A 3D mixing-based portable magnetic device for fully automatic immunofluorescence staining of gamma-H2AX in UVC-irradiated CD4(+)cells

Abstract

Immunofluorescence (IF) is a common method used in cell biology. The conventional protocol for IF staining is time and labor-intensive, operator dependent and reagent-consuming. Magnetic Bead (MB)-based microdevices are frequently utilized in cellular assays, but integration of simple and efficient mixing with downstream multi-step manipulation of MBs for automatic IF staining is still challenging. We herein present a portable, inexpensive and integratable device for MB-based automatic IF staining. First, a front-end cell capture step is performed using a 3D-mixing module, which is built upon a novel mechanism named ec-2MagRotors and generates periodically changing 3D magnetic fields. A 5-fold enhancement of cell capture efficiency was attained even with a low bead-to-cell concentration ratio (5 : 1), when conducting magnetic 3D mixing. Second, a 1D-moving module is employed downstream to automatically manipulate MB-cell complexes for IF staining. Further, a simplified protocol for staining of gamma-H2AX, a biomarker widely used in evaluation of cell radiation damage, is presented for proof-of-principle study of the magnetic device. Using UVC-irradiated CD4(+)cells as samples, our device achieved fully automatic gamma-H2AX staining within 40 minutes at room temperature and showed a linear dose-response relationship. The developed portable magnetic device is automatic, efficient, cost-effective and simple-to-use, holding great potential for applications in different IF assays.