A 3D mixing-based portable magnetic device for fully automatic immunofluorescence staining of γ-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 γ-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 γ-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.

Fig. 1. The IF staining fluidic chip: (A) picture showing the structures, which contains a channel with one capture chamber, five reaction chambers, and five smaller isolation chambers between neighboring reaction chambers; (B) three layers of thermoplastic polymer plates (PMMA) bonded with double-sided adhesive films between PMMA plates.

Fig. 2. Schematic diagram of the portable magnetic device including a 3D-mixing module (based on the novel mechanism of ec-2MagRotors) and a 1D-moving module.

Fig. 3. Design (A) and picture for apperance (B) of the portable magnetic device. (a) The upper and the lower circular plates of the 3D-mixing module; (b) fluidic chip support and moving motor; (c) magnet support and moving motor.

Fig. 4. (A) Schematic diagram of the protocol for IF staining of γ-H2AX in CD4⁺ cells on a fluidic chip after UVC irradiation. The stained cells are detected using flow cytometry. (B) Pictures of the fluidic chip before and after sample/reagent loading.

Fig. 5. Movement of MBs under control of the rotating 3D magnetic field: (A) in the capture chamber, (B) the trajectory.

Fig. 6. Evaluation of capture efficiency of CD4⁺ cells based on the 3D mixing module: (A) pictures of the MB–CD4⁺ cell complex at magnification factors of 10×, 20×, 40×, respectively. (B) Effect of on-chip 3D mixing on the capture efficiency of CD4⁺ cells to MBs with diameters of 2.8 μm and 4.5 μm, respectively. (C) Effect of MB size and bead-to-cell concentration ratio on the capture efficiency of CD4⁺ cells. This study tested two sizes of MBs (with diameters of 2.8 μm and 4.5 μm) over bead-to-cell concentration ratios varying from 50 : 1 to 400 : 1.

Fig. 7. Characterization of the portable magnetic device for on-chip automatic manipulation of MBs: (A) images of the movement of the MB–cell complexes from chamber 1 to chamber 6 (a–f); (B) effect of MB size on the recovery rate of CD4⁺ cells after on-chip automatic 3D mixing and 1D moving.

Fig. 8. Relationship between UVC radiation dose and relative fluorescence intensity of γ-H2AX in CD4⁺ cells stained using both manual in-tube protocol and automatic on-chip protocol.