NDLI: Covalent immobilisation of antibodies in Teflon-FEP microfluidic devices for the sensitive quantification of clinically relevant protein biomarkers

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Covalent immobilisation of antibodies in Teflon-FEP microfluidic devices for the sensitive quantification of clinically relevant protein biomarkers

Content Provider	Royal Society of Chemistry (RSC)
Author	Edwards, Alexander D. Pereira, Filipa M. Barbosa, Ana I. Reis, Nuno M. Pivetal, Jeremy Castanheira, Ana P.
Copyright Year	2017
Abstract	This study reports for the first time the sensitive colorimetric and fluorescence detection of clinically relevant protein biomarkers by sandwich immunoassays using the covalent immobilisation of antibodies onto the fluoropolymer surface inside Teflon®-FEP microfluidic devices. Teflon®-FEP has outstanding optical transparency ideal for high-sensitivity colorimetric and fluorescence bioassays, however this thermoplastic is regarded as chemically inert and very hydrophobic. Covalent immobilisation can offer benefits over passive adsorption to plastic surfaces by allowing better control over antibody density, orientation and analyte binding capacity, and so we tested a range of different and novel covalent immobilisation strategies. We first functionalised the inner surface of a 10-bore, 200 μm internal diameter FEP microcapillary film with high-molecular weight polyvinyl alcohol (PVOH) without changing the outstanding optical transparency of the device delivered by the matched refractive index of FEP and water. Glutaraldehyde immobilisation was compared with the use of photoactivated linkers and NHS-ester crosslinkers for covalently immobilising capture antibodies onto PVOH. Three clinically relevant sandwich ELISAs were tested against the cytokine IL-1β, the myocardial infarct marker cardiac troponin I (cTnI), and the chronic heart failure marker brain natriuretic peptide (BNP). Overall, glutaraldehyde immobilisation was effective for BNP assays, but yielded unacceptable background for IL-1β and cTnI assays caused by direct binding of the biotinylated detection antibody to the modified PVOH surface. We found NHS-ester groups reacted with APTES-treated PVOH coated fluoropolymers. This facilitated a novel method for capture antibody immobilisation onto fluoropolymer devices using a bifunctional NHS-maleimide crosslinker. The density of covalently immobilised capture antibodies achieved using PVOH/APTES/NHS/maleimide approached levels seen with passive adsorption, and sensitive and quantitative assay performance was achieved using this method. Overall, the PVOH coating provided an excellent surface for controlled covalent antibody immobilisation onto Teflon®-FEP for performing high-sensitivity immunoassays.
Starting Page	959
Ending Page	968
Page Count	10
File Format	HTM / HTML PDF
ISSN	00032654
Volume Number	142
Issue Number	6
Journal	The Analyst
DOI	10.1039/c6an02622b
Language	English
Publisher	Royal Society of Chemistry
Access Restriction	Open
Subject Keyword	Myocardial infarction Analyte Polytetrafluoroethylene BNP Fluorescence Glutaraldehyde Polyvinyl alcohol Heart failure Antibody Maleimide Cytokine (3-Aminopropyl)triethoxysilane N-Hydroxysuccinimide Hydrophobe Thermoplastic Protein Interleukin 1 beta Sandwich compound Adsorption Troponin I Natriuretic peptide Direct binding Micrometre Covalent bond Fluoropolymer
Content Type	Text
Resource Type	Article
Subject	Spectroscopy Environmental Chemistry Analytical Chemistry Biochemistry Electrochemistry

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1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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