University of Rhode Island DigitalCommons@URI Open Access Master's Theses 2013 Development of Enhanced Lateral Flow test Devices for Point-of- Care Diagnostics Roman Gerbers University of Rhode Island, [email protected] Follow this and additional works at: https://digitalcommons.uri.edu/theses Recommended Citation Gerbers, Roman, "Development of Enhanced Lateral Flow test Devices for Point-of-Care Diagnostics" (2013). Open Access Master's Theses. Paper 123. https://digitalcommons.uri.edu/theses/123 This Thesis is brought to you for free and open access by DigitalCommons@URI. It has been accepted for inclusion in Open Access Master's Theses by an authorized administrator of DigitalCommons@URI. For more information, please contact [email protected]. DEVELOPMENT OF ENHANCED LATERAL FLOW TEST DEVICES FOR POINT-OF-CARE DIAGNOSTICS BY ROMAN GERBERS A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN MECHANICAL ENGINEERING UNIVERSITY OF RHODE ISLAND 2013 MASTER OF SCIENCE THESIS OF ROMAN GERBERS APPROVED: Thesis Committee: Major Professor Dr. Mohammad Faghri Dr. Constantine Anagnostopoulos Dr. Jason R. Dwyer Dr. Nasser H. Zawia DEAN OF THE GRADUATE SCHOOL UNIVERSITY OF RHODE ISLAND 2013 ABSTRACT Lateral flow Imunoassays (LFIA) are common, simple to use point-of-care devices for the diagnostic market. Conventionally LFIAs are limited in their complexity since they are optimized for minimally trained operators. Paper-based analytical devices (PAD) are advanced sensors based on a wide range of recently developed techniques for complex analytical methods. In this research, a point-of-care (POC) immunosensor was developed based on techniques adapted from lateral flow and paper-based analytical devices. Alternating layers of paper and tape were used to expand the common 2D design of lateral flow tests to 3D in order to enable complex fluid flow control. Four fluidic valves were integrated for automatic sequential loading of three different fluids to a detection area. Fabrication processes, reagent concentrations, materials and device geometries were optimized and a chip-yield of 92% was achieved. A three step alkaline phosphatase (ALP)-based enzyme-linked immunosorbent assay (ELISA) procedure with Rabitt IgG as model analyte was used to prove the working principle of the sensor. After optimization of crucial assay parameters practicability was verified by visual detection of signal development on nitrocellulose membrane after reaction of ALP and NBT/BCIP with a good detection limit of 4.8 fm. Keywords: Lateral Flow Imunoassay (LFIA) Paper-based Analytical device (PAD), Alkaline Phosphatase (ALP), Enzyme Linked Immunosorbent Assay (ELISA), Point-of- Care (POC) ACKNOWLEDGMENTS It has been a wonderful and unforgettable experience spending the exchange year on completing my Master’s thesis in the Department of Mechanical Engineering, University of Rhode Island. First of all, I would like to thank Dr. Mohammad Faghri for his continuous guidance, encouragement and support throughout the course of this research. It has been a real pleasure to work with him, on this promising field. I am grateful to Dr. Constantine Anagnostopoulos for his invaluable suggestions and continuous help. Furthermore, I would also like to thank Dr. Hong Chen, and Jeremy Cogswell. I learned a lot from them, such as sophisticated laboratory skills and the ability of developing new techniques to tackle the challenges in research. Great thanks to my lab colleague and good friend Wilke Föllscher without him it would not have been the same unforgettable experience. Special thanks to Nadine Madanchi, who has been supporting and encouraging me from the beginning till the end, especially in the time of difficulties. Finally, I want to thank my parents Kirsten Gerbers and Björn Gerbers. Without their enduring love, care and support in any situations of my life I could not have gone this path and reached the point where I am today. iii TABLE OF CONTENTS ABSTRACT ........................................................................................................................ ii ACKNOWLEDGMENTS .................................................................................................... iii TABLE OF CONTENTS ...................................................................................................... iv LIST OF TABLES ............................................................................................................. viii LIST OF FIGURES ............................................................................................................. ix LIST OF ABBREVIATIONS............................................................................................... xiii LIST OF SYMBOLS ......................................................................................................... xiv CHAPTER 1 - INTRODUCTION ......................................................................................... 1 1.1 Point of Care Diagnostics and Diagnostic Devices ......................................... 1 1.2 Justification .................................................................................................... 4 1.3 Paper Based Analytical Devices ..................................................................... 5 1.4 Immunoassays ............................................................................................... 6 1.4.1 Antibodies and Recognition Reaction ................................................ 6 1.5 Lateral Flow Test Strips .................................................................................. 9 1.5.1 Membrane ....................................................................................... 10 1.5.2 Sample Pad ...................................................................................... 12 1.5.3 Conjugate Pad .................................................................................. 14 1.5.4 Absorbent Pad ................................................................................. 16 1.5.5 Housing ............................................................................................ 16 1.6 Objective and Outline of the Thesis ............................................................ 16 CHAPTER 2 – LITERATURE REVIEW .............................................................................. 18 2.1 Analytical Methods Based on Immunoassays ............................................. 18 2.1.1 Labeling-Detection Systems ............................................................. 18 2.1.2 Immunoassay Designs ..................................................................... 21 iv 2.1.3 Surface Binding Techniques ............................................................. 23 2.1.4 Assay Validation ............................................................................... 25 2.2 Limitations of Lateral Flow Immunoassay ................................................... 31 2.3 Sensing Approaches on Paper-based Devices ............................................. 32 2.3.1 Optical Detection: Methods and Detector Systems ........................ 33 2.3.2 Electrochemical Detection: Methods and Detector Systems .......... 35 2.3.3 Energetic Principles ......................................................................... 37 2.3.4 Analytical Principles ......................................................................... 37 2.4 Fabrication Methods for PADs .................................................................... 39 2.4.1 Three-dimensional PADs .................................................................. 40 2.5 Control of Fluid Flow in Paper-based Devices ............................................. 42 2.5.1 Single-use Buttons ........................................................................... 43 2.5.2 Fluidic Timers ................................................................................... 44 2.5.3 Mechanical Switch ........................................................................... 44 2.5.4 Fluidic Valve ..................................................................................... 45 2.6 Modelling of Fluid Flow in Cellulose Substrates .......................................... 47 2.6.1 Paper Wet-out.................................................................................. 47 2.6.2 Fully wetted Flow ............................................................................. 48 2.6.3 Abundant vs. Constricted Flow ........................................................ 49 2.6.4 Electrical Circuit Analogy ................................................................. 50 CHAPTER 3 - METHODOLOGY ...................................................................................... 52 3.1 Chip Fabrication Method ............................................................................. 52 3.2 Valve Fabrication Methods .......................................................................... 53 3.2.1 Disk Punching ................................................................................... 55 3.2.2 Disk-holding Layers .......................................................................... 57 3.2.3 Surfactant Cellulose-Powder ........................................................... 59 v 3.2.4 Optimization of Surfactant .............................................................. 61 3.3 Material Selection and Processing .............................................................. 62 3.4 3D Lateral Flow Test Strip