Elsevier

Biosensors and Bioelectronics

Volume 68, 15 June 2015, Pages 508-515
Biosensors and Bioelectronics

An electrochemical immunosensing method for detecting melanoma cells

https://doi.org/10.1016/j.bios.2015.01.022Get rights and content

Highlights

  • A label-free, highly sensitive immunosensor was fabricated to detect melanoma cells.

  • The immunosensor performance characteristics have been optimized.

  • This method can selectively detect melanoma cells in the presence of non-melanoma cells.

  • The immunosensor was successfully applied to detect melanoma cells in blood samples (i.e., in the presence of peripheral blood mononuclear cells).

Abstract

An electrochemical immunosensing method was developed to detect melanoma cells based on the affinity between cell surface melanocortin 1 receptor (MC1R) antigen and anti-MC1R antibody (MC1R-Ab). The MC1R-Abs were immobilized in amino-functionalized silica nanoparticles (n-SiNPs)-polypyrrole (PPy) nanocomposite modified on working electrode surface of screen-printed electrode (SPE). Cyclic voltammetry was employed, with the help of redox mediator ([Fe(CN)6]3−), to measure the change in anodic oxidation peak current arising due to the specific interaction between MC1R antigens and MC1R-Abs when the target melanoma cells are present in the sample. Various factors affecting the sensor performance, such as the amount of MC1R-Abs loaded, incubation time with the target melanoma cells, the presence of interfering non-melanoma cells, were tested and optimized over different expected melanoma cell loads in the range of 50–7500 cells/2.5 mL. The immunosensor is highly sensitive (20 cells/mL), specific, and reproducible, and the antibody-loaded electrode in ready-to-use stage is stable over two weeks. Thus, in conjunction with a microfluidic lab-on-a-chip device our electrochemical immunosensing approach may be suitable for highly sensitive, selective, and rapid detection of circulating tumor cells (CTCs) in blood samples.

Introduction

Cancer is a leading cause of death around the world. Melanoma is widely prevalent and the number of melanoma cases and associated mortality have rapidly increased in the United States (US) and worldwide over the past several years (Desmond and Soong, 2003, Jemal et al., 2001, Linos et al., 2009). On average, metastatic melanoma patients survive six to nine months, with an overall survival rate of 40% (Shivers et al., 1998). When a cancer metastasizes, the tumor cells begin to circulate in blood and lymphatic system (Joosse and Pantel, 2013, Williams, 2013). During the early phase of metastasis, only few circulating tumor cells (CTCs) are present in blood along with millions of leukocytes and billions of erythrocytes (Joosse and Pantel, 2013, Williams, 2013). Thus, quantification and enumeration of CTCs at an early stage of cancer progression is of significant prognostic value.

Based on real-time polymerase chain reaction (RT-PCR) analyses of peripheral blood, specific mRNA for tyrosinase (Kunter et al., 1996, Smith et al., 1991), MelanA/MART-1 (Kiyohara et al., 2014, Schittek et al., 1999), and glycoprotein (gp)100 (Tsukamoto et al., 2000) are considered as indicative of the presence of circulating melanoma cells (CMCs). However, the detection methods based on these markers lack sensitivity or selectivity and often produce false-positive results. Therefore, immunological methods are pursued for identification and detection of CTCs based on cancer cell surface protein markers. The US FDA has approved the CELLSEARCH® CTC Test Kit (Janssen Diagnostics, Raritan, NJ) for detecting CTCs in blood using immunomagnetic separation (Paterlini-Brechot and Benali, 2007, Riethdorf et al., 2007). This method detects CTCs expressing epithelial cell-adhesion molecule (EpCAM) and cytokeratins only. However, the ability of CELLSEARCH® CTC Test to detect other cell surface markers has been questioned (Joosse and Pantel, 2013), and if the cells express low or no EpCAM, this test may fail to detect the tumor altogether (Riethdorf et al., 2007). A lab-on-a-chip method was developed for detecting CTCs based on the affinity between EpCAM and its antibody (Maheswaran et al., 2008, Nagrath et al., 2007, Stott et al., 2010, Yoon et al., 2013), which is somewhat complicated and time-consuming. A semi-integrated electrical biosensor was also developed for CTCs detection in blood via immunomagnetic and size-based separation (Chung et al., 2011). Zhao et al. (2013) developed an ensemble decision aliquot ranking method to detect CTCs. Hou et al. (2013) reported a nanovelcro CTCs assay for isolation of single tumor cell in addition to effectively capturing CMCs. However, their method of detection requires several steps of manipulation of the blood prior to detection.

Several label-free immunosensing methods have been reported for the detection of cancer cells including surface-enhanced Raman spectroscopy (Wang et al., 2011) and electrochemical methods (Hu et al., 2013, Moscovici et al., 2013). Among these, the electrochemical methods have many advantages such as simple, rapid, inexpensive, unaffected by sample turbidity, and ultrasensitive for detecting various target analytes in complex biological samples (Drummond et al., 2003, Karimi-Maleh et al., 2013, Karimi-Maleh et al., 2014a, Karimi-Maleh et al., 2014b, Moradi et al., 2013). Therefore, electrochemical immunosensors are being developed incorporating a variety of nanoparticles, such as gold and silica, as labels to dramatically enhance the signal intensity (Chikkaveeraiah et al., 2012, Cui et al., 2014, Gao et al., 2013, Ho et al., 2010, Liu and Jiang, 2006, Rusling, 2012, Wang et al., 2014a, Wu et al., 2013). The nano-functionalized electrode surface also affords effective immobilization of antibody with good stability and bioactivity. Wilson (2005) reported an electrochemical immunosensor for simultaneous detection of colorectal cancer and liver cancer markers. Liu and Jiang developed an electrochemical immunosensor (anti-carcinoembryonic antigen (CEA) antibody immobilized on colloidal silica nanoparticles/titania sol–gel composite membrane on gold electrode) for detecting CEA markers with a detection limit of 0.5 ng/mL (Liu and Jiang, 2006). Domnanich et al. (2011) used a protein immobilized on xanthan/chitosan-modified gold-chip microarray for detecting melanoma-relevant markers in picomolar range. Kelly group designed an aperture sensor array for electrochemical detection of prostate cancer cells in 15 min (Moscovici et al., 2013). Many have investigated label-free, multistep sandwich assays to detect cancer markers by measuring signal changes due to the affinity between two antibodies that recognize a cell surface antigen on an electrode surface (Chikkaveeraiah et al., 2012, Cui et al., 2014, Rusling, 2012, Wang et al., 2014a). Wang et al. (2014b) used S100B as a serum biomarker to detect melanoma in blood by an electrochemical assay; however, S100B is not specific to melanoma. Thus, in general, many of the existing methods lack in one or more of the following important attributes: sensitivity, selectivity, simplicity, and rapidity.

The MC1R is a G-protein-coupled receptor, expressed highly selectively by melanocytes and melanoma cells. It is expressed in a majority (>80%) of melanoma lines and also primary and metastatic cutaneous melanomas compared to other cell surface markers (Raposinho et al., 2010, Salazar-Onfray et al., 2002, Schwahn et al., 2001, Xia et al., 1996). It is also useful marker for the diagnosis of uveal melanoma. Therefore, we selected MC1R as target marker for the detection of melanoma cells. Herein, we report an electrochemical immunosensing system for highly sensitive and specific detection of melanoma cells in complex environments using the melanoma-specific cell surface protein melanocortin 1 receptor (MC1R) as a target marker. We immobilized anti-MC1R antibodies (MC1R-Abs) on amino-functionalized silica nanoparticles (n-SiNPs)-polypyrrole (PPy) nanocomposite thin film modified screen-printed electrode (SPE) and used as an immunosensor. The change in the anodic oxidation current obtained via cyclic voltammetry (CV) resulting from the affinity between MC1R antigens and MC1R-Abs on the immunosensor was used for detecting the melanoma cells.

Section snippets

Material and methods

MC1R-Abs (200 µg/mL) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Tetraethoxysilane, (3-aminopropyl) triethoxysilane, pyrrole, carbonyldiimidazole, ammonium hydroxide (28–30%), and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma-Aldrich, Alfa Aesar, and Acros Organics. All chemicals were of analytical grade and used as received, unless otherwise stated. Highly pure deionized (DI) water (resistivity=18.2  cm, Milli-Q gradient system, Millipore) was used for

Results and discussion

According to the reported methods, PPy on the electrode surface offers many advantages such as microporous structure, fast electron transfer rate, large surface area, good electrochemical properties, enhanced conducting pathways, entrapment of large number of nanoparticles (Lyons, 1994, Rajesh et al., 2010a, Raveh et al., 2013, Razola et al., 2002). Therefore, we incorporated n-SiNPs in the microporous PPy matrix deposited on the SPE surface and immobilized MC1R-Abs on n-SiNPs using CDI as a

Conclusions

We have described the fabrication of a novel immunosensor for selective and sensitive detection of melanoma cells in both 1× PBS and blood sample. The working electrode surface of the SPE was modified with n-SiNPs/PPy nanocomposite thin film, obtained via controlled in-situ electrochemical polymerization, which offers good electrical conductivity and affords effective immobilization of antibodies without denaturation. Using MC1R-Abs immobilized on n-SiNPs/PPy nanocomposite modified SPE, we

Acknowledgments

Research reported in this project was supported by the Skin Diseases Research Core Center (SDRC) Grant 1P30AR066524 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health. Authors gratefully acknowledge receiving PBMCs from Dr. David Beebe's Microtechnology and Medicine Biology Laboratory, University of Wisconsin-Madison.

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