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

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 (R(2) = 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.

Figure 1. Inkjet printing of antibody array and assay format.

(A) Schematic of the two-stage antibody patterning using the PEI and GA cross linker system. PEI ink was first deposited by piezoelectric inkjet printing in discrete regions of a PMMA foil. After activating the PEI patterned surface with glutaraldehyde, anti-CRP antibody was deposited locally in a second inkjet printing step. (B) A multichannel R2R hot embossed PMMA microfluidic chip is aligned and bonded permanently with the antibody patterned PMMA foil by solvent bonding lamination. Blocking reagent, mixture of sample and detection antibody, and wash buffer are sequentially introduced at one end of the microchip by pipette and drawn using syringe.

Figure 2. Basic properties of PEI inks at different polymer concentrations.

(A) Viscosity, (B) surface tension, (C) effect of PEI concentration in the ink on anti-CRP antibody binding capacity of PMMA substrate (5µg/ml anti-CRP, 500 ng/ml CRP, 0.0132 mg/ml secondary antibody; n = 2).

Figure 3. AFM images of (A) native PMMA (1 µm×1 µm) (B) PEI patterned native PMMA (20 µm×20 µm) (C) PEI patterned plasma treated PMMA (20 µm×20 µm) (D) antibody patterned PEI-GA film on plasma treated PMMA (20 µm×20 µm).

Figure 4. XPS spectra of native PMMA, PEI patterned native PMMA and PEI patterned plasma activated PMMA surface.

Figure 5. Comparison of performance of biochips for detection of CRP antigen where anti-CRP antibody inkjet printed on PMMA substrate modified under different conditions (500 ng/ml CRP and 0.0132 ng/ml secondary antibody; n = 3).

Figure 6. Microfluidic biochip responses for detection of CRP antigen.

(A) Fluorescence image of the biochip after immunoassay. Each microfluidic channel was patterned with three replicate CRP capture antibody spots. Samples containing CRP at concentration of 0, 10, 50, 100, 200, 500, 1000, 1500 ng/ml were infused through separate sample channels (rows 1–8, respectively). In each channel the bound antigen was detected with fluorescently labelled secondary antibody (0.0132 mg/ml). (B) Dose response curves for CRP antigen. Fluorescence values were plotted for each CRP concentration. Each data point represents the mean ± SD for the three spots within each microchannel.