Development of an integrated microsystem for the multiplexed detection of protein markers in serum using electrochemical immunosensors

Recent advances in the fabrication of microfluidic platforms initiated during the late 90s have facilitated the realisation of micro total analysis systems. The integration of miniaturised fluidic handling and delivery systems with chemical and biochemical sensors provide applied scientists with powerful tools for in-field measurements away from central laboratories. Amongst the various classes of elements able to transduce a chemical or biochemical events into a measurable signal, electrochemical platforms undoubtedly present the most promising advantages. Electrodes of all type, sizes and geometries can easily be integrated within a microfluidic platform and provide excellent sensitivity and versatility in comparison to other transduction techniques based on for example optical or mass sensing. Furthermore, the associated electronics used to drive the electrochemical detection and signal processing can also be easily miniaturised and integrated onto the same platform by carefully designing application specific integrated circuits. We have recently reported a simple and rapid approach for prototype microfluidics and sensor assembly to perform complex protein and genetic electrochemical assays with excellent reproducibility. The microfluidic platform was realized by high precision milling of polycarbonate sheets, which offers flexibility and rapid turn over of the desired designs. Sixteen-electrode sensor arrays were fabricated using photolithographic deposition technologies in order to realize three-electrodes cells comprising of gold counter and working electrodes as well as silver reference electrode. Fluidic chips and electrode arrays were assembled via a laser machined double-sided adhesive gaskets, creating the microchannels necessary for sample and reagent delivery. Surface chemistry methodologies were evaluated in order to achieve the double function of eliminating non-specific binding and optimal spacing of the anchor biocomponents for maximum accessibility- - to the target proteins. Storage conditions were optimized, demonstrating a long-term stability of the reporter conjugates jointly stored within a single reservoir in the microsystem. The final system has been optimized in terms of incubation times, temperatures and simultaneous, multiplexed detection of the protein markers was achieved in less than 10 minutes with less than ng/mL detection limits. The microsystem has been validated using real patient serum samples and excellent correlation with ELISA results obtained.