Elsevier

Biosensors and Bioelectronics

Volume 116, 30 September 2018, Pages 100-107
Biosensors and Bioelectronics

Mannosyl electrochemical impedance cytosensor for label-free MDA-MB-231 cancer cell detection

https://doi.org/10.1016/j.bios.2018.05.002Get rights and content

Highlights

  • A label-free, specific, and ultrasensitive electrochemical impedance sensor was developed for breast cancer cells MDA-MB-231.

  • Cell was recognized via the interaction between mannosyl electrode and overexpressed mannose receptors on the cell surface.

  • The robust mannosyl electrode was prepared via electrografting amino-functional mannose on carbon surface.

  • Expensive antibodies are not needed for preparing the sensor that showed good biocompatibility for viable captured cells.

  • Linearity was seen between 1.0 × 10 and 1.0 × 105 cells mL−1 of MDA-MB-231 cell with the detection limit of 10 cells mL −1.

Abstract

A label-free and ultrasensitive electrochemical impedance cytosensor was developed to specifically detect the breast cancer cells MDA-MB-231 via the interaction between the mannosyl glassy carbon electrode (GCE) and the overexpressed mannose receptors on the target cell surface. The mannosyl GCE was prepared through electrografting of the amino-functionalized mannose derivatives on GCE surface in which a covalent bond was formed between carbon of the electrode and the amino group of the mannose derivative. The fluorescent microscopy indicated that the electrode is specific for MDA-MB-231 cells, with good biocompatibility for viable captured cells. The derivative with a shorter alkyl linker, mannose-C2NH2, showed a better sensitivity than that with a longer linker, mannose-C6NH2. GCE modified with amino-functionalized galactose derivative, galactose-C2NH2, shows no function to the detection of MDA-MB-231 cells. The specific interaction between the mannosyl GCE and Con A (a mannose-binding lectin) or MDA-MB-231 breast cancer cells with overexpressed mannose receptors was determined through the change of peak separation in the cyclic voltammogram or the change of charge transfer resistance in the electrochemical impedance spectra (Nyquist plot) in the electrolytes containing a reversible redox couple [Fe(CN)6]3−/[Fe(CN)6]4−. The charge transfer resistance in the Nyquist plots linearly depended on the concentration of MDA-MB-231 cells (1.0 × 10–1.0 × 105 cells mL−1, with 10 cells mL−1 being the lower detection limit). Introducing 0.1% polyethylene glycol-200 (PEG-200) was able to prevent the interference caused by 1.0 × 103 HEK-293T cells mL−1, a non-cancer cell line (control).

Introduction

Circulating tumor cells (CTCs) are a primary cancer biomarker and play a crucial role in early cancer diagnosis and prognosis (Chaffer and Weinberg, 2011, Yap et al., 2014). CTCs are detached from a primary tumor or during metastasis in the vasculature and deposited into the blood stream, resulting in potential establishment of secondary foci of disease. CTCs have been used as a minimally invasive multifunctional biomarker in >270 clinical trials (Alix-Panabieres and Pantel, 2014). However, an extraordinarily rare fraction of CTCs cancer cells reside in the blood vessels, and few as one out of billions of cells may be present in the blood of cancer patients (Alix-Panabieres and Pantel, 2014, Green et al., 2016). Various detection techniques, including polymerase chain reaction (PCR) based method, the cytometric method, the fluorescent microfluidic chip method or immunomagnetic separation method, the centrifugal force method, size-based enrichment, the invasive capacity method, and the density-based method, have been developed (Joosse et al., 2015). Many of these methods are either time-consuming or expensive and require a further fluorescence-label or other sophisticated instruments. Early detection of cancer can considerably increase the chances for effective treatment patient survival. Accurate quantitative detection of cancer cells is vital to cancer diagnosis. Therefore, developing an accurate label-free method for cancer cell determination is highly desirable (Chen et al., 2016).

Electrochemical impedance spectroscopy (EIS) is a suitable analytical technique for label-free cancer cell determination (Han et al., 2016, Liu et al., 2016, Yang et al., 2013, Zhang et al., 2010, Zheng et al., 2012), because EIS is extremely sensitive at the electrode interface to impedance changes, which are induced by the specific combination between the target cell and cell recognition element, which is usually immobilized on the electrode surface. Therefore, developing a facile, universal, and robust immobilization method is crucial to the successful fabrication of an electrochemical impedance cytosensor. Self-assembled monolayers (SAMs), particularly for the SAM formation of thiol (R−SH) on gold (Au) surfaces through stable covalent bond formation of Au−S−R, have become a standard immobilization method for electrode modification. In some situations, this standard approach has some disadvantages. A gold surface is essential and makes this method relatively expensive, although electroplating, sputtering plating, and vapor deposition can considerably reduce the amount of required gold. Another disadvantage is that it is difficult to distinguish the effect of gold within observed electrochemical behavior, especially in the study of electrocatalysis. Because of these concerns, a low-cost and universal method is required. The electrografting method for immobilization of amino compounds on the electrode surface has been well established (Belanger and Pinson, 2011, Kim et al., 2010, Lee et al., 2013). The method involves the formation of an initial radical cation that deprotonates the ɑ of the amino group to yield a carbon radical, and a hydrogen shift provides the aminyl radical that binds to the electrode surface (Belanger and Pinson, 2011). Taking advantage of the high sensitivity of EIS, immobilization of a new recognition molecule is highly desired for specific cancer cell detection on the electrode to achieve reliable analytical selectivity.

Cell surface molecular recognition between carbohydrates and proteins mediates several vital physiological and pathophysiological processes, such as cellular growth, adhesion, bacterial and viral infections, cancer metastasis, inflammation, and immune surveillance (Lai et al., 2010). This weak single-recognition event limitation can be overcome by multivalent carbohydrate–lectin interaction, resulting in high binding affinities and specificities (Gestwicki et al., 2002, Mathai Mammen and George, 1998). In this study, the surface of a mannosyl glassy carbon electrode (GCE) cytosensor was investigated as a suitable multivalent platform that can be combined with EIS to detect particular proteins or cells with high sensitivity and selectivity. Concanavalin A (Con A), a plant lectin extracted from jack bean seeds, can specifically bind to mannose and glucose but exhibits no obvious affinity toward galactose (Huang et al., 2015). Con A was selected as the model lectin for investigating the specific interaction between lectin and the carbohydrate-modified electrode surface. Bovine serum albumin (BSA) was used as a negative control protein. MDA-MB-231 breast cancer cells, which overexpress mannose receptors (MRs) on their surface (Ye et al., 2016, Zhang et al., 2018), are used as a model for CTCs. The HEK-293T cell was used as a control non-cancer cell line without MR expression (Zhang et al., 2018). This concept is shown in Fig. 1. The electrode surface modifications were then confirmed through X-ray photoelectron spectroscopy (XPS) and surface contact angle measurements on a glassy carbon (GC) sheet. The fabricated cytosensor and its application for the detection of CTCs were investigated through cyclic voltammetry, EIS, and fluorescence microscopy. In this paper, we developed a carbohydrate-based mannosyl electrochemical impedance cytosensor for highly sensitive and selective detection of MDA-MB-231 breast cancer.

Section snippets

Materials and apparatus

Concanavalin A (Con A), bovine serum albumin (BSA), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), Triton X-100, paraformaldehyde (PFA) were obtained from Sigma Aldrich. PBS buffer (10 mM PBS + 150 mM NaCl, pH 7.4) and HBS buffer (HEPES buffered saline, 10 mM HEPES + 2 mM MgCl2, 2 mM CaCl2, and 150 mM NaCl, pH 7.4) were prepared, respectively. A conventional three-electrode electrochemical cell was used for all

Characterization of carbohydrate-modified electrodes

The experimental designs were based on the mannoside on the GCE surface that interacts with the overexpressing MR receptor on the MDA-MB-231 cell. This concept is shown in Fig. 1. First, the carbohydrate moieties were immobilized on the GCE surface. To elucidate whether the carbohydrate-modified GCEs can recognize the targeting proteins and cells and to understand how the length of the alkyl side-chain with amino-terminal carbohydrate derivatives determines the interaction between the modified

Conclusions

A label-free and ultrasensitive electrochemical impedance cytosensor has been successfully fabricated via a facile electrografting technique to specifically detect breast cancer cells, MDA-MB-231. A linear concentration range from 10 cells mL−1 to 105 cells mL−1 was observed with a detection limit as low as 10 cells mL−1. An interference from HEK-293T cell probably induced by nonspecific adsorption was observed but it was significantly suppressed by introducing 0.1% PEG-200 to the electrolytes.

Acknowledgments

We thank National Chung Hsing University, Kaohsiung Medical University and Ministry of Science and Technology of Taiwan for the financial support under contracts MOST 105–2113-M-005–016-MY2 and MOST 106–2113-M-037-014.

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