Fig 1.
Schematic diagram of flexible sensor sheet.
During the measurement, the device is attached to the bottom of the culture dish to form a temporarily closed microspace around the target cells, hence enabling the short-time evaluation of oxygen consumption rate. The device comprises a transparent EVOH/PDMS sheet and an array of microchamber structures (φ 90 μm, 50 μm depth) that contain a 1-μm-thick sensing layer at their bottom.
Fig 2.
(a) Self-aligned hot embossing method used to fabricate the device. First, a 50-μm-thick EVOH film was laminated onto a PDMS sheet, which was followed by the spin-coating of PtOEP/polystyrene. Subsequently, a silicon micromold was embossed onto the layered polymer under optimized conditions, typically a temperature of 130°C and a force of 10 kN, to form microhole structures containing a self-aligned phosphor sensor layer at their bottom. Finally, the residual phosphor layer outside the hole features was peeled off to obtain the sensor sheet. (b) Experimental setup for in situ measurement of cell oxygen consumption. Both phosphorescence lifetime measurement and cultivated cell imaging were automatically performed. The data acquisition speed of the automated sequential measurement system was 100 points/min.
Fig 3.
(a) Photograph of the flexible sensor device. The device comprises a transparent EVOH/PDMS sheet and an array of microchamber structures. (b) Bright-field image and phosphorescence image of the sheet. (c) SEM image of the sheet. Each microchamber has a 90 μm diameter and a 50 μm depth, and contains a 1-μm-thick sensing layer at its bottom. (d) Phosphorescence lifetime τ measurement at three different dissolved oxygen concentrations (Cox = 0.00, 0.08, and 0.24 mmol/L). In the presence of oxygen, τdecreased as a result of the quenching of the excited triplet state by the collision of oxygen molecules with phosphor molecules. (e) Oxygen sensor calibration. τ and Cox exhibited a linear relationship in accordance with the Stern-Volmer equation.
Fig 4.
(a) Dependence of oxygen consumption rate (Rox) on pressure. Rox converges at pressures of 4.5–12.7 kPa because of the high sealing performance of the microchamber. (b) Results of trypan blue cell viability assay. Each point represents the mean with standard deviation (S.D.) of 3 measurements. The cell viability on 30 min was statistically different from that on 0, 10, 20 min (p<0.05, respectively, the Tukey test).
Fig 5.
(a) Cross-sectional image of MCF7 and flexible sensor sheet. Scale bar: 50 μm. (b)-(e) Bright-field images of MCF7 and flexible sensor sheet. Scale bar: 50 μm. The numbers of cells in the chambers are n = 18, 20, 15, and 13, respectively. (f) Rox measurement performed using the device. The linear decrease in oxygen concentration was measured as a result of cell respiration.
Fig 6.
(a) Bright-field image of rat hippocampus slice. Scale bar: 500 μm. (b) Oxygen concentration rate mapping of rat hippocampus slice indicated with blue box in a. (c) Oxygen concentration rate mapping indicated with yellow box in a. CA1 and CA3 are pyramidal cells of the hippocampus and DG is the dentate gyrus. Broken lines show neuronal cell bodies. This result indicates that the oxygen consumption rate of DG is higher than those of CA3 and CA1, and the oxygen consumption rate around neuronal cell bodies is higher than in the molecular layers. Scale bar: 200 μm.