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

Abstract

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.

Improved Enumeration of Weakly Fluorescent CD4+ T-lymphocytes by Confining Cells in a Spinning Sample Cartridge with a Helical Minichannel

“…To conform to the ASSURED criteria (affordable, sensitive, specific, user friendly, rapid and robust, equipment free and deliverable to end users) set out by the WHO for microfluidic POC devices [15], we aim to integrate and automate all the necessary functional modules in a mobile platform for sample loading, reagent mixing, transport, detection and analysis by adopting a single DC motor that controls the rotation of the sample cartridge in various prescribed modes. We have implemented sample mixing inside the cartridge through bidirectional rotation of the cartridge by motor control and enhanced the efficiency of antigen-antibody reaction without lysing a finger-pricked whole blood sample [30]. The reacted sample can be transported from the mixing chamber to the helical minichannel by rotating the motor at lower speeds in one direction, which generates a pressure inside the reaction chamber based on the nut and bolt mechanism.…”

Integrated Point-of-Care Immune Cell Analyzer with Rapid Blood Sample Reaction and Wide Field-of-View Detection

“…Moreover, most POC devices rely heavily on chemical or biological processes, typically in parallel assay procedures that involve different reagents and materials. The POC devices should be able to accomplish adjustable and quick mixing that accelerates a reaction to decrease the response time and increase the sample analysis and processing speed. − …”

Universal Pre-Mixing Dry-Film Stickers Capable of Retrofitting Existing Microfluidics

“…When integrated into lab-on-a-chip devices, microfluidic mixers have the potential to automate many laboratory steps including dilutions, extractions, addition of reagents or drugs, and particle synthesis [1][2][3][4][5][6][7] . To that end, numerous stand-alone, highly-efficient microfluidic mixers have utilized clever design principles to overcome diffusion limitations that hinder microscale mixing [8][9][10] .…”