Fluorescence and electrochemical microfluidic biosensors were developed for the detection of cholera toxin subunit B (CTB) as a model analyte. The microfluidic devices were made from polydimethylsiloxane (PDMS) using soft lithography from silicon templates. The polymer channels were sealed with a glass plate and packaged in a polymethylmethacrylate housing that provided leakproof sealing and a connection to a syringe pump. In the electrochemical format, an interdigitated ultramicroelectrode array (IDUA) was patterned onto the glass slide using photolithography, gold evaporation and lift-off processes. For CTB recognition, CTB-specific antibodies were immobilized onto superparamagnetic beads and ganglioside GM(1) was incorporated into liposomes. The fluorescence dye sulforhodamine B (SRB) and the electroactive compounds potassium hexacyanoferrate (II)/hexacyanoferrate (III) were used as detection markers that were encapsulated inside the liposomes for the fluorescence and electrochemical detection formats, respectively. Initial optimization experiments were carried out by applying the superparamagnetic beads in microtiter plate assays and SRB liposomes before they were transferred to the microfluidic systems. The limits of detection (LoD) of both assay formats for CTB were found to be 6.6 and 1.0 ng mL(-1) for the fluorescence and electrochemical formats, respectively. Changing the detection system was very easy, requiring only the synthesis of different marker-encapsulating liposomes, as well as the exchange of the detection unit. It was found that, in addition to a lower LoD, the electrochemical format assay showed advantages over the fluorescence format in terms of flexibility and reliability of signal recording.

Clinical analyses benefit world-wide from rapid and reliable diagnostics tests. New tests are sought with greatest demand not only for new analytes, but also to reduce costs, complexity and lengthy analysis times of current techniques. Among the myriad of possibilities available today to develop new test systems, amperometric biosensors are prominent players—best represented by the ubiquitous amperometric-based glucose sensors. Electrochemical approaches in general require little and often enough only simple hardware components, are rugged and yet provide low limits of detection. They thus offer many of the desirable attributes for point-of-care/point-of-need tests. This review focuses on investigating the important integration of sample preparation with (primarily electrochemical) biosensors. Sample clean up requirements, miniaturized sample preparation strategies, and their potential integration with sensors will be discussed, focusing on clinical sample analyses.

Sample preparation and processing steps are the most critical assay aspects that require our attention in the development of diagnostic devices for analytes present in complex matrices. In the best scenarios, diagnostic devices should use only simple sample processing. We have therefore investigated minimal preparation of stool samples and their effect on our sensitive microfluidic immunosensor for the detection of cholera toxin. This biosensor was previously developed and tested in buffer solutions only, using either fluorescence or electrochemical detection strategies. The microfluidic devices were made from polydimethylsiloxane using soft lithography and silicon templates. Cholera toxin subunit B (CTB)-specific antibodies immobilized onto superparamagnetic beads and ganglioside GM1-containing liposomes were used for CTB recognition in the detection system. Quantification of CTB was tested by spiking it in human stool samples. Here, optimal minimal sample processing steps, including filtration and centrifugation, were optimized using a microtiter plate assay owing to its high-throughput capabilities. Subsequently, it was transferred to the microfluidic systems, enhancing the diagnostic characteristic of the biosensor. It was found that the debris removal obtained through simple centrifugation resulted in an acceptable removal of matrix effects for the fluorescence format, reaching a limit of detection of only 9.0 ng/mL. However, the electron transfer in the electrochemical format was slightly negatively affected (limit of detection of 31.7 ng/mL). Subsequently, cross-reactivity using the heat-labile Escherichia coli toxin was investigated using the electrochemical microfluidic immunosensors and was determined to be negligible. With minimal sample preparation required, these microfluidic liposome-based systems have demonstrated excellent analytical performance in a complex matrix and will thus be applicable to other sample matrices.

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