A micro-chamber free digital bio-detection for multiplexed and ultrasensitive immunoassay based on encoded magnetic microbeads and tyramide signal amplification strategy

Digital bio-detection has become one of the most appealing methods in recent years due to its excellent performance with ultra-sensitivity in detection of low-abundance targets. Traditional digital bio-detection needs the utilization of micro-chambers for physical isolation of targets, while the recently developed beads-based micro-chamber free one is attracting extensive attention, although there exist the disadvantages of overlaps between positive ("1") and negative ("0") signals as well as the decreased detection sensitivity in multiplexed mode. Here we propose a feasible and robust micro-chamber free digital bio-detection for multiplexed and ultrasensitive immunoassay based on encoded magnetic microbeads (EMMs) and tyramide signal amplification (TSA) strategy. An EMMs-based multiplexed platform is constructed by using a fluorescent encoding method, then a puissant signal amplification of positive events in TSA procedure is achieved via systematical revelation of key factors influences. For proof of concept, a three-plexed tumor markers detection is performed to evaluate our established platform. The detection sensitivity is comparable to the corresponding single-plexed assays and is also approximately 30-15,000 times improvement compared to the conventional suspension chip. Therefore, this multiplexed micro-chamber free digital bio-detection paves a promising way to be an ultrasensitive and powerful tool for clinical diagnosis.