Bi-Module Sensing Device to <i>In Situ - Fig 1 </i> Quantitatively Detect Hydrogen Peroxide Released from Migrating Tumor Cells

<p>(A) 3D image of the bi-module device that consists of an electrochemical detection module and cell migration module (<i>a</i>); photograph of a device for experiment (<i>b</i>); section view of a bi-module devise (<i>c</i>); (B) Schematic diagram of the micro-fabrication processes: patterning and fabrication of electrodes on a ITO glass (<i>a</i>); bonding of a PDMS ring (5 mm in diameter, 1 mm in height) with ITO glass (<i>b</i>); assemble of a polycarbonate membrane on top of the PDMS right (<i>c</i>); assemble of a PDMS ring (5 mm in diameter, 5 mm in height) on top of the membrane (<i>d</i>). ITO: indium tin oxide, PDMS: Poly(dimethylsiloxane).</p

Bi-Module Sensing Device to <i>In Situ - Fig 5 </i> Quantitatively Detect Hydrogen Peroxide Released from Migrating Tumor Cells

<p>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 (<i>a</i>), cells pre-treated by DPI (10 μM) (<i>b</i>), cells pre-treated by catalase (5 μg mL^-1) (<i>d</i>), cells pre-treated by DMSO (0.5%, V/V) (<i>e</i>), serum-starved cells in a device containing serum free medium in top and bottom chambers (<i>c</i>), and fresh polycarbonate membrane after (H&E) staining (<i>f</i>). 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 <i>p</i><0.01, n = 3.</p

Experimental protocol for the electrochemical detection of H₂O₂ production during cell migration.

<p>(<i>a</i>) MWCNT/graphene/MnO₂ functional material is casted on working electrode (right one) surface following a layer of Nafion coating; (<i>b</i>) 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; (<i>c</i>) cell suspension is injected into the upper cell seeding chamber using a micropipette; (<i>d</i>) 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).</p

Cell migration quantified by bi-module device.

<p>(A) <i>in situ</i> 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 <i>p</i><0.05, n = 3.</p

Performance comparison of bi-module device with standard biological migration assay and lab-on-chip migration assay.

<p><b><i>a</i></b>: this work;</p><p>H&E: hematoxylin and eosin; ECIS: electrical cell−substrate impedance sensing; 3D: three dimension</p><p>Performance comparison of bi-module device with standard biological migration assay and lab-on-chip migration assay.</p

Bi-Module Sensing Device to <i>In Situ</i> Quantitatively Detect Hydrogen Peroxide Released from Migrating Tumor Cells

<div><p>Cell migration is one of the key cell functions in physiological and pathological processes, especially in tumor metastasis. However, it is not feasible to monitor the important biochemical molecules produced during cell migrations <i>in situ</i> by conventional cell migration assays. Herein, for the first time a device containing both electrochemical sensing and trans-well cell migration modules was fabricated to sensitively quantify biochemical molecules released from the cell migration process <i>in situ</i>. The fully assembled device with a multi-wall carbon nanotube/graphene/MnO₂ nanocomposite functionalized electrode was able to successfully characterize hydrogen peroxide (H₂O₂) production from melanoma A375 cells, larynx carcinoma HEp-2 cells and liver cancer Hep G2 under serum established chemotaxis. The maximum concentration of H₂O₂ produced from A375, HEp-2 and Hep G2 in chemotaxis was 130±1.3 nM, 70±0.7 nM and 63±0.7 nM, respectively. While the time required reaching the summit of H₂O₂ production was 3.0, 4.0 and 1.5 h for A375, HEp-2 and Hep G2, respectively. By staining the polycarbonate micropore membrane disassembled from the device, we found that the average migration rate of the A375, HEp-2 and Hep G2 cells were 98±6%, 38±4% and 32 ±3%, respectively. The novel bi-module cell migration platform enables <i>in situ</i> investigation of cell secretion and cell function simultaneously, highlighting its potential for characterizing cell motility through monitoring H₂O₂ production on rare samples and for identifying underlying mechanisms of cell migration.</p></div

Amperometric performance (<i>i-t</i> curve) of fully assembled device.

<p>(A) <i>i</i>-<i>t</i> curves of successive additions of H₂O (<i>a</i>) or 4 μM H₂O₂ (<i>b</i>) into RPMI 1640 at an applied potential of -0.4 V <i>vs</i> ITO RE/CE; (B) <i>i-t</i> 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 <i>vs</i> ITO RE/CE; Inset of (B) <i>i-t</i> 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</p