In Situ Electrochemical Sensing and Real-Time Monitoring Live Cells

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In-Situ Electrochemical Sensing and Real-Time Monitoring Live Cells Based on Freestanding Nanohybrid Paper Electrode Assembled from 3D Functionalized Graphene Framework Yan Zhang, Jian Xiao, Qi-Ying Lv, Lu Wang, Xulin Dong, Muhammad Asif, Jinghua Ren, Wenshan He, Yimin Sun, Fei Xiao, and Shuai Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b08781 • Publication Date (Web): 20 Jul 2017 Downloaded from http://pubs.acs.org on August 1, 2017

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ACS Applied Materials & Interfaces

In-Situ Electrochemical Sensing and Real-Time Monitoring Live Cells Based on Freestanding Nanohybrid Paper Electrode Assembled from 3D Functionalized Graphene Framework Yan Zhang,† Jian Xiao,† Qiying Lv,† Lu Wang,† Xulin Dong,† Muhammad Asif,† Jinghua Ren,‡ Wenshan He,‡ Yimin Sun,§ Fei Xiao,†,* Shuai Wang†,* †

Key laboratory of Material Chemistry for Energy Conversion and Storage, Ministry

of Education, Hubei Key Laboratory of Material Chemistry and Service Failure, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, P. R. China ‡

Union Hospital, Tongji Medical College, Huazhong University of Science and

Technology, Wuhan, 430022, P. R. China §

Hubei key Laboratory of Plasma Chemistry and Advanced Materials, School of

Materials Science and Engineering, Wuhan Institute of Technology, Wuhan 430073, P. R. China

ABSTRACT: In this work, we develop a new type of freestanding nanohybrid paper electrode assembled from 3D ionic liquid (IL) functionalized graphene framework (GF) decorated by gold nanoflowers (AuNFs), and explore its practical application in in-situ electrochemical sensing of live breast cell samples by real-time tracking biomarker H2O2 released from cells. The AuNFs modified IL functionalized GF (AuNFs/IL–GF) was synthesized via a facile and efficient dopamine assisted one-pot self-assembly strategy. The as-obtained nanohybrid assembly exhibits a typical 3D 1

ACS Paragon Plus Environment


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hierarchical porous structure, where the highly active electrocatalyst AuNFs are well dispersed on IL–GF scaffold. And the graft of hydrophilic IL molecules (i.e., 1-butyl-3-methylimidazolium tetrafluoroborate, BMIMBF4) on graphene nanosheets not only avoids their agglomeration and disorder stacking during the self-assembly, but also endows the integrated IL–GF monolithic material with unique hydrophilic properties, which enables it to be readily dispersed in aqueous solution and processed into freestanding paper-like material. Owing to the unique structural properties and the combinational advantages of different components in the AuNFs/IL–GF composite, the resultant nanohybrid paper electrode exhibits good nonenzymatic electrochemical sensing performance towards H2O2. When used in real-time tracking H2O2 secreted from different breast cells attached to the paper electrode without or with radiotherapy treatment, the proposed electrochemical sensor based on freestanding AuNFs/IL–GF paper electrode can distinguish the normal breast cell HBL-100 from the cancer breast cells MDA-MB-231 and MCF-7 cells, and assess the radiotherapy effects to different breast cancer cells, which opens a new horizon in real-time monitoring cancer cells by electrochemical sensing platform.

KEYWORDS: Freestanding paper electrode; Three-dimensional functionalized graphene framework; Gold nanoflower;

Electrochemical sensing; Real-time

monitoring live cells

1. Introduction Cancer diseases have become a major and unmet challenge to healthcare all over the world due to their prevalence, high rate of recurrence, and potential lethality.1 The currently available methods in clinic diagnosis of cancer are primarily based on 2


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imaging techniques and morphological analysis of cells (i.e., cytology) or tissues (i.e., histopathology) using computed tomography, magnetic resonance imaging, X-ray, mammography, endoscopy and ultrasound,2,3 which are lack of sensitivity in detecting cancer until the cancer cells have invaded surrounding tissues and metastasized throughout the body.4,5 Therefore, early cancer diagnosis and therapeutic treatment are of critically importance to improve cancer patient survival. Up to now, great efforts have been focused on qualitative and quantitative determination of biomarkers for the early primary cancer screening, classification, prognosis, and therapeutic guidelines.6 Cancer biomarkers are specific molecular probes secreted by tumor or responses of the body to the presence of carcinomas in oncology.7 Hydrogen peroxidase (H2O2), a byproduct of many reactions by most oxidases in mitochondria, is related with the regulation of cellular growth and proliferation in vivo.8 Recent researches have demonstrated that live cells excrete a certain amount of H2O2 induced by oxidative stress reaction under stimulation, which diffuses out through the membranes to keep the intracellular H2O2 concentration at the normal level.9 Emerging evidence indicate that several types of tumor cells release more H2O2 than normal cells due to the disorganization and proliferation of the tumor.10-13 Therefore, the efflux amount of H2O2 from live cells are highly desirable targets and represent an exciting class of cancer biomarkers, which spark great research interest in developing high-performance analytical techniques for the rapid and accurate monitoring of H2O2 level in cellular environment. Electrochemical sensors have been touted as a particularly promising class of effective analytical technologies for real-time in vitro and in vivo detection of clinically relevant targets because of their striking aspects such as high sensitivity and accuracy, rapid response, low cost, user-friendliness, simple instrumentation and easy 3



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miniaturization.14 The electrochemical H2O2 sensors are mainly based on catalytic reduction of H2O2 by natural enzyme, i.e., horseradish peroxidase or hemoglobin. 16 However, the insufficient stability, unsatisfactory reproducibility and complicated immobilization procedure of enzymatic H2O2 sensors have seriously limited their application in practice. In this case, the non-enzymatic electrochemical H2O2 sensors based on several nanostructured materials have been considered as an alternative to enzymatic H2O2 sensor in light of the intrinsic catalytic activity of thesenano materials to the electrochemical redox of H2O2, the low cost and easy preparation of non-enzymatic electrode, and their good operational and long-term stability.17-20 In particular, noble metal nanomaterials, such as Au,21-23 Pt,24, 25 Pd,10 and their alloy nanoparticles,11,26-28, have gained great research interest due to their excellent catalytic properties, high-efficiency electronic transmission, and good chemical stability and biocompatibility. When applied in non-enzyme sensor system, they can dramatically improve the sensitivity, selectivity, and stability of the resultant sensors, and provide exciting opportunities and directions in the development of high-performance electrochemical sensor for real-time sensitive detection of H2O2 in live cell samples. In parallel with the remarkable progress in nanostructured electrocatalyst, the development of nanocatalyst support material has also gained ever-increasing attention due to its important role in controlling the morphology, size and distribution of the nanocatalyst on it, which has a tremendous influence on the catalytic activity and stability of the nanocatalyst.29 The 3D graphene-assembled monolithic porous materials such as graphene foam, graphene hydrogel and graphene aerogel, possess high electronic conductivity and good chemical stability originated from their building block graphene nanosheests, and high surface-area-to-volume ratio and good mechanical stability ascribed to their unique 3D porous structure, which have been 4


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employed as an ideal support platform to anchor a wide spectrum of functional nanomaterials, and thus hold technological promise for a variety of sustainable applications in energy storage and conversation, adsorption, separation, catalysis and sensing.30-34 However, due to the lack of the functional group on graphene surface, the loading amount of nanocatlyst on graphene support is insufficient. Furthermore, these graphene-based macroscopic assemblies possess undesirable poor processability, which limit their large-scale applications. In this work, we present the preparation of a new type of 3D porous imidazolium-based ionic liquid (IL) functionalized graphene framework (GF) supported gold nanoflowers (AuNFs) by a facile and efficient one-pot self-assembly, the resultant AuNFs decorated IL–GF monolithic material (AuNFs/IL–GF) can be processed into freestanding and flexible paper under the filtration procedure. The proposed one-pot self-assembly approach for the synthesis of AuNFs/IL–GF with the assistance of reducing agent dopamine (DA) enables the formation of well-defined 3D functionalized graphene hydrogel by hydrothermal reaction at a low temperature of 90oC and the chemical reduction of Au species at the same time. The as-obtained nanohybrid material exhibits a typical hierarchical pore structure, where the highly dense AuNFs are well dispersed on IL–GF scaffold. More importantly, our finding demonstrates that during the hydrothermal self-assembly, the imidazolium-based IL molecules have grafted on graphene surface by the possible cation–π interaction. ILs possess several fascinating properties such as high conductivity, low toxicity, high thermal, chemical and electrochemical stabilities, and wide solubility range. The graft of IL moieties effectively avoids the agglomeration and disorder stacking of graphene nanosheets during the assembly, and endows the IL–GF material with more surface functional groups and tailored dispersion properties.35-38 Herein, the introduction of 5



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hydrophilic IL molecules (i.e., 1-butyl-3-methylimidazolium tetrafluoroborate, BMIMBF4) allows AuNFs/IL–GF to be well dispersed in aqueous solution, which can be readily processed into freestanding and flexible AuNFs/IL–GF paper by filtration. The paper-based analytical system not only simplifies sample handling and processing, minimizes the sample amount and reduces the cost, but also offers the advantages of flexibility, portability and disposability for the precise and sensitive point-of-care measurement and on-the-spot diagnosis. The practical application of the freestanding AuNFs/IL–GF paper electrode has been explored in in-vitro electrochemical detection of biomarker H2O2 in breast cell samples. Under the optimized conditions, the AuNFs/IL–GF paper electrode exhibits good sensing performance towards H2O2 in terms of high selectivity, large linear range, low detection limit, fast response time, and high stability and reproducibility. When used in in-vitro detection of H2O2 level secreted from different breast cells, the proposed electrochemical sensor based AuNFs/IL–GF paper electrode can distinguish the normal breast cell HBL-100 from the cancer breast cells MCF-7 and MDA-MB-23, as well as assess the radiotherapy effects to cancer cells by evaluating secreted trace amount of H2O2 under different conditions, which offers the possibility for real-time monitoring live cancer cells by the proposed nanohybrid paper electrode, and will be of great significance in the early-stage diagnosis and management of cancer diseases.

2. EXERIMENTAL SECTIONS 2.1. Regents and Materials. Graphite powder, sulfuric acid (AR grade, 98 wt%), N-N-dimethyl-formamide, DA and H2O2 solution (30 wt% aqueous) were purchased from Sinopharm Chemical Reagent Co., Ltd (China). Teflon membrane filter (0.22 6


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u/50 mm), HAuCl4·3H2O and BMIMBF4 were obtained from Aladdin Chemistry Co., Ltd (China). N-formylmethionyl-leucyl-phenyl-alanine (fLMP, ≥99.5%) and catalase (come from bovine liver, Lyophilized, ≥3000 units mg-1) were obtained from Sigma-Aldrich Co., Ltd (USA). Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), trypsine-EDTA (0.25%) and penicillinstreptomycin were purchased from HyCl one (Waltham, USA). The chemotherapeutic agent cisplatin (DDP), propidium iodide (PI) and calcein AM viability dye (UltraPure Grade) were purchased from Sigma (USA). All other chemicals used were of analytical reagent grade. All solutions were prepared using deionized water (resistivity: 18.25 Ω cm-1).

2.2. Preparation of Electrode. Graphene oxide (GO) was synthesized according to a modified Hummer method from graphite powder and purified by centrifugation and washing. Then DA aqueous solution (80 mg mL-1, 0.5 mL) and HAuCl4 aqueous solution (a mass ratio of 1% GO) were added to the GO aqueous suspension (2 mg mL-1, 40 mL) under sonication for 30 min. After that, 0.2 mL BMIMBF4 was dispersed into the as-prepared solution to form a homogenous suspension after 1 h sonication. The mixture was sealed in glass bottle and maintained at 90oC for 6 h, and the nanohybrid hydrogel cylinder was obtained. After naturally cooled to room-temperature and freeze-dried under vacuum, the nanohybrid hydrogel cylinder was converted into aerogel cylinder. For the preparation of nanohybrid paper, the as-prepared nanohybrid hydrogel was redispersed in aqueous solution and filtrated through Teflon membrane filter. For comparison, the IL–GF material was prepared under the same condition without the addition of HAuCl4. After peeled off from Teflon membrane and freeze-dried under vacuum, the freestanding AuNFs/IL–GF paper and IL–GF paper were obtained. 7



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2.3. Instruments. Scanning electron microscopy (SEM) measurements were taken using a FESEM instrument (SIRION 200, FEI, Nederland). Transmission electron microscopy (TEM) images were obtained by a HITACHI H-8100 EM with an accelerating voltage of 200 kV. X-Ray diffraction (XRD) patterns were performed on a Shimadzu X-6000 X-ray diffractometer. Fourier transform infrared (FTIR) spectra

of

the

samples

were

collected

on

Bruker

VERTEX

70

FTIR

spectrophotometer. XPS measurements were performed on VG ESCALAB 250 spectrometer fitted with monochromatic Al Kα (1486.6 eV) X-ray radiation as the X-ray source for excitation. The fluorescence microscope (Olympus BX41F, Japan) equipped with a DP73 camera was used to examine sericin scaffolds under the light with the different wavelengths. The images were taken with the software cellSens standard 1.7 (Olympus, Japan). The radiation treatment were performed with 1 6 MV X-ray linear accelerator (Primus, Siemens AG, Erlangen, Germany). All electrochemical measurements were performed by a three-electrode cell system at room temperature. Ag/AgCl (saturated KCl) electrode and a platinum wire were acted as reference and counter electrode, respectively. The working electrode was the paper electrode with an average area of 0.5 cm2. The cyclic voltammetry (CV) and amperometric response were performed using a CHI 660E electrochemical workstation (shanghai CH Instruments Co., China).

2.4. Cell Culture and Treatment. The human breast cells HBL-100, MCF-7 and MDA-MB-23 obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) were cultured at 37°C, 5% CO2 in DMEM supplemented with 10% FBS, 100 IU mL-1 penicillin and 100 mg mL-1 streptomycin, which were then seeded into plates at a density of 5×106 cells per well and maintained in a culture 8


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medium consisting of DMEM at 37°C and subcultured every 3 days. Calcein-AM viability dye was dissolved in dimethyl sulphoxide (DMSO), and the concentration is 1 mM. PI was dissolved in ddH2O, and the concentration is 1.5 mM. The calcein-AM and PI solutions were stored at -20oC. The dye solution was prepared by mixing 10 µL calcein-AM and 15 µL PI solutions with 5 mL phosphate-buffered saline (PBS) solution, the final concentrations of calcein-AM and 15 PI are 2 µM and 4 µM, respectively. To assess cell viability, 100 µL dye solution was added into 200 µL washed cells suspension (105~106/mL), which was incubated for 15 min at 37 °C to stain viable cells green and dead cells red. The samples were then rinsed twice in PBS solution and observed by a fluorescent microscope. For chemotherapy, the cells were treated with DDP (0.01 M, pH 7.4) with final concentration of 0.2 µg ml-1. And after 24 h, serum-free DMEM containing CFSE (2.5 µM) was added into each well. For radiation treatment, the cells were irradiated using 16 MV X-ray linear accelerator at a dose rate of 200 cGy/min using a collimator with a 40×40 cm2 radiation field at treatment isocenter. The dose of radiation was 2 Gy.

3. RESULTS AND DISCUSSION 3.1. Characterization of AuNFs/IL–GF paper. Figure 1A illustrates the preparation procedures of freestanding AuNFs/IL–GF paper. First, the AuNFs/IL–GF was prepared by one-pot hydrothermal self-assembly of GO nanosheets, IL molecules and HAuCl4 in the assistance of reducing agent DA, where DA molecules can react with oxygen-containing functional groups of GO nanosheets and make them crosslinked with each other to form 3D assembled reduced GO architecture.39 DA molecules can also spontaneously polymerize into polydopamine (PDA) and adhesively coat on graphene,40 which act as the reducing and capping agent for in situ 9



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synthesis of AuNFs on 3D GF scaffold. During the hydrothermal self-assemble procedure, the dark brown precursor solution transfers into black hydrogel cylinder, and converted to aerogel cylinder by freeze-drying under vacuum. The surface functionalization of graphene assembly by hydrophilic BMIMBF4 molecules via cation–π interaction makes it readily dispersed in aqueous solution.41 After filtrated and peeled off from the substrate, the freestanding AuNFs/IL–GF paper is obtained, which is highly conductive, mechanically strong and flexible, and can be used as paper electrode in electrochemical sensor system. Figure 1B–1D show the representative SEM images of IL–GF materials with different magnification. Under the hydrothermal reaction with the assistance of DA, the IL functionalized GO nanosheets are stacked to form a stable 3D framework with interpenetrated nano- and micro-pores. N2 adsorption-desorption measurement has been carried out to analyzed the porous characteristics of IL–GF. The calculated Brunauer–Emmett–Teller surface area of IL–GF is 255.12 m2 g-1. On the basis of the Barrett–Joyner–Halenda (BJH) model, the pore sizes of IL–GF are centered from 2 to 8 nm (Supporting Information, Figure S1). In the presence of HAuCl4, the surface of 3D IL–GF skeleton is decorated with highly dense and uniformly dispersed Au nanomaterials (Figure 1E and 1F), which exhibit typical nanoflower structure (Figure 1F inset). The microstructure of AuNFs/IL–GF has further been characterized by high-resolution TEM image. As shown in Figure 1G, the AuNFs units on graphene consist of several hexagonal Au nanoplates. The well-resolved lattice fringes with interplanar distances of 0.204 and 0.235 nm are indexed to Au (200) and (111) planes, respectively.42 And the lattice index of 0.340 nm is assigned to graphene (002) of IL–GF substrate.43 Furthermore, SEM elemental mapping shown in Figure 1H demonstrates the homogeneous distribution of C, O, N, F, B and Au elements in 10


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AuNFs/IL–GF, which are originated from the PDA polymer, IL–GF substrate and AuNFs decorated on it.

Figure 1 (A) Photographs illustrate the preparation process of freestanding AuNFs/IL–GF paper. The homogenous suspension of IL, AuCl4- and GO precursor (a), AuNFs/IL–GF hydrogel (b), AuNFs/IL–GF aerogel (c), AuNFs/IL–GF aqueous suspension (d), and freestanding AuNFs/IL–GF paper (e). Step i: DA assisted one-pot hydrothermal self-assembling. Step ii: Freeze-drying under vacuum. Step iii: 11



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Redispersed in aqueous solution. Step iv: Filtrating. (B)–(D) SEM images of IL–GF with different magnifications. (E) and (F) SEM images of AuNFs/IL–GF with different magnifications. (G) TEM image of AuNFs anchored on IL–GF support. (H) Elemental mapping images of C, O, N, B, F and Au for AuNFs/IL–GF.

The FT–IR spectrum has been employed to analyze the component of AuNFs/IL–GF sample, with pristine GF and GO precursor as the control (Figure 2A). For GO sample, the peaks at 3425, 1732, and 1401~1050 cm-1 are corresponded to the –O-H, –C=O and –C-O-H stretching from carboxylic acid and aliphatic functional groups, respectively.44 After hydrothermal treatment in the presence of DA, GO nanosheets are reduced and self-assemble into GF architecture. Compared with that of GO, the intensity of stretching peaks corresponding to oxygen containing functional groups remarkably decrease for GF, indicating the effective reduction of GO by DA under hydrothermal treatment. And for AuNFs/IL–GF sample, other absorption bands at 500~1000 cm-1, 1300 cm-1 and 1498 cm-1 are assigned to the stretching vibration of –CH3-N, –CH2-N, and –C-H on imidazolium ring, and the strong absorption peak at 1020 cm-1 is originated from B-F of BMIMBF4.45 These demonstrate the successful graft of IL molecules on GF. The surface composition of AuNFs/IL–GF and precursor GO are further characterized by Raman spectroscopy (Figure S2). The GO sample exhibits two prominent peaks at 1355 cm-1 and 1595 cm-1, which correspond to the well-documented D and G bands, respectively. In comparison, the intensity ratio of these two peaks (ID/IG) increases for AuNFs/IL–GF sample, confirming the partial restoration of well-ordered graphite structure in AuNFs/IL–GF sample.46

12


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XRD patterns of AuNFs/IL–GF and IL–GF samples are shown in Figure 2B. Evidently, AuNFs/IL–GF and IL–GF samples display a broad diffraction peak at 25o fitting in the graphite-like structure (002) of GF.47 And the diffraction peak assigned to GO at 2θ = 10.0o disappears for both AuNFs/IL–GF and IL–GF samples, which indicates the decrease of layer-to-layer distance due to the effective removal of oxygen-containing groups on GO nanosheets during the hydrothermal self-assembly process using the reducing agent DA. Moreover, there are four distinct Bragg peaks at 38.22°, 44.33°, 64.54° and 77.52° observed for AuNFs/IL–GF sample, which are assigned to (111), (200), (220) and (311) planes of face-centered-cubic (fcc) Au (JCPDS 04-0784),48 where the Au (111) plane is the dominating orientation in AuNFs.

13



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Figure 2 (A) FI-IR spectra of AuNFs/IL–GF, GF and GO samples. (B) XRD spectra of AuNFs/IL–GF and IL–GF samples.

The surface composition and bonding configuration of AuNFs/IL–GF sample have been examined by XPS measurement. The XPS survey spectrum also shows the characteristic peaks corresponding to C, O, N, B, F and Au elements in AuNFs/IL–GF (Figure 3A). The core-level XPS signal of C 1s exhibits a predominant peak of C-C/C=C at 284.50 eV corresponding to the carbon bonds, and two weak peaks of C-O (~287.3 eV) and C=O (~289.0 eV) assigned to the carbon in epoxy/ether groups (Figure 3B), which reveals the considerable deoxygenation for AuNFs/IL–GF sample.49 In addition, there is a characteristic peak of C–N group in the core-level XPS curve of C 1s, corresponding to the imidazolium ring of IL (i.e, BMIMBF4) in AuNFs/IL–GF.50 The core-level XPS spectrum of N 1s has been shown in Figure 3C, which can be deconvoluted into –NH– and –NH2 at 399.2 and 401.3 eV, respectively.51 And the appearance of B 1s XPS peaks at 192 and 198 eV (Figure 3D) and F 1s peak at 686 eV (Figure 3E) derived from the B–F groups in BMIMBF4. These results together confirm the successful graft of IL molecules on graphene to form the IL–GF hybrid material. Furthermore, as shown in Figure 3F, the core-level investigation of Au 4f displays two characteristic binding energies corresponding to Au 4f

7/2

and Au 4f

5/2

at 87.45 and 83.95 eV, respectively, indicating the effective

loading of Au species on IL–GP.52 These are in line with the FT–IR and XRD results.

14


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Figure 3 (A) XPS survey of AuNFs/IL–GF sample. High-resolution XPS spectra of (B) Au C1s, (C) N1s, (D) B1s, (E) F1s and (F) Au 4f of AuNFs/IL–GF sample.

3.2. Electrochemical Sensing of H2O2 on AuNFs/IL–GF Paper Electrode. The proposed AuNFs/IL–GF paper has been used as freestanding electrode in electrochemical sensor system, and the electrocatalytic properties of Au/IL–GF paper electrode towards H2O2 redox has been investigated by CV measurements at the potential range from -1.0 to 0.6 V. As shown as Figure 4A, the CV curve of AuNFs/IL–GF paper electrode demonstrates a remarkable reduction peak at -0.6 V in 0.1 M PBS solution containing 2 mM H2O2. However, there are no any distinct redox peaks for H2O2 on IL–GF paper electrode in the same scan potential range. This suggests that the electrocatalytic activity of AuNFs/IL–GF paper is originated from the high-loading AuNFs on IL–GF substrate, which facilitate the electron transfer and improve the conductivity of the nanocomposites. As the concentration of H2O2 increases, the reduction peak current significantly increases, implying that AuNFs/IL–GF paper electrode possesses excellent electrocatalytic activity to H2O2,

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which has great application potential in the electrochemical non-enzymatic H2O2 sensing. The effect of mass ratio of HAuCl4 to GO in precursor suspension on electrochemical sensing performance of resultant AuNFs/IL–GF electrode has been investigated by amperometric measurement (Figure S3). The results show that with the increase of the mass ratio of HAuCl4 to GO from 0.2% to 1%, the amperometric current densities of AuNFs/IL–GF electrode upon the addition of 0.1 mM H2O2 increase significantly. This is because the higher content of HAuCl4 causes the larger surface coverage and higher loading amount of AuNFs catalysts on IL–GF support, which leads to an increased electrocatalytic activity of AuNFs/IL–GF electrode. However, when the mass ratio of HAuCl4 to GO increases to more than 1%, the amperometric current densities trend to decrease. This is due to the aggregation of AuNFs on IL–GF support, which decreases their electrocatalytic activity. Therefore, the mass ratio of HAuCl4 to GO has been fixed at 1% in the following tests. Figure 4B illustrates the amperometric response of AuNFs/IL–GF paper electrode upon the successive addition of H2O2 at a working potential of -0.6 V in continuously stirred PBS solution. The AuNFs/IL–GF paper electrode rapidly responds to the addition of H2O2, which reaches a steady-state current within 3 s, indicative of the fast electron transfers and diffusion of H2O2 to the electrode. And a linear relationship between the amperometric current density and H2O2 concentration is obtained in the range from 0.5 µM to 2.3 mM, with a sensitivity of 425.6 µA cm-2 mM-1 and a detection limit of 100 nM (S/N = 3). These analytical properties are superior to those of recent published works (Table S1),20,

21, 24, 25, 53-59

which is

benefited from the combinational advantages of different components in AuNFs/IL–GF nanobybrid material. In this work, the use of the reducing agent DA 16


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enables the mild-condition reduction and self-assembly of GO to form GF, the surface modification of IL molecules on GF, and the in-situ growth of AuNFs on IL–GF support at the same time. The as-prepared 3D porous IL–GF assembly with large surface area, abundant surface functional groups from ILs, and good chemical and mechanical stability serves as an ideal support platform to anchor AuNFs. The AuNFs loaded on IL–GF support exhibit high density, uniform distribution, and well-defined nanoflower morphology, which possess high electrocatalytic activities to H2O2 reduction. More importantly, the functionalization of GF by hydrophilic IL molecules BMIMBF4 favors the dispersion of the monolithic GF based nanohybrid material in aqueous solution. The as-formed suspension can be assembled into freestanding paper-like structure by filtrating, which expands its large-scale application as flexible paper electrode in miniaturized electrochemical sensing system. The sensor performances of AuNFs/IL–GF paper based assay system with selectivity, stability, and reproducibility have further been examined. The results show that the addition of foreign electroactive species including ascorbic acid (AA, 100 µM), uric acid (UA, 100 µM), glucose (100 µM), and acetamidophenol (AAP, 100 µM) cause negligible interferences to the amperometric response of 100 µM H2O2 (Figure 4C), indicating its good anti-interference ability. Moreover, after storage at room temperature for six weeks, the AuNFs/IL–GF paper electrode possesses 95% of its initial activity (Figure 4D), and the amperometric responses of six different electrodes provide relative standard deviation (R.S.D.) values less than 4% for successive addition of 100 µM H2O2 (Figure 4D inset). These results suggest good long-term stability and reproducibility of AuNFs/IL–GF paper electrode.

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Figure 4 (A) CV curves of AuNFs/IL–GF paper electrode with different concentration of H2O2 in PBS solution (pH 7.0). (B) Amperometric response of AuNFs/IL–GF paper electrode for the addition of different H2O2 concentrations into N2-saturated PBS solution (pH 7.0). Applied potential: -0.6 V. Inset shows the amperometric response of low concentrations of H2O2, and the plot of H2O2 concentration vs current in the linear range. (C) Amperometric response of AuNFs/IL–GF paper electrode under the addition of 0.1 mM H2O2 followed by some common interfering species into 0.1 M PBS solution (pH 7.0). Applied potential: -0.6 V. (D) Amperometric current responses of AuNFs/IL–GF paper electrode to 100 µM H2O2 for 6 weeks. Inset is the current response of six different electrodes to 100 µM H2O2.

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3.3 Detection of extracellular H2O2 released from breast cancer cells. The practical application of AuNFs/IL–GF paper electrode has been explored in sensitive electrochemical detection of cancer biomarker H2O2 released from women breast cancer cells. Herein, two types of breast cancer cells, i.e., MDA-MB-231 and MCF-7 were chosen as the model, with a non-tumorigenic human breast epithelial cell line HBL-100 cell as the control. Before electrochemical testing, the biocompatibility of AuNFs/IL–GF paper has also been investigated by standard cell counting Kit-8 (CCK-8) assay and fluorescent images. As shown in Figure 5A, when attached to and incubated with AuNFs/IL–GF paper electrode for over 4 h, the cell viability determined by CCK-8 assay maintains about 98% and 99% for MDA-MB-231 and MCF-7 cells, respectively. After that, the states of different live cells have been investigated by bright-field and dark-field fluorescent images, which show that all live cells are healthy when incubated with AuNFs/IL–GF paper electrode for over 4 h (Figure 5B–5G). The live-dead numbers of cells is calculated by dual-fluorescent calceinAM/PI assay, and the live-to-dead cell ratio is 135 (Figure S4). These results indicate that AuNFs/IL–GF paper electrode has low cytotoxicity to live cells. Moreover, after soaking in DMEM supplemented with 10% FBS, 100 IU mL-1 penicillin and 100 mg mL-1 streptomycin for 168 hours (7 days), the amperometric current responses of AuNFs/IL–GF paper electrode to 100 µM H2O2 reserve 92.4% of its initial value (Figure S5). When the AuNFs/IL–GF paper electrode was undergoing high temperature sterilization (i. e., 102 kPa and 120 oC) in

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autoclave for 30 min, its electrochemical activity decreases less than 5%, indicating its good chemical and thermal stability.

Figure 5 (A) Quantitative cell viability results by CCK-8 assay for different cells attached to and incubated with AuNFs/IL–GF paper electrode for 0 h (control), 1 h, 2 h, 3 h and 4 h. Bright-field microscope images of (B) HBL-100, (D) MCF-7 and (F) MDA-MB-231 cells, and dark-filed microscope images of (C) HBL-100, (E) MCF-7 and (G) MDA-MB-231 cells incubated with AuNFs/IL–GF paper electrode.

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The electrochemical sensing performances in tracking H2O2 secretion from different live breast cells that attached to AuNFs/IL–GF paper electrode have further been investigated. And the amperometric current responses of AuNFs/IL–GF paper electrode in tested well containing 5×106 cells were recorded. As illustrated in Figure 5A, the amperometric current density increases by 0.50 µA cm-2 upon the addition of a well-documented artificial stimulator N-formylmethionyl-leucyl-phenylalanine (fMLP) into the tested well to stimulate non-tumorigenic breast epithelial cell line HBL-100 cell to secrete H2O2. While after adding a selective scavenger of H2O2, i.e., catalase, the amperometric current response gradually decreases to a background level. In comparison, the control testing preformed under the same condition but without live cells does not show any detectable changes in the amperometric current responses to the addition of either fMLP or catalase (Figure 6A). These results collectively demonstrate that the increased amperometric current response in tested well is originated from H2O2 secreted from HBL-100 cells under the stimulation. Moreover, the increased amperometric current densities of H2O2 released from breast cancer cells under the same condition are shown in Figure 6B and 6C, which are 0.72 µA cm-2 for MCF-7 cell and 0.79 µA cm-2 for MDA-MB-231 cell. And the relative responses originated from 24 cell samples grown in a 24-well plate to 80% are in the range from 92.4% to 108.5%, with R.S.D. values less than 4.5%, indicating the good cycling stability of the AuNFs/IL–GF paper electrode (Figure S6). The number of extracellular H2O2 molecule released per cell (No) can be calculated according to the equation: No = [(∆j/S)×NA)]/Ncell,11 where ∆j is the increased amperometric current density of H2O2 released from different live cells under the stimulation, S is sensitivity of the proposed AuNFs/IL–GF paper electrode, which is 425.6 µA cm-2 mM-1 from the standard calibration curve, NA is the Avogadro constant (6.02×1023 M-1), and Ncell 21



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is the cell number of 5×106. Accordingly, the No values are calculated to be 1.14×1011, 2.04×1011 and 2.23×1011 for HBL-100, MCF-7 and MDA-MB-231, respectively. Significantly, the secreted amount of H2O2 per cell varies according to different types of live cells. Compared with normal breast cell line HBL-100, breast cancer cell line MCF-7 and MDA-MB-231 secrete much more H2O2 of 144% and 158%, respectively (Figure 5D), which is probably due to their higher H2O2 production and/or lower ROS scavenging capacities originating from the rapid uncontrolled growth of cancer cells. As a result, the level of H2O2 generated from live cells can be utilized an effective biomarker to identify the cancer cell line and its normal counterparts. The proposed electrochemical sensor based on AuNFs/IL–GF paper electrode has further been used in assessing radiotherapy effect to cancer cells. For in-vitro radiotherapy experiments, both cancer cell lines MCF-7 and MDA-MB-231 were irradiated using 16 MV X-ray linear accelerator at a dose rate of 200 cGy/min using a collimator with a 40×40 cm2 radiation field at treatment isocenter. After radiotherapy treatment, MCF-7 cells secrete less amount of H2O2 than that without treatment upon being stimulated by fMLP. After 1 h, 2 h, 3 h and 4 h from the radiotherapy treatment, the increased amperometric current densities of H2O2 secreted from MCF-7 cell are 84.3%, 81.3%, 75.1% and 72.7% of that without treatment, which are roughly consistent with the cell viability determined by CCK-8 assay (Figure 6E), indicating that the lower signal should be correlated to lowered numbers of viable cells after radiotherapy treatment, undergoing which the living cancer cells were killed and released less H2O2 upon being simulated. This confirms that the radiotherapy has high therapeutic activity against MCF-7 cells. This trend has also been observed for MDA-MB-231 cells under treatment. After 1 h, 2 h, 3 h and 4 h from the radiotherapy treatment, the secreted amounts of H2O2 per cell are 70.4%, 62.9%, 58.0% and 52.9% 22


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of that without treatment (Figure 6F). Evidently, the levels of H2O2 secreted per cell for MDA-MB-231 cells under radiotherapy are much lower than that of MCF-7 cells under the same condition, to some extent demonstrating that MDA-MB-231 cells are more sensitive to radiotherapy.

Figure 6 (A) Amperometric responses of AuNFs/IL–GF paper electrode to the addition of 0.1 mM fMLP and 500 U/mL catalase in tested well with HBL-100 cells (a) and without cells (b). Amperometric responses of AuNFs/IL–GF paper electrode to the addition of 0.1 mM fMLP in tested well containing (B) MCF-7 cells and (C) MDA-MB-231 cells without treatment (a) and under radiotherapy treatment (b). (D) Relative responses of the secreted H2O2 for HBL-100, MCF-7 and MDA-MB-7 cells. And relative increased amperometric current responses of the secreted H2O2 and quantitative cell viability results of CCK-8 assay for (E) MCF-7 cells and (F) MDA-MB-231 cells after 1 h, 2 h, 3 h and 4 h from radiotherapy treatment, using the live cells without treatment as control.

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4. Conclusion In summary, we reported the preparation of 3D porous AuNFs/IL–GF nanohybrid assembly via a facile and efficient one-pot self-assembly. This strategy possesses several advantages including: i) The use of DA enables the reduction and assembly of well-defined 3D IL–GF material by hydrothermal reaction at a low temperature and the simultaneous in situ growth of AuNFs on IL–GF scaffold. ii) The as-obtained nanohybrid assembly exhibits a typical 3D hierarchical pore structure and large surface area, which served as an ideal support to anchor electrocatalysts. iii) Au species on IL–GF exhibit unique nanoflower structure, high loading and well distribution, which possesses high electrocatalytic activity to H2O2 reduction. iv) The graft of hydrophilic IL molecules on graphene enables the integrated IL–GF monolithic material to be possessed into freestanding and flexible AuNFs/IL–GF paper-like material, which offers the advantages of flexibility, portability and disposability for the precise and sensitive point-of-care measurement and on-the-spot diagnosis. When used in real-time tracking H2O2 secreted from different breast cells, the proposed electrochemical sensor based AuNFs/IL–GF paper electrode can distinguish the normal breast cell from the cancer breast cells, as well as assess radiotherapy effects to different cancer cells, which hold great promise in pathological diagnose and management of cancer.

ASSOCIATED CONTENT Supporting Information Raman spectra of GO and AuNFs/IL−GF samples, N2 adsorption–desorption isotherms of IL–GF and its corresponding pore size distribution, amperometric current density of 0.1 mM H2O2 on AuNFs/IL–GF paper electrode obtained from 24


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precursor suspension containing GO (2 mg mL-1) and HAuCl4 with different mass ratio of HAuCl4 to GO, live-dead image of MCF-7 cells by calceinAM/PI assay to stain the viable cells green by calcein-AM and dead cells red by PI, relative increased amperometric responses originated from H2O2 secreted from 24 live cancer cell MCF-7 samples grown in a 24-well plate to 80%, relative amperometric current responses of AuNFs/IL–GF paper electrode after soaking in DMEM supplemented with 10% FBS, 100 IU mL-1 penicillin and 100 mg mL-1 streptomycin for 168 hours and Table S1. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (F. Xiao) *E-mail: [email protected] (S. Wang)

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work is supported by National Natural Science Foundation of China (No. 51572094 and No. 51504168), and Natural Science Foundation of Hubei Province (No. 2015CFB230). We thank the Analytical and Testing Center of Huazhong University of Science and Technology, and the Wuhan National Laboratory for Optoelectronics. 25



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