For the miniaturization of biological assays, especially for the fabrication of microarrays, immobilization of biomolecules at the surfaces of the chips is the decisive factor. Accordingly, a variety of binding techniques have been developed over the years to immobilize DNA or proteins onto such substrates. Most of them require rather complex fabrication processes and sophisticated surface chemistry. Here, a comparatively simple immobilization technique is presented, which is based on the local generation of small spots of surface attached polymer networks. Immobilization is achieved in a one-step procedure: probe molecules are mixed with a photoactive copolymer in aqueous buffer, spotted onto a solid support, and cross-linked as well as bound to the substrate during brief flood exposure to UV light. The described procedure permits spatially confined surface functionalization and allows reliable binding of biological species to conventional substrates such as glass microscope slides as well as various types of plastic substrates with comparable performance. The latter also permits immobilization on structured, thermoformed substrates resulting in an all-plastic biochip platform, which is simple and cheap and seems to be promising for a variety of microdiagnostic applications.

In this article, we present a concept which uses liquid plugs as reaction volumes for heterogeneous assay reactions to facilitate time-resolved analysis of biomolecular reactions. For this purpose, the reaction is first compartmentalized to a train of many identical plugs. Therefore, we established a simple fluidic setup build from off-the-shelf available tubing and connectors. It permits reliable formation of plugs and successive dosing of further assay reagents to these compartments (plug volume <5% CV). The time course of the reaction is obtained by routing the plugs successively through a detector. Thereby, the arrival time of a given plug at the detector represents the reaction time of the overall reaction at that moment. Thus, each analyzed plug represents a discrete state of the overall reaction. With this approach, we can achieve a temporal resolution as small as one second, which hardly can be met by conventional analytical methods for analysis of endogenous biological compounds. For analysis of the content of the plugs, we developed a method which allows for heterogeneous assays in two-phase flow. For this purpose, functionalized superparamagnetic beads are enclosed in the plugs for specific binding of the assay product. Purification from supernatant species is achieved by transferring the beads with bound analyte across the phase boundary between aqueous plugs and water-immiscible carrier fluid. We demonstrate this assay principle exemplarily for a sandwich immunoassay (cytokine IL-8). Time-resolved analysis is validated by monitoring a cell-free in vitro expression reaction (turboGFP) in plugs and conventionally in bulk solution. We show that our approach allows for analyzing the entire course of a reaction in a single run. It permits kinetic studies of biological processes with significantly reduced experimental effort and consumption of costly reagents.

Two-phase flow systems are increasingly popular for miniaturized, high-throughput performance of analytical or chemical reactions. In this contribution, we extend a previously described method that allows to increase the range of applications of heterogeneous reactions in two-phase flow, i.e., reactions that rely on isolation and purification of the compound of interest for downstream analysis. Our concept is based on liquid plugs, which serve as miniaturized compartments for the analytical reactions. Purification of the target compound is achieved by extracting the analyte from the aqueous compartments using magnetic beads as solid carriers. In the present paper, we elucidate the influence of parameters such as the polarity of the liquid/liquid and solid/liquid interfaces, the magnetic forces and the fluidic conditions onto the extraction performance. The conditions for reliable extraction and purification of the target compounds are determined. Furthermore, we investigate how to facilitate breaking of the plugs through reduction of the surface tension of the solid/liquid interface. When a lower surface tension is employed, a smaller number of beads is required for the extraction process, which implies a higher sensitivity of the device. In addition, we generate channels with different surface chemistries, which are able to manipulate the flow of the two immiscible liquids. We describe a very simple way to generate such devices and show that we can achieve a transition from segmented flow of plugs to a side-by side flow of the two immiscible liquids, a key requirement for the purification of the compounds.