Fig 1.
(A) 3D image of the bi-module device that consists of an electrochemical detection module and cell migration module (a); photograph of a device for experiment (b); section view of a bi-module devise (c); (B) Schematic diagram of the micro-fabrication processes: patterning and fabrication of electrodes on a ITO glass (a); bonding of a PDMS ring (5 mm in diameter, 1 mm in height) with ITO glass (b); assemble of a polycarbonate membrane on top of the PDMS right (c); assemble of a PDMS ring (5 mm in diameter, 5 mm in height) on top of the membrane (d). ITO: indium tin oxide, PDMS: Poly(dimethylsiloxane).
Fig 2.
Experimental protocol for the electrochemical detection of H2O2 production during cell migration.
(a) MWCNT/graphene/MnO2 functional material is casted on working electrode (right one) surface following a layer of Nafion coating; (b) RPMI 1640 medium is injected into the PDMS ring (bottom) using a micropipette; then polycarbonate membrane is placed on top of the PDMS ring (bottom), and another piece of PDMS ring (upper) is aligned over the membrane; (c) cell suspension is injected into the upper cell seeding chamber using a micropipette; (d) the cell-loaded device is incubated in a cell incubator for 12h and the electrochemical signal is recorded. MWCNT: mutil-wall carbon nanotube, PDMS: Poly(dimethylsiloxane).
Fig 3.
Amperometric performance (i-t curve) of fully assembled device.
(A) i-t curves of successive additions of H2O (a) or 4 μM H2O2 (b) into RPMI 1640 at an applied potential of -0.4 V vs ITO RE/CE; (B) i-t curves of PMA injection (0.5mg mL-1) without cells loading (control 1), DMSO (0.5%, v/v%) injection with cell loading (control 2), PMA injection (0.5 mg mL-1) with cells loading, and followed by catalase injection (5 μg mL-1)-cell response at an applied potential of -0.4 V vs ITO RE/CE; Inset of (B) i-t curves of PMA injection with cells loaded in top and bottom chamber of assembled device. RE/CE: reference electrode/ counter electrode; PMA: phorbol 12-myristate-13-acetate, DMSO: dimethyl sulfoxide
Fig 4.
(A) Amperometric responses of fully functionalized bi-module device during melanoma A375 cell migration. No cell: device without cell loading; No serum: medium without serum in bottom chamber; DPI: RPMI 1640 containing 10% serum (conditioned medium) in bottom chamber, cell in upper chamber was incubated with H2O2 generation inhibitor, DPI; Catalase: conditioned medium in bottom chamber, cell in upper chamber was incubated with H2O2 decomposer, catalase; Cell migration: conditioned medium in bottom chamber; DMSO: conditioned medium in bottom chamber, cell in upper chamber was incubated with DMSO (solvent of DPI). (B) The corresponding current response obtained from amperometric curves of three independent experiments, (n = 3, * denotes p<0.05). DPI: diphenyleneiodonium.
Fig 5.
Cell migration quantified by hematoxylin and eosin (H&E) staining the polycarbonate membrane disassembled from device (A) photographs captured under microscopy: RPMI 1640 containing 10% serum in bottom chamber and serum-starved cells in top chamber (a), cells pre-treated by DPI (10 μM) (b), cells pre-treated by catalase (5 μg mL-1) (d), cells pre-treated by DMSO (0.5%, V/V) (e), serum-starved cells in a device containing serum free medium in top and bottom chambers (c), and fresh polycarbonate membrane after (H&E) staining (f). Red arrow points the migrating cells. (B) histogram of migrating cell percentage using cells loaded in a device without serum in top and bottom chambers as a reference, * denotes p<0.01, n = 3.
Table 1.
Performance comparison of bi-module device with standard biological migration assay and lab-on-chip migration assay.
Fig 6.
Cell migration quantified by bi-module device.
(A) in situ amperometric signal of melanoma A375 cell, larynx HEp-2 cell and liver cancer Hep G2 cell seeded in device containing conditional medium (B) histogram of maximum current signal during 12h of cells; (C) H&E staining of migrating A375, Hep G2 and HEp-2 cells; (D) histogram of increased migrating cell percentage using cells loaded in a device without serum in bottom chamber as a reference, * denotes p<0.05, n = 3.
Fig 7.
Bi-module device can in situ detects the generation of biochemical molecules in the process of cell migration.