Patterned immobilization of antibodies within roll-to-roll hot embossed polymeric microfluidic channels.

Belachew Feyssa,Christina Liedert,Liisa Kivimaki,Leena-Sisko Johansson,Heli Jantunen,Leena Hakalahti,Katerina Kourentzi

doi:10.1371/journal.pone.0068918

Belachew Feyssa, Christina Liedert + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0068918

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Abstract

This paper describes a method for the patterned immobilization of capture antibodies into a microfluidic platform fabricated by roll-to-roll (R2R) hot embossing on poly (methyl methacrylate) (PMMA). Covalent attachment of antibodies was achieved by two sequential inkjet printing steps. First, a polyethyleneimine (PEI) layer was deposited onto oxygen plasma activated PMMA foil and further cross-linked with glutaraldehyde (GA) to provide an amine-reactive aldehyde surface (PEI-GA). This step was followed by a second deposition of antibody by overprinting on the PEI-GA patterned PMMA foil. The PEI polymer ink was first formulated to ensure stable drop formation in inkjet printing and the printed films were characterized using atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). Anti-CRP antibody was patterned on PMMA foil by the developed method and bonded permanently with R2R hot embossed PMMA microchannels by solvent bonding lamination. The functionality of the immobilized antibody inside the microfluidic channel was evaluated by fluorescence-based sandwich immunoassay for detection of C-reactive protein (CRP). The antibody-antigen assay exhibited a good level of linearity over the range of 10 ng/ml to 500 ng/ml (R2 = 0.991) with a calculated detection limit of 5.2 ng/ml. The developed patterning method is straightforward, rapid and provides a versatile approach for creating multiple protein patterns in a single microfluidic channel for multiplexed immunoassays.

Highlights

The analysis of biomarkers directly at the side of a patient, which is known as point-of-care testing (POCT), is a continuously expanding trend in the practice of medical diagnosis [1,2,3]
The ink viscosity should be below 20 cps at least and ideally below 10 cps, in piezoelectric inkjet printing systems [32,33]
The surface tension of the ink for piezoelectric inkjet printing should be in the range of 30–70 mN/m, which is high enough to prevent dripping of the ink on the nozzle and low enough to spread over the substrate [32]

Summary

Introduction

The analysis of biomarkers directly at the side of a patient, which is known as point-of-care testing (POCT), is a continuously expanding trend in the practice of medical diagnosis [1,2,3]. The above-mentioned covalent linkage chemistry was used and the immobilization process was optimized for the large-scale fabrication of antibody patterned foil-based immunoassay chips with printing method. To examine the effect of PEI concentration on the binding capacity of the capture antibody, a fluorescence sandwich immunoassay was performed on the PEI-coated PMMA films in a FAST Frame multi slide plate.

Results

Conclusion