Detection of immunoglobulins in a laser induced fluorescence system utilizing polydimethysiloxane microchips with advanced surface and optical properties

Abstract

We developed an automated laser induced fluorescence system utilizing microfluidic chips for detection and quantification of immunoglobulins. Microchips were fabricated from polydimethysiloxane ͑PDMS͒ using the so-called "prepolymerization technique." The microchip structure helped minimize the effects of PDMS autofluorescence and light scattering. Furthermore, a thin and uniform PDMS layer forming the top of the microchip enabled proper focusing and collection of the excitation beam and the emitted fluorescence, respectively. The developed system was tested for the detection of mouse immunoglobulins. The capturing antibodies were immobilized on internal microchannel walls in the form of a polyelectrolyte. We clearly show that this immobilization technique, if correctly realized, gives results with high reproducibility. After sample incubation and washing, secondary antibodies labeled by fluorescein isothiocyanate were introduced into microchannels to build a detectable complex. We show that mouse antibodies can be quantified in a wide concentration range, 0.01-100 g ml −1 . The lower detection limit was below 0.001 g ml −1 ͑6.7 pM͒. The developed laser induced fluorescence ͑LIF͒ apparatus is relatively cheap and easy to construct. The total cost of the developed LIF detector is lower than a typical price of plate readers. If compared to classical ELISA ͑enzyme linked immunosorbent assay͒ plate systems, the detection of immunoglobulins or other proteins in the developed PDMS microfluidic device brings other important benefits such as reduced time demands ͑10 min incubation͒ and low reagent consumption ͑less than 1 l͒. The cost of the developed PDMS chips is comparable with the price of commercial ELISA plates. The main troubleshooting related to the apparatus development is also discussed in order to help potential constructors.

Surface Modification of PDMS in Microfluidic Devices

“…Th is method has many advantages such as simplicity, universality and thickness control in nanoscale. Schrott and coworkers reported the production of multilayers of dextran sulfate/ human IgG on an IgG-pretreated PDMS surface through a combination of the LBL technique and protein adsorption [16]. In order to immobilize the Polyvinyl alcohol (PVA) and glycerol, Luo et al immersed PDMS into an aqueous solution of PVA and glycerol [17].…”

Silicones for Microfluidic Systems

“…• Immobilization of biomolecules for biomolecular detection using immunoassays [35,75,[177][178][179][180] and enzymatic microreactors [181,182]. • Cell culture under controlled fl ow conditions on surfaces modifi ed with ligands important for cell adhesion and proliferation [35,73,76,105].…”

Development of a microplate reader compatible microfluidic chip for ELISA

“…The antibody or antigen coating layers can be applied as arrays of microspots on different substrates such as silanized glass (Knecht et al 2004), PDMS (Schrott et al 2011), silicon (Yakovleva et al 2002) or as arrays immobilized in hydrogels (Rubina et al 2005). Various immunoassay microfluidic devices have been reported (Murakami et al 2004;Kang and Park 2005;Zhao et al 2008), among which the plastic one is the most economical approach and thus attracts a great research interest.…”

“…Due to their small scale, microdevices have many practical advantages such as large specific volume, very low reagent consumption, and short diffusion length. Several developmental advances have been realized in miniaturization of capillary electrophoresisbased immunoassays (Ou et al 1999;Roper et al 2003;Herr et al 2005;Chen and Zhang 2008;Hui and Ma 2011) and microchip-based ELISA (Rossier and Girault 2001;Murakami et al 2004;Kang and Park 2005;Zhao et al 2008;Weng et al 2011;Schrott et al 2011). The capillary electrophoresis-based immunoassays utilize an electric field to separate the antigenantibody complex from the rest of the molecules in the assay solutions.…”