PMC

Finite element simulation of MIPA device (a) Three dimensional velocity field and flow stream line profile of MIPA device. Slice figures show the detailed velocity field in the (b) y–z plane and (c) x–y plane. The model sets the inlet and outlet velocities to 0.001 m s⁻¹ and zero, respectively. (d) Time lapse of the TNF-α diffused concentration in MIPA device. Diffusion coefficient D = 10⁻¹⁰ m² s⁻¹. Initial THP-1 cell secreted TNF-α concentration C₀ = 1.0 nM mm⁻³. The model sets the both inlet and outlet velocities to zero.

Detection of TNF-α secreted from LPS-stimulated THP-1 cells using the MIPA device (a) Standard curve for TNF-α detection. TNF-α with a known concentration (0–10 000 pg mL⁻¹) was spiked in the complete cell growth medium and detected using AlphaLISA and the customized optical setup. (b) Plot of TNF-α concentration secreted by LPS-stimulated THP-1 cells as a function of cell number and LPS concentration. (c and d) Plots of average TNF-α concentration secreted by individual cells as a function of LPS concentration (c) or LPS concentration per cell (d). (e) Plot of TNF-α concentration secreted by normal and LPS-deactivated THP-1 cells trapped on the PMM (n = 20 000 cells). P-values calculated using the paired Student’s t-test are indicated for significant differences (P < 0.05 (*) and P < 0.005 (**)). NS, statistically not significant.

Isolation and enrichment of THP-1 cells using the MIPA device (a) A photograph of the MIPA device. The MIPA device was injected with dyed solutions for visualization of the cell culture chamber and the immunoassay chamber. The device dimension is 9 mm L × 7 mm W × 4 mm H. (b) SEM image showing the PMM. Scale bar, 10 μm. (c) Temporal sequence of false-colored brightfield images showing isolation and enrichment of THP-1 cells on the PMM. The cell loading concentration was 5 × 10⁵ cells mL⁻¹ at 5 μL min⁻¹ flow rate. Scale bar, 100 μm. (d) Plot of density of trapped cells on the PMM as a function of injection volume, using three different cell loading concentrations as indicated.

Functional immunophenotyping using the MIPA device (a) Schematic of the multi-layered MIPA device consisting of a cell culture chamber, a PDMS microfiltration membrane (PMM), and an immunoassay chamber. The size of both the cell culture and immunoassay chambers is 3.7 mm (L: length) × 3 mm (W: width) × 100 μm (H: height). The inset shows the pre-filter structure (300 μm L × 50 μm W × 100 μm H) to block particles larger than 50 μm in diameter (e.g. aggregated cells) and the supporting posts to prevent deformation of the PMM. The supporting post diameter is 50 μm with a center-to-center distance of 200 μm. A fiber probe was attached underneath the immunoassay chamber to collect AlphaLISA emission signal. (b) Schematic showing the immunophenotyping assay protocol used in this study: (1) Isolation and enrichment of THP-1 cells on the PMM; (2) LPS-stimulation of cells; (3) Loading and incubation of AlphaLISA beads in the immunoassay chamber; (4) TNF-α detection using the AlphaLISA assay, in which the streptavidin-coated donor (blue) and acceptor beads (orange) are both conjugated with TNF-α antibodies. The beads are brought into close proximity (<200 nm) through binding simultaneously to TNF-α. Using 680 nm laser for excitation, the singlet oxygen released by the donor bead diffuses to the nearby acceptor bead and triggers it to emit 615 nm fluorescent light.