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

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 in situ 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 in situ. 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 in situ 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.


Introduction
Cell migration plays a role in many physiological and pathological processes, including tumor metastasis. [1][2][3] It is a physical and chemical multistep cycle including extension of a protrusion, formation of stable attachments near the leading edge of the protrusion, translocation of the cell body forward, and release of adhesions and retraction at the cell rear. [4][5][6] Cell endeavours have focused on establishing a microenvironment that mimics in vivo conditions for cell migration and analysis of migration at a single cell level. [9,[42][43][44] But, those achievements mainly illustrated the morphology and functional changes of cells during cell migration. No studies have been reported to study biochemical molecule generation during the cell migration process.
In this work, an electrochemical sensor embedded poly(dimethylsiloxane) (PDMS) device was developed to monitor H 2 O 2 in situ during tumor cell migration process. To achieve this goal, a multi-wall carbon nanotube (MWCNT)/graphene/MnO 2 composite functionalized indium tin oxide (ITO) glass electrode was fabricated as a H 2 O 2 sensing module. This H 2 O 2 sensing module was assembled with a cell migration module that is a PDMS chamber/ polycarbonate membrane/PDMS chamber sandwich structure. The fully assembled bi-module device in situ sensed H 2 O 2 production of human melanoma cell migration under a serum established chemotaxis field. The effect of the cell H 2 O 2 production inhibitor, diphenyleneiodonium (DPI), and H 2 O 2 decomposition enzyme, catalase, on cell migration was also investigated on assembled devices. H 2 O 2 generation and migration capability measured with assembled devices were interpreted with standard Boyden transwell assays and the results confirmed that the fully assembled bi-module device could indeed monitor H 2 O 2 in situ during cell migration.

Materials and Methods Materials
Graphite, multi-walled carbon nanotubes (MWCNT), ascorbic acid, 30% hydrogen peroxide, potassium hexacyanoferrate (III) (K 3 [Fe(CN) 6 ]), Nafion were purchased from Aladdin, China. Phosphate buffered saline (PBS), potassium permanganate (KMnO 4 ) were from Chongqing co. Indium tin oxide (ITO) glass and silver paste were obtained from Jieshen Electronics Technology CO. Ltd (China). Printed circuit broad (PCB) UV photosensitive dry film (40 μm) was obtained from IC Machinery Equipment Group (China). Human melanoma cells, A375, were obtained from ATCC. Human liver carcinoma cell line Hep G2 and human Larynx carcinoma cell line HEp-2, gifts from Dr. Yuan Li (Chongqing Medical University), were originally purchased from China Center for Type Culture Collection. The cells were maintained in RPMI 1640 medium (Gibco) with 10% fetal bovine serum (Gibco), 100 μg mL -1 penicillin and 100 μg mL -1 streptomycin. Phorbol 12-myristate-13-acetate (PMA), di-phenyleneiodonium (DPI), catalase and Whatman Cyclopore polycarbonatemembrane (cyclopore PC circles, 5.0 μm) were purchased from Sigma Aldrich. PMA and DPI were dissolved with dimethyl sulfoxide (DMSO) to a concentration of 5 mg mL -1 and 10 mM, respectively, as a stock solution. All other chemicals used in this study were analytical grade. The deionized (DI) water used in all experiments was produced by a Q-Grad1 system, Millipore Corporation.

Bi-module device design and fabrication
As depicted in Fig 1A, the device consists of two modules: an electrochemical sensor for H 2 O 2 detection and a micro trans-well platform for cell migration. From bottom to top, there are an electrochemical sensor, a PDMS chamber, a polycarbonate micropore membrane and another PDMS chamber. The micropores on the polycarbonate membrane are the channel for cells to transport from the upper chamber to the bottom chamber. Fig 1B describes the schematic diagram of the micro-fabrication process for the bi-module device. A two-electrode system was chosen to build the electrochemical sensor. The size of the working electrode and reference/ counter electrode is 4.91 mm 2 and 12.8 mm 2 , respectively. To fabricate electrodes using ITO glass, a dry photosensitive film (40 μm) was coated on the ITO glass and patterned following a UV photolithography process. ITO layer that was not covered by photosensitive film was dissolved by immersing the chips in etchant solution (37%HCl: H 2 O:FeCl 3 •6H 2 O = 3L:1L:25g) for 30min. Finally, the patterned electrodes were recovered by removing the residue photosensitive film (a). A PDMS ring with a diameter of 5 mm and a height of 1mm was treated by plasma cleaner (Harrick, PDC-002) for 60 second and then bonded with ITO electrodes (b). To assemble a transwell chamber for assaying cell migration, a polycarbonate membrane was placed on top of the PDMS ring (c). Finally, another PDMS ring with a diameter of 5 mm and height of 5 mm was assembled on top of the polycarbonate membrane (d).

Electrochemical device for hydrogen peroxide analysis
The sensing material for hydrogen peroxide (H 2 O 2 ) detection used in this study was a MWCNT/graphene/MnO 2 aerogel. This functional material was synthesized according to our previous study. [41] In brief, a mixture containing MWCNT (1mg mL -1 ), graphene oxide (1mg mL -1 ) and KMnO 4 (10mg mL -1 ) was prepared and stirred at room temperature for 16 h. Then, the reaction mixture was centrifuged to collect the precipitate. Next, the re-suspended precipitate was mixed with ascorbic acids solution (100 mg mL -1 ) at 50°C for 15h to form a MWCNT/ graphene/MnO 2 hydrogel, and then freeze-dried for 24 h to completely remove water. The obtained aerogel (MWCNT/graphene /MnO 2 ) was dispersed in 500 μL of ethanol (5 mg/mL) and casted onto the surface of ITO working electrodes. The MWCNT/graphene/MnO 2 -functionalized electrode was characterized by cyclic voltammetry (CV) in 0.5 M KCl solution containing 50 mM K 3 Fe(CN) 6 at the scan rate of 10 mVs -1 . Then the amperometric response of the fully assembled electrochemical sensor to H 2 O 2 was characterized with RPMI 1640 medium according to the literature. [41] To analyse the stability of the MWCNT/graphene/MnO 2 functionalized electrode, the electrode was immersed in the cell culture medium for 24 h. The CV response of the electrode was recorded when adding H 2 O 2 (4 μM) in to the cell culture medium at 0, 12, 18 and 24 h and the changes of reduction peak current of the CV curve was compared.
Quantification of H2O2 production from migrating cell in a fully assembled device Human melanoma A375 cells, liver cancer Hep G2 and larynx carcinoma HEp-2 cells were cultured in RPMI 1640 medium supplemented with 10% FCS under standard conditions (37°C, 5% CO 2 ). Fig 2 illustrates the measurement settings. A functional sensing material, MWCNT/graphene/MnO 2 in ethanol (5 mg/mL), was cast on the working electrode. Then one microliter of Nafion that was diluted in ethanol (1:30, V/V) was casted (a). 50 μL of RPMI 1640 medium, with or without serum, was placed in the bottom PDMS chamber. A polycarbonate membrane and another PDMS ring were assembled on top of the bottom chamber in order (b). Next, serumstarved tumor cells (1×10 6 ) in 100 μL serum-free RPMI 1640 medium were placed into the top chamber (c). All steps were conducted in a biological hood with caution to avoid microorganism contamination. Finally, the device with cell-loading was placed in a cell culture incubator maintaining stable temperature and CO 2 atmosphere (37°C, 5% CO 2 ). The copper wires were linked to an electrochemical station (CHI 760) and the amperometric signal (i-t curve) was recorded for 12 h (d). The signal from the device without cell loading was recorded as a basal control. Cells incubated with a H 2 O 2 generation inhibitor DPI (10 μM) and a H 2 O 2 decomposer catalase (5 μg mL -1 ) were measured in parallel. Since the DPI and PMA were dissolved in DMSO, the impact of this organic solvent (0.5%, V/V) on H 2 O 2 production was evaluated. After electrochemical measurement, the polycarbonate membrane in the device was disassembled and the migrated cells were visualized by hematoxylin and eosin (H&E) staining. In brief, the membrane was immersed in 4% paraformaldehyde solution for 10 min and then stained by hematoxylin and eosin solution for 10 and 2 min, respectively. Finally, loosely attached cells on topside of the membrane were removed by scrubbing twice with cotton tipped swab. [48] The cell visualized on the bottom side of the membrane was defined as migrating cell. Six randomly selected fields per membrane were imaged (Olympus IX73, Japan) and the number of the purple-stained cells was counted. The percentage of migrated cells was calculated using medium without serum in the bottom chamber as a reference. All experiments were repeated three times independently.

Statistical analysis
Results are expressed as means ± the standard error of the mean (SEM). The data were analyzed by Student's t-test using Origin Statistic software (OriginLab Corporation, USA). A pvalue < 0.05 was considered significant.

Electrochemical characterization of the assembled device
Our previous study demonstrated that MWCNT/graphene/MnO 2 specifically responses to H 2 O 2 . [41] To evaluate the stability of the sensor that immersed in cell culture medium for 24 h, 1 mM H 2 O 2 was added into the medium at 0, 12, 18 and 24 h, and the CV response was recorded. The H 2 O 2 induced peak current change (S1 Fig) shows that immersing the MWCNT/ graphene/MnO 2 decorated electrode in cell culture medium for 24 h would not attenuate the function of the sensor. To realize H 2 O 2 production in situ, attention has been paid on the amperometric response of the MWCNT/graphene/MnO 2 functionalized device to subsequent additions of H 2 O 2 in cell culture medium (RPMI 1640). First, the choice of the applied potential at the working electrode is optimized to achieve a higher sensitivity. The amperometric i-t curves under potentials between -0.3 V and −0. . With the addition of catalase that can decompose H 2 O 2 to water and oxygen, the reduction peak current increase caused by PMA injection decreases sharply (line: cell response). It has been reported that H 2 O 2 can diffuse through cellular membranes to a distance even nearly 1mm because of its solubility in both lipid and aqueous environments and comparatively low reactivity. [33,49] To investigate the effect of the cell location on H 2 O 2 detection, we measured the production of H 2 O 2 from cells growing in the upper and bottom PDMS chamber of the assembled device. A similar current intensity was observed from cells seeded in upper and bottom chamber upon PMA challenge (inset of Fig 3B), indicating the electrochemical sensor located at the bottom chamber can in situ sense H 2 O 2 secreted from cells seeding in the top chamber.
Quantification of H 2 O 2 generation during cell migration by electrochemical devices  cells that was monitored for 12 h at 37°C. The current baseline of the electrochemical device during a 12 h incubation time was recorded without cell loading (control 1). No visible current change was observed indicating the H 2 O 2 will not automatically be generated from the medium during 12h incubation. The signal from cells that were seeded in a device with serum free medium in the bottom chamber was characterized as a migration control (cell 1, no serum in top and bottom chamber). The amperometric track shows a current increase (16-28 nA) at the time course of 2-4 h, while the current gradually flows back during 5-7 h and stays stable during the rest of the assay time. We tested the H 2 O 2 generated from cells seeded in a device in which RPMI 1640 medium plus 10% FBS (conditioned medium) was placed in the bottom chamber. The in situ measurement shows a cathode current increase trend and the current change reaches the maximum 84 ±1 nA at 3h. For the rest of the time, the current gradually traced back to baseline (cell 2, migration). To specify that the amperometric signal was indeed given by H 2 O 2 production during cell migration, NADH oxidase inhibitor DPI (10 μM) and H 2 O 2 decomposer catalase (5 μg mL -1 ) were used to pre-treat cells loaded in the upper chamber. Cell response 3 is the current signal from DPI pre-treated cells that were seeded in a device containing conditioned medium in the bottom chamber. A maximum current increase (23 ±1 nA) can be read from the i-t curve. While, for cells incubated with catalase, a similar i-t curve was recorded. The impact of DMSO (solvent of DPI) on H 2 O 2 productionwas measured in a cell migration section. Fig 4B shows the histogram of current change at a time point of 3h. The highest current change (84±1 nA) is given by cells responding to medium containing 10% serum. The current value obtained from serum-starved cells incubated with DPI and catalase in devices that contained conditional medium (RPMI 1640 plus 10% FBS) in the bottom chamber are 23±1 nA and 32±2 nA, respectively, which are significantly lower than the non-pretreated cells. Previous studies argued that DPI shows paradoxical effect in inducing DNA damage, mitochondria dysfunctional and even apoptosis. [50][51][52] To investigate if the small current increase was caused by DPI impaired cell growth, we compared the viability of cells pre-treated by DPI, catalase and DMSO using MTT method (S3 Fig). The results show that DPI (10 μM) or catalase (5 μg mL -1 ) does not reduce the viability of melanoma A375 cell.
In addition, according to the sensitivity of the electrochemical device as characterized in Fig  3A, H  To examine if the H 2 O 2 production is associated with cell migration in the 12 h period, a migration experiment was conducted parallel to quantifying H 2 O 2 with bi-module devices in situ. The polycarbonate membrane disassembled from the device was stained by hematoxylin and eosin (H&E) solution. Fig 5A shows the representative H&E staining images of polycarbonate membranes that was placed on a glass slide. The rod-like objects in all images are the micropore of the membrane. The migrating cells are characterized as purple-staining spot. More cells are observed from polycarbonate membrane that was disassembled from device containing serum-starved cell in the upper chamber and conditioned medium in the bottom chamber. Purple-staining cell sharply reduced on membrane from DPI and catalase pre-treatment groups. By counting the purple-staining cells from six random recorded microscopy images, the migration percentage was calculated using the group with no serum medium in bottom and top chamber as a reference. The results show that serum established chemotaxis induces 133±5% and 143±6% cell migration at 3, and 6 h (Fig 5B). Cells incubated with H 2 O 2 generation inhibitor DPI can significantly reduce the chemotaxis triggered migration. The catalase pre-treated cells also show less migration (-17±2% and 14±2% cell migrated at time of 3 and 6 h). Collectively, cell migration experiment on the bi-module device showed that serum-starved cells under serum-established chemotaxiscan produce H 2 O 2 , while the production of H 2 O 2 is associated with cell motility.
Next, a Boyden chamber assay was conducted side-by-side to quantify cell migration. The results demonstrate that cells loaded with different cell densities can develop cell migration under serum-established chemotaxis (S4 Fig). Comparing with standard Boyden chamber assays, wound healing assay and previous reported on-chip cell migration platforms (Table 1), the bi-module device not only capable for study the morphology and functional changes of cells during cell migration, but monitor the generation of H 2 O 2 , an important reactive oxygen species having pathology and physiology significance.
Since cell motility is an important factor associated with tumor metastasis, we studied three types of tumor cells with bi-module devices. A375 cells are a well-recognized malignant  (Fig 6B) and the corresponding H 2 O 2 concentration was 130±1.3 nM, 70±0.7 nM and 63±0.7 nM, respectively. The number of H 2 O 2 molecule produced per cell is 6.5×10 10 , 3.6×10 10 and 3.2×10 10 for A375, HEp-2 and Hep G2 cell, respectively, calculating from the amperometric signal according to literatures. [33,41] Comparing to PMA triggered H 2 O 2 production [33,41], the H 2 O 2 molecule produced per cell under serum established chemotaxis is one order smaller. In a parallel experiment, we examined the migrating cells by counting the purplestaining cells on the polycarbonate membrane of the device. As shown in Fig 6C, more A375 cells can be observed from the H&E stained polycarbonate membrane. The average increased migrating cell of A375, HEp-2 and Hep G2 cells after 12 h incubation are 98±7%, 38±4% and 32±3%, respectively (Fig 6D). The quantitative analysis in Fig 6B and 6D confirms that a H 2 O 2 production corresponds to cell motility. As characterized in supplementary S5 Fig, CV response of serum-starved cell in serum-free RPMI 1640 and RPMI 1640 are nearly identical. While adding of serum (10% FBS) into the serum-free medium leads to a reduction peak current increasing, indicating that serum would induce production of H 2 O 2 from serum-starved cell. The phenomena is in line with documented information that growth factors can stimulate NADPH oxidase leading to the production of H 2 O 2 . [53][54][55] The elevated endogenous H 2 O 2 might trigger the activation of ERK and FAK signalling transduction pathways. The phosphorylation of ERK and FAK can lead to enhanced cell migration by activation downstream signalling proteins. [14,53,54] As illustrated in Fig 7, by using this device, for the first time, we quantified the H 2 O 2 production in a transwell cell migration setting. We anticipate the combination of electrochemical sensing with trans-well module can quantify other important biochemical molecules in situ, providing key information for depicting the relationship between biochemical signalling and cell function.

Conclusions
Hydrogen peroxide is believed to modulate signalling pathways that control cell motility. However, little is known about H 2 O 2 generation during the cell migration process. A novel bimodule device was fabricated to characterize H 2 O 2 production in situ while monitoring cell migration capability. For the first time, we quantified H 2 O 2 molecule generation from cells under a serum established chemotaxis is~6.5×10 10 per melanoma A375 cells,~3.6×10 10 per liver carcinoma Hep G2 cells and~3.2×10 10 per larynx carcinoma HEp-2 cells. In addition, a parallel migration assay with H 2 O 2 generation inhibitor and decomposer demonstrated that the H 2 O 2 generation is associated to cell migration. The bi-module cell migration platform enables in situ investigation for monitoring H 2 O 2 production and cell function simultaneously, highlighting its potential for characterizing cell motility through monitoring cell secretion with rare samples and for investigation of mechanism of cell migration.  production from serum-starved cells by direct serum stimulation. Melanoma A375 cells were serum-starved for 8 h and then collected. RPMI 1640 medium was placed in the PDMS chamber and CV response was recorded. Then serum-starved cell (4×10 5 ) was pipetted into the chamber. After 10 min, the CV response was recorded. Finally, serum (10% FBS) was added into the chamber. The CV response was recorded after 30 min incubation. (TIF)