions assume that “pH-sensor” chips are cured and ready to be used for demonstration. 2. To motivate this demonstration, questions such as “What are acids and bases?” and “How can we determine the pH of solu- tions using pH papers?” can be presented to the class. Obtain aluminum pans with “pH-sensor” chips and proceed with experimental demonstration. These instructions explain the procedure for working with one chip (see Note 7). 3. Using a round drinking straw, puncture two inlet holes at the top of both channels, and two outlet holes at the bottom of both channels with a gentle twisting motion (to form two sep- arate channels). 4. Use double-sided tape to attach the small pieces of pH paper onto the aluminum pan, within the boundaries of the chan- nels. In both of the channels, tape a piece of acid-sensing pH paper near the inlet and tape a piece of base-sensing pH paper near the outlet. 5. Carefully prepare small vials of 1 M HCl and 1 M NaOH (each with volume of ~30 mL) (see Note 18). Obtain two disposable transfer pipets, and load one with NaOH and the other with HCl. 6. Simultaneously inject NaOH into the left channel and HCl into the right channel, and directly visualize the resulting color changes in the pH papers. An example of the results produced is shown in Fig. 6. 7. Alternatively, safer acidic or basic solutions, such as cooking vinegar and dissolved antacid solution, could be used for this experiment (see Note 19). 3.5. “pH-Sensor Chip” Demonstration
- 55. Another Random Document on Scribd Without Any Related Topics
- 56. There, too, the gentle, great black ox will stand: Folk say he knelt at night in strawy stall; Perchance he knows these kings from Eastern land, For now he lifts his head with lowing call! [1] Weihnachten—Christmas [2] In many parts of Southern Germany it is a custom to place on the outer door the initials of the three kings—C. M. B. [3] Esel—German for “donkey,”
- 57. MELCHIOR’S RIDE Melchior rides from door to door, Large Christmas doles he seeks; A pannier wide receives the store, Yet never a word he speaks! The nougat bells so merrily ring Yet never a note he hears; He gathers the gifts the good folk bring, And onward still he steers. The children laugh, and the children chaff, He sits so stiff and straight, And grandpère waves, with his thorn-tree staff, A greeting at the gate! Olives and almonds, and cheese and bread, And the pack on his back grows stout! Let the hungry poor to their fill be fed, While the nougat bells ring out. Thus, Melchior rides from door to door, Seeking of all his fee; And their presents into his pannier pour, Yet never a whit cares he! For a wicker-work man is Melchior droll,
- 58. A wicker-work man, and no more; But the people love him, with heart and soul, As he rides from door to door!
- 59. ONE OF THE TWELVE A Christmas Carol From the Provençal of Roumanille “Great stir among the shepherd folk; To Bethlehem they go, To worship there a God whose head On straw is laid full low; Upon the lovely newborn Child Their gifts will they bestow. “But I, who am as poor as Job— A widowed mother I, Who for my little son’s sweet sake For alms to all apply— Ah, what have I that I can take The Child of Love most high? “Thy cradle and thy pillow, too, My little lamb forlorn, Thou sorely needest them—no, no, I cannot leave thee shorn! I cannot take them to the God That in the straw was born.” Oh, miracle! The nursing babe— The babe e’en as he fed— Smiled in his tender mother’s face,
- 60. And, “Go, go quick!” he said; “To Jesus, to my Saviour, take My kisses and my bed.” The mother, all thrilled through and through, To heaven her hands did raise; She gave the babe her breast, then took The cradle—went her ways,... And now, at Bethlehem arrived, To Mary Mother says: “O Mary, Pearl of Paradise, That heaven on earth hath shed, O Virgin Mother, hear the word My little babe hath said: To Jesus, to my Saviour, take My kisses and my bed. “Here, Mary, here the cradle is; Thy need is more than mine; Receive, and in it lay thy Son, Messiah all-divine! And let me kiss, upon my knees, That darling Babe of thine!” The blessed Virgin, then, at once, Right glad of heart, bent low, And in the cradle laid her Child, And kissed him, doing so. Then with his foot St. Joseph rocked The cradle to and fro.
- 61. “Now, thanks to thee, good woman, thanks, For this that thou hast done.” Thus say they both, with friendly looks. “Of thanks I merit none; Yet, holy Mother, pity me, For sake of thy dear Son.” Since then a happy soul was hers; God’s blessing on her fell; One of the Twelve her child became, That with our Lord did dwell. Thus was this story told to me, Which I afar would tell.
- 62. THE WITCH’S CHILD ’Tis Elfinell—a witch’s child, From holy minster banned.... Again the old glad bells ring out Through all the Christmas land. No gift might she receive or give, Nor kneel to Mary’s child: She watched from far the joyous troop That past the Crib defiled; Far in the shadow of the porch, Yet even there espied: “Now, hence away, unhallowed Elf!” The sacristan did chide. “Hence, till some witness thou canst bring Of gift received from thee, In His dear name, whose birth we sing, But this shall never be!” Poor Elfinell—she turned away: “Though none for me may speak, Yet there be those may take my gift; And them I go to seek!” So, flitting light through lonesome fields
- 63. By summer long forgot, She crossed the valley drifted deep— The brook in icy grot; And gained, at last, a still, white wood All hung with flowers of snow: There, down she sat, and quaintly called In tender tones and low. They heard and came—the doe and fawn, The squirrel and the hare, And dwellers shy in earthy homes, And wanderers of the air! To these she gave fresh leaves of kale. To those the soft white bread, Or filberts smooth, or yellow corn; So each and all she fed. She fed them from her hand—she sighed; “Might you but speak for me, And say, ye took my Christmas gift, Then, I the Crib might see!” At this, those glad, wild creatures join, And close the child around; They draw her on, she scarce knows how, Across the snowy ground! They crowd with soft, warm, furry touch; They stoop with frolic wing: Grown strangely bold, to haunts of men
- 64. The elfin child they bring! They reach the town, the minster door; The door they straightway pass; And up the aisle and by the priest That saith the holy mass. Nor stay, until they reach the Crib With all its wreathen greens; And there above, with eyes of love, The witch-child looks and leans! Spake, then, the priest to all his flock: “Forbid no more this child! To speak for her, God sendeth these, His loved ones of the wild! “’Twas God that made them take her gift, Our stubborn hearts to shame! Melt, hearts of ours; and open, hands, And give in Christ’s dear name.” Thus, Elfinell with gifts was showered, Upon a Christmas Day; The while, beside the altar’s font, The ban was washed away. A carven stall the minster shows, Whereon ye see the priest priest— The kneeling child—and clustering forms Of friendly bird and beast.
- 66. BABUSHKA (A Russian Legend) Babushka sits before the fire Upon a winter’s night; The driving winds heap up the snow, Her hut is snug and tight; The howling winds,—they only make Babushka’s more bright! She hears a knocking at the door: So late—who can it be? She hastes to lift the wooden latch, No thought of fear has she; The wind-blown candle in her hand Shines out on strangers three. Their beards are white with age, and snow That in the darkness flies; Their floating locks are long and white, But kindly are their eyes That sparkle underneath their brows, Like stars in frosty skies. “Babushka, we have come from far, We tarry but to say, A little Prince is born this night, Who all the world shall sway. Come, join the search; come, go with us,
- 67. Who go our gifts to pay.” Babushka shivers at the door: “I would I might behold The little Prince who shall be King, But ah! the night is cold, The wind so fierce, the snow so deep, And I, good sirs, am old.” The strangers three, no word they speak, But fade in snowy space! Babushka sits before her fire, And dreams, with wistful face: “I would that I had questioned them, So I the way might trace! “When morning comes with blessèd light, I’ll early be awake; My staff in hand I’ll go,—perchance, Those strangers I’ll o’ertake; And, for the Child some little toys I’ll carry, for His sake.” The morning came, and, staff in hand, She wandered in the snow. She asked the way of all she met, But none the way could show. “It must be farther yet,” she sighed; “Then farther will I go.” And still, ’tis said, on Christmas Eve, When high the drifts are piled,
- 68. With staff, with basket on her arm, Babushka seeks the Child: At every door her face is seen,— Her wistful face and mild! Her gifts at every door she leaves; She bends, and murmurs low, Above each little face half-hid By pillows white as snow: “And is He here?” Then, softly sighs, “Nay, farther must I go!”
- 69. A CHRISTMAS OFFERING (Florence, Italy) I shall never forget Cimabue’s Madonna, No, nor the niche close by in the wall, Where, on the straw, the Bambino was lying, While the oxen knelt in the stall. Rude are the images, tinsel the flowers; But a tear to the eye unconsciously starts, Beholding the tribute the children have rendered, In the votive gift of “hearts”! Among them a little gold watch was hanging, That told of some sick child’s treasured wealth, Sent with a prayer that his Christmas present Might be the good gift of health!
- 70. CHRISTMAS POST In Sulz-am-Neckar, when night shuts down, And the Christmas Eve has come, All through the little snow-white town There’s a joyous stir and hum. Now here and now there, along the street, From windows wide open flung, Float childish laughter and prattle sweet In the kindly German tongue. For the happy moment at last is here, When each child a letter sends, Directed to Christkindlein dear— The Children’s Friend of Friends! Then, out at the window—strung on a thread, The precious letter is cast; Though far and high on the night wind sped, ’Twill be found and read at last! In Sulz-am-Neckar, prompt as the day, The children awake to find Among the Christmas branches gay Christkindlein’s answer kind!
- 72. THE CHRISTMAS SHEAF (Provençal) It was a gleaner in the fields,— The fields gleaned long ago: The evening wind swept down from heights Already brushed with snow. The gleaner turned to right, to left, With searching steps forlorn; The stubble-blade beneath her feet Was sharp as any thorn. But as she stooped, and as she searched, Half blind with gathering tears, Beside her in the field stood One Whose voice beguiled her fears: “What seek ye here, this bitter eve, The harvest long gone by?” She lifted up her weary face, She answered with a sigh: “I seek but some few heads of wheat To nail against the wall, To feed at morn the blessed birds, When with loud chirps they call.
- 73. “Poor ever have I been, God knows! Yet ne’er so poor before, But they might taste their glad Noël Beside my cottage door.” Then answer made that Presence sweet, “Go home, and trust right well The birds beside your cottage door Shall find their glad Noël.” And so it was—from soundest sleep The gleaner woke at morn, To see, nailed up beside her door, A sheaf of golden corn! And thereupon the birds did feast,— The birds from far and wide: All know it was Our Lord Himself That goodly sheaf supplied!
- 74. THE BIRDS ON THE CHRISTMAS SHEAF “And wherefore,” the finch to the starling said, On the Christmas sheaf, as they hungrily fed, “Wherefore do now the children of men Open their hands, when, again and again, They drove us away from their plenteous store, From the corn in the field, from the threshing-floor?” “That,” said the starling, “I’ll try to explain: They are feasting, themselves, and they spare us this grain; For oft, as they feast and make merry, they sing, ‘Peace upon earth and good will’——” “But this thing” (Said the finch), “we birds have been singing all year, Then, why not before have they shared their good cheer?”
- 75. WHAT THE PINE TREES SAID I heard the swaying pine trees speak, As I went down the glen: “Next year,” said one, “the wind shall seek, But find me not again!” “I shall go forth upon the seas, A mast, or steering-beam; On me shall breathe the tropic breeze, Above, strange stars shall gleam.” “And I—the ax shall cleave my grain, And many times divide; From my dear brood I’ll shed the rain, And roof their ingleside.” Then up and spake a slender shaft, That like an arrow grew; “No breeze my leafless stem shall waft, No ax my trunk shall hew— But though a single hour is mine, How happy shall I be! Young hearts shall leap, young eyes shall shine To greet their Christmas tree!”
- 77. TWO CHILD ANGELS Two Child Angels on Christmas Night, They stood on the brow of Heaven’s hill; The stars beneath them were glancing bright, And the air was clear and still. “That is the Earth that dazzles so— That shines with a glad and a radiant light— That is the Earth where, long ago, I was born on the Christmas Night!” Thus said the one, and the other replied, “Forever dear is the Earth in my sight; For there, full long ago, I died, On the holy Christmas Night!”
- 78. THE OLD DOLL (Just after Christmas) Little one, little one, open your arms, Now are your wishes come true, come true! Here is a love with a thousand charms, And see! she is reaching her hands out to you! Put the old doll by, asleep let her lie, And open your arms to welcome the new. Little one, little one, play your sweet part, Mother-love lavishes treasure untold. Whisper fond words, and close to your heart, Your warm little heart, the new idol enfold. (’Tis so with us all,—to worship we fall Before the new shrine, forgetting the old!) Little one, little one, wherefore that sigh? Weary of playing the long day through? But there’s something that looks like a tear in your eye, And your lips—why, your lips are quivering, too! Do I guess aright?—it is coming night, And you cry for the old—you are tired of the new? Little one, little one, old loves are best; And the heart still clings though the hands loose their hold! Take the old doll back, in your arms she shall rest,
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