Efficient electrochemical detection of cancer cells on in situ surface-functionalized MoS2 nanosheets

Yulin Guo a, Yijin Shu a, Aiqun Li a, Baole Li a, Jiang Pi b, Jiye Cai ac, Huai-hong Cai *a and Qingsheng Gao *a
aDepartment of Chemistry, College of Chemistry and Materials Science, Jinan University, Guangzhou 510632, China. E-mail: tqsgao@jnu.edu.cn; thhcai@jnu.edu.cn
bDepartment of Microbiology and Immunology, University of Illinois, Chicago 60612, USA
cState Key Laboratory of Quality Research in Chinese Medicines, Macau University of Science and Technology, Macau 999078, China

Received 14th April 2017 , Accepted 14th June 2017

First published on 17th June 2017


Surface engineering is crucial to improve the biocompatibility and sensing response of two-dimensional (2D) nanomaterials. For nanostructured MoS2 biosensors, post functionalization via cumbersome procedures unfortunately leads to inevitable structural damage and thus reduced functionalities. Herein, in situ surface functionalization by the reactant thiourea (TU) was employed to one-step fabricate TU-capped MoS2 (TU-MoS2) nanosheets. The amino-group terminated surface of TU-MoS2 favours immobilization of the GE11 peptide that can specifically recognize cancer cells. The resulting sensor shows high sensitivity and selectivity in detecting cancer cells, relying on the varied expression of the epidermal growth factor receptor (EGFR) on cell membranes. In the case of human liver cancer cells, it is featured by a wide linear range (50–106 cells mL−1) and a low detection limit (50 cells mL−1) in electrochemical impedance spectroscopy, as the variation of charge-transfer resistance is plotted against cell concentration. Furthermore, it exhibits good efficiency in monitoring the dynamic variation of EGFR expression on living cells in response to drug treatment, which is promising for clinical diagnosis and drug screening in miniaturization. By elucidating an efficient biosensing platform on the basis of surface engineered MoS2 nanosheets, this work sheds some light on the development of biosensing technology and relevant materials.


Introduction

Two-dimensional (2D) nanomaterials have emerged as important segments to construct biosensing platforms, owing to their large surface areas, tuneable electronic/optical properties, comparatively high electron mobility, liquid media stability, and intercalatable structures.1–4 For example, graphene and graphene oxides can be used as optical and electrochemical biosensors.5–9 However, they have either no or a small band gap, which intrinsically inhibits their response to excitations.10 2D transition metal dichalcogenides (TMDs) possess suitable band gaps associated with atomic layers,11,12 and therefore can deliver much higher sensitivity than graphene based materials.10,13–17 As a typical 2D TMD, MoS2 with an open layered structure allows guest molecule intercalation,18,19 facilitating the regulation of band configuration towards further improved responses.1,20 Meanwhile, MoS2 presents low toxicity in sensing organic analytes, ensuring the viability of targets.21–24 Recently, MoS2-based biosensors have demonstrated high sensitivity for detecting proteins,25–27 DNA,14,28–30 and cellular metabolism products (e.g., H2O2, hormone, etc.).31–33 However, direct sensing of cells, especially the cytosensing of cancer cells, has been rarely reported to the best of our knowledge.

To construct a biosensor, surface functionalization on MoS2 after material preparation is necessary to respond to specific bio-targets.34–37 Regarding the 2D crystalline structure constituted by S–Mo–S layers,11 MoS2 mainly exposes basal planes in which no dangling bonds are presented. This means that guest molecules tend to lie flat on the surface of MoS2via physisorption in the post surface functionalization, rather than establishing covalent or strong ionic bonds. Such unselective and weak physisorption leads to poor sensitivity and reproducibility in biosensing. Efforts have been made to functionalize MoS2via silylation and thiolation,10 which rely on the sulfur deficiencies created by exfoliation or partial reduction. Unfortunately, such functionalization is limited by cumbersome procedures, inevitable structure damage and consequently reduced sensitivity. Therefore, an in situ functionalization during MoS2 preparation is required to accomplish well-controlled surface architectures and properties.

Recently, we have found that both edge sites and basal planes of MoS2 can strongly bond with sulfur-rich reactants under microwave (MW) irradiation,38 which is herein extended as an in situ functionalization to construct biosensors. As displayed in Scheme 1, thiourea-capped MoS2 (TU-MoS2) nanosheets provide an amino-group-rich surface for easy immobilization of the GE11 peptide, via establishing an amide linkage. The GE11 peptide can specifically recognize the epidermal growth factor receptor (EGFR) on cell membranes, which is over expressed in cancer cells.39 In this way, the GE11 modified TU-MoS2 (GE11/TU-MoS2) can selectively capture cancer cells, giving varied signals in an electrochemical redox system. Electrochemical impedance spectroscopy (EIS) shows a sensitive response in charge transfer resistance (Rct), which increases with cell concentration because of the restricted diffusion of probing molecules by the captured cells. HepG2 cells, a human liver cancer cell line, are chosen as a reliable model. The biosensor shows a wide linear range (50–106 cells mL−1) with a low detection limit of 50 cells mL−1 in the plot of Rct variation vs. cell concentration. Moreover, such a response depends on the different EGFR expression on various cells, resulting in good selectivity for sensing. This new sensing platform is further applied to monitor the evolution of the EGFR on living cells in response to drugs, indicating the promise for clinical diagnosis and drug screening in miniaturization.


image file: c7tb01024a-s1.tif
Scheme 1 Schematic illustration for (a) the fabrication of in situ surface functionalized TU-MoS2 nanosheets, and (b) the construction of the electrochemical biosensor for cell detection.

Results and discussion

Thanks to the unique heating via dipolar polarization and ionic conduction,40 MW irradiation shortens the synthetic procedure of TU-MoS2 to only 10 minutes. The presence of TU in the as-obtained sample is identified by Fourier Transform Infrared (FT-IR) spectra and thermogravimetric analysis (TGA). In the FT-IR spectra (Fig. 1a), TU-MoS2 presents absorption bands associated with vN–H (3140 cm−1), vC[double bond, length as m-dash]S (1400 cm−1), vC–N (1108 cm−1) and δN–H (619 cm−1). The obvious vN–H band indicates the rich amino groups on the MoS2 surface. And the TGA result displays a visible weight loss related to TU decomposition at 280–350 °C (Fig. 1b), which is delayed in comparison with free TU at 175–245 °C (Fig. S1 ESI), implying the strong interactions between TU and MoS2. In a control experiment, TU-MoS2 was treated with 0.05 M H2SO4 at 150 °C under MW irradiation to remove TU. Afterward, the absorption bands of vN–H, vC[double bond, length as m-dash]S, vC–N and δN–H disappear in the FT-IR spectra (Fig. 1a), verifying the removal of TU by H2SO4 (CS(NH2)2 + H2SO4 = 2SO2 + N2 +CO2 + 3H2O).38 Accordingly, the weight loss associated with TU decomposition becomes weak in TGA (Fig. 1b). X-ray diffraction (XRD) analysis further confirms the crystallographic variation in TU-MoS2 (Fig. 1c). Besides the characteristic peaks assigned to the (100), (102) and (110) planes of hexagonal MoS2 (JCPDS No. 37-1492), TU-MoS2 displays a shifted peak (2θ = 9.64°, d = 9.16 Å) corresponding to the expanded (002) d-spacing. Such a peak disappears in MoS2 after removing TU, and instead of that, a peak attributed to typical MoS2(002) emerges. Noticeably, the expanded (002) d-spacing indicates the intercalation of TU into S–Mo–S interlayers.
image file: c7tb01024a-f1.tif
Fig. 1 (a) FT-IR spectra, (b) TGA curves and (f) XRD patterns of TU-MoS2 and bare MoS2 after removing TU. (c) SEM, (d) TEM, and (e) HR-TEM images of TU-MoS2.

Scanning electron microscopy (SEM) and transition electron microscopy (TEM) were conducted to investigate the structures of TU-MoS2. Typically, nanosheets with a lateral size of 200–300 nm are observed in the product (Fig. 1d). The TEM image illustrates thicknesses ranging from 10 to 20 nm (Fig. 1e), consistent with the observation by atomic-force microscopy (AFM, Fig. S2 ESI). In the high-resolution TEM (HR-TEM), the visible lattice fringes of 0.27 nm can be indexed to the (100) or (010) planes of hexagonal MoS2 (Fig. 1f). It's noticed that an interlayer spacing of 0.92 nm corresponding to the (002) is visible in TU-MoS2, which is larger than that of bare MoS2 (0.62 nm, Fig. S3 ESI). This observation is in good accordance with the XRD analysis (Fig. 1c).

Obviously, TU molecules exist in the as-synthesized TU-MoS2. Because of the missing coordination at edges, MoS2(100) and MoS2(010) usually exhibit a high surface energy, and the basal plane of MoS2(002) is relatively inert.41,42 In this regard, (100) and (010) should be the adoptable sites for TU adsorption. However, such planes are minor in comparison with the (002) plane in a typical 2D nanostructure. Their limited contribution does not match the high TU content in TU-MoS2, which is estimated as ∼15 wt% by TGA (Fig. 1b). Moreover, the visibly expanded interlayer spacing in TU-MoS2 suggests TU intercalation into S–Mo–S interlayers. This should be driven by the strong interactions between TU and S defects on MoS2(002), latter of which are generated during the fast nanocrystal growth under MW irradiation. The X-ray photoelectron spectra (XPS) of the N element (Fig. S4, ESI) clearly identify the strong interactions between MoS2 and TU, in which the peaks for N 1s are obviously blue-shifted as compared with free TU. The above results indicate that such in situ surface functionalization can effectively modify the major surface of MoS2, i.e., the (002), which endows the original inert basal planes with abundant amino groups. In view of the delayed weight loss in TGA, the modification of TU on MoS2 should be established via strong bonds with miss-coordinated Mo atoms, which will improve the sensitivity and reproducibility in biosensing.

Via a simple immersion process, the EGFR-targeting GE11 can be immobilized on the TU-MoS2 surface. The FT-IR investigation (Fig. 2a) clearly identifies the successful immobilization of GE11 on TU-MoS2, in which the characteristic absorption bands of GE11 are clearly observed in GE11/TU-MoS2. The rich amino groups on the TU-MoS2 surface easily link with GE11 in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl), via establishing a strong amide linkage. Furthermore, the immobilization makes no obvious influence on the morphology of TU-MoS2, as confirmed by SEM (Fig. 2b). Because GE11 recognizes the EGFR that is highly expressed on cancer cells, the GE11/TU-MoS2 can specifically capture HepG2, as shown in Fig. 2c. Obviously, the bulky cells on the TU-MoS2 surface would prohibit the diffusion of probing molecules to catalytically active sites, leading to a sensitive electrochemical response as an available electrochemical redox is employed.


image file: c7tb01024a-f2.tif
Fig. 2 (a) FT-IR spectra of TU-MoS2, GE11/TU-MoS2 and GE11, and SEM images of GE11/TU-MoS2 (b) before and (c) after capturing HepG2 cells. Insets of b and c are the corresponding SEM images with high magnification. The HepG2 cells in c are highlighted by red circles.

To study electrochemical sensing, a [Fe(CN)6]3−/4− redox probe was selected, which can be effectively catalysed by MoS2. The cyclic voltammograms (CVs) on various MoS2-modified GCEs in 5.0 mM K3Fe(CN)6 and 0.5 M KCl solution show the quasi-reversible one-electron redox behaviour of ferricyanide ions (Fig. 3a). TU-MoS2 presents a high peak current and narrow ΔE in comparison with MoS2 free from TU, indicating the amplified currents due to the enhanced catalytic turnover on TU-MoS2. This should be ascribed to the surface functionalization by TU molecules. Possessing rich –NH2 and C[double bond, length as m-dash]S groups on the surface, the TU-MoS2 nanosheets would have strong coordination interactions with Fe3+/Fe2+ in [Fe(CN)6]3−/4−, promoting the relevant catalytic reduction and oxidation. A similar promoted catalysis was ever observed on MW-synthesized MoS2 intercalated by N,N-dimethylformamide species, which was ascribed to the modified electronic properties of MoS2.20 Different from this work, the XPS investigation on our TU-MoS2 and MoS2 nanosheets shows a negligible difference in Mo 3d and S 2p profiles (Fig. S5, ESI), excluding the variation in the nature of catalytic sites. Fig. 3b further displays the CVs of [Fe(CN)6]3−/4− on TU-MoS2 with varied scanning rates. The increase in scanning rate (v) from 10 to 100 mV s−1 results in correspondingly increasing currents. The plots of the anodic (ipa) and cathodic (ipc) peak currents vs. v1/2 clearly show a linear relationship (inset in Fig. 3b), in good accordance with the Randles–Sevcik equation. It's suggested that the [Fe(CN)6]3−/4− reduction/oxidation on TU-MoS2 is a diffusion-controlled reaction, and adsorbates (e.g., biomolecules or cells) on MoS2 will hinder the diffusion of [Fe(CN)6]3−/4−, resulting in a variation in the electrochemical response.


image file: c7tb01024a-f3.tif
Fig. 3 (a) Cyclic voltammograms (CVs) of GCEs modified by various MoS2 materials, and (b) CVs with varied scanning rates (v) and (inset of b) plots of peak current (ipa and ipc) vs. v1/2 on TU-MoS2 modified GCEs. (c) EIS profiles of GCEs modified by various MoS2 materials.

After GE11 conjugation, the TU-MoS2 modified GCE displays a visibly reduced current (Fig. 3a). It further decreases after the treatment with cell culture medium containing HepG2 (1 × 104 cells mL−1), indicating the restricted diffusion by cells. However, the variation in CV analysis is not much sensitive. By contrast, EIS technology can provide a more sensitive measurement, which is also an available tool to study the interface properties of electrodes.43 The charge-transfer resistance (Rct) at the electrode surface can be easily determined from EIS fitting data (inset of Fig. 3c), and represents the reaction rate of the redox couple, in reciprocal proportion. As shown in Fig. 3c, cell loading on TU-MoS2 results in an obvious increase in Rct, suggesting the high sensitivity for cell detection. The EIS response with diffusion-controlled features, consistent with ipc and ipcvs. v1/2 (Fig. 3b), identifies the sensing mechanism, in which the varied [Fe(CN)6]3−/4− diffusion by cells alters the reaction rate and thus the representative Rct. Therefore, EIS is adopted as a feasible measurement technique for cell detection in this work.

Herein, HepG2 cells, a human liver cancer cell line, are adopted as a model. As shown in Fig. 4a, the GE11/TU-MoS2 modified GCEs (GE11/TU-MoS2/GCEs) show the increasing Rct for [Fe(CN)6]3−/4− redox proportionally with HepG2 concentration. The relative electron-transfer resistance (ΔRct), determined by the difference between the assay system (Rct) and the blank (Rct,o) without cell capture, is taken to study the relationship with cell concentration. In the range from 50 to 1 × 106 cells mL−1, a linear calibration plot of ΔRctvs. log[thin space (1/6-em)]Ccell is observed (Fig. 4b). The wide linear range is associated with the efficient electrocatalysis on active TU-MoS2. The good linear relationship further implies the first-order reaction involved in electrochemical sensing, which is in good accordance with the plots of ipa and ipcvs. v1/2 (Fig. 3b). These consistent observations well confirm the electrochemical response arising from the varied diffusion of probing molecules by cell obstruction. In the repeated three tests, a linear equation of ΔRct = 37.51 log[thin space (1/6-em)]CCell − 47.94 with a correlation coefficient of 0.991 was obtained, and the detection limit was estimated to be 50 cells mL−1. This cytosensor shows superiority over recently reported HepG2 sensors in analytical performance (Table S1, ESI),44–47 which is ascribed to the excellent electrocatalytic capability of TU-MoS2 nanosheets, and effective peptide conjugation on amino-group terminated MoS2 surfaces. Additionally, excellent stability and reproducibility were also obtained using the same GE11/TU-MoS2/GCEs for 3 repeated tests with different cell concentrations (Fig. S6, ESI).


image file: c7tb01024a-f4.tif
Fig. 4 EIS response of [Fe(CN)6]3−/4− redox on (a) GE11/TU-MoS2/GCEs and (c) GE11/MoS2/GCEs after the incubation of HepG2 cells with varied concentration, and (b, inset of c) the corresponding plots of ΔRctvs. log[thin space (1/6-em)]Ccell. (d) Plots of ΔRctvs. log[thin space (1/6-em)]Ccell on GE11/TU-MoS2/GCE after incubation with human red cells, A549 cells and HepG2 cells. Error bars in (b)–(d) represent the standard deviations.

A control test was conducted on MoS2 nanosheets that were obtained via removing TU by H2SO4. After the same GE11 modification, the obtained material was applied as an electrochemical sensor for HepG2. As displayed in Fig. 4c, the EIS signal doesn't follow the increasing cell concentration in the range of 1 × 101–1 × 105 cells mL−1, and with a high cell concentration (106 cells mL−1), the Rct suddenly increases. When ΔRct is plotted as a function of log[thin space (1/6-em)]Ccell, a poor linear relationship is observed (inset of Fig. 4c). Owing to the removal of TU from the MoS2 surface, the linkage with GE11 is absent, and MoS2 cannot specifically recognize HepG2. The disproportionate variation in the EIS analysis should be attributed to the physical adsorption of cells on the electrode during their Brownian movement.

Furthermore, GE11/TU-MoS2/GCEs deliver a distinguishable recognition of cells relying on their varied EGFR expression. For comparison, the red blood cells and lung cancer cells (A549) were taken into investigation, which show none or low expression of EGFR,48,49 respectively. In the case of red blood cells, no obvious relationship is detected by GE11/TU-MoS2/GCEs (Fig. 4d), because of the absence of EGFR on their cell membrane. Although Rct increases with the concentration of A549, ΔRct does not follow a linear relationship at high concentration, which is ascribed to the low EGFR expression on A549 in comparison with HepG2.48 Obviously, the GE11/TU-MoS2 cytosensor demonstrates high sensitivity and selectivity for detecting cancer cells, on the basis of the varied EGFR expression. More importantly, it allows us to further estimate the expression levels of EGFR on different types of cancer cells.

The efficient cancer-cell detection on TU-MoS2 provides a facile evaluation of the dynamic change of EGFR expression on living cells, in response to drug treatment. This is of great importance to construct an efficient analysis platform for drug screening. Oridonin, a Chinese medicine, was used as a model medicine to effectively kill cancerous HepG2.50 It could inhibit EGFR expression and block EGFR-relative signal pathways, resulting in cell apoptosis (Fig. 5a).51 Drug-treated HepG2 cells are easily analysed using GE11/TU-MoS2/GCEs, which reveals a progressively decreasing Rct in comparison with untreated cells (Fig. 5b). This strongly indicates the effective inhibition of EGFR expression by oridonin. The concentration of living HepG2 cells after oridonin treatment can be obtained according to the formula of ΔRctvs. log[thin space (1/6-em)]Ccell, which clearly shows the agreement with the results of the conventional but inconvenient MTT colorimetric assay (Fig. 5c).


image file: c7tb01024a-f5.tif
Fig. 5 (a) Schematic illustration for oridonin-induced cancer cell apoptosis, and HepG2 viability determined by (b) EIS analysis on the TU-MoS2 based electrochemical sensor and (c) conventional MTT assay after oridonin treatment with different concentrations. Inset of b shows the EIS profiles responding to oridonin treatment. Error bars in (b) and (c) represent the standard deviations.

In comparison with conventional methods for cell detection, our TU-MoS2 biosensor shows the following advantages. Firstly, the in situ surface functionalization by TU reactant molecules and the consequent facile conjugation of GE11 on TU-MoS2 eliminate the harsh conditions and cumbersome procedures for surface-functionalization and sensor fabrication, thereby maintaining the high sensitivity of sensors. And secondly, the excellent electrocatalytic activity of TU-MoS2 contributes to the sensitive detection, which provides new opportunities for sensor design learning from the established catalytic principles.52–57 And thirdly, the obviation of cell lysis and cell labelling successfully avoids the destruction and disturbing of cell activity.

Conclusion

In summary, in situ surface functionalized MoS2 (TU-MoS2) was developed to construct a sensitive biosensor system for cancer cell detection. The designed TU-MoS2 nanosheets provide amino-group terminated surfaces for immobilizing antigens, such as the GE11 peptide, accomplishing selective recognition of cancer cells. In the case of HepG2, this MoS2-based electrochemical biosensor shows a wide linear range (50–106 cells mL−1) and a low detection limit of 50 cells mL−1, in the plot of ΔRctvs. log[thin space (1/6-em)]Ccell. Moreover, such a response depends on the different expression of EGFR on various cells, resulting in the striking selectivity for sensing. Further application to monitor the EGFR variation on living cells, in response to drugs, clearly verifies the good promise for clinical diagnosis and drug screening in miniaturization. As for real samples, control over the TU-MoS2 surface architecture and optimized immobilization of peptides (e.g. GE11) are highly required in future research. By elucidating biosensing platforms constituted by surface engineered 2D materials, this work opens up new opportunities for the development of sensing technology and relevant materials.

Experimental section

Materials and reagents

Ammonium heptamolybdate tetrahydrate ((NH4)6Mo7O24·4H2O), thiourea (CS(NH2)2), and phosphate-buffered saline (PBS) were purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). Nafion solution (5 wt% in lower aliphatic alcohols and water) was purchased from Sigma-Aldrich. The GE11 peptide was purchased from GL Biochem Ltd (Shanghai). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) was purchased from Sigma-Aldrich. All chemicals were of analytical grade and used as received without further purification. All aqueous solutions were prepared using ultrapure water (>18 MΩ).

Synthesis of TU-MoS2 and bare MoS2 nanosheets

According to our previous report,38 a MW-assisted hydrothermal route was adopted to synthesize TU-MoS2 nanosheets. Typically, ammonium heptamolybdate tetrahydrate ((NH4)6Mo7O24·4H2O) and thiourea (CS(NH2)2) were dissolved in 15.0 mL of distilled water, and then the solution was turned to a microwave reactor (Preekem WX-8000, Shanghai). After microwave irradiation at 220 °C for 10 min, the solid was collected by centrifugation, thoroughly washed with distilled water three times. TU-MoS2 was finally obtained after drying overnight at 50 °C. As for bare MoS2, the above TU-MoS2 was treated with diluted H2SO4 (0.05 mol L−1) under microwave conditions at 150 °C for 2 h. This procedure ensured the removal of TU capping, and resulted in bare MoS2 with similar sheet-like nanostructures.

Physical characterization

SEM and TEM investigations were conducted on a ZEISS ULTRA55 and a JEOL JEM 2100F, respectively. XRD was performed on a Bruker D8 diffractometer using Cu Kα radiation (λ = 1.54056 Å). FT-IR spectra were collected on a Nicolet 6700 FTIR spectrometer. XPS was processed on a PerkinElmer PHI5000c XPS, using C 1s (B.E. = 284.6 eV) as a reference. TGA was performed on a NETZSCH STA449F3 under N2 flow. AFM investigation was conducted on a Bruker Innova. To prepare the samples, TU-MoS2 was sonicated in H2O for 30 minutes, and was then dropped onto fresh mica.

Preparation of TU-MoS2/GCEs and GE11/TU-MoS2/GCEs

The detailed procedure for the preparation of TU-MoS2/GCEs is as follows: 4 mg of TU-MoS2 nanosheets, 40 μL of 5 wt% Nafion solution and 30 μL of 5 wt% PVDF solution were dispersed in 1 mL water–ethanol with a Vwater/Vethanol ratio of 4[thin space (1/6-em)]:[thin space (1/6-em)]1, and then the mixture was vigorously sonicated for at least 30 min to achieve a homogeneous ink. The as-obtained solution (5 μL) was loaded onto pre-treated GCE (3 mm diameter), and dried naturally in air. In order to make a comparison, MoS2/GCEs were prepared via the same procedure.

For GE11 conjugation, the above TU-MoS2/GCEs and MoS2/GCEs were immersed into 0.5 mL PBS (pH 7.5) containing 1.0 mg of GE11 and 0.1 mM EDC·HCl, at 4 °C for 2.5 hours. The obtained cytosensor was stored at 4 °C prior to use.

Cell culture

Human liver cancer HpeG2 cells were obtained from the Life Science Research Institute of the Cell Resource Centre (Shanghai, China). Cells were seeded in a 24-well plate with cover glasses and cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco, USA), supplemented with 10% fetal bovine serum (FBS), 100 μg mL−1 penicillin, and 100 μg mL−1 streptomycin in a 5% CO2-humidified chamber at 37 °C. The cell density was determined using a hemocytometer prior to any experiments.

Blood samples of healthy volunteers were from The First Affiliated Hospital of Jinan University (Guangzhou, China). All the experiments were performed in compliance with the law prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press (1996). The Institutional Animal Care and Use Committee of National Jinan University (Guangzhou, China) approved the study protocol. Red blood cells were separated by centrifugation at 2000 rpm for 10 min, washed with PBS, and red blood cells were stored at 4 °C prior to use.

Analytical procedures by the electrochemical cytosensor

The modified GCE was immersed in the cell solution for cell capture at 37 °C for 3 h. After careful rinsing with 0.01 M PBS (pH 7.4), the obtained cytosensor was ready for electrochemical measurements. CV and EIS profiles were recorded in 0.01 M pH 7.4 phosphate-buffered saline (PBS) buffer solution containing 10 mM [Fe(CN)6]3−/4− and KCl, with a CHI650E electrochemical analyzer. The three-electrode system was composed of a platinum wire as the auxiliary, a saturated calomel electrode as the reference and a GCE as the working electrode. EIS measurements were obtained at open circuit voltage from 1 × 105 to 0.1 Hz with an AC voltage of 5 mV. The electron-transfer resistance (Rct) was obtained from EIS fitting data. And, the relative electron-transfer resistance (ΔRct) was determined by the difference between the assay system (Rct) and the blank (Rct,o) without cell capture, namely, ΔRct = RctRct,o.

Evaluation of EGFR expression after drug treatment

EGFR expression was measured by EIS analysis. Cells were cultivated in 96-well plates containing DMEM at 37 °C and 5% CO2 for 24 h. Subsequently, the medium was substituted for fresh medium supplemented with different concentrations of oridonin for 6 h. After drug treatment, the EIS test was conducted, in which Rct and ΔRct were obtained from the fitting data. And then, the cell concentration (Ccell) after oridonin treatment was calculated according to the established relationship between ΔRct and CcellRct = 37.51 log[thin space (1/6-em)]CCell − 47.94). For comparison, the conventional MTT colorimetric assay was also conducted.

Acknowledgements

This work is financially supported by the National Natural Science Foundation of China (Grant No. 21373102 and 21433002), the Natural Science Foundation of Guangdong Province (Grant No. 2015A030306014 and S2012040006713), and the Guangdong Program for Support of Top-notch Young Professionals (Grant No. 2014TQ01N036). J. Y. Cai also thanks the support from the Macao Science and Technology Development Fund (Grant No. 028/2014/A1).

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Footnote

Electronic supplementary information (ESI) available: TGA of bare TU, SEM and TEM images of bare MoS2, XPS profiles of TU-MoS2 and MoS2, and reproducibility of TU-MoS2 based biosensors. See DOI: 10.1039/c7tb01024a

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