Biomimetic Graphene-Based 3D Scaffold for Long-Term Cell Culture

Subscriber access provided by UNIV OF MISSOURI ST LOUIS

Article

Biomimetic Graphene-Based 3D Scaffold for Long-Term Cell Culture and Real-Time Electrochemical Monitoring Xue-Bo Hu, Yan-Ling Liu, Wen-Jie Wang, Hai-Wei Zhang, Yu Qin, Shan Guo, Xin-Wei Zhang, Lei Fu, and Wei-Hua Huang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03324 • Publication Date (Web): 18 Dec 2017 Downloaded from http://pubs.acs.org on December 19, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Biomimetic Graphene-Based 3D Scaffold for Long-Term Cell Culture and Real-Time Electrochemical Monitoring Xue-Bo Hu,† Yan-Ling Liu,*† Wen-Jie Wang,# Hai-Wei Zhang,† Yu Qin,† Shan Guo,† Xin-Wei Zhang,† Lei Fu,# and Wei-Hua Huang*† †Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China #College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China * E-mail: [email protected] * E-mail: [email protected]

1

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Current achievements on electrochemical monitoring of cells are often gained on two-dimensional (2D) substrates, which fail in mimicking the cellular environments and accurately reproducing the cellular functions within a three-dimensional (3D) tissue. In this regard, 3D scaffold concurrently integrated with the function of cell culture and electrochemical sensing is conceivably a promising platform to real time monitor cells under their in vivo-like 3D microenvironments. However, it is particularly challenging to construct such a multifunctional scaffold platform. Herein, we developed a 3-aminophenylboronic acid (APBA) functionalized graphene foam (GF) network, which combines the biomimetic property of APBA with the mechanical and electrochemical properties of GF. Hence, the GF network can serve as a 3D scaffold to culture cells for a long period with high viability and simultaneously as an electrode for highly sensitive electrochemical sensing. This allows monitoring of gaseous messengers H2S released from the cells cultured on the 3D scaffold in real time. This work represents considerable progress in fabricating 3D cell culture scaffold with electrochemical properties, thereby facilitating future studies of physiologically relevant processes.

2


Page 2 of 21

Page 3 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Electrochemical sensing has attracted extensive attention in real-time monitoring of biochemical molecules released from living cells, owing to its unique characteristics, such as high sensitivity, easy miniaturization and high temporal-spatial resolution.1-7 Studies on electrochemical monitoring of cell relevant biomolecules have notably promoted the understanding of basic cellular and physiological biology.1-7 However, present researches in this field are often achieved on two-dimensional (2D) substrates (i.e. cells are cultured to adjust to an artificial flat, rigid surface),6-9 which fail in mimicking the complex and dynamic environments of the cells, and are some way off in accurately reproducing the cellular functions within a 3D tissue.10-12 To overcome the limitations of 2D culture, great efforts have been made on developing three-dimensional (3D) culture models.10-18 But the materials, known as matrices or scaffolds, in these 3D models often suffer poor electrical conductivity and fail in electrochemical sensing of biochemical molecules released from living cells. To be the matrix or scaffold for 3D cell culture and electrochemical monitoring, the materials should not only have biocompatible porous structures to facilitate effective

transportation

of

nutrients,11-12,18-21

but

also

demonstrate

high

electrochemical performance towards biochemical molecules. In this regard, a pioneering work has demonstrated the feasibility by photolithographically fabricating pillar arrays of pyrolysed carbon for inducing differentiation of human neural stem cells and sensing of released dopamine.22 Recent development in tissue engineering show that graphene is also a promising cell scaffolds in numerous bioapplications, 3



owing to its high surface area, excellent electrical conductivity, strong mechanical strength, remarkable biocompatibility and ease of functionalization.8-9,23-29 In particular, graphene foam (GF), synthesized by convenient chemical vapor deposition,30 is an excellent 3D porous structure with a high specific surface area that allows the growth of cells, and the interconnected pores also support the cellular communication and ensure the efficient mass transport of nutrients for cells.31-32 Moreover, GF possesses micro-scale topographic features such as curvatures or anisotropic microstructures, which is different from 2D graphene-based materials so that cell adhesion might be enhanced by these various surface properties.31,33 Therefore, GF is a promising candidate for a 3D cell scaffold as it incorporates structural (3D porous structure) and electrical (electrochemical property) cues in the same scaffold to provide a 3D environment for cell culture. But to be a scaffold for long-term cell culture and electrochemical monitoring, further functionalization of GF surface is still required to improve the cell affinity of GF.31, 33-34 Among the materials that can be functionalized on the surface to fabricate high cell affinity surface, small cell-adhesive molecules demonstrate to be appropriate choices owing to advantages of controlled modification on irregular shaped electrode and introducing less sensitivity drop in electrochemical sensing.35-39 As a small cell-adhesive molecule, 3-aminophenylboronic acid (APBA) can react with the 1,2- or 1,3-diols in carbohydrate moieties on cell membranes and thus fabricate a more cytocompatible surface for cell adhesion and proliferation.40-43 Herein, we report a APBA functionalized GF networks for cell culture and electrochemical sensing. The 4


Page 4 of 21

Page 5 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


synergetic properties of the 3D porous networks and cell-adhesive molecule endow the networks with superior properties to be a multifunctional scaffold. Cells can grow and proliferate along the skeletons of the 3D porous scaffold and exhibited high viability after the cells were cultured for a long period. Meanwhile, the 3D electrode demonstrates high sensitivity and fast response in electrochemical sensing. As a proof of concept, the release of gaseous messenger H2S from HeLa cells cultured on the 3D scaffold for different time period has been successfully monitored in real time. Taken together, this work demonstrates that a 3D scaffold for long-term cell culture and an electrode for real-time electrochemical sensing can be integrated into a single biomimetic 3D device, indicating its great potential in better comprehending the physiological processes.

EXPERIMENTAL SECTION Materials and Instruments. Nickel foam (1.65 mm in thickness and ~ 320 g m-2 in areal density) was obtained from Alantum Advanced Technology Materials (Shenyang, China). Cysteine (Cys), DL-Propargylglycine (cystathionine γ-lyase inhibitor, PAG), 1-pyrenebutyric

acid

N-hydroxysuccinimide

ester

(denoted

as

PBA)

and

3-aminophenylboronic acid monohydrate (denoted as APBA) were purchased from Sigma-Aldrich. HeLa cells and GFP-HeLa cell (constitutive expression of green fluorescent protein in the cytoplasm of the HeLa cells) lines were obtained from China Center for Type Culture Collection. The cell culture medium and the related supplements

were

obtained

from

3',6'-Di(oacetyl)-4',5'-bis[N,N-bis(carboxymethyl)-aminomethyl] 5


Gibco

Corp. fluorescein,


tetraacetoxymethylester

Page 6 of 21

(Calcein-AM)

and

3,8-diamino-5-[3-(diethylmethylammonio)propyl]-6-phenylphenanthridinium diiodide (PI) for cell staining were obtained from Dojindo laboratory (USA). For all the experiments, deionized water (Millipore, 18.0 MΩ) was used. Other chemicals unless specified were reagent grade and used as obtained without further purification. Scanning electron microscopy (SEM) images were obtained by using a field-emission

microscope

(ZEISS

SIGMA)

and

Energy dispersive

X-ray

spectroscopy (EDX) images were obtained by using an INCAPentalFETx3 Oxford EDX spectrometer. Raman spectroscopy was performed with a laser micro-Raman spectrometer (Renishaw in Via, Renishaw, 532 nm excitation wavelength). X-ray photoelectron spectroscopy (XPS) measurements were conducted by ESCALAB 250Xi photoelectron spectrometer (Thermo Fisher) with Al Kα X-ray radiation as the X-ray source for excitation. The measuring spot size was 500 µm, and the binding energies were calibrated by referencing the C 1s peak (284.6 eV). Fourier-transform infrared spectroscopy (FTIR) was measured on a NICOLET 5700 FTIR Spectrometer (Thermo, USA). Inverted fluorescent microscopes (AxioObserver Z1 and Axiovert 200M, Zeiss) and confocal microscope (Perkin Elmer UltraView Vox System, USA) were used for the observation of cells. Electrochemical measurements were carried out on a CHI 660A electrochemical workstation (CHI Instruments) at room temperature. Fabrication of GF/PBA/APBA. Graphene foam (GF) was grown on nickel foam by the atmospheric pressure chemical vapor deposition (CVD) process. Firstly, the nickel 6


Page 7 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


foam was heated to 1000 °C in 30 min with a mixd gas of Ar (500 sccm) and H2 (50 sccm) in a CVD chamber (HTF 55322C Lindberg/Blue M), and kept the temperature at 1000 °C for 10 min without changing the gas flow. Then a small amount of CH4 (5 sccm) was brought into the reaction chamber for 5 min. After the furnace was cooled to room temperature naturally with the atmosphere of Ar (500 sccm), graphene was deposited on the surface of the nickel foam. To remove the nickel scaffold, the sample was placed into a diluted nitric acid solution (1M) for 24 h and then washed the GF by ultrapure water and ethanol for several times. In order to obtain the 1-pyrenebutyric acid N-hydroxysuccinimide ester modified GF composite (denoted as GF/PBA), GF was immersed in a solution of 0.5 mg/mL PBA for 24 h. After the composite was washed with ultrapure water for several times, it was immersed in a solution of 1 mg/mL 3-aminophenylboronic acid monohydrate for another 24 h. At last the final composite (denoted as GF/PBA/APBA) was put in an oven at 75 ºC for 2 hours. Cell culture and manipulation. HeLa and GFP-HeLa cells were cultured by Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin in a humidified incubator (37 ºC and 5% CO2, HERACELL 150i, Thermo Scientific). For SEM and electrochemical detection, HeLa cells with a density of ~1×106 cell/cm2 were seeded on the scaffolds. GFP-HeLa and HeLa cells with a density of 2×105 cell/cm2 were seeded on the scaffold for the observation of proliferation behavior and the viability of long-time cell culture, respectively. To measure the H2S released from the HeLa cells, the cells were stimulated with cysteine. In control experiments, the cells were stimulated with PBS, 7



or the cells were incubated with PAG for 30 min before stimulation by cysteine.

RESULTS AND DISCUSSION Preparation and characterization of the graphene-based 3D biomimetic scaffold. To fabricate the graphene-based 3D biomimetic scaffold, 3D graphene macroscopic structure with a foam-like network was firstly synthesized by template-directed chemical vapor deposition and subsequent template removal.30 Then, 1-pyrenebutyric acid N-hydroxysuccinimide ester with an aromatic structure, was assembled onto the surface of GF to further covalently couple small cell-adhesive molecule APBA via amide bond. As illustrated in Figure S1a, GF possesses a 3D seamlessly interconnected porous structure with freestanding ability,30 and the high-magnification SEM shows the smooth surface of GF without cracks. We can confirm from the inset of Figure S1a-b that after immobilization of PBA, the surface of GF is covered by a continuous film, which lay the foundation for the further functionalization of APBA. After APBA was covalently bonded on GF/PBA, a uniform film was formed. The high-magnification SEM image of the GF/PBA/APBA (Figure S1c, generated by the existence of uncovered parts during the two-step functionalizations) reveals that the surface of scaffold is composed of three layers (1st layer: GF, 2nd layer: PBA, 3rd layer: APBA). The whole macrostructure of GF (Figure S1a), GF/PBA (Figure S1b) and GF/PBA/APBA (Figure 1b) demonstrated to be the 3D interconnected porous networks, indicating the layer-by-layer modification procedure caused no structural collapse and the freestanding ability of GF could be maintained. EDX, XPS and FTIR characterization was then conducted to further confirm that 8


Page 8 of 21

Page 9 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the APBA had been successfully immobilized on the scaffold. Figure 1c shows a SEM image of the skeleton of GF/PBA/APBA and the corresponding EDX elemental mapping images of C (red), B (blue) and N (yellow), which suggest the immobilization and the homogeneous distribution of APBA. The wide survey XPS spectrum of GF/PBA/APBA, as displayed in Figure 1d, shows predominant signals of oxygen, nitrogen, carbon and boron (red rectangle and magnified as the inset). The spectrum of B 1s is fit with three components at 189.7 eV, 191.7 eV, and 192.7 eV, assigned to B–C, C–B–O and B–O bond, respectively.43-44 The core spectrum of C 1s (Figure 1e) clearly clarify five typical separated peak components, corresponding to C–H/C–C (284.6 eV), C–B (285.3 eV), C–N (285.8 eV), C–O (286.6 eV) and C=O (288.8 eV) groups in GF/PBA/APBA, respectively.45-46 The surface property of GF/PBA/APBA was further investigated by FTIR spectroscopy and the result is presented in Figure S2. The presence of vibration mode at 1633 cm-1 belongs to the C=O and C–N stretching induced by the formation of amide linkages between the carboxylic groups of PBA and the amine groups of APBA.47 Other peaks centered at 1096, 1340, 1578, 1553 and 1646 cm-1 can be assigned to the B–C stretching mode, B–O stretching mode, carboxyl stretching mode and amine group, respectively.47-48

9



Figure 1. (a) Low-magnification SEM image of GF/PBA/APBA scaffolds. (b) High-magnification SEM image of single GF/PBA/APBA skeleton and the inset shows the enlarged SEM images of the red rectangle. (c) SEM image of GF/PBA/APBA and the corresponding EDX elemental mapping images of C (red), B (blue) and N (yellow). (d-e) XPS of GF/PBA/APBA, in which (d) corresponds to the wide survey spectrum of the composites (inset: B 1s states) and (e) corresponds to C 1s states. (f) Raman spectra of GF (red line) and GF/PBA/APBA (black line).

The Raman spectra of GF (red line) and GF/PBA/APBA (black line) are presented in Figure 1f. The intensity of the G (1580 cm-1) and 2D (2720 cm-1) band demonstrates the multilayer property of GF,30 which makes it sufficiently rigid to serve as a scaffold for cell culture. In the Raman spectrum of GF/PBA/APBA, the small D peak located at 1350 cm-1 (ID/IG = 0.059) indicates that very few defects are introduced to the GF during the functionalization of PBA and APBA. This makes GF/PBA/APBA appropriate to serve as a high conductivity scaffold for electrochemical sensing. Taken together, the above results demonstrate the advantages of this mild fabrication procedure in obtaining GF-based composites, maintaining the mechanical and electrical properties of the scaffold. 10


Page 10 of 21

Page 11 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Electrochemical behaviors of GF, GF/PBA and GF/PBA/APBA. To test the electrochemical properties of the obtained 3D electrodes, the electrochemical responses of the electrodes towards [Ru(NH3)6]2+/3+ were investigated. We can confirm from Figure 2a that the layer-by-layer functionalization did not induce too much negative effects on electron transport, demonstrating the good electrochemical property. As a signaling molecule in biology, H2S is involved in the regulation of physiological and pathological processes, such as neuromodulation, regulation of vascular tone and reduction of metabolic rate.49-52 In order to comprehensively unravel its diverse functions, real-time monitoring of H2S released from living cells is essential but less works has been conducted on this field. Herein, the electrochemical behavior of the electrode toward H2S was investigated, which shows an oxidation peak centered at about 0.25 V versus Ag/AgCl (red line in Figure 2b). No obvious oxidation peak appeared in the cyclic voltammograms of GF/PBA/APBA in PBS solution without H2S (black dash line in Figure 2b), confirming that the current was generated by the oxidation of H2S. The amperometric results of GF/PBA/APBA (Figure 2c), GF/PBA and GF (Figure S3) electrode towards H2S were investigated and compared. The GF/PBA/APBA electrode reveals stable current responses to a series of H2S concentration and the responses to 200 nM H2S can be clearly observed from the inset of Figure 2c. In addition, the fast response of the electrode towards H2S arisen from the highly conductive pathway for electrons can be also confirmed. The detection limit of GF/PBA/APBA electrode was calculated to be 50 nM, which is the lowest of 11



those previously reported.53-56 The sensor could response sensitively and reproducibly to the oxidation of H2S in the concentration range of 0.2 µM to 10 µM, only with slight degradation for high concentration (10 µM) probably owing to the adsorption of oxidation products.57 The sensitivity of the GF/PBA/APBA electrode in the linear range of 0.2 µM to 10 µM was calculated to be 19.2 nA/µM (Figure 2d). The results indicate that the GF/PBA/APBA electrode shows good electrochemical performance towards H2S, making it appropriate to serve as a 3D electrode for real-time monitoring of H2S from cells.

Figure 2. (a) Cyclic voltammograms of GF (blue line), GF/PBA (red line) and GF/PBA/APBA (black line) electrode obtained in 1 mM [Ru(NH3)6]2+/3+ in 1M KCl at a scan rate of 100 mV/s. (b) Cyclic voltammograms of GF/PBA/APBA electrode in the absence (black dash line) and presence (red line) of 2 mM H2S in deaerated PBS solution at a scan rate of 100 mV/s. (c) Amperometric response of GF/PBA/APBA electrode to a series of H2S concentration increases in a stirred deaerated PBS solution at a potential of +0.50 V (vs. Ag/AgCl). The response of GF/PBA/APBA to 200 nM H2S is magnified in the inset. (d) The corresponding calibration curve of GF/PBA/APBA electrode with increasing H2S concentration.

Cell adhesion and proliferation on GF, GF/PBA and GF/PBA/APBA scaffold. In order to demonstrate the performance of the biomimetic 3D scaffold for cell adhesion, 12


Page 12 of 21

Page 13 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


GFP-HeLa cells were seeded on GF and GF/PBA/APBA scaffold. After the cells were cultured for 12 h, almost no attached cells were observed on the skeletons of the GF scaffold (Figure 3a). While for the GF/PBA/APBA scaffold, a large number of spindle-shaped cells adhered on the surface and edge of the skeletons (Figure 3b), indicating the ability of the cell-binding molecule (APBA) in promoting cell adhesion. To demonstrate that cells grew on a 3D scaffold, confocal microscopy was used to observe the cells cultured for another 12 h. As shown in Figure 3c, cells adhered along the skeletons of the scaffold and the inset illustrates that the attached cells distributed all around the surface (on different heights). The numerous spindle-shaped cells observed by SEM (Figure 3d) further clearly demonstrated that cells attached and grown very well on the 3D scaffold. The hanging cells on the edge of the skeleton (Figure 3d, inset) indicate the strong pseudopodia/scaffold interaction. In general, the above results obviously reveal the favorite cell-adhesive ability of the biomimetic scaffold introduced by APBA. The proliferation behavior of the cells adhered on the scaffold was also investigated. For the GFP-HeLa cells cultured on the same skeleton for 48 h and 72 h (Figure S4), the number of cells increased obviously with the extension of culture time though there was difficulty in precisely counting cell numbers on a complex 3D scaffold to obtain proliferation curve. The larger microscopic field shown in Figure S5 also confirmed the proliferation behavior of the cells adhered on the scaffold (cells cultured for 2, 5 and 10 days). The good cell proliferation ability may be attributed to the 3D porous structure to facilitate mass transport of nutrition for cell 13



metabolism.31-32 Cell viability was then characterized by the fluorescent live/dead cell markers Calcein-AM and PI after HeLa cells being cultured for up to 11 Days (the culturing time is dependent on the initial density of cells seeded on the scaffold). The results show that the live cells covered all parts of the scaffold (Figure 3e and S6) and almost no dead cells were found. The corresponding confocal microscopy images (Figure S7 and Movie S1) also illustrated the high viability of cells on the scaffold, further demonstrating the prominent biocompatibility of GF/PBA/APBA scaffold for long-time cell culture.

Figure 3. Microscopic images of GFP-HeLa cells cultured on (a) GF and (b) GF/PBA/APBA

composites for 12 h. (c) Representative 3D reconstruction of constructs from confocal microscopy images for GFP-HeLa cells cultured on GF/PBA/APBA composites for 24 h. The inset shows the z-plane cross-section in the direction of the white arrows. d) SEM images of cells cultured on GF/PBA/APBA composites for 24 h and the inset shows the enlarged image of single cell attached on the skeleton (red rectangle). (e) Microscopic images of HeLa cells cultured for 11 days and labeled with Calcein-AM (green) and PI (red). The bright field (BF) and merge of bright field and fluorescence field (Merge) images are presented. 14


Page 14 of 21

Page 15 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Real-time monitoring of H2S released from HeLa cells cultured on the 3D graphene-based biomimetic scaffold. Motivated by the good electrochemical performances and biocompatibility of GF/PBA/APBA, the biomimetic GF-based scaffold was applied to real-time monitor H2S released from HeLa cells cultured on the scaffold. By stimulating HeLa cells with cysteine which can be enzymatically catalyzed by cystathionine γ-lyase (CSE) to produce H2S (Figure 4a).49-52 Owing to the close distance between cells and electrode, H2S rapidly diffuses to the electrode surface to be amperometrically detected, resulting in the fast current rise (blue line in Figure 4b). This attributes to the 3D seamlessly interconnected network with high conductivity that ensures a 3D multiplexing and highly conductive pathways for electron transport (Figure 4a). After cells were incubated with PAG (CSE inhibitor)58-59 for 30 min, no obvious current increase was observed when cells were stimulated by cysteine (red line in Fig. 4b). Cells stimulated by sham stimulus (PBS solution) also led no obvious current rise (black line in Figure 4b). These results indicate that the amperometric signal was produced by H2S release from HeLa cells. In order to in situ monitor H2S produced during cell proliferation, H2S released from cells cultured for different time (6, 12 and 24 hours) were monitored, and the currents increase with the extension of culture time (Figure 4c). The current rise can be attributed to the increased H2S concentration produced by the growing number of cells, which further confirms that cells attached on the electrode show good proliferation behavior. Besides, the results also illustrate the long-term chemical stability of the electrode in cell culture environment. Taken together, the overall 15



results indicate the capability of GF/PBA/APBA electrode for real-time monitoring of H2S released from living cells growing on the 3D scaffold.

Figure 4. (a) Scheme showing real-time monitoring of H2S released from the attached cells on

GF/PBA/APBA scaffold. (b) Amperometric response of HeLa cells cultured on GF/PBA/APBA electrode for 12 hours in deaerated PBS solution under different conditions: stimulating with 2 mM cysteine (blue line), stimulating with 2 mM cysteine after the incubation of 0.2 mM PAG (red line), and stimulating with PBS solution (black line). (c) Amperometric response of HeLa cells cultured on GF/PBA/APBA electrode for different times: 6 hours (black line), 12 hours (blue line) and 24 hours (red line).

CONCLUSION In summary, we have fabricated a 3D biomimetic GF/PBA/APBA scaffold for long-term cell culture with high viability and real-time monitoring of H2S released from the cells cultured on the 3D scaffold. The incorporation of structural (3D porous structure), chemical (π-π and covalent interactions), and electrical (electrochemical property) cues in this scaffold has made considerable progress on integration of a multifunctional 3D cell culture platform. In view of the great significance and tremendous demand in real-time monitoring of cellular behaviors under 3D vivo-like 16


Page 16 of 21

Page 17 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


microenvironments to reflect in vivo cell functions, it is anticipated that this platform will open a new route to fulfil this challenge. Further functionalization on this 3D biomimetic scaffold with rational design will also promote study on various aspects such as tissue engineering, drug screening and clinical delivery. ASSOCIATED CONTENT Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org. SEM images of GF, GF/PBA and GF/PBA/APBA, FTIR spectrum of GF/PBA/APBA, amperometric response of GF and GF/PBA, microscopic images of GFP-HeLa cells cultured on GF/PBA/APBA for different times, and confocal microscopy images for HeLa cells cultured on GF/PBA/APBA composites for 11 days and labeled with Calcein-AM (green) and PI (red). AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] * E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. 17



Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (Nos. 21725504, 21675121, 21575110, 21375099) and the Open Funds of the State Key Laboratory of Electroanalytical Chemistry (SKLEAC201706).

18


Page 18 of 21

Page 19 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


REFERENCES (1) Wightman, R. M. Science 2006, 311, 1570–1574. (2) Schulte, A.; Schuhmann, W. Angew. Chem. Int. Ed. 2007, 46, 8760−8777. (3) Amatore, C.; Arbault, S.; Guille, M.; Lemaître, F.; Chem. Rev. 2008, 108, 2585−2621. (4) Huang, Y.; Cai, D.; Chen, P. Anal. Chem. 2011, 83, 4393−4406. (5) Zhang, M.; Yu, P.; Mao, L. Acc. Chem. Res. 2012, 45, 533−543. (6) Privett, B. J.; Shin, J. H.; Schoenfisch, M. H. Chem. Soc. Rev. 2010, 39, 1925−1935. (7) Bedioui, F.; Griveau, S. Electroanalysis 2013, 25, 587−600. (8) Liu, Y.; Dong, X.; Chen, P. Chem. Soc. Rev. 2012, 41, 2283–2307. (9) Vashist, S. K.; J. Luong, H. T. Carbon 2015, 84, 519–550. (10) Lee, J.; Cuddihy, M. J.; Kotov, N. A. Tissue Eng., Part B: Rev 2008, 14, 61–86. (11) Haycock, J. W. Methods Mol. Biol. 2011, 695, 1–15. (12) Page, H.; Flood, P.; Reynaud, E. G. Cell Tissue Res. 2013, 352, 123–131. (13) Imai, K.; Takeya, S.; Haruki, S. Cell Struct. Funct. 2000, 25, 329–336. (14) Cukierman, E.; Pankov, R.; Stevens, D. R; Yamada, K. M. Science 2001, 294, 1708–1712. (15) Schindler, M.; Nur-E-Kamal, A.; Ahmed, I.; Kamal, J.; Liu, H. Y.; Amor, N.; Ponery, A. S.; Crockett, D. P.; Grafe, T. H.; Chung, H. Y.; Weik, T.; Jones, E.; Meiners, S. Cell Biochem. Biophys. 2006, 45, 215–227. (16) Tibbitt, M. W.; K. Anseth, S.; Biotechnol. Bioeng. 2009, 103, 655–663. (17) Feng, Z. Q.; Chu, X.; Huang, N. P.; Wang, T.; Wang, Y.; Shi, X.; Ding, Y.; Gu, Z. Z. Biomaterials 2009, 30, 2753-2763. (18) Akhavan, O. J. Mater. Chem. B 2016, 4, 3169–3190. (19) Glicklis, R.; Shapiro, L.; Agbaria, R.; Merchuk, J. C.; Cohen, S. Biotechnol. Bioeng. 2000, 67, 344–353. (20) Yang, J.; Chung, T. W.; Nagaoka, M.; Goto, M.; Cho, C. S. Biotechnol. Lett. 2001, 23, 1385–1389. (21) Yang, F.; Murugan, R.; Ramakrishna, S.; Wang, X.; Ma, Y. X.; Wang, S. Biomaterials 2004, 25, 1891–1900. (22) Amato, L.; Heiskanen, A.; Caviglia, C.; Shah, F.; Zór, K.; Skolimowski, M.; Madou, M.; Gammelgaard, L.; Hansen, R.; Seiz, E. G.; Ramos, M.; Moreno, T. R.; Martínez-Serrano, A.; Keller, S. S.; Ramos, M. Adv. Funct. Mater. 2014, 24, 7042–7052. (23) Shao, Y.; Wang, J.; Wu, H.; Liu, J.; Aksay, I. A.; Lin, Y. Electroanalysis 2010, 22, 1027–1036. (24) Chen, D.; Tang, L.; Li, J. Chem. Soc. Rev. 2010, 39, 3157–3180. (25) Feng, L.; Liu, Z. Nanomedicine 2011, 6, 317-324. (26) Yang, Y.; Asiri, A. M.; Tang, Z.; Du, D.; Lin, Y. Mater. Today 2013, 16, 365–373. (27) Orecchioni, M.; Moyon, C. M.; Delogu, L. G.; Bianco, A. Adv. Drug Deliver. Rev. 2016, 105, 163–175. (28) Lin, J.; Chen, X.; Huang, P. Adv. Drug Deliver. Rev. 2016, 105, 242–254. (29) Shin, S. R.; Li, Y. C.; Jang, H. L.; Khoshakhlagh, P.; Akbari, M.; Nasajpour, A.; Zhang, Y. S.; Tamayol, A.; Khademhosseini, A. Adv. Drug Deliver. Rev. 2016, 105, 255−274. (30) Chen, Z.; Ren, W.; Gao, L.; Liu, B.; Pei, S; Cheng, H. M. Nat. Mater. 2011, 10, 424−428. (31) Li, N.; Zhang, Q.; Gao, S.; Song, Q.; Huang, R.; Wang, L.; Liu, L.; Dai, J.; Tang, M.; Cheng, G.; Sci. Rep. 2013, 3, 1604. (32) Severino, F. P. U.; Ban, J.; Song, Q.; Tang, M.; Bianconi, G.; Cheng, G.; Torre, V. Sci. Rep. 2016, 6, 29640. (33) Song, Q.; Jiang, Z.; Li, N.; Liu, P.; Liu, L.; Tang, M.; Cheng, G. Biomaterials 2014, 35, 6930−6940. (34) Li, Z.; Wang, H.; Yang, B.; Sun, Y.; Huo, R. Mater. Sci. Eng., C 2015, 57, 181−188. 19



(35) Li, L. M.; Wang, W.; Zhang, S. H.; Chen, S. J.; Guo, S. S.; Français, O.; Cheng, J. K.; Huang, W. H. Anal. Chem. 2011, 83, 9524–9530. (36) Robinson, J. T.; Jorgolli, M.; Shalek, A. K.; Yoon, M. H.; Gertner, R. S.; Park, H. Nat. Nanotechnol. 2012, 7, 180–184. (37) Guo, C. X.; Ng, S. R.; Khoo, S. Y.; Zheng, X.; Chen, P.; Li, C. M. ACS Nano 2012, 6, 6944–6951. (38) Wang, J.; Trouillon, R.; Lin, Y.; Svensson, M. I.; Ewing, A. G. Anal. Chem. 2013, 85, 5600–5608. (39) Mendes, P. M. Chem. Soc. Rev. 2013, 42, 9207–9218. (40) Monzo, A.; Bonn, G. K.; Guttman, A. TrAC-Trends Anal. Chem. 2007, 26, 423–432. (41) Jin, S.; Cheng, Y.; Reid, S.; Li, M.; Wang, B. Med. Res. Rev. 2010, 30, 171–257. (42) Zhong, X.; Bai, H. J.; Xu, J. J.; Chen, H. Y.; Zhu, Y. H. Adv. Funct. Mater. 2010, 20, 992–999. (43) Park, S.; Gildersleeve, J. C.; Blixt, O.; Shin, I. Chem. Soc. Rev. 2013, 42, 4310–4326. (44) Zhong, S.; Zhou, L.; Wu, L.; Tang, L.; He, Q.; Ahmed, J. J. Power Sources, 2014, 272, 344–350. (45) Chu, Z.; Kang, Y.; Jiang, Z.; Li, G.; Hu, T.; Wang, J.; Zhou, Z.; Li, Y.; Wang, X. RSC Adv. 2014, 4, 26855–26860. (46) Deng, Y.; Zhang, X.; Zhao, X.; Li, Q.; Ye, Z.; Li, Z.; Liu, Y.; Zhou, Y.; Ma, H.; Pan, G.; Pei, D.; Fang, J.; Wei. S. Acta Biomater. 2013, 9, 8840–8850. (47) Xing, M.; Fang, W.; Yang, X.; Tian, B.; Zhang, J. Chem. Commun. 2014, 50, 6637–6640. (48) Liu, Y. L.; Wang, X. Y.; Xu, J. Q.; Xiao, C.; Liu, Y. H.; Zhang, X. W.; Liu, J. T.; Huang, W. H. Chem. Sci. 2015, 6, 1853–1858. (49) Kimura, H. Mol. Neurobiol. 2002, 26, 13–19. (50) Szabó, C. Nat. Rev. Drug Discov. 2007, 6, 917–935. (51) Yang, G.; Wu, L.; Jiang, B.; Yang, W.; Qi, J.; Cao, K.; Meng, Q.; Mustafa, A. K.; Mu, W.; Zhang, S.; Snyder, S. H.; Wang, R. Science 2008, 322, 587–590. (52) Li, L.; Moore, P. K.; Trends Pharmacol. Sci. 2008, 29, 84–90. (53) Lawrence, N. S.; Jiang, L.; Jones, T. G. J.; Compton, R. G.; Anal. Chem 2003, 75, 2054–2059. (54) Lawrence, N. S.; Deo, R. P.; Wang, J. Anal. Chim. Acta 2004, 517, 131–137. (55) Li, D. W.; Qu, L. L.; Hu, K.; Long, Y. T.; Tian, H. Angew. Chem. Int. Ed., 2015, 54, 12758–12761 (56) Hu, X. B.; Liu, Y. L.; Zhang, H. W.; Xiao, C.; Qin, Y.; Duo, H. H.; Xu, J. Q.; Guo, S.; Pang, D. W.; Huang, W. H. ChemElectroChem, 2016, 3, 1998–2002. (57) Wang, S.; Liu, X.; Zhang M. Anal. Chem. 2017, 89, 5382−5388. (58) Abeles, R. H.; Walsh, C. T. J. Am. Chem. Soc. 1973, 95, 6124–6125. (59) Washtien, W.; Abele, R. H. Biochemistry 1977, 16, 2485–2491.

20


Page 20 of 21

Page 21 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Graphic Table of Contents:

21


Biomimetic Graphene-Based 3D Scaffold for Long-Term Cell Culture

Recommend Documents