From Sample to Smartphone: Consumer-Operable Analytical Devices for Multiplex Allergen Detection

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From Sample to Smartphone: Consumer-Operable Analytical Devices for Multiplex Allergen Detection From sample to smartphone: consumer-operable analytical devices for multiplex allergen detection Georgina M.S. Ross Thesis committee Promotor Prof. Dr M.W.F. Nielen Special Professor Analytical Chemistry with a special emphasis on the detection of food contaminants Wageningen University & Research Co-promotor Dr G. IJ. Salentijn Assistant Professor at the Laboratory of Organic Chemistry Wageningen University & Research Other members Prof. Dr HJ. Wichers, Wageningen University & Research Prof. Dr C.T. Elliott, Queen’s University Belfast, United Kingdom Prof. Dr E.M.J. Verpoorte, University of Groningen Dr Bert Pöpping, FOCOS, GbR, Alzenau, Germany This research was conducted under the auspices of the Graduate School VLAG (Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences). From sample to smartphone: consumer-operable analytical devices for multiplex allergen detection Georgina M.S. Ross Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus, Prof. Dr A.P.J. Mol, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 12th March 2021 at 1 p.m. in the Aula. Georgina M.S. Ross From sample to smartphone: consumer-operable analytical devices for multiplex allergen detection 274 pages PhD thesis, Wageningen University, Wageningen, NL (2021) With references, with summary in English DOI: 10.18174/541188 ISBN: 978-94-6395-701-4 Science and everyday life cannot and should not be separated Rosalind Franklin Table of Contents 1 Introduction 11 Consumer-friendly food allergen detection: moving towards 39 2 smartphone-based immunoassays Rapid antibody selection using surface plasmon resonance for 97 3 high-speed and sensitive hazelnut lateral flow prototypes A critical comparison between flow-through and lateral flow immunoassay formats for visual and smartphone-based 125 4 multiplex allergen detection Interconnectable solid-liquid protein extraction unit and chip- 161 5 based dilution for multiplexed consumer immunodiagnostics Unraveling the hook effect: a comprehensive study of high antigen concentration effects in sandwich lateral flow 191 6 immunoassays 7 General discussion 229 Summary 257 Curriculum Vitae 261 Acknowledgements 265 Overview of completed training activities 271 Abbreviations (c)BCPI (corrected)Blue Channel Pixel Intensity ALARA As Low as Reasonably Achievable ASSURED Affordable, Sensitive, Specific, User-friendly, Rapid, Equipment-free, Deliverable AuNPs Gold Nanoparticles BB Borate Buffer BC Blank Cookie BCA Bicinchoninic acid assay BSA Bovine Serum Albumin CAD Computer Aided Design CCD Charge Coupled Device CIP Clean in Place CMOS Complementary Metal Oxide Semiconductor CMYK Cyan, Magenta, Yellow, Key CNP-mAb Carbon nanoparticle-labelled monoclonal antibodies CNPs Carbon Nanoparticles CPUs Central Processing Units DF Dilution Factor EC European Commission ECSA European Citizen Science Association ELISA Enzyme Linked Immunosorbent Assay EU European Union EW Evanescent Wave FARRP Food Allergy Research and Resource Program Fc-IgG Fc-receptor specific antibody FDA Food & Drug Administration FDM Fused Deposition Modelling FO Fiber Optic fps Frames per second GAMaB Goat anti-mouse antibody GPS Global Positioning System GUI Graphical User Interface HA Hazelnut Assay HC Hazelnut Cookie HPC Hazelnut, Peanut, Control; LFIA IgG Immunoglobulin LED Light Emitting Diode LFIA Lateral Flow Immunoassay LOAEL Lowest Observable Adverse Effect Limit LOC Lab on a Chip LOD Limit of Detection mAb Monoclonal Antibody MFC Miniaturized Flow Cytometry MIP Molecularly Imprinted Polymers MQ MilliQ Water NOAEL No Observable Adverse Effect Limit PA Peanut Assay pAb Polyclonal Antibody PAL Precautionary Allergen Labeling PBS Phosphate Buffered Saline PC Peanut Cookie PDMS Polydimethylsiloxane PHC Peanut, Hazelnut, Control; LFIA PoC Point of Care PPE Personal Protective Equipment ppm parts per million QD Quantum Dots RASFF Rapid Alert System Food & Feed RB Running Buffer RGB Red, Green, Blue RIU Refractive Index Unit ROI Region of Interest RSD Relative Standard Deviation RU Response Unit SB Storage Buffer SEM Scanning Electron Microscopy SLA Stereolithography SPR Surface Plasmon Resonance THP Total Hazelnut Protein TIR Total Internal Reflection TLC Thin Layer Chromatography TPP Total Peanut Protein ULOC Unibody Lab on a Chip USB Universal Serial Bus UV-VIS Ultra-Violet Visible Spectrophotometry VITAL Voluntary Incidental Trace Allergen Labeling WB Washing Buffer WHO World Health Organization µPAD Micro Paper Analytical Device CHAPTER 1 1 Introduction 12 | Chapter 1 Introduction | 13 1 Introduction Food allergies represent a significant and increasingly prevalent health concern affecting the lives of over 220 million people worldwide1,2. Allergies are typically caused by proteins that are naturally present in many foods, but they can also be towards food additives (e.g., sulphites). These so-called food allergens are capable of eliciting an adverse immune response in sensitized individuals. While people can become or sensitized, to almost any food, the majority of food allergies are caused by 14 allergens: milk, eggs, peanuts, tree- nuts, fish, shellfish, soy, wheat celery, mustard, sesame, sulphur dioxide/sulphites, lupine 1 and mollusk (see Figure 1.1). Allergic symptoms can range from mild, such as rashes, itching, stomach pains and diarrhea, to severe including shortness of breath, loss of consciousness and even fatal anaphylaxis3. The 14 Allergens Figure 1.1. Icons of the 14 regulated allergens. Under European Commission (EC) legislation (Directive 2003/89/EC), food products containing any of the 14 allergens must explicitly state so on the food packaging4. In 2014 a further amendment specified the requirement for allergen labeling of non-packaged foods containing the 14 legislated allergens. This regulatory framework safeguards individuals from exposure to known allergenic foods. However, it is the presence of undeclared allergens that have been inadvertently introduced into a food product that are the biggest risk for the allergic consumer; traces of allergens that occur due to cross-contamination are not regulated by the EU5. To prevent allergen cross-contact, manufacturers must stick to strict clean-in-place (CIP) procedures, especially when allergen-containing and allergen- 14 | Chapter 1 free foods or foods containing different allergens are manufactured/processed in the same facility6. Even with dedicated sanitation procedures, trace amounts of allergenic proteins can still infiltrate other food products. To reduce the risk of unintentional exposure to allergens, food manufacturers can voluntarily incorporate precautionary allergen labeling (PAL) on pre-packaged food (e.g., ‘May Contain X’ labels)7; but this comes with its own risks such as, consistently changing ingredient formulations8, global labeling differences9, lack of agreed regulatory threshold levels10 and inconsistent terminology/readability11 of the label12. Even with regulatory frameworks, voluntary PAL statements, mandatory allergen declarations and CIP procedures, a rising number of international food-related recalls are due to mislabeling of food products containing undeclared allergens5,13. These allergens are infiltrating our global food supply-chain, putting the allergic community at constant risk. In the past, most people trusted the food industry and governments to maintain food safety and to control for the presence of allergens, but with the increasing number of food-allergen related recalls, that perception is shifting. This growing consumer distrust is understandable, especially when government agencies responsible for public food safety (e.g., the FDA) announce temporary ingredient and food labeling alterations that leave allergic individuals inadequately protected8,12. Providing consumers with the analytical tools to test for trace allergens themselves could give them and additional layer of security and assurance that their food is safe to eat14. This would require the development of consumer-operable devices that can detect allergens in a safe and accessible way. For too long, industrial quality assurance/control procedures, labeling initiatives and governmental regulations have fell short of providing protection for food allergic individuals. As consumers become more aware of the potential risks of undeclared allergen presence, we are witnessing a paradigm shift in food allergen testing with the emergence of inexpensive, sensitive and portable citizen-science focused tests capable of on-the-go allergen detection, taking the analysis out of the lab and literally into the hands of allergic individuals. Portable analytical systems capable of executing simplified sample extraction, preparation, and multi-allergen detection, with minimal user-input will enable consumers to carry out their own allergen analysis15. The World Health Organization (WHO) outlined the necessary criteria for decentralized testing as being affordable, sensitive, specific, user-friendly, rapid, equipment-free and deliverable (ASSURED)16,17. Combining disposable, paper-based colorimetric tests, such as lateral flow immunoassays (LFIAs) that are already readable by the naked eye, with a digital device capable of recording photos or videos can empower consumers to perform their own food safety testing and automate data analysis15. Scientific developments that make on-site screening for trace allergens accessible, allowing
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