Abstract
Introduction
For the last years, low-cost microfluidics and microarrays on polyester substrates have gained applications in a variety of fields, including analytical Point-of-Care systems. The use of a 2D platforms brings a significant reduction in assay costs, both due to a lower consumption of materials and reagents, as well as a simplicity of manufacturing process in comparison to current approaches [1]. Commercial applicability of immunoassays requires the development of efficient strategies for antibody immobilization in bulk quantities on a wide variety of readily available substrates. Other factors, which influence the usefulness of antibody immobilization approach are scalability, reproducibility and cost-efficiency. Simple, one-step methods such as passive adsorption on hydrophobic surfaces or direct protein immobilization with the use of functionalized silanes are particularly appreciated in fabrication of immunosensing platforms [2]. The use of masking properties of cured toner as an intermediate for passive and covalent immobilization of antibodies on poly(ethylene terephtalate) (PET) seems to be an attractive and still undiscovered path for development of immunoassays on versatile, printable substrates [3].
Fabrication of toner-basedplatforms for Ab-capturing and immunosensing
Throughout presented research two general approaches for immobilization of antibody and antibody-binding protein (protein A/G) were examined: 1) adsorption on pristine PET@toner foil via hydrophobic interactions, 2) amine-dependent, covalent binding on glycidyloxypropyl tromethoxysilane (GTPMS)- coated surface. Print pattern (at various grayscale levels) was designed in ChemSketch 2.0 and printed on PET surface using HP 100 LaserJet P1006 printer at resolution of 1200 dpi. Pristine PET@toner foils were used for passive adsorption. For GTPMS coating, hydrophilization of toner was performed by a 10-minute treatment with the use of UV/ozone cleaner. Then, 3% GTPMS solution in 50% ethanol was dispensed on freshly-oxidized foil for 30 min. Both multi-purpose substrates characterized by different surface properties were then incubated with rabbit anti-CRP antibody or protein A/G followed by surface blocking.
Method
To fully characterize the analytical performance in a role of immunosensing platforms, two types of immunoreactions were carried out on prepared 2D Ab/protein arrays. Direct immuno-labelling of rabbit anti-CRP antibody with anti-rabbit IgG-alkaline phosphatase (ALP) conjugate aimed at comparative evaluation of specificity and efficiency of antibody coating. Indirect sandwich immunoassay for C-reactive protein was selected as a model to determine basic working parameters of toner-based immunoassay. Antibody-capturing properties of protein A/G surface were evaluated by determination of monoclonal antibody-ALP conjugate binding capacity. For quantitative analysis, enzymatic reaction was carried out by dispensing p-nitrophenyl phosphate as chromogenic ALP substrate.
Results and Conclusions
Results of immunolabeling confirmed the usefulness of cured toner as an intermediate for efficient antibody and protein A/G coating via hydrophobicity-driven adsorption. Due to an increase of surface area and a hydrophobicity higher than for pristine PET, the densest layers were observed for fully printed surfaces (100% black level). The differences in the surface properties were also reflected in kinetics of antibody binding. UV/ozone treatment resulted in permanent oxidation and thus hydrophilization of toner surface. The introduction of hydroxyl groups favored reaction with GTPMS. Epoxy-silane coating of oxidized toner enabled a rapid fabrication of protein-reactive surface characterized by a high binding capacity in a single step immobilization. Thanks to good adhesion and thus tight bonding of laser-cut adhesive tape to PET@toner, the microchannel architecture was obtained. It opened up the possibility to carry out immunoassays in microfluidic systems. It was proven that presented methodologies, due to the design and fabrication simplicity, use of commonly available materials and up-scalability, could be successfully applied in immunodiagnostics or flexible microfluidics for Lab-on-a-Foil technology.
References
[1] E.F.M.Gabriel, B.G.Lucca, G.R.M.Duarte, W.K.T.Coltro, Recent advances in toner-based microfluidic
devices for bioanalytical applications, Anal. Methods. 10 (2018) 2952-2962. doi:10.1039/c8ay01095a
[2] A.I.Barbosa, A.s.Barreto, N.M.Reis, Transparent, Hydrophobic Fluorinated Ethylene Propylene Offers Rapid, Robust, and Irreversible Passive Adsorption of Diagnostic Antibodies for Sensitive Optical Biosensing, ACS Appl. Bio. Mater. 2 (2019) 2780–2790. 10.1021/acsabm.9b00214
[3] B.L.Thompson, C.Birch, J.Li, J.A.DuVall, D.LeRoux, D.A. Nelson, A-C.Tsuei, D.L.Mills, S.T. Krauss, B.E.Root. J.P.Landers, Microfluidic enzymatic DNA extraction on a hybrid polyester-toner-PMMA device, Analyst. 141 (2016) 4667-4675 doi:10.1039/c6an00209a
Acknowledgement
This work has been financially supported by the National Centre for Research and Development in Poland (grant no. POIR.04.01.04-00-0027/17).