RESUMEN
Immunoagglutination assay is a promising approach for the detection of waterborne analytes like virus, cells, proteins with its advantages such as a smaller amount of reagents and easier operation. This paper presents a microfluidic agglutination assay on which all the assay processes including analyte capture, agglutination, and detection are performed. The chip integrates an on-chip pump for sample loading, a dynamic magnetic bead (MB) clump for analyte capture and agglutination, and a sheath-less flow cytometry for particle detection, sizing, and counting. The chip is tested with streptavidin-coated MBs and biotinylated bovine serum albumin as a model assay, which realizes a limit of detection (LOD) of 1 pM. Then, an antigen/antibody assay using rabbit IgG and goat anti-rabbit IgG coated MBs is tested and a LOD of 5.5 pM is achieved. At last, human ferritin in 10% fetal bovine serum is tested with Ab-functionalized MBs and the detection achieves a LOD of 8.5 pM. The whole procedure takes only 10 min in total.
RESUMEN
Microfiltration is a compelling method to separate particles based on their distinct size and deformability. However, this approach is prone to clogging after processing a certain number of particles and forming bubbles in the separation procedure, which often leads to malfunctioning of devices. In this work, we report a bubble-free and clogging-free microfluidic particle separation platform with high throughput. The platform features an integrated bidirectional micropump, a hydrophilic microporous filtration membrane and a hydrophobic porous degassing membrane. The bidirectional micropump enables the fluid to flow back and forth repeatedly, which flushes the filtration membrane and clears the filtration micropores for further filtration, and to flow forward to implement multi-filtration. The hydrophobic porous membrane on top of the separation channel removes air bubbles forming in the separation channel, improving the separation efficiency and operational reliability. The microbead mixture and undiluted whole blood were separated using the microfluidic chip. After 5 cycles of reverse flushing and forward re-filtration, a 2857-fold enrichment ratio and an 89.8% recovery rate of 10 µm microbeads were achieved for microbead separation with 99.9% removal efficiency of 2 µm microbeads. After 8 cycles, white blood cells were effectively separated from whole blood with a 396-fold enrichment ratio and a 70.6% recovery rate at a throughput of 39.1 µl min-1, demonstrating that the platform can potentially be used in biomedical applications.
RESUMEN
Rapid separation of white blood cells from whole blood sample is often required for their subsequent analyses of functions and phenotypes, and many advances have been made in this field. However, most current microfiltration-based cell separation microfluidic chips still suffer from low-throughput and membrane clogging. This paper reports on a high-throughput and clogging-free microfluidic filtration platform, which features with an integrated bidirectional micropump and commercially available polycarbonate microporous membranes. The integrated bidirectional micropump enables the fluid to flush micropores back and forth, effectively avoiding membrane clogging. The microporous membrane allows red blood cells passing through high-density pores in a cross-flow mixed with dead-end filtration mode. All the separation processes, including blood and buffer loading, separation, and sample collection, are automatically controlled for easy operation and high throughput. Both microbead mixture and undiluted whole blood sample are separated by the platform effectively. In particular, for white blood cell separation, the chip recovered 72.1% white blood cells with an over 232-fold enrichment ratio at a throughput as high as 37.5 µl/min. This high-throughput, clogging-free, and highly integrated platform holds great promise for point-of-care blood pretreatment, analysis, and diagnosis applications.
RESUMEN
Homogeneous assays possess important advantages that no washing or physical separation is required, contributing to robust protocols and easy implementation which ensures potential point-of-care applications. Optimizing the detection strategy to reduce the number of reagents used and simplify the detection device is desirable. A method of homogeneous bead-agglutination assay based on micro-chip sheathless flow cytometry has been developed. The detection processes include mixing the capture-probe conjugated beads with an analyte containing sample, followed by flowing the reaction mixtures through the micro-chip sheathless flow cytometric device. The analyte concentrations were detected by counting the proportion of monomers in the reaction mixtures. Streptavidin-coated magnetic beads and biotinylated bovine serum albumin (bBSA) were used as a model system to verify the method, and detection limits of 0.15 pM and 1.5 pM for bBSA were achieved, using commercial Calibur and the developed micro-chip sheathless flow cytometric device, respectively. The setup of the micro-chip sheathless flow cytometric device is significantly simple; meanwhile, the system maintains relatively high sensitivity, which mainly benefits from the application of forward scattering to distinguish aggregates from monomers. The micro-chip sheathless flow cytometric device for bead agglutination detection provides us with a promising method for versatile immunoassays on microfluidic platforms.