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Investigating the effect of substrate materials on wearable immunoassay performance Khai Tuck Lee, Jacob W. Coffey, Kye J. Robinson, David A. Muller, Lisbeth Grondahl, Mark A. F. Kendall, Paul R. Young, and Simon Robert Corrie Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b03933 • Publication Date (Web): 22 Dec 2016 Downloaded from http://pubs.acs.org on January 5, 2017
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Langmuir
Investigating the effect of substrate materials on wearable immunoassay performance. Khai T. Lee1#, Jacob W. Coffey1#, Kye J. Robinson1, David A. Muller1,2, Lisbeth Grøndahl2, Mark A. F. Kendall1,4,5, Paul R. Young2,3,4, Simon R. Corrie1,2,4,6* #
These authors contributed equally to this work. Australian Institute for Bioengineering and Nanotechnology, Delivery of Drugs and Genes Group (D2G2), ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, The University of Queensland, St Lucia, Queensland 4072, Australia 2 School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Queensland, 4072, Australia 1
3
Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland, 4072, Australia 4
Australian Infectious Diseases Research Centre, St. Lucia, Queensland, 4067, Australia
5
Faculty of Medicine and Biomedical Sciences, Royal Brisbane and Women’s Hospital, Herston, Queensland, 4029, Australia. 6
Department of Chemical Engineering, ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash University, Clayton, Victoria, 3800, Australia
Wearable, immunoassay, polycarbonate, gold, poly(ethylene glycol), PEG, microneedle, antibody, dengue ABSTRACT: Immunoassays are ubiquitous across research and clinical laboratories, yet little attention is paid to the effect of the substrate material on the assay performance characteristics. Given the emerging interest in “wearable” immunoassay formats, investigations into substrate materials that provide an optimal mix of mechanical and bioanalytical properties is paramount. In the course of our research in developing “wearable immunoassays” which can penetrate skin to selectively capture disease antigens from the underlying blood vessels, we recently identified significant differences in immunoassay performance between gold and polycarbonate surfaces, even with a consistent surface modification procedure. We observed significant differences in PEG density, antibody immobilization, and non-specific adsorption between the two substrates. Despite a higher PEG density formed on gold-coated surfaces in comparison to amine-functionalised polycarbonate, the latter revealed a higher immobilized capture antibody density and lower non-specific adsorption, leading to improved signal-to-noise ratios and assay sensitivities. The major conclusion from this study is that in designing wearable bioassays or biosensors, the and its effect on the anti-fouling polymer layer can significantly affect the assay performance in terms of analytical specificity and sensitivity.
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INTRODUCTION Solid-phase immunoassays involving affinity-based capture/detection of a protein underpin a range of bioassays (multi-step, high-affinity, endpoint procedures; e.g. ELISA) and biosensors (real-time monitoring of antigen capture/detection; e.g. Biacore) used in life sciences research, drug discovery and development, and clinical diagnostics.1-6 While a variety of configurations have been developed based on flat surfaces,13,14 nanoparticles,7-9 porous media,10-12 etc, the substrate material is usually chosen based on the detection method employed for the assay. For example, “label-free” immunoassays using optical detection based on surfaceplasmon resonance technology are often paired with gold surfaces,15 whereas colorimetric assays are commonly performed on modified polystyrene. A range of new immunoassays and sensors are emerging for “wearable” applications,16 and it is likely that non-traditional immunoassay substrates will be required to meet the selection criteria, covering aspects of surface chemistry, mechanical suitability, and biocompatibility. Common to all assays is the need for low detection limits, high signal-to-noise ratio, and a dynamic range that is sufficient to detect the target analyte(s) in biological media. The key question is then the relative merits of different surfaces in terms of immunoassay performance. Our group is interested in developing “wearable immunoassays” that can be used to capture and detect diseaserelated proteins from the richly perfused dermal vasculature of the skin, using microprojection array (MPA) substrates manufactured from common materials. We have demonstrated that both gold-coated silicon17,18 and polycarbonate19,20 MPAs can be applied to the skin of live mice for the purposes of extracting antibodies and proteins, raised in response to vaccination17,18,21 or viral infection.19 Given that this method requires both high efficiency capture of proteins from a native biological fluid, and minimizing deposition of capture probes into the skin,22 we have developed surface modification strategies to coat the MPAs with poly(ethylene glycol) layers (PEG). Using hetero-bifunctional PEG molecules, we can immobilize capture antibodies at high density onto the MPA surfaces, while also reducing the non-specific protein adsorption during application to mouse skin, creating wearable immunoassays that can selectively accumulate proteins over 24 hours on live mice.17 Anti-fouling polymer films are commonly employed on biomedical and biosensor devices in order to reduce protein adsorption and provide attachment points for specific biomolecular probes. The most commonly employed polymer in this context is poly(ethylene glycol) (PEG). The composition of thiolated PEG monolayers formed on gold substrates, and the resulting protein resistance, has been studied in detail. A range of groups have shown that by independently varying the molecular weight and chain density of PEG layers (via solubility differences under “cloud point” conditions23), the protein resistance can be tuned accordingly.24-29 As the PEG density on a surface increases, the molecular conformation of the PEG molecules changes from the “random coil” regime (S>>2RF), through to the “mushroom” (S~2RF) and “extended chain” (S
