Au nanoparticle decorated resin microspheres: synthesis and application in electrochemical cytosensors for sensitive and selective detection of lung cancer A549 cells

Abstract

In this article, for the first time, we report a facile method for the synthesis and immobilization of AuNPs on m-aminophenol based resin (MAPR) microspheres via a simple reduction route. The AuNP-decorated MAPR (AuNPs/MAPR) microspheres, with a large number of active groups (amino and hydroxy groups), can not only act as suitable immobilization carriers for antibodies (EGFR antibody), but also play an important role in facilitating electron transfer. Moreover, the AuNPs/MAPR microspheres possess a high surface-enhanced Raman scattering (SERS) activity and have great potential as a SERS-active substrate. A novel electrochemical cytosensor which can sensitively differentiate lung cancer cells (A549 cells) from normal ones (AT II cells) by making use of the advantages of EGFR antibodies and AuNPs/MAPR microspheres has been designed. EGFR antibodies are immobilized on the outmost layer of the electrode surface to selectively recognize EGFR receptors that are over-expressed on lung cancer cells. The confocal microscopy images and cytotoxicity assays of the AuNPs/MAPR microspheres confirm that the prepared cytosensors exhibit good biocompatibility, high sensitivity and selectivity for the detection of A549 cells. To the best of our knowledge, this is the first cytosensor using AuNPs/MAPR microspheres as a carrier. It exhibits a broad linear range with a detection limit as low as 5 cells per mL, even in the presence of a large number of normal cells. Our study demonstrates that the proposed cytosensors can also be used to successfully determine A549 cells in diluted blood samples.

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1. Introduction

At the beginning of the 21^st century, cancer has become one of the most likely causes of death worldwide.^1–4 The ever-increasing tendency to die from cancer has made it a top threat to human beings.¹ Diagnosing and treating cancer can be very complicated because it is not recognised as a extraneous body by our immune system.^1,5 Once cancer evolves into the phase of metastasis, other tissues of the body can be invaded and affected. However, the chance of being cured of cancer increases with early detection and treatment of the disease.¹ Therefore, the study of sensitive and accurate identification and detection of cancer cells plays a vital role in early diagnosis and rapid, targeted therapy. Up to now, a number of conventional approaches have been developed for cancer cell detection, including fluorescent imaging, cytological testing, radiography, magnetic resonance imaging, and so on.² Nevertheless, these methods require expensive instrumentation and multi-step preparation. Accordingly, developing a simple and rapid method for early detection of cancer is highly desirable.^6,7 To address these requirements, electrochemical cytosensors have attracted considerable attention because of their high sensitivity, rapidity, simple operation, and reproducibility.^8–12 Recently, the “target-binding” technology has been widely applied to develop electrochemical cytosensors with selectivity.^3,12–14 Wang’s group has constructed an electrochemical cytosensor to selectively detect cancerous cells amongst normal ones based on the high affinity of folic acid for the folate receptor.¹² Due to the highly specific recognition between antibody and antigen, Hu’s group has reported an ultrasensitive electrochemical cytosensor for the determination of carcinoembryonic antigen (CEA)-positive cancer cells.³ Takahashi’s group has developed an electrochemical cytosensor for the detection of cancer cells based on the specific recognition between epidermal growth factor (EGFR) receptors and EGFR antibody (anti-EGFR).¹³ However, all these above-mentioned methods suffer from various drawbacks such as the requirement of an extra immobilization agent^3,12 and the involvement of special enzymes.¹³ Accordingly, the development of a new preparation strategy that can directly immobilize the antibody on a nanomaterial is highly desirable.

Recently, colloidal resin microsphere-based composites have attracted more and more attention because of their unique properties, such as rapid electron transfer, catalytic activity, specificity, and biocompatibility.^15–17 These functional resin microspheres can not only act as suitable immobilization carriers for biomacromolecules, but also have active effects on the transduction of biosensors.¹⁷ More recently, research on the application of Au nanoparticle (AuNP) decorated resin microspheres has attracted great attention.^17,18 For example, Yang’s group has designed multifunctional AuNP-decorated resin microspheres for killing tumor cells through photothermal conversion.¹⁸ Our group has reported functional resin microspheres loaded with Au nanoparticles that are applied for the detection of tumor markers in human serum.¹⁷ However, the application of AuNP-decorated resin microspheres for the selective detection of cancer cells is rarely reported. Accordingly, the development of a new facile strategy for the application of AuNP-decorated resin microspheres in cancer cell detection is highly desirable.

In this study, we report for the first time a facile method for the synthesis and immobilization of AuNPs on m-aminophenol based resin (MAPR) microspheres via a simple reduction route. The AuNP-decorated MAPR (AuNPs/MAPR) microspheres, with a large number of active groups (amino and hydroxy groups), can not only act as suitable immobilization carriers for antibodies (EGFR antibody), but also play an important role in facilitating electron transfer. Moreover, the AuNPs/MAPR microspheres possess a high surface-enhanced Raman scattering (SERS) activity and have great potential as a SERS-active substrate. A novel electrochemical cytosensor which can sensitively differentiate lung cancer cells from normal ones by making use of the advantages of EGFR antibodies and AuNPs/MAPR microspheres has been designed. EGFR antibodies are immobilized on the outermost layer of the electrode surface to selectively recognize EGFR receptors that are over-expressed on lung cancer cells (A549 cells). It is found that the prepared cytosensors exhibit good biocompatibility, high sensitivity and selectivity for the detection of A549 cells. To the best of our knowledge, this is the first cytosensor using AuNPs/MAPR microspheres as a carrier.

2. Materials and methods

2.1 Materials

6-(Ferrocenyl)hexanethiol (6-Fc-HT) was purchased from Sigma-Aldrich. Nile blue A (NBA) was purchased from Alfa Aesar (USA). Rhodamine 6G (R6G), ferrocenecarboxylic acid (Fc-COOH), m-aminophenol, bovine serum albumin (BSA), ammonium hydroxide (NH₃·H₂O) (28 wt% in water), dopamine and formaldehyde (37 wt%) were purchased from Aladin Ltd. (Shanghai, China). Chloroauric acid solution (HAuCl₄·4H₂O) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). NaH₂PO₄, Na₂HPO₄, and ethanol were purchased from Beijing Chemical Reagent (Beijing, China). Anti-EGFR polyclonal antibody standard grade antigens were purchased from Shanghai Sangon Biotech Co., LTD (Shanghai, China). All chemicals were used as received without further purification. The water used throughout all experiments was purified through a Millipore system. Phosphate buffered saline (PBS) was prepared by mixing stock solutions of NaH₂PO₄ and Na₂HPO₄. Human blood samples were kindly provided by the Hospital of Southeast University (Sipailou 2, Nanjing, China). The blood collection was approved by the Institutional Review Board (IRB). The blood samples (healthy, non-pregnant adults who weigh at least 50 kg) were taken by venipuncture. The human serum was separated from the blood samples by centrifugation. Human serum samples were diluted to different concentrations with a PBS solution of pH 6.5, and each sample was analyzed three times.

2.2 Instruments

Fourier Transform Infrared (FT-IR) spectroscopy measurements were carried out on a FT-IR Spectrometer TENSOR 27 (Bruker Optik GmbH, Ettlingen, Germany). Transmission electron microscopy (TEM) measurements were carried out on a HITACHI H-8100 EM (Hitachi, Tokyo, Japan) with an applied accelerating potential of 200 kV. The sample for TEM characterization was prepared by placing a drop of the dispersion on a carbon-coated copper grid and drying at room temperature. Scanning electron microscopy (SEM) measurements were carried out on a XL30 ESEM FEG scanning electron microscope at an applied accelerating potential of 20 kV. The sample for SEM characterization was prepared by placing a drop of the dispersion on a bare Si substrate and air-drying at room temperature. Electrochemical measurements were performed with a CHI 660D electrochemical analyzer (CH Instruments, Inc., Shanghai). A conventional three electrode cell was used, including a glassy carbon electrode (GCE, geometric area = 0.07 cm²) as the working electrode, a Ag/AgCl (3 M KCl) electrode as the reference electrode, and platinum foil as the counter electrode. All potentials given in this work were referenced to the Ag/AgCl electrode. All the experiments were carried out at ambient temperature.

2.3 Synthesis of MAPR microspheres

MAPR microspheres were prepared according to the reported method with a little modification.¹⁹ In a typical synthesis, 0.051 g of m-aminophenol was added to 13 mL of ethanol and 5 mL of water, followed by adding 56.5 μL of aqueous ammonia solution (NH₄OH, 28 wt%) to form a homogeneous solution, and left for 30 min at 35 °C. After the addition of 46 μL of formaldehyde solution (37 wt%), the resulting mixture was stirred at 40 °C for 200 min and subsequently heated at 100 °C for 24 h in a Teflon-lined autoclave. The resin spheres were purified with distilled water and ethanol by centrifugation 3 times at a speed of 5000 rpm. The MAPR microspheres were collected by centrifugation and washed with doubly distilled water to remove some small spheres in the mixture. The obtained precipitates (MAPR microspheres) were redispersed in 20 mL of water for characterization and further use.

2.4 Synthesis of AuNP-decorated MAPR microspheres

In a typical synthesis, 200 μL of 24.3 mM HAuCl₄ solution was first added into 8 mL of MAPR microsphere dispersion, and then the mixture was stirred for 30 min. Next, 100 μL of 0.10 M NaBH₄ aqueous solution was added into the previous solution under stirring, and left for 30 min at room temperature. The precipitate was collected by centrifugation and washed with water twice. The resultant AuNP/MAPR microspheres were redispersed in 1 mL of H₂O for characterization and further use.

2.5 SERS measurements

The AuNP/MAPR microspheres were immobilized on ITO surfaces as SERS active substrates by using electrostatically assisted 3-aminopropyltriethoxysilane (APTES)-functionalized surface-assembly. The ITO glass slips were washed with ultrapure water at least three times. The slips were further cleaned three times in ethanol with sonication and dried at 60 °C for 2 h in an air oven. The cleaned ITO glass slips were vertically immersed in a 1% (v/v) solution of APTES in anhydrous ethanol for 2 h, rinsed three times in ethanol with sonication to remove excess silane and dried in an air oven.

Then, the functional ITO glass slips were dipped overnight into the stirred colloidal suspension of AuNP/MAPR microspheres in order to form AuNP/MAPR layers. For subsequent SERS experiments, NBA and R6G were used as the analytes. The AuNP/MAPR layer decorated ITO substrates previously described were first vertically immersed in each aqueous analyte solution, with a series of NBA or R6G concentrations, for 6 h and then left to dry at room temperature for SERS measurements. The Raman experiments were carried out with a Renishaw Invia Reflex system equipped with Peltier-cooled charge-coupled device (CCD) detectors and a Leica microscope. Samples were excited with a 785 nm diode laser under line-focus mode and the laser power was adjust to 0.5%, which was about 0.06 mW. The corresponding laser was focused onto the sample surface using a 50× long working distance objective. Spectra were collected in continuous mode with a 10 s exposure time, accumulated twice, and a grating of 1200 mm⁻¹ was used. Every SERS spectrum was averaged from 5 measurements. All experiments were performed in triplicate and values were averaged.

2.6 Fabrication of the cytosensor

1 mL AuNPs/MAPR microsphere dispersion was added to 5 mL water containing 2 mM 6-Fc-HT solution, and then the mixture was stirred for 2 h. AuNPs are covalently attached to the 6-Fc-HT with the thiol-terminated surface; Au–S bonding can be formed between the AuNPs/MAPR microspheres and the surface thiol-groups of 6-Fc-HT. The precipitate was collected by centrifugation in order to remove unreacted 6-Fc-HT and redispersed in 1 mL of H₂O. The GCE was firstly immersed into 1% chitosan solution for 1 h. Then, 2 μL of 6-Fc-HT/AuNPs/MAPR microspheres was dropped on the surface of the pretreated GCE and left to dry at room temperature. The 6-Fc-HT/AuNPs/MAPR microsphere-modified GCE (6-Fc-HT/AuNPs/MAPR/GCE) was immersed into a mixture of 20 mM NHS (N-hydroxysuccinimide) and 100 mM EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) in water for 2 h at room temperature to activate the terminal carboxylate groups of the microspheres and were then rinsed with water. After washing, anti-EGFR was immediately dropped on the surface of the modified electrode and then incubated for 100 min to form the anti-EGFR/6-Fc-HT/AuNPs/MAPR/GCE. Following that, this electrode was washed and incubated in 1 wt% BSA solution for 60 min to eliminate nonspecific binding. The cytosensor, anti-EGFR/6-Fc-HT/AuNPs/MAPR/GCE, was thus fabricated and used for cell detection. Scheme 1 displays the electrochemical cytosensor fabrication procedure.

Scheme 1 Schematic illustration of the stepwise electrochemical cytosensor fabrication process.

2.7 Cell culture and detection

Human lung adenocarcinoma cell line (A549) and human type II alveolar epithelial cell line (AT II) were obtained from the Medical School of Southeast University. They were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin solution, and incubated at 37 °C in a 5% CO₂ environment. The cells were trypsinized and subcultured every two days. The cell number was detected using a Petroff Hausser cell counter.

For immobilization on the surface of the cytosensor, the cells were separated from the medium by centrifugation at 1500 rpm for 3 min and then washed three times with PBS. The precipitate was carefully redispersed in PBS to obtain a homogeneous cell suspension at a certain concentration. Finally, the anti-EGFR/AuNPs/MAPR microsphere-electrode was immersed into the cell suspensions for 30 min. Square wave voltammetry (SWV) was performed for cell detection because it is very sensitive to current signal change. The SWV spectra were collected from the prepared cytosensor after it was incubated in A549 cell suspensions of different concentrations.

2.8 Confocal imaging

ITO slices coated with AuNPs/MAPR microsphere layer/anti-EGFR films were prepared and then placed in a 10 cm cell culture plate. Then A549 or AT II cells were introduced into the plate and cultured in DMEM medium supplemented with 10% FBS at 37 °C. At times of 15, 30 and 60 h, one of the ITO slices was taken out and the cells on it were stained with Rhodamine 123. The fluorescent images of the cells were quickly observed under a confocal laser-scanning microscope (CLSM, Leica TCS-SP8).

2.9 Cytotoxicity of AuNPs/MAPR microspheres

The cytotoxicity of AuNPs/MAPR microspheres to A549 cancer cells and AT II normal cells was evaluated using the MTT assay. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 100 IU mL⁻¹ penicillin–streptomycin at 37 °C in a humid atmosphere with 5% CO₂. For the MTT assay, cells were seeded onto 96-well plates at a density of 5 × 10³ cells per well in 100 μL complete culture medium. After culturing for one day, when the cells grew to 80% confluence in each well, the medium was replaced with 100 μL fresh medium containing different concentrations of AuNPs/MAPR microspheres and further cultured for 24 h. Then, 10 μL MTT solution (5 mg mL⁻¹ in cell PBS) was added directly into each well and the cells were incubated for an additional 4 h at 37 °C. After that, the medium was carefully aspirated. The cells were solubilized in 150 μL DMSO, and then the absorbance at 492 nm was measured against a background control using a microplate reader (Thermo-scientific, Multiskan FC, USA).

3. Results and discussion

The structure of the as-prepared MAPR microspheres was first characterized using SEM. Fig. 1A shows a representative SEM image of the as-prepared product, which consists exclusively of a large number of sub-microspheres. The magnified SEM image of these sub-microspheres demonstrates that the MAPR microspheres are about 300 to 400 nm in diameter, as shown in Fig. 1B. Fig. 1C shows the low magnification SEM image of the AuNPs/MAPR microspheres, which consist exclusively of a large number of sub-microspheres. A large number of small nanoparticles are adsorbed on the surface of the MAPR microspheres, as shown in Fig. 1C. The high magnification SEM image shown in Fig. 1D further reveals that the AuNPs/MPAR microspheres have a behavior of very dense nanoparticles. The TEM image shows that a large number of AuNPs are deposited onto the surface of the exterior of the MAPR microspheres, as shown in Fig. S1.† The MAPR microspheres have the branched chain of amino groups and hydroxyl groups, due to the raw materials used in the synthesis (m-aminophenol). The existence of amino groups and hydroxyl groups will be further proved by FT-IR spectroscopy. These functional groups of the MAPR microspheres can act as suitable adsorption carriers for Au³⁺ ions. The reduction of these Au³⁺ ions by sodium borohydride allows these AuNPs to attach onto the MAPR microspheres. Meanwhile, these functional groups of the MAPR microspheres can also favor the immobilization of antibodies (EGFR antibody) by a carboxyl-to-amine crosslinking reaction.

Fig. 1 (A) Low and (B) high magnification SEM images of the MAPR microspheres. (C) Low and (D) high magnification SEM images of the AuNPs/MAPR microspheres.

SERS spectroscopy has received a great deal of attention for its utility as a sensitive technique for biomedical analysis.^20,23,24 Metallic nanomaterials exhibit high SERS activity due to an increased field at the metallic nanoparticle surface, which is a consequence of the interaction of the incoming laser radiation with electrons in the metal surface or collective oscillations of the metal electrons.²¹ To study the SERS activity of the AuNPs/MAPR microspheres, two organic molecules, NBA and R6G, were used to testify the SERS activities of the substrates. The latter molecule, R6G, is often employed in SERS studies because of its large Raman cross-section. The AuNPs/MAPR microspheres are adopted as the standard probe to enhance the Raman signals in the following experiments. Fig. 2A shows the obtained SERS spectra when different concentrations (1 × 10⁻⁹ M up to 1 × 10⁻⁵ M, line a to line e in Fig. 2A) of R6G solutions were tested. As shown in Fig. 2A, the typical SERS spectra of R6G are characterized by prominent strong bands at 613, 776, 1181,1315, 1364, and 1511 cm⁻¹, assigned to the typical aromatic benzene ring breathing and stretching vibrational modes.²² The SERS signals increased proportionally with the increasing concentration of R6G, which can be utilized as a quantitative measurement of R6G concentration. As shown in Fig. 2C, the calibration plots show a good linear relationship between the SERS signals and the logarithmic values of the concentrations of R6G. The peak at 1511 cm⁻¹ of the R6G molecule is chosen as the reference peak in the study. It is clearly seen that there is a linear relationship between the SERS signals and the concentration of R6G over the range of 1 × 10⁻⁹ M up to 1 × 10⁻⁵ M with a correlation coefficient of 0.993, as shown in Fig. 2C. The spectra obtained when the substrate was immersed in NBA solutions of different concentrations are shown in Fig. 2B (line a to line e), following the same procedure from low to high concentration as that in the R6G measurements. The peak at 592 cm⁻¹ of the NBA molecule is chosen as the reference peak in the study. It is clearly seen that there is a linear relationship between the SERS signals and the concentration of NBA over the range of 1 × 10⁻⁷ M up to 5 × 10⁻⁵ M with a correlation coefficient of 0.995, as shown in Fig. 2D. All the above observations indicate that the as-prepared AuNPs/MAPR microspheres exhibit high SERS activity.

Fig. 2 (A) Representative 785 nm excited SERS spectra with different concentrations of R6G, (a) 10⁻⁵ M, (b) 10⁻⁶ M, (c) 10⁻⁷ M, (d) 10⁻⁸ M and (e) 10⁻⁹ M, on AuNPs/MAPR microspheres. (C) A logarithmic plot of R6G concentration and the intensity of SERS signal for the band at 1511 cm⁻¹. (B) Representative 785 nm excited SERS spectra with different concentrations of NBA, (a) 5 × 10⁻⁵ M, (b) 5 × 10⁻⁶ M, (c) 1 × 10⁻⁶ M, (d) 5 × 10⁻⁷ M and (e) 1 × 10⁻⁷ M, on AuNPs/MAPR microspheres. (D) A logarithmic plot of NBA concentration and the intensity of SERS signal for the band at 592 cm⁻¹.

To investigate whether the AuNPs/MAPR microspheres are suitable for practical applications, the cytotoxicity of the AuNPs/MAPR microspheres is evaluated against A549 cells (Fig. 3A) and AT II cells (Fig. 3B). The effect of the AuNPs/MAPR microspheres on A549 and AT II cell survival is investigated by MTT assay. As shown in Fig. 3, A549 cells and AT II cells are treated with different concentrations (0, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8 and 25.6 mg mL⁻¹) of AuNPs/MAPR microsphere dispersions for 24 h and then the viability is tested. Some of the concentrations of AuNP/MAPR microspheres result in viability values of more than 100% (100% is the viability of the control), indicating that AuNP/MAPR microspheres show no toxicity at a certain concentration and promote cell proliferation. As a result, the MTT assay results shown in Fig. 3 reveals that no cytotoxicity is observed after 24 h incubation.

Fig. 3 Cytotoxicity of the tested AuNPs/MAPR microspheres against A549 cells (A) and AT II cells (B).

Fig. 4A shows the FT-IR results for the MAPR microspheres. The peak at 1225 cm⁻¹ is assigned to the C–OH stretching.²⁵ The peak observed at 1624 cm⁻¹ can be attributed to a stretching of the secondary amine of the m-aminophenol in the MAPR microspheres.²⁶ The peak of the –N–H absorption bands is also observed at 3367 cm⁻¹,²⁷ as shown in Fig. 4A. The stretching peak of the –OH of the carboxylic group is also observed at 3740 cm⁻¹.²⁸ It is observed that the bonds of the –OH groups, the carboxylic (–COOH) groups and the amines (–NH), are present in the MAPR microspheres, suggesting that the AuNPs can easily adsorb on the exterior of the MAPR microspheres. Fig. 4B shows the FT-IR results for the AuNPs/MAPR microspheres. The characteristic absorption bands observed at 1624 cm⁻¹, 3367 cm⁻¹ and 3740 cm⁻¹ are also observed in the FT-IR spectrum of the AuNPs/MAPR microspheres.^26–28 It is observed that the bonds of the –OH groups and the carboxylic (–COOH) group are present in the AuNPs/MAPR microspheres, which can provide a favorable microenvironment for immobilization of anti-EGFR without additional immobilized reagent. The FT-IR spectrum in Fig. 4C of the 6-Fc-HT/AuNPs/MAPR microspheres shows several peaks. A series of characteristic vibrational modes centered around 887, 1002 and 1710 cm⁻¹ are the characteristic absorption peaks of ferrocene, assigned as the D_5d symmetry,²⁹ revealing that the 6-Fc-HT/AuNPs/MAPR microspheres are successfully prepared. Fig. 4D shows the FT-IR spectrum of anti-EGFR/6-Fc-HT/AuNPs/MAPR microspheres. The peak at 1649 cm⁻¹ is assigned to the C=O symmetric stretching band of the C=O group.³⁰ The peaks of the characteristic absorption bands of ferrocene are also observed at 1002 and 1105 cm⁻¹, as shown in Fig. 4D. The peak at 1551 cm⁻¹ is the characteristic asymmetric stretching mode in the –CONH– of the antibody,³⁰ indicating that the anti-EGFR antibodies have successfully decorated the 6-Fc-HT/AuNPs/MAPR microspheres.

Fig. 4 FT-IR spectra of MAPR microsphere (A), AuNPs/MAPR microspheres (B), 6-Fc-HT/AuNPs/MAPR microspheres (C) and anti-EGFR/6-Fc-HT/AuNPs/MAPR microspheres (D).

Electrochemical impedance spectroscopy (EIS) is an effective tool for monitoring the interfacial properties of an electrode during the modification process.³¹ In the Nyquist diagram, the linear portion at the low frequencies and the semicircle portion at the high frequencies correspond to the diffusion-limited process and the electron transfer-limited process, respectively. The electron transfer resistance (R_et) can be estimated from the diameter of the semicircle in an impedance spectrum. Fig. 5A shows the complex impedance plots of the different layer modified electrodes in 5.0 mM [Fe(CN)₆]^4−/3− solution. It is observed that the bare GCE exhibits a small semicircle at high frequencies with a diameter of 143 ohm and a linear part at low frequencies (line a of Fig. 5A), which is characteristic of a diffusion-limiting step of the electrochemical process on a bare GCE. After deposition of 6-Fc-HT/AuNPs/MAPR microspheres (line b of Fig. 5A), the R_et of 97 ohm is much smaller than that of the bare GCE, indicating that the 6-Fc-HT/AuNPs/MAPR composites can promote electron transfer. When the anti-EGFR is adsorbed onto the 6-Fc-HT/AuNPs/MAPR/GCE (line c of Fig. 5A), the value of R_et increases to 182 ohm. This result is ascribed to the nonconductive properties of anti-EGFR, which insulates the conductive support and blocks electron transfer. Similarly, after the capture of BSA and A549 cells, the values of R_et increase to 219 ohm and 311 ohm, as shown by line d and line e of Fig. 5A, respectively. It is suggested that the formation of a nonconductive and hydrophobic immunocomplex layer hinders electron transfer. All these observations demonstrate that the 6-Fc-HT/AuNPs/MAPR microspheres, anti-EGFR, BSA and A549 cells have been successively assembled onto the GCE.

Fig. 5 (A) Nyquist plots of the EIS for each immobilization step, recorded from 0.1 to 10⁵ Hz for bare GCE (a), 6-Fc-HT/AuNPs/MAPR/GCE (b), anti-EGFR/6-Fc-HT/AuNPs/MAPR/GCE (c), BSA/anti-EGFR/6-Fc-HT/AuNPs/MAPR/GCE (d), and A549 cells/BSA/anti-EGFR/6-Fc-HT/AuNPs/MAPR/GCE (e) in 1 mM [Fe(CN)₆]^3−/4− solution. (B) Cyclic voltammetry (CV) of the anti-EGFR/6-Fc-HT/AuNPs/MAPR/GCE (a) and the anti-EGFR/Fc-COOH/MAPR/GCE (b) in supporting electrolyte at a scan rate of 100 mV s⁻¹. (C) CV of the modified electrodes at different scan rates (from inner to outer): 10, 20, 50, 80, 100, 150 and 200 mV s⁻¹. (D) The calibration plots between the dependence of redox peak currents and the potential scan rates.

Fig. 5B shows the current responses of the anti-EGFR/6-Fc-HT/AuNPs/MAPR/GCE in supporting electrolyte. It is observed that a pair of redox peaks with high magnitude are shown in line a of Fig. 5B. The apparent formal potential was calculated as 0.35 V from the average of the oxidation and reduction peak potentials. A control experiment was carried out, where Fc-COOH decorated MAPR microspheres were used to modify the GCE, producing an anti-EGFR/Fc-COOH/MAPR/GCE. However, the current response of the anti-EGFR/Fc-COOH/MAPR/GCE (line b of Fig. 5B) is lower than that of the anti-EGFR/6-Fc-HT/AuNPs/MAPR/GCE (line a of Fig. 5B). This can be attributed to the magnification function of the assembled AuNPs coated on the MAPR microspheres, because the AuNPs can facilitate electron transfer between surface immobilized protein and the underlying electrode.³² The relationship between scan rate and peak current is very important in understanding the electrochemical mechanism. The cyclic voltammetry (CV) of the proposed cytosensor (the anti-EGFR/6-Fc-HT/AuNPs/MAPR/GCE) in 5.0 mM [Fe(CN)₆]^4−/3− solution at different scan rates are investigated in the range of 20–200 mV s⁻¹. It is clearly seen in Fig. 5C that the potentials and peak currents are dependent on the scan rate. The peak current is enhanced when the scan rate is increased. As shown in Fig. 5D, linear relationships with good correlation coefficients are observed between the peak current and the scan rate in the range between 10 and 200 mV s⁻¹, suggesting that the redox process of the ferrocene of the 6-Fc-HT/AuNPs/MAPR microspheres on the electrode surface is a fast and surface-confined process.

The evaluation of A549 and AT II cells with electrochemical methods is also investigated using fibered confocal fluorescence microscopy. The A549 cells are adhered to the cytosensor surface. The viability of the cells on the cytosensor is easily affected by the physical and chemical properties of the cytosensor. The biocompatibility of the cytosensor is investigated by monitoring A549 cell proliferation on the anti-EGFR/6-Fc-HT/AuNPs/MAPR decorated ITO surface, as shown in Fig. 6A–C. The density of cultured A549 cells on the anti-EGFR/6-Fc-HT/AuNPs/MAPR decorated ITO surface increases with extended incubation time, confirming that A549 cells can grow and proliferate very well on the anti-EGFR/6-Fc-HT/AuNPs/MAPR decorated ITO surface. It is clearly seen that the proliferated A549 cells have good viability after incubation for 60 h, due to the morphology of the distinguishable filopodia,¹⁴ as shown in Fig. 6C. We also performed one control experiment by monitoring AT II cell proliferation on the anti-EGFR/6-Fc-HT/AuNPs/MAPR decorated ITO surface, as shown in Fig. 6D and E. As is well known, cancer cells divide more rapidly than normal cells. As a result, AT II cell proliferation (Fig. 6E) is slower than that of A549 cells (Fig. 6C) after incubation for 60 h. Proliferated AT II cells with good viability are shown in Fig. 6E. All the above observations indicate that our prepared cytosensor exhibits excellent biocompatibility.

Fig. 6 Fibered confocal fluorescence microscopy images of A549 cells on the anti-EGFR/6-Fc-HT/AuNPs/MAPR decorated ITO surface, incubated for 15 h (A), 30 h (B) and 60 h (C). Images of AT II cells on the modified ITO surface, incubated for 30 h (D) and 60 h (E). (F) SWV responses of the AuNPs/MAPR/GCE (a) and anti-EGFR/AuNPs/MAPR/GCE (b) after incubation in 1 × 10⁻³ cells per mL of A549 cell solution and 1 × 10⁻³ cells per mL of AT II cell solution, respectively.

The specificity of anti-EGFR, which can possibly cross-react with normal cells, is investigated by carrying out two control experiments. As shown in Fig. 6F (line b), when the cytosensor is decorated with anti-EGFR, the peak current (ΔI) of the A549 cells is obvious, while the peak current (ΔI) of the AT II cells is quite small. When anti-EGFR is absent, both of the corresponding responses are quite weak, as shown in Fig. 6F (line a). These observations indicate that the cross-reactivity can be ignored.

Fig. 7A shows the square wave voltammetry (SWV) responses of the cytosensor after being incubated in the different concentrations of A549 cell suspension. A549 cells are attached to the electrode surface through the immunoreaction with anti-EGFR, which was immobilized on the cytosensor previously. The insulating A549 cell layer acting as a nonconductor obstructed the electron transfer between the electrolyte and electrode surface. Therefore, the SWV peak currents decreased proportionally with the increasing concentration of A549 cells, which can be utilized as a quantitative measurement of A549 cell concentration. Fig. 7A shows that the peak current decreases with the increasing concentration of A549 cells. As shown in Fig. 7B, the calibration plot shows a good linear relationship between the peak currents and the logarithmic values of the A549 cell concentrations. It is clearly seen that there is a linear relationship between the peak current and the concentration of A549 cells over the range of 5–10⁶ cells per mL with a correlation coefficient of 0.994, suggesting a wide detection range and a detection limit as low as 5 cells per mL. Table S1 (ESI†) compares the sensing performance of different cytosensors for the detection of cancer cells, showing that our proposed cytosensor exhibits a lower detection limit than those of previously reported sensing systems.

Fig. 7 (A) SWV responses of the anti-EGFR/6-Fc-HT/AuNPs/MAPR/GCE after being incubated with different concentrations of A549 cells in 0.1 M PBS (pH: 7.4) solution: 0, 5, 10, 10², 10³, 10⁴, 10⁵, 10⁶ cells per mL (from bottom to top). (B) The calibration plot of the changes of the SWV current responses versus the logarithmic values of A549 cell concentrations. (C) SWV responses of the anti-EGFR/6-Fc-HT/AuNPs/MAPR/GCE after being incubated with different concentrations of A549 cells in the presence of 10⁴ cells per mL of AT II cells (from bottom to top: 0, 5, 10, 10², 10³, 10⁴, 10⁵, 10⁶ cells per mL of A549 cells). (D) The calibration plot of the changes of the SWV peak current intensity versus the logarithmic values of A549 cell concentrations in 0.1 M PBS (pH: 7.4) solution.

The selectivity of the prepared cytosensor is further evaluated in this study. SWV is also applied to monitor A549 cells in the presence of normal cells [human type II alveolar epithelial cell line (AT II cells)]. It is well-known that EGFR receptors can act as a tumor indicator. The EGFR receptors are overexpressed on lung cancer cells (A549 cells) while being expressed in limited numbers on normal lung cells.¹³ As a result, the proposed cytosensor can selectively detect A549 cells in the presence of AT II cells, due to the special recognition between anti-EGFR and EGFR receptors. Fig. 7C shows the SWV responses of the cytosensor after being incubated in different concentrations of A549 cell suspension in the presence of AT II cells. It is clearly seen in Fig. 7C that the SWV peak current intensity proportionally decreases with the A549 cell concentration. The calibration plot shows a good linear relationship between the peak currents and the logarithmic values of the A549 cell concentration in the range from 0 to 10⁶ A549 cells per mL with a correlation coefficient of 0.993, as shown in Fig. 7D. A detection limit as low as 5 cells per mL is obtained in the presence of a large number of AT II cells (10⁴ cells per mL), indicating that the prepared cytosensors exhibit high selectivity as well as high sensitivity. The detection limit is better than the previously reported cytosensors.^9,10,12 All these observations confirm that the cytosensors show a wider detection range and the detection can be finished quickly within half a minute, avoiding possible contamination of the cells.

Direct determination of cancer cell levels in blood serum plays an important role in the early detection of cancer. To testify the feasibility of the cytosensor in practical analysis, we employed the cytosensor to measure A549 cell concentrations in human serum samples. The electrolyte solution containing 5 mL of serum sample is used for the SW voltammetry experiment. The calibration plot showed a linear relationship between the peak currents and the logarithmic values of the A549 cell concentrations in Fig. S2 (ESI†). It is clearly seen that there is a linear relationship between the peak currents and the concentration of A549 cells over the range of 0–10⁶ cells per mL with a correlation coefficient of 0.98, as shown in Fig. S2.† A detection limit as low as 10 cells per mL is obtained in human serum samples. All the above observations indicate that the biosensor offers a method to detect A549 cell concentration in human serum samples.

4. Conclusions

In summary, a facile method for the synthesis and immobilization of small AuNPs on MAPR microspheres via a simple reduction route is reported for the first time. The AuNPs/MAPR microspheres not only possess high surface enhanced Raman scattering (SERS) activity and have great potential as a SERS-active substrate, but also play an important role in facilitating electron transfer. A novel electrochemical cytosensor which can sensitively differentiate lung cancer cells from normal ones by making use of the advantages of EGFR antibodies and AuNPs/MAPR microspheres has been designed. The prepared cytosensors exhibit good biocompatibility, high sensitivity and selectivity for the detection of A549 cells, and exhibit great potential for application in the development of biosensors.

Acknowledgements

We gratefully acknowledge support from the Chinese 973 Project (Grant: 2012CB933302), the National Natural Science Foundation of China (Grant: 21175022), the Ministry of Science & Technology of China (Grant: 2012AA022703), the Scientific Research Foundation of Graduate School of Southeast University (no. YBJJ1410), the Fundamental Research Funds for the Central Universities (no. 2242014Y10054) and Graduate Students’ Scientific Research Innovation Project of Jiangsu Province Ordinary University (no. CXZZ13_0124).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra00444f

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