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A novel aptasensor based on MUC-1 conjugated CNSs for ultrasensitive detection of tumor cells† Hongmei Cao, Daixin Ye, Qianqian Zhao, Juan Luo, Song Zhang and Jilie Kong* A novel strategy for the quantitative determination of human colon cancer DLD-1 cells utilizing an electrochemical aptasensor was developed by effective surface recognition between Mucin 1 glycoprotein over-expressed on the cell membrane and MUC-1 aptamer (MUC-1) bound on carbon nanospheres (CNSs). An MTT assay revealed that the as-prepared CNSs by green route exhibited satisfactory biocompatibility for cell viability, providing a suitable platform for the cell adhesion study. Furthermore, using CNSs as a sensing layer accelerated electron transfer and provided a highly stable matrix for the convenient conjugation of target MUC-1 aptamer, considerably amplifying the electrochemical signals. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were applied to assess the optimal conditions and detection performance of the as-fabricated aptasensor. The attachment of colon cancer DLD-1 cells onto the MUC-1 aptamer immobilized CNSs led to increased EIS responses, which changed linearly in cell concentration ranging from 1.25 102 to 1.25 106 cells per mL with a lower detection limit of 40 cells per mL. With this method, colon cancer DLD-1 cells can be easily distinguished from normal cells, Human astrocytes 1800. The novel aptasensor
Received 10th May 2014 Accepted 6th July 2014
revealed high specificity to DLD-1 cells. Furthermore, the aptasensor described here showed good reproducibility and high stability because of the CNSs of high stability and biocompatibility. The
DOI: 10.1039/c4an00844h
proposed protocols are a promising technique for the early monitoring of human colon cancer, and
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might have potential clinical applications such as cancer diagnosis, drug screening.
Introduction At present, cancer with a high mortality ratio is the greatest challenge in human healthcare and global economy. Moreover, cancer accounts for 13% of all deaths worldwide (2007 data), and this trend is expected to continue rising with an estimated 12 million deaths per year by 2030. Thus, the early detection of tumor cells and their activity monitoring is very signicant for public health protection, clinical diagnostics and life science research. Glycoproteins play key roles in a wide variety of cell activities such as cellular adhesion, immune response, cell signaling, differentiation, etc.1 Furthermore, the change in glycoproteins on the cell surface has been demonstrated to be associated with several cancers. Mucin 1 is a glycoprotein containing a hydrophobic membrane-spanning domain of 31 amino acids, a cytoplasmic domain of 69 amino acids, and an extracellular domain consisting of a region of nearly identical repeats of 20 amino acids per repeat.2 It presents in a variety of malignant tumors, and has been conrmed as a tumor marker for the Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, PR China. E-mail: [email protected]; Fax: +86 21 65641740; Tel: +86 21 65642138 † Electronic supplementary 10.1039/c4an00844h
information
(ESI)
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DOI:
diagnosis of early cancers. It is over-expressed in almost all human epithelial cell adenocarcinomas, including colorectal,3,4 lung,5 prostate,6 ovarian,7 pancreatic8,9 and bladder carcinomas;10 whereas in normal cells, its expression is signicantly low. Thus, the sensitive determination of cell surface MUC-1 glycoprotein expression is crucial for achieving early cancer detection. In recent years, there have been some reports regarding mucin 1 glycoprotein based on MUC-1 aptamer (MUC-1). Aptamers possess unique advantages compared to other targeting agents such as small size, stability in harsh biological environments, and high affinity for targets, easy for chemical modication as well as reduced immunogenicity and toxicity.11 Therefore, these properties make MUC-1 the mostvaluable molecular probe for the sensitive recognition of MUC-1 glycoprotein or specic cancer cells at very low concentrations. MUC-1 aptamers have been widely utilized as a sensing element for cancer cells. For instance, Savla et al. took an advantage of the mucin 1 aptamer–doxorubicin conjugate for the imaging and treatment of ovarian cancer.12 Pang et al. utilized uorescent mucin 1 aptamer for the turn-on detection of epithelial tumor marker mucin.13 Cai et al. detected human breast cancer by aptamer–silver–gold nanostructures with surface-enhanced Raman scattering (SERS).14 However, these methods are not simple and rapid enough for detecting MUC-1 glycoprotein or cancerous cells because of the complex FRET, SERS and
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uorescent techniques. Therefore, it is important to develop an efficient, rapid, low-cost, ease of operation method, and fabricate a device to detect tumor cells. Electrochemical impedance spectroscopy (EIS) has been developed to study and detect various biological substances, ranging from DNA and proteins to viruses and bacteria.15,16 This technique has emerged as an effective approach to monitor cellular processes, including cell spreading, adhesion, toxicology and motility17,18 because of its cost efficiency, easy-tooperate, rapid response and high sensitivity, particularly, without the need for labelling species. For example, Hu et al. reported the preparation of [email protected] and [email protected] electrochemical biosensors for the sensitive detection of KB and BXPC cells, respectively.19,20 Wang et al. fabricated an enhanced impedance cytosensor based on folate conjugated-polyethylenimine carbon nanotubes for tumor targeting.21 Ju et al. prepared gold nanoparticles–chitosan nanocomposite gel for the electrochemical investigation of K562 leukemia cells.22 Several electrochemical biosensors have been applied for advanced tumor-targeting.23,24 Recently, with the advent of nanoscience and nanotechnology, carbon nanomaterials as a sensing interface have attracted considerable attention in electrochemical biosensors. Carbon nanospheres (CNSs) have also been demonstrated as an ideal candidate for applications in electrochemical sensing platforms because of their high chemical stability, good signal amplication ability, and convenient and absolutely ‘green’ synthetic method. In this work, the synthetic approach is an entirely ‘green’ method by a controllable hydrothermal synthetic route in aqueous glucose solutions,25 and the synthetic procedure does not involve any organic solvents. In particular, the as-prepared CNSs inherit numerous functional groups and reactive surfaces, which conjugate with target biomolecules. On the basis of these properties, we attempted to immobilize MUC-1 aptamer on CNSs for the sensitive detection of DLD-1 cells. In this paper, based on MUC-1 aptamer with specicity against Mucin 1 glycoprotein over-expressed on a DLD-1 membrane, we successfully constructed an electrochemical impedance aptasensor by MUC-1 aptamer conjugated nanomaterials of CNSs as a sensing platform for tumor targeting. We rst report a MUC-1 aptamer-based quantitative detection protocol for human colon cancer DLD-1 cells. Furthermore, we investigated the performance of this impedance aptasensor with a detection limit of approximately 40 cells per mL, as well as a linear detection range from 1.25 102 to 1.25 106 cells per mL. The CNSs-based EIS aptasensor exhibited a broad detection range with a fairly low detection limit for DLD-1 cells. Therefore, our proposed aptasensor would have signicant potential applications in the early diagnosis of human colorectal cancer.
Experimental section Materials and reagents Amino MUC1 DNA aptamer (HPLC puried) were manufactured by Sangon Biotech (Shanghai) Co., Ltd. The sequence was 50 NH2-GCA GTT GAT CCT TTG GAT ACC CTG G-30 . D-(+)-Glucose
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(analytical purity) was purchased from Sinopharm Chemical Reagent Co., Ltd (China). N-Hydroxysuccinimide sodium salt (NHS, 98%) was obtained from Aladdin industrial corporation (Shanghai), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, C8H17N3$HCl), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT, C18H16N5SBr, ultrapure grade) and dimethyl sulfoxide (DMSO) were acquired from Sigma-Aldrich Company (China). 10 mM phosphate buffer solution (PBS, pH 7.4) was used as the rinsing solution and the diluent of MUC-1 aptamer. [Fe(CN)6]3/4 solution containing 10 mM K3Fe(CN)6, 10 mM K4Fe(CN)6 and 0.1 M KCl (as the supporting electrolyte) was prepared as a redox probe in the measuring system. Doubly distilled water was used throughout the experiments. All the other reagents were of analytical grade and used without further purication. Instrumentation and electrochemical measurements EIS was carried out on Autolab PGSTST 30 analyzer (Metrohm Autolab B.V., Switzerland). Impedance spectra were recorded within the frequency range of 101 to 105 Hz with a signal amplitude of 5 mV. Cyclic voltammetry (CV) were performed on a CHI 1030 electrochemical workstation with a conventional three-electrode system consisting of a working electrode, a Pt foil auxiliary electrode and a saturated calomel electrode (SCE) as the reference. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images were obtained using a JEOL 2011 microscope (Japan) operated at 200 kV and a Philips XL30 electron microscope (The Netherlands) operated at 10 kV, respectively. The MTT assay was performed on an enzyme-labeled instrument (SUNOSTIK SPR-960). Cell culture and collection Human colon cancer DLD-1 cells were purchased from the Cell Bank of The Chinese Academy of Sciences. Human DLD-1 colorectal cancer cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 units per penicillin streptomycin and incubated at 37 C in a humidied atmosphere containing 5% wt/vol CO2. Aer 72 h, DLD-1 cells were trypsinized in a 0.25% trypsin solution, collected from the culture medium by centrifugation at 1000 rpm for 5 min, and then washed twice with pH 7.4 sterile PBS. The sediment was resuspended in PBS to obtain a 0.5 mL homogeneous cell suspension. Cell suspensions with different concentrations were prepared from this stock. Cell number was determined using a Petroff-Hausser counter. Synthesis of CNSs The CNSs used in this work were synthesized by a hydrothermal route according to a literature report. A “green” synthetic approach has been developed that involves the transformation of sugars into homogeneous and stable colloidal CNSs, which are hydrophilic.26 Briey, 4 g of a-D-glucose was dissolved in 40 mL of ultrapure water to form a clear solution. The suspension was then placed in a 100 mL Teon-sealed reactor, and the temperature was maintained at 180 C for 6 h. The obtained black suspension was isolated by centrifugation (12 000 rpm, 15 min). The CNSs
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were obtained aer repeatedly washing with ethanol and water and drying under vacuum at 60 C overnight.
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Surface modication of CNSs The carboxylation procedure for the CNSs are as follows. The obtained CNSs were added to an acid mixture (the volume ratio of H2SO4 to HNO3 was 3), and treated by stirring for 2 h for generating carboxylic groups on the surface of CNSs. The resulting dispersion was puried by centrifugation (18 000 rpm, 15 min). Aer washing to achieve a neutral pH, the carboxylated CNSs were dried at 80 C for 12 h. Fabrication of the aptasensor for DLD-1 cells The fabrication and recognition process of this electrochemical aptasensor is illustrated in Scheme 1. Briey, the EIS aptasensor was assembled by the following process. First, 3 mL of carboxylated CNSs (0.5 mg mL1) was dropped on the surface of the pretreated GCE and dried at room temperature. EDC and NHS were used to activate the carboxyl groups of CNSs to the formation of the NHS ester. In detail, CNSs/GCE was immersed in a solution containing 2 mM EDC and 5 mM NHS for 1 h to form a stable active ester followed by thorough rinsing with deionized water. MUC-1 aptamer with various concentrations as the capture probe was immediately dropped on the surface of the activated CNSs/GCE and incubated for 0.5 h to form an MUC-1 aptamer/CNSs/GCE. During this process, the active NHS esters were replaced by the amino groups of the aptamer and the acid group of CNSs was conjugated through the amide
Scheme 1
bond. Finally, 5 mL of different concentrations of a DLD-1 cell suspension was dropped onto the modied electrodes and incubated at 37 C for several hours to achieve cell capture. Before use, pH 7.4 PBS was used to rinse the electrodes thoroughly to remove nonspecic adsorbed biomolecules. In this manner, the proposed aptasensor was eventually produced by a layer-by-layer (LBL) technique with excellent capability for the special recognition of tumor cells.
Results and discussion Characterization of the CNSs–COOH nanospheres sensing layer Fig. 1 shows typical transmission electron microscopy (TEM A.B) and eld-emission scanning electron microscopy (FESEM C.) images of the carboxylated carbon nanosphere material. The obtained functionalized carbon nanospheres with an average diameter of 500 nm were uniform in size and morphology. Typical TEM image in Fig. 1B (inset) clearly revealed a thin layer (COOH) wrapped outside the CNSs (inset). The FT-IR spectrum (Fig. 2) was used to identify the functional groups on CNSs. Two absorption peaks observed at 1710 cm1 and 1620 cm1 were ascribed to C]O and C]C vibrations, respectively. The bands at 3417 cm1 and 1396 cm1 were attributed to O–H stretching vibration and bending vibrations, and the peak at 1240 cm1 was assigned to the C–OH stretching vibration. The FT-IR spectrum results are consistent with the TEM image (inset), demonstrating that the carboxylated CNSs were successfully prepared. COOH groups imparted CNS–COOH with good
Schematic representation of carboxylated CNSs-based aptasensing strategy for human colon cancer DLD-1 cell detection by the EIS
technique.
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Fig. 1 TEM images of carboxylated CNSs (A and B), FESEM images of carboxylated CNSs (C), DLD-1 cells viability exposure to CNSs–COOH at various concentrations for 24 and 48 h (D).
water-solubility and easy conjugation. These monodispersed carboxylated CNSs were vital for conjugating targeting amino MUC-1 aptamer on each nanosphere, which would affect the sensitivity and analytical performance of the fabricated aptasensor. EIS characteristics of aptasensor fabrication Because of the immediate and sensitive response from electrode surface changes,27 EIS has been popularly developed to monitor the layer by layer (LBL) assembly process. EIS includes a semicircle portion corresponding to the electron-transfer limited process at higher frequencies and a linear portion representing the diffusion-limited process at lower frequencies. The semicircle diameter equals the electron-transfer resistance (Ret). Fig. 3A illustrates the Nyquist diagrams of the electrochemical impedance spectra recorded from 0.1 to 105 Hz for different modied glassy carbon electrodes (GCE) in the presence of [Fe(CN)6]3/4 (10 mM, 1 : 1) containing 0.1 M KCl solutions. At a bare GCE, the redox process of the probe showed low electron-transfer impedance (Ret) value (curve a). To obtain the covalent conjugation of amino MUC-1 aptamer, the abovementioned modied carboxylated CNSs/GCE was then immersed in a solution containing EDC and NHS to activate the carboxy group of CNSs. Aer activation, the Ret (curve b) increased compared with the bare GCE (curve a) because the stable active ester of CNSs could hinder electron transmission to some extent. Upon the conjugation of MUC-1 aptamer on the surfaces of CNSs, the Ret value increased signicantly (curve c), implying that the assembly of the MUC-1 aptamer on the electrode surface inhibited interfacial electron transfer. When the
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DLD-1 cells were attached to the MUC-1/CNSs/GCE by a special interaction between the MUC-1 aptamer and MUC-1 glycoprotein on the cellular membrane, the Ret value signicantly increased (curve d), which was attributed to the fact that the cell membrane can further hamper the redox probe close to the electrode surface. On the basis of the equivalent circuit model (inset in Fig. 3B), the successively changing impedance data were tted to Ret values, which were illustrated by the histogram in Fig. 3B, and the EIS tting parameters of different modied electrodes are provided in Table S1 (ESI).† Evaluation of cytotoxicity of CNSs Cell viability was determined by an MTT assay to evaluate the cytotoxicity of CNSs with various concentrations for 24 h and 48 h, as shown in Fig. 1D (detailed data of MTT in Table S3 and S4†). Briey, the cells were plated in 96-well at-bottomed plates at 5 104 cells per well and allowed to grow overnight prior to exposure to CNSs with different concentrations. Controls were incubated under the same conditions without the addition of CNSs. Aer 24 h or 48 h further incubation, the MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) was added for 4 h at 37 C to allow the conversion of MTT to a purple formazan product by active mitochondria. The formazan product was then dissolved in DMSO to solubilize the precipitate of the formazan crystals. Finally, the plate was shaken for 15 min before measuring optical absorbance at 492 nm by an automatic ELISA analyzer (SPR-960). The absorbance of formazan is proportional to the number of living cells.28 With increasing CNSs concentration from 0 to 1.25 mg mL1, the DLD-1 cell viability showed a slightly decreasing trend
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Fig. 2
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FT-IR spectrum of carboxylated CNSs.
compared to the controls at 24 h. Aer 48 h, the MTT assay exhibited only slightly lower absorbance and approximately 80% of the DLD-1 cells were still alive. The MTT results indicated that the CNSs shows excellent biocompatibility and become a nontoxic support for the immobilization of cells.
Optimization of experimental conditions For aptasensor assay, the incubation time of DLD-1 cells was an important parameter in the performance of cell-capture. To evaluate the effects of incubation time on the response signal, the fabricated aptasensors were incubated at 37 C for different times from 30 to 150 min. The results showed that the EIS response revealed a increasing trend with increasing incubation time (Fig. 4A). It was benecial for MUC-1 aptamer special capturing more and more DLD-1 cells with time. When the incubation time was over 120 min, there was no apparent change in the Ret value, suggesting that the amount of DLD-1 cells captured on the surface of the aptasensor was saturated. Thus, 120 min was used as the optimal incubation time for capturing cells. Moreover, the concentration of MUC-1 aptamer is a critical factor to recognize DLD-1 cells. The capturing
efficiency of the aptasensor was directly related to the concentration of the MUC-1 aptamer on the electrode surface. Thus, an investigation of different concentrations of MUC-1 aptamer from 0.1 to 80 mM was carried out in the presence of [Fe(CN)6]3/4 couple (Fig. 4B). Then cyclic voltammograms of MUC-1 aptamer at different concentrations were recorded to choose the optimal condition for this experiment. For example, the CV of the MUC-1 aptamer concentration at 10 mM is recorded in ESI Fig. S1.† With increasing concentration of the MUC-1 aptamer, the CV peak current at potential of 0.36 V of the cell adhered aptasensor decreased, demonstrating that an increasing number of DLD-1 cells were being captured. A minimum CV response obtained at 40 mM then increased slightly, indicating that the cells captured on the modied electrodes had been saturated. Accordingly, an optimal concentration of MUC-1 aptamer at 40 mM was recommended for further research. EIS for quantitative detection of DLD-1 cells EIS, an electrochemical technique, has become an alternative to developing electrochemical biosensors for the quantitative detection of tumor cells. Herein, using [Fe(CN)6]3/4 as the redox probe, EIS was applied to evaluate the performance of the fabricated aptasensor under optimized conditions. The diameter of the semicircle on the Nyquist diagram gradually increased with increasing DLD-1 cell concentration ranging from 1.25 102 to 1.25 106 cells per mL, implying that a higher amount of DLD-1 cells immobilized on the electrode with a correlation coefficient of 0.994 (Fig. 5). The linear regression equation was DRet (U) ¼ 390.14 log C[DLD-1] (cells per mL) 405.68 with a detection limit of 40 cells per mL (S/N ¼ 3). The proposed assay displayed a lower detection limit compared to other reported electrochemical cell sensors, such as 90 cells per mL at an impedance cytosensor based on folate conjugatedpolyethylenimine carbon nanotubes for HeLa cells,21 8.71 102 cells per mL at a bio-inspired support of gold nanoparticles– chitosan nanocomposites gel for K562,22 100 cells per mL at aptamer-quantum dots for MCF-7.29 The proposed aptasensor indicating a lower detection limit and high sensitivity could be attributed to the following reasons. On one hand, CNSs with
EIS of the electrode at different fabrication stages (a) GCE, after (b) activated CNS–COOH, (c) immobilization of MUC-1 aptamer, and (d) capture of DLD-1 cells in 10 mM [Fe(CN)6]3/4 with 1.0 M KCl. The equivalent circuit model inset in the histogram was applied to fit the impedance data of the assembly process to Ret values (B). Fig. 3
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Fig. 4 Effects of (A) incubation time of DLD-1 cells on EIS response and (B) the concentration of MUC-1 aptamer on cyclic voltammetry response in [Fe(CN)6]3/4 (10 mM, 1 : 1) containing 1.0 M KCl.
excellent conductivity and outstanding biocompatibility was suitable for the immobilization of MUC-1 aptamer with high stability and bioactivity, providing an ideal interface for cell capture and improving the sensitivity of the aptasensor. On the other hand, high affinity between MUC-1 aptamer and MUC-1 glycoprotein, which is over-expressed on the cell membrane of DLD-1 cells, would further enhance the sensitivity of the present strategy. Specicity, stability and reproducibility of the aptasensing strategy The aptasensor showed an excellent specic response against DLD-1 cells. In our experiment, Human astrocytes 1800 cells and DLD-1 cells at the same concentration (1.25 104 cells per mL) were selected to evaluate the specicity of this aptasensor under optimized conditions. As shown in Fig. 6, the DPV peak current of the aptasensor changed less for 1800 cells, while DLD-1 cells lead to a signicant change in DPV peak current. The DPV peak current alteration of the aptasensor in the presence of DLD-1 cells was 7 mA, which is considerably higher than 1.5 mA for 1800 cells. Although nonspecic adsorption and slight expression of the MUC-1 glycoprotein on the surface of Human astrocytes 1800 cells may also result in a small DPV peak current change, a comparison of the peaks current can clearly indicate that the proposed method can efficiently distinguish DLD-1 cells from control cells with high specicity. The good selectivity of this aptasensor was attributed to the
Fig. 6 Specificity of the proposed aptasensor. Typical DPV response change in peak current with the modified electrode after respective incubation with DLD-1 cells and 1800 cells in 10 mM [Fe(CN)6]3/4(10 mM, 1 : 1) containing 0.1 M KCl. Error bars are the standard deviation of three replicate determinations.
high specicity of the aptamer. We further studied the intraassay precision of this aptasensor by detecting DLD-1 cells at two levels. At DLD-1 cell concentrations of 1.25 105 and 1.25 104 cells per mL, the aptasensor displayed the relative standard deviations (RSD) of 4.2% and 4.9% for ve determinations, respectively, signifying an acceptable precision. The
Fig. 5 Nyquist diagrams of electrochemical impedance spectra recorded at aptamer/CNSs–COOH fabricated aptasensor after capturing different concentrations of DLD-1 cells: (a) 0, (b) 1.25 102, (c) 1.25 103, (d) 1.25 104, (e) 1.25 105, and (f) 1.25 106 cells per mL from 0.1– 105 Hz in [Fe(CN)6]3/4 (10 mM, 1 : 1) containing 1.0 M KCl. Calibration curve of the aptasensor for DLD-1 cells (B).
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reproducibility of the aptasensor for DLD-1 cells was estimated using ve replicate measurements from the batch, the results showed an RSD of 3.5%, identifying the excellent fabrication reproducibility of this strategy. Moreover, it was necessary to observe this stability because it is a crucial property for fabricated aptasensors. When the modied aptasensor was stored in the refrigerator at 4 C for 15 days, EIS decreased by 10.9% from its initial signal response (ESI Fig. S2†), and the DPV response still retained 90.3% of its initial signal response. EIS and DPV results are consistent, suggesting satisfactory stability, which can be attributed to the strong interactions between CNSs and aptamer and the high stability of CNSs as a matrix.
Conclusions In summary, this work has successfully proposed a CNSs-based aptasensing strategy for ultrasensitive human colon cancer DLD-1 cells determination by a recognition process of the MUC1 aptamer to MUC-1 glyprotein on the cellular membrane that can be used for monitoring the adhesion of DLD-1 cells by electrochemical impedance spectroscopy, producing a sensitive impedance aptasensor for human colon cancer DLD-1 cells. The proposed aptasensor showed a wide linear detection range, and high sensitivity for the quantication of DLD-1 cells attributed to the conjugation of targeting MUC-1 aptamer to the conductivity and biocompatibility of CNSs. Furthermore, the developed aptasensor displayed high specicity to tumor cells overexpressing the MUC-1 glycoprotein, acceptable reproducibility and excellent stability, providing a feasible technique for the early diagnosis of colon cancer.
Acknowledgements This work was supported by The National Natural Science Foundation of China (21335002, 21175029) and the Shanghai Leading Academic Discipline Project (B109).
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A novel aptasensor based on MUC-1 conjugated CNSs for ultrasensitive detection of tumor cells† Hongmei Cao, Daixin Ye, Qianqian Zhao, Juan Luo, Song Zhang and Jilie Kong* A novel strategy for the quantitative determination of human colon cancer DLD-1 cells utilizing an electrochemical aptasensor was developed by effective surface recognition between Mucin 1 glycoprotein over-expressed on the cell membrane and MUC-1 aptamer (MUC-1) bound on carbon nanospheres (CNSs). An MTT assay revealed that the as-prepared CNSs by green route exhibited satisfactory biocompatibility for cell viability, providing a suitable platform for the cell adhesion study. Furthermore, using CNSs as a sensing layer accelerated electron transfer and provided a highly stable matrix for the convenient conjugation of target MUC-1 aptamer, considerably amplifying the electrochemical signals. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were applied to assess the optimal conditions and detection performance of the as-fabricated aptasensor. The attachment of colon cancer DLD-1 cells onto the MUC-1 aptamer immobilized CNSs led to increased EIS responses, which changed linearly in cell concentration ranging from 1.25 102 to 1.25 106 cells per mL with a lower detection limit of 40 cells per mL. With this method, colon cancer DLD-1 cells can be easily distinguished from normal cells, Human astrocytes 1800. The novel aptasensor
Received 10th May 2014 Accepted 6th July 2014
revealed high specificity to DLD-1 cells. Furthermore, the aptasensor described here showed good reproducibility and high stability because of the CNSs of high stability and biocompatibility. The
DOI: 10.1039/c4an00844h
proposed protocols are a promising technique for the early monitoring of human colon cancer, and
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might have potential clinical applications such as cancer diagnosis, drug screening.
Introduction At present, cancer with a high mortality ratio is the greatest challenge in human healthcare and global economy. Moreover, cancer accounts for 13% of all deaths worldwide (2007 data), and this trend is expected to continue rising with an estimated 12 million deaths per year by 2030. Thus, the early detection of tumor cells and their activity monitoring is very signicant for public health protection, clinical diagnostics and life science research. Glycoproteins play key roles in a wide variety of cell activities such as cellular adhesion, immune response, cell signaling, differentiation, etc.1 Furthermore, the change in glycoproteins on the cell surface has been demonstrated to be associated with several cancers. Mucin 1 is a glycoprotein containing a hydrophobic membrane-spanning domain of 31 amino acids, a cytoplasmic domain of 69 amino acids, and an extracellular domain consisting of a region of nearly identical repeats of 20 amino acids per repeat.2 It presents in a variety of malignant tumors, and has been conrmed as a tumor marker for the Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, PR China. E-mail: [email protected]; Fax: +86 21 65641740; Tel: +86 21 65642138 † Electronic supplementary 10.1039/c4an00844h
information
(ESI)
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DOI:
diagnosis of early cancers. It is over-expressed in almost all human epithelial cell adenocarcinomas, including colorectal,3,4 lung,5 prostate,6 ovarian,7 pancreatic8,9 and bladder carcinomas;10 whereas in normal cells, its expression is signicantly low. Thus, the sensitive determination of cell surface MUC-1 glycoprotein expression is crucial for achieving early cancer detection. In recent years, there have been some reports regarding mucin 1 glycoprotein based on MUC-1 aptamer (MUC-1). Aptamers possess unique advantages compared to other targeting agents such as small size, stability in harsh biological environments, and high affinity for targets, easy for chemical modication as well as reduced immunogenicity and toxicity.11 Therefore, these properties make MUC-1 the mostvaluable molecular probe for the sensitive recognition of MUC-1 glycoprotein or specic cancer cells at very low concentrations. MUC-1 aptamers have been widely utilized as a sensing element for cancer cells. For instance, Savla et al. took an advantage of the mucin 1 aptamer–doxorubicin conjugate for the imaging and treatment of ovarian cancer.12 Pang et al. utilized uorescent mucin 1 aptamer for the turn-on detection of epithelial tumor marker mucin.13 Cai et al. detected human breast cancer by aptamer–silver–gold nanostructures with surface-enhanced Raman scattering (SERS).14 However, these methods are not simple and rapid enough for detecting MUC-1 glycoprotein or cancerous cells because of the complex FRET, SERS and
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uorescent techniques. Therefore, it is important to develop an efficient, rapid, low-cost, ease of operation method, and fabricate a device to detect tumor cells. Electrochemical impedance spectroscopy (EIS) has been developed to study and detect various biological substances, ranging from DNA and proteins to viruses and bacteria.15,16 This technique has emerged as an effective approach to monitor cellular processes, including cell spreading, adhesion, toxicology and motility17,18 because of its cost efficiency, easy-tooperate, rapid response and high sensitivity, particularly, without the need for labelling species. For example, Hu et al. reported the preparation of [email protected] and [email protected] electrochemical biosensors for the sensitive detection of KB and BXPC cells, respectively.19,20 Wang et al. fabricated an enhanced impedance cytosensor based on folate conjugated-polyethylenimine carbon nanotubes for tumor targeting.21 Ju et al. prepared gold nanoparticles–chitosan nanocomposite gel for the electrochemical investigation of K562 leukemia cells.22 Several electrochemical biosensors have been applied for advanced tumor-targeting.23,24 Recently, with the advent of nanoscience and nanotechnology, carbon nanomaterials as a sensing interface have attracted considerable attention in electrochemical biosensors. Carbon nanospheres (CNSs) have also been demonstrated as an ideal candidate for applications in electrochemical sensing platforms because of their high chemical stability, good signal amplication ability, and convenient and absolutely ‘green’ synthetic method. In this work, the synthetic approach is an entirely ‘green’ method by a controllable hydrothermal synthetic route in aqueous glucose solutions,25 and the synthetic procedure does not involve any organic solvents. In particular, the as-prepared CNSs inherit numerous functional groups and reactive surfaces, which conjugate with target biomolecules. On the basis of these properties, we attempted to immobilize MUC-1 aptamer on CNSs for the sensitive detection of DLD-1 cells. In this paper, based on MUC-1 aptamer with specicity against Mucin 1 glycoprotein over-expressed on a DLD-1 membrane, we successfully constructed an electrochemical impedance aptasensor by MUC-1 aptamer conjugated nanomaterials of CNSs as a sensing platform for tumor targeting. We rst report a MUC-1 aptamer-based quantitative detection protocol for human colon cancer DLD-1 cells. Furthermore, we investigated the performance of this impedance aptasensor with a detection limit of approximately 40 cells per mL, as well as a linear detection range from 1.25 102 to 1.25 106 cells per mL. The CNSs-based EIS aptasensor exhibited a broad detection range with a fairly low detection limit for DLD-1 cells. Therefore, our proposed aptasensor would have signicant potential applications in the early diagnosis of human colorectal cancer.
Experimental section Materials and reagents Amino MUC1 DNA aptamer (HPLC puried) were manufactured by Sangon Biotech (Shanghai) Co., Ltd. The sequence was 50 NH2-GCA GTT GAT CCT TTG GAT ACC CTG G-30 . D-(+)-Glucose
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(analytical purity) was purchased from Sinopharm Chemical Reagent Co., Ltd (China). N-Hydroxysuccinimide sodium salt (NHS, 98%) was obtained from Aladdin industrial corporation (Shanghai), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, C8H17N3$HCl), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT, C18H16N5SBr, ultrapure grade) and dimethyl sulfoxide (DMSO) were acquired from Sigma-Aldrich Company (China). 10 mM phosphate buffer solution (PBS, pH 7.4) was used as the rinsing solution and the diluent of MUC-1 aptamer. [Fe(CN)6]3/4 solution containing 10 mM K3Fe(CN)6, 10 mM K4Fe(CN)6 and 0.1 M KCl (as the supporting electrolyte) was prepared as a redox probe in the measuring system. Doubly distilled water was used throughout the experiments. All the other reagents were of analytical grade and used without further purication. Instrumentation and electrochemical measurements EIS was carried out on Autolab PGSTST 30 analyzer (Metrohm Autolab B.V., Switzerland). Impedance spectra were recorded within the frequency range of 101 to 105 Hz with a signal amplitude of 5 mV. Cyclic voltammetry (CV) were performed on a CHI 1030 electrochemical workstation with a conventional three-electrode system consisting of a working electrode, a Pt foil auxiliary electrode and a saturated calomel electrode (SCE) as the reference. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images were obtained using a JEOL 2011 microscope (Japan) operated at 200 kV and a Philips XL30 electron microscope (The Netherlands) operated at 10 kV, respectively. The MTT assay was performed on an enzyme-labeled instrument (SUNOSTIK SPR-960). Cell culture and collection Human colon cancer DLD-1 cells were purchased from the Cell Bank of The Chinese Academy of Sciences. Human DLD-1 colorectal cancer cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 units per penicillin streptomycin and incubated at 37 C in a humidied atmosphere containing 5% wt/vol CO2. Aer 72 h, DLD-1 cells were trypsinized in a 0.25% trypsin solution, collected from the culture medium by centrifugation at 1000 rpm for 5 min, and then washed twice with pH 7.4 sterile PBS. The sediment was resuspended in PBS to obtain a 0.5 mL homogeneous cell suspension. Cell suspensions with different concentrations were prepared from this stock. Cell number was determined using a Petroff-Hausser counter. Synthesis of CNSs The CNSs used in this work were synthesized by a hydrothermal route according to a literature report. A “green” synthetic approach has been developed that involves the transformation of sugars into homogeneous and stable colloidal CNSs, which are hydrophilic.26 Briey, 4 g of a-D-glucose was dissolved in 40 mL of ultrapure water to form a clear solution. The suspension was then placed in a 100 mL Teon-sealed reactor, and the temperature was maintained at 180 C for 6 h. The obtained black suspension was isolated by centrifugation (12 000 rpm, 15 min). The CNSs
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were obtained aer repeatedly washing with ethanol and water and drying under vacuum at 60 C overnight.
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Surface modication of CNSs The carboxylation procedure for the CNSs are as follows. The obtained CNSs were added to an acid mixture (the volume ratio of H2SO4 to HNO3 was 3), and treated by stirring for 2 h for generating carboxylic groups on the surface of CNSs. The resulting dispersion was puried by centrifugation (18 000 rpm, 15 min). Aer washing to achieve a neutral pH, the carboxylated CNSs were dried at 80 C for 12 h. Fabrication of the aptasensor for DLD-1 cells The fabrication and recognition process of this electrochemical aptasensor is illustrated in Scheme 1. Briey, the EIS aptasensor was assembled by the following process. First, 3 mL of carboxylated CNSs (0.5 mg mL1) was dropped on the surface of the pretreated GCE and dried at room temperature. EDC and NHS were used to activate the carboxyl groups of CNSs to the formation of the NHS ester. In detail, CNSs/GCE was immersed in a solution containing 2 mM EDC and 5 mM NHS for 1 h to form a stable active ester followed by thorough rinsing with deionized water. MUC-1 aptamer with various concentrations as the capture probe was immediately dropped on the surface of the activated CNSs/GCE and incubated for 0.5 h to form an MUC-1 aptamer/CNSs/GCE. During this process, the active NHS esters were replaced by the amino groups of the aptamer and the acid group of CNSs was conjugated through the amide
Scheme 1
bond. Finally, 5 mL of different concentrations of a DLD-1 cell suspension was dropped onto the modied electrodes and incubated at 37 C for several hours to achieve cell capture. Before use, pH 7.4 PBS was used to rinse the electrodes thoroughly to remove nonspecic adsorbed biomolecules. In this manner, the proposed aptasensor was eventually produced by a layer-by-layer (LBL) technique with excellent capability for the special recognition of tumor cells.
Results and discussion Characterization of the CNSs–COOH nanospheres sensing layer Fig. 1 shows typical transmission electron microscopy (TEM A.B) and eld-emission scanning electron microscopy (FESEM C.) images of the carboxylated carbon nanosphere material. The obtained functionalized carbon nanospheres with an average diameter of 500 nm were uniform in size and morphology. Typical TEM image in Fig. 1B (inset) clearly revealed a thin layer (COOH) wrapped outside the CNSs (inset). The FT-IR spectrum (Fig. 2) was used to identify the functional groups on CNSs. Two absorption peaks observed at 1710 cm1 and 1620 cm1 were ascribed to C]O and C]C vibrations, respectively. The bands at 3417 cm1 and 1396 cm1 were attributed to O–H stretching vibration and bending vibrations, and the peak at 1240 cm1 was assigned to the C–OH stretching vibration. The FT-IR spectrum results are consistent with the TEM image (inset), demonstrating that the carboxylated CNSs were successfully prepared. COOH groups imparted CNS–COOH with good
Schematic representation of carboxylated CNSs-based aptasensing strategy for human colon cancer DLD-1 cell detection by the EIS
technique.
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Fig. 1 TEM images of carboxylated CNSs (A and B), FESEM images of carboxylated CNSs (C), DLD-1 cells viability exposure to CNSs–COOH at various concentrations for 24 and 48 h (D).
water-solubility and easy conjugation. These monodispersed carboxylated CNSs were vital for conjugating targeting amino MUC-1 aptamer on each nanosphere, which would affect the sensitivity and analytical performance of the fabricated aptasensor. EIS characteristics of aptasensor fabrication Because of the immediate and sensitive response from electrode surface changes,27 EIS has been popularly developed to monitor the layer by layer (LBL) assembly process. EIS includes a semicircle portion corresponding to the electron-transfer limited process at higher frequencies and a linear portion representing the diffusion-limited process at lower frequencies. The semicircle diameter equals the electron-transfer resistance (Ret). Fig. 3A illustrates the Nyquist diagrams of the electrochemical impedance spectra recorded from 0.1 to 105 Hz for different modied glassy carbon electrodes (GCE) in the presence of [Fe(CN)6]3/4 (10 mM, 1 : 1) containing 0.1 M KCl solutions. At a bare GCE, the redox process of the probe showed low electron-transfer impedance (Ret) value (curve a). To obtain the covalent conjugation of amino MUC-1 aptamer, the abovementioned modied carboxylated CNSs/GCE was then immersed in a solution containing EDC and NHS to activate the carboxy group of CNSs. Aer activation, the Ret (curve b) increased compared with the bare GCE (curve a) because the stable active ester of CNSs could hinder electron transmission to some extent. Upon the conjugation of MUC-1 aptamer on the surfaces of CNSs, the Ret value increased signicantly (curve c), implying that the assembly of the MUC-1 aptamer on the electrode surface inhibited interfacial electron transfer. When the
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DLD-1 cells were attached to the MUC-1/CNSs/GCE by a special interaction between the MUC-1 aptamer and MUC-1 glycoprotein on the cellular membrane, the Ret value signicantly increased (curve d), which was attributed to the fact that the cell membrane can further hamper the redox probe close to the electrode surface. On the basis of the equivalent circuit model (inset in Fig. 3B), the successively changing impedance data were tted to Ret values, which were illustrated by the histogram in Fig. 3B, and the EIS tting parameters of different modied electrodes are provided in Table S1 (ESI).† Evaluation of cytotoxicity of CNSs Cell viability was determined by an MTT assay to evaluate the cytotoxicity of CNSs with various concentrations for 24 h and 48 h, as shown in Fig. 1D (detailed data of MTT in Table S3 and S4†). Briey, the cells were plated in 96-well at-bottomed plates at 5 104 cells per well and allowed to grow overnight prior to exposure to CNSs with different concentrations. Controls were incubated under the same conditions without the addition of CNSs. Aer 24 h or 48 h further incubation, the MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) was added for 4 h at 37 C to allow the conversion of MTT to a purple formazan product by active mitochondria. The formazan product was then dissolved in DMSO to solubilize the precipitate of the formazan crystals. Finally, the plate was shaken for 15 min before measuring optical absorbance at 492 nm by an automatic ELISA analyzer (SPR-960). The absorbance of formazan is proportional to the number of living cells.28 With increasing CNSs concentration from 0 to 1.25 mg mL1, the DLD-1 cell viability showed a slightly decreasing trend
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Fig. 2
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FT-IR spectrum of carboxylated CNSs.
compared to the controls at 24 h. Aer 48 h, the MTT assay exhibited only slightly lower absorbance and approximately 80% of the DLD-1 cells were still alive. The MTT results indicated that the CNSs shows excellent biocompatibility and become a nontoxic support for the immobilization of cells.
Optimization of experimental conditions For aptasensor assay, the incubation time of DLD-1 cells was an important parameter in the performance of cell-capture. To evaluate the effects of incubation time on the response signal, the fabricated aptasensors were incubated at 37 C for different times from 30 to 150 min. The results showed that the EIS response revealed a increasing trend with increasing incubation time (Fig. 4A). It was benecial for MUC-1 aptamer special capturing more and more DLD-1 cells with time. When the incubation time was over 120 min, there was no apparent change in the Ret value, suggesting that the amount of DLD-1 cells captured on the surface of the aptasensor was saturated. Thus, 120 min was used as the optimal incubation time for capturing cells. Moreover, the concentration of MUC-1 aptamer is a critical factor to recognize DLD-1 cells. The capturing
efficiency of the aptasensor was directly related to the concentration of the MUC-1 aptamer on the electrode surface. Thus, an investigation of different concentrations of MUC-1 aptamer from 0.1 to 80 mM was carried out in the presence of [Fe(CN)6]3/4 couple (Fig. 4B). Then cyclic voltammograms of MUC-1 aptamer at different concentrations were recorded to choose the optimal condition for this experiment. For example, the CV of the MUC-1 aptamer concentration at 10 mM is recorded in ESI Fig. S1.† With increasing concentration of the MUC-1 aptamer, the CV peak current at potential of 0.36 V of the cell adhered aptasensor decreased, demonstrating that an increasing number of DLD-1 cells were being captured. A minimum CV response obtained at 40 mM then increased slightly, indicating that the cells captured on the modied electrodes had been saturated. Accordingly, an optimal concentration of MUC-1 aptamer at 40 mM was recommended for further research. EIS for quantitative detection of DLD-1 cells EIS, an electrochemical technique, has become an alternative to developing electrochemical biosensors for the quantitative detection of tumor cells. Herein, using [Fe(CN)6]3/4 as the redox probe, EIS was applied to evaluate the performance of the fabricated aptasensor under optimized conditions. The diameter of the semicircle on the Nyquist diagram gradually increased with increasing DLD-1 cell concentration ranging from 1.25 102 to 1.25 106 cells per mL, implying that a higher amount of DLD-1 cells immobilized on the electrode with a correlation coefficient of 0.994 (Fig. 5). The linear regression equation was DRet (U) ¼ 390.14 log C[DLD-1] (cells per mL) 405.68 with a detection limit of 40 cells per mL (S/N ¼ 3). The proposed assay displayed a lower detection limit compared to other reported electrochemical cell sensors, such as 90 cells per mL at an impedance cytosensor based on folate conjugatedpolyethylenimine carbon nanotubes for HeLa cells,21 8.71 102 cells per mL at a bio-inspired support of gold nanoparticles– chitosan nanocomposites gel for K562,22 100 cells per mL at aptamer-quantum dots for MCF-7.29 The proposed aptasensor indicating a lower detection limit and high sensitivity could be attributed to the following reasons. On one hand, CNSs with
EIS of the electrode at different fabrication stages (a) GCE, after (b) activated CNS–COOH, (c) immobilization of MUC-1 aptamer, and (d) capture of DLD-1 cells in 10 mM [Fe(CN)6]3/4 with 1.0 M KCl. The equivalent circuit model inset in the histogram was applied to fit the impedance data of the assembly process to Ret values (B). Fig. 3
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Fig. 4 Effects of (A) incubation time of DLD-1 cells on EIS response and (B) the concentration of MUC-1 aptamer on cyclic voltammetry response in [Fe(CN)6]3/4 (10 mM, 1 : 1) containing 1.0 M KCl.
excellent conductivity and outstanding biocompatibility was suitable for the immobilization of MUC-1 aptamer with high stability and bioactivity, providing an ideal interface for cell capture and improving the sensitivity of the aptasensor. On the other hand, high affinity between MUC-1 aptamer and MUC-1 glycoprotein, which is over-expressed on the cell membrane of DLD-1 cells, would further enhance the sensitivity of the present strategy. Specicity, stability and reproducibility of the aptasensing strategy The aptasensor showed an excellent specic response against DLD-1 cells. In our experiment, Human astrocytes 1800 cells and DLD-1 cells at the same concentration (1.25 104 cells per mL) were selected to evaluate the specicity of this aptasensor under optimized conditions. As shown in Fig. 6, the DPV peak current of the aptasensor changed less for 1800 cells, while DLD-1 cells lead to a signicant change in DPV peak current. The DPV peak current alteration of the aptasensor in the presence of DLD-1 cells was 7 mA, which is considerably higher than 1.5 mA for 1800 cells. Although nonspecic adsorption and slight expression of the MUC-1 glycoprotein on the surface of Human astrocytes 1800 cells may also result in a small DPV peak current change, a comparison of the peaks current can clearly indicate that the proposed method can efficiently distinguish DLD-1 cells from control cells with high specicity. The good selectivity of this aptasensor was attributed to the
Fig. 6 Specificity of the proposed aptasensor. Typical DPV response change in peak current with the modified electrode after respective incubation with DLD-1 cells and 1800 cells in 10 mM [Fe(CN)6]3/4(10 mM, 1 : 1) containing 0.1 M KCl. Error bars are the standard deviation of three replicate determinations.
high specicity of the aptamer. We further studied the intraassay precision of this aptasensor by detecting DLD-1 cells at two levels. At DLD-1 cell concentrations of 1.25 105 and 1.25 104 cells per mL, the aptasensor displayed the relative standard deviations (RSD) of 4.2% and 4.9% for ve determinations, respectively, signifying an acceptable precision. The
Fig. 5 Nyquist diagrams of electrochemical impedance spectra recorded at aptamer/CNSs–COOH fabricated aptasensor after capturing different concentrations of DLD-1 cells: (a) 0, (b) 1.25 102, (c) 1.25 103, (d) 1.25 104, (e) 1.25 105, and (f) 1.25 106 cells per mL from 0.1– 105 Hz in [Fe(CN)6]3/4 (10 mM, 1 : 1) containing 1.0 M KCl. Calibration curve of the aptasensor for DLD-1 cells (B).
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reproducibility of the aptasensor for DLD-1 cells was estimated using ve replicate measurements from the batch, the results showed an RSD of 3.5%, identifying the excellent fabrication reproducibility of this strategy. Moreover, it was necessary to observe this stability because it is a crucial property for fabricated aptasensors. When the modied aptasensor was stored in the refrigerator at 4 C for 15 days, EIS decreased by 10.9% from its initial signal response (ESI Fig. S2†), and the DPV response still retained 90.3% of its initial signal response. EIS and DPV results are consistent, suggesting satisfactory stability, which can be attributed to the strong interactions between CNSs and aptamer and the high stability of CNSs as a matrix.
Conclusions In summary, this work has successfully proposed a CNSs-based aptasensing strategy for ultrasensitive human colon cancer DLD-1 cells determination by a recognition process of the MUC1 aptamer to MUC-1 glyprotein on the cellular membrane that can be used for monitoring the adhesion of DLD-1 cells by electrochemical impedance spectroscopy, producing a sensitive impedance aptasensor for human colon cancer DLD-1 cells. The proposed aptasensor showed a wide linear detection range, and high sensitivity for the quantication of DLD-1 cells attributed to the conjugation of targeting MUC-1 aptamer to the conductivity and biocompatibility of CNSs. Furthermore, the developed aptasensor displayed high specicity to tumor cells overexpressing the MUC-1 glycoprotein, acceptable reproducibility and excellent stability, providing a feasible technique for the early diagnosis of colon cancer.
Acknowledgements This work was supported by The National Natural Science Foundation of China (21335002, 21175029) and the Shanghai Leading Academic Discipline Project (B109).
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