Investigating Electrochemiluminescence (ECL) as highly sensitive and effective signaling means for microfluidic biosensors

The conception and realization of microfluidic Total Analysis Systems (microTAS) has revolutionized the analytical process by integrating the whole breadth of analytical techniques into miniaturized systems. Paramount for efficient and competitive microTAS are integrated detection strategies, which lead to low limits of detection while reducing the sample volume. The concept of electrochemiluminescence (ECL) has been especially intriguing ever since the introduction of a version based on Ru(bpy)32+ by Alan Bard in 1972, due to its immense sensitivity, non-existent auto-luminescent background signal, and simplicity in experimental design. Therefore, integrating ECL detection into microTAS is a logical consequence to achieve simple, yet highly sensitive sensors. ECL follows complex electron transfer pathways, and its efficiency can be enhanced, but also hindered, by numerous factors. Our studies identified the novel combination of the coreactant N-butyldiethanolamine (NBEA) with the surfactant Zonyl FSN as an optimal signal enhancer for Ru(bpy)32+-based ECL. This combination of coreactant and surfactant led to a limit of detection (LOD) for Ru(bpy)32+ of 2.2 nM, compared to 0.59 µM for the commonly used Tripropylamine/Triton X-100 system, and a 50-fold increase in sensitivity. Investigations under different buffer conditions revealed that the ECL signal was significantly influenced by buffer composition and pH values. Furthermore it was possible to generate an ECL signal at a potential well below 1.2 V vs. Ag/AgCl, the common potential for Ru(bpy)32+-based ECL. The low oxidation potential (LOP) signal was significantly increased under the use of the coreactant NBEA with Tris buffer at pH 8.5, and was about three times higher than for the standard coreactant, TPA, in phosphate-based buffer at pH 7. Such low potential ECL signals are desirable for electrode lifetime enhancement and prevention of possible DNA damage in bioassays. However, to truly extend a sensor’s limit of detection, one must go beyond a mere one-to-one labeling approach, especially when dealing with DNA, which, by its nature, is mostly present at low concentrations in real-life samples. Liposomes, molecules capable of encapsulating large quantities of analyte and of being DNA-specific, offer a convenient way of enhancing detection capabilities. Therefore, Ru(bpy)32+encapsulating liposomes were successfully synthesized and linked to Cryptosporidium parvum (C. parvum) DNA. ECL detection of the DNA was achieved inside a microfluidic chip with a microfabricated three electrode system. After identification of the appropriate assay and flow parameters, it was possible to achieve on-chip ECL detection in less than ten minutes, while the microfluidic chip was also capable of fluorescent and electrochemical detection. The study not only presents a novel ECL-based microfluidic biosensor, but functioning strategies that are urgently required to increase its usability and sensitivity