Microfluidic Immunodetection System Based on Asymmetric Particle Aggregation

Abstract

Micro- and nanoparticles are mobile substrates for capturing, transporting and detecting biomolecules or cells via surface functionalization and are used in bioanalytical researches. A large surface area of the suspension of such particles enables effective interaction with the target molecule in the sample solution. Controlled manipulation of these solid particles provides a multifaceted opportunity in the microfluidic format for on-chip bioanalysis. Thus, it is an important component of Point-of-Care applications or Lab-on-a-Chip devices. In this thesis, we propose asymmetric immunoaggregated particles (AIPs) between two micro-particles of different size and magnetism, and propose methods to detect such aggregates including a microfluidic device. The number of particle aggregates formed via antigen-antibody reaction is known to be an indicator of the quantity of target analyte. As with the conventional symmetric particle aggregation (single type particle aggregation), asymmetric aggregation also follows similar reaction behavior. Also, one can easily distinguish the aggregates using not only the size discrimination methods but also magnetic separation. Utilizing an additional physical property can simplify the transducer design. We first developed an optical detection method that reads contours of particles/AIPs to investigate the behavior of aggregation. We, then, developed a magnet integrated sensor to image AIBs selectively, followed by a microchannel-based rapid detection device using a syringe pump. In the microfluidic device, AIPs were detected by optical monitoring in a flow under an external magnetic field. AIPs are attracted to the top surface of the channel by a magnetic field and made to slide along the upper surface by flow drag. This sliding behavior is in contrast with other particles such as magnetic (MG) and polystyrene particles (PS)

while attracted MG hardly slide (or roll) due to their small size, PS quickly move with the flow due to the absence of magnetism. Sliding AIPs are optically monitored in a designated sensing area in the microchannel. A custom-built program code is used for counting the AIPs and further analysis such as number and velocity distributions that are correlated with target concentration. Furthermore, we analyzed the trajectory of each AIP inside the microchannel through force analysis for system optimization. The proposed system shows a detection range of 40 pg/mL to 54 ng/mL for influenza type A H1N1 nucleoprotein (NP). The non-specific aggregation ratio was obtained at 2.47 ± 0.59% in the absence of antigen (BSA 0.1% w/v included) and the dynamic range was over 1000-fold. The detection takes 6 min, much faster than conventional methods (~10 min to several hours). This method uses microscopic power not more than 100×, so optical requirements are not strict and fluorescence are not required. Simple structure makes our sensor reusable, cheap, and robust.