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A nut-and-bolt microfluidic system was previously developed for a point-of-care (POC) human immunodeficiency virus (HIV) test and was able to acquire images of CD4 (cluster of differentiation 4) + T-lymphocytes in a sample drop of blood followed by image analysis. However, as the system was not fully integrated with a sample reaction module, the mixing of the sample with the antibody reagent was carried out manually. To achieve a rapid reaction with a reduced amount of costly reagent in a POC diagnostic system, an efficient sample mixing function must be implemented. Here, we propose a novel method to drastically accelerate the process of sample mixing and increase the reaction rate in the nut-and-bolt microfluidic system, where the sample is mixed with the reagent in a reaction chamber formed by connecting a nut with a bolt-like sample cartridge. The mixing is facilitated by rotating the sample cartridge bidirectionally using a DC motor, which agitates the sample in a chaotic manner. A microbead complex formed by the avidin-biotin interaction was used as a model reaction system to examine the feasibility of our mixing module. We found that the reaction time for the avidin-biotin binding by mixing was 7.5 times shorter than in the incubation method, achieving a reaction efficiency of over 95%. The performance of our mixing system was further demonstrated by measuring the concentration of CD4 cells labeled with a fluorescent antibody in the blood sample. The antigen-antibody reaction mixing was faster by a factor of 20, reaching a reaction efficiency comparable to the conventional incubation method.

A Nut-and-Bolt Microfluidic Mixing System for the Rapid Labeling of Immune Cells with Antibodies