A Renewable and Ultrasensitive Electrochemiluminescence Immunosenor Based on Magnetic RuL@SiO2-Au∼RuL-Ab2 Sandwich-Type Nano-Immunocomplexes

An ultrasensitive and renewable electrochemiluminescence (ECL) immunosensor was developed for the detection of tumor markers by combining a newly designed trace tag and streptavidin-coated magnetic particles (SCMPs). The trace tag (RuL@SiO2-Au∼RuL-Ab2) was prepared by loading Ru(bpy)32+(RuL)-conjuged secondary antibodies (RuL-Ab2) on RuL@SiO2 (RuL-doped SiO2) doped Au (RuL@SiO2-Au). To fabricate the immunosensor, SCMPs were mixed with biotinylated AFP primary antibody (Biotin-Ab1), AFP, and RuL@SiO2-Au∼RuL-Ab2 complexes, then the resulting SCMP/Biotin-Ab1/AFP/RuL@SiO2-Au∼RuL-Ab2 (SBAR) sandwich-type immunocomplexes were absorbed on screen printed carbon electrode (SPCE) for detection. The immunocomplexes can be easily washed away from the surface of the SPCE when the magnetic field was removed, which made the immunosensor reusable. The present immunosensor showed a wide linear range of 0.05–100 ng mL−1 for detecting AFP, with a low detection limit of 0.02 ng mL−1 (defined as S/N = 3). The method takes advantage of three properties of the immunosensor: firstly, the RuL@SiO2-Au∼RuL-Ab2 composite exhibited dual amplification since SiO2 could load large amount of reporter molecules (RuL) for signal amplification. Gold particles could provide a large active surface to load more reporter molecules (RuL-Ab2). Accordingly, through the ECL response of RuL and tripropylamine (TPA), a strong ECL signal was obtained and an amplification analysis of protein interaction was achieved. Secondly, the sensor is renewable because the sandwich-type immunocomplexes can be readily absorbed or removed on the SPCE’s surface in a magnetic field. Thirdly, the SCMP modified probes can perform the rapid separation and purification of signal antibodies in a magnetic field. Thus, the present immunosensor can simultaneously realize separation, enrichment and determination. It showed potential application for the detection of AFP in human sera

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