Cancer is one of the leading causes of death worldwide, and the number of newly diagnosed patients is expected to rise. Hence, early detection of cancer is sought to increase the chances for successful treatment and survival rates [1
]. Currently, most research efforts in cancer diagnosis focus on developing biosensors with high selectivity and sensitivity, and inexpensive, rapid and easy operation. Current routine techniques to detect cancer include polymerase chain reaction (PCR), immunohistochemistry, and flow cytometry. Although these methods are effective in cancer diagnosis, they are expensive, time-consuming, and require highly skilled operators [3
]. By contrast, electrochemical methods offer many advantages compared with other techniques, such as rapidity, low cost, ease of operation, use of small reagent/sample volumes, and, most importantly, high selectivity and sensitivity in the detection of specific antigens [7
]. Several researchers have incorporated electrochemical biosensors in a microfluidic system to perform chemical and biomedical analysis [10
]. There are many significant contributions of microfluidic technology that enhance clinical and biological assays and that involve the ability to isolate rare cells from blood on the single-cell scale as an alternative to bulk isolation/detection methods. Devices using continuous flow allow the easy control of physical and chemical environments and provide high levels of experimental automation. Moreover, since rare cells are low in number, continuous-flow devices allow samples to be exposed to the sensor surface continuously, which enhances the capability of capturing the rare cells [12
Diagnosis of cancer at an early stage is the key aim in the fabrication of novel biosensors to diagnose patients with not only the metastatic disease but also screen patients with relapse after treatment [15
]. In recent years, rare circulating tumor cells (CTCs) have attracted more attention for improving cancer diagnosis and therapy. However, these cells represent a small percentage of the total blood cells in circulation; among >109
red and white blood cells, there are approximately 1–100 CTCs/mL blood [18
]. Therefore, targeting CTCs and cancer stem cells (CSCs) requires efficient techniques to capture these rare cells and exclude other cell types [20
]. Capturing CSCs can be achieved by the detection of cell surface biomarkers, such as CD13, CD24, CD44, CD90, CD133, EpCAM, and OV6, that are highly expressed by cancer cells but not by normal cells [25
]. In hepatocellular carcinoma (HCC), the third leading cause of cancer mortality worldwide, CSCs can be recognized by several surface antigens. Hepatic oval cells (HOCs) are defined as liver stem/progenitor cells in the liver Herring pipe; they are considered one of the most important origins of liver stem cells [27
]. Among many markers, OV6 is widely chosen as the best marker to capture and quantify HOCs.
High-efficiency fabricated biosensors can be achieved through careful surface architecture design. Nanomaterials such as carbon nanotubes and graphene have been widely exploited in biosensing applications [29
]. Among these nanostructured materials, multi-wall carbon nanotubes (MWCNTs) can serve as scaffolds that provide a self-supported structure and possess several features that can be functionalized through conjugation for detection purposes. MWCNTs are long, thin, hollow cylinders of carbon with additional graphene tubes around the core. MWCNTs are strong, stiff fibers due to their well-ordered arrangement of carbon atoms linked via sp2
]. These tubes are in high demand for engineering new devices in different fields because of their unique combination of chemical, optical, electrical, mechanical, and magnetic properties. To design sensitive biosensors, MWCNTs offer several advantages including high surface area, promotion of electron transfer reactions between electroactive compounds and electrodes, minimization of fouling of the electrode surface, enhancement of electro-catalytic activity and immobilization of molecules on their surfaces. Modification of MWCNT platforms with a polymer enables the generation of an efficient immobilization matrix for biosensing technology [33
]. Chitosan (CS) is a naturally occurring polysaccharide that is derived by the partial deacetylation of chitin. Chitosan provides several advantages such as low cost, non-toxicity, biodegradability, biocompatibility and excellent film-forming ability [35
]. Its multiple functional groups and capability to be chemically modified make chitosan a promising matrix for biosensors.
In the present work, MWCNT electrodes covered by a chitosan film and functionalized with anti-OV6 antibody were developed to target HOCs (Figure 1
). The fabricated sensor was embedded with a 3D-printed flow cell to allow for continuous exposure of the cancer cells over the sensor architecture. The electrochemistry of the functionalized sensor was examined using cyclic voltammetry (CV) and square wave voltammetry (SWV). The fabricated system exhibited an excellent cell capture response and may be an effective tool for tumor biomarker detection.
2. Materials and Methods
2.1. 3D-Printing Process of the Flow Cell
The flow cell was manufactured using a digital light processing (DLP)-3D-printer (Fab 12, procedure medical GmbH, Dortmund, Germany). Digital light processing is an additive manufacturing method based on selective exposure (UV-light) of a photo polymeric system. Due to the use of a digital mirror device (DMD-chip) a whole layer can be printed at one time. Digital light processing is characterized by its high printing speed, good resolution in x, y and z directions as well as the good surface quality of the printed parts. The used material was an acrylate-based photopolymer optimized for cell-based medical products. The photopolymer is characterized by its good biocompatibility. In several cytotoxicity tests according to German Institute of Standardization—European Standards (DIN-EN) ISO 10993-5, no cytotoxic effects could be observed. The layer thickness during the printing process was 100 µm. After the printing process, the flow cell was cleaned in a 99% isopropanol solution using an ultrasonic bath for 15 min. After the cleaning process, the flow cell was post-cured in a UV-light chamber for 7 min.
2.2. Preparation of the Functionalized Chitosan Matrix on the Multiwall Carbon Nanotubes Electrode
As shown in Figure 1
, 5 µL of 0.5% w
chitosan (Sigma Aldrich, San Luis, MI, USA) in 1% v
acetic acid was dropped on an MWCNT electrode and dried at room temperature for 3 h. After rinsing with water, the modified electrode was incubated with 5 µL of 2.5% v
glutaraldehyde (GA) (Sigma Aldrich) in phosphate-buffered saline (PBS) for 2 h and then washed with water. Five µL of 200 mg/mL human/rat OV-6 antibody (R&D Systems, Abingdon, UK) in PBS was dropped onto the activated surface and incubated at 4 °C overnight. Excess antibodies were removed by washing with PBS before the modified electrode surface was blocked with 1% bovine serum albumin (BSA) and incubated at room temperature for 90 min to prevent any unspecific adsorption and block any remaining active sites. After a final washing step with PBS, the developed sensors were used immediately or stored at 4 °C.
2.3. Contact Angle Measurements
The contact angles of water on the modified film were measured using a goniometer (Easy Drop, Krüss, Hamburg, Germany) at room temperature. Three µL of Milli-Q water was deposited onto the surface, and the angle was measured immediately. All contact angle measurements were repeated at least in triplicate.
2.4. Cell Lines and Cell Culture
The liver and breast cancer cells were cultured according to standard mammalian tissue protocols with a sterile technique. Briefly, human liver hepatocellular carcinoma cell line (HepG2) and human breast adenocarcinoma cell line (MCF-7) (American Type Culture Collection) were cultured in DMEM (PAA Laboratories GmbH, Pasching, Austria) supplemented with 10% fetal bovine serum (FBS) or 10 µg/mL insulin, respectively, and a 1% antibiotic/antimycotic solution at 37 °C in 5% CO2 and 95% air humidified atmosphere as adherent monolayers in 25 cm2 cell culture flasks. After 48 h, the cells were detached from the flask using Trypsin, separated from the medium via centrifugation and counted using an automated cell counter (NanoEntek, Waltham, MA, USA). Trypan blue was used to count and discriminate between viable and non-viable cancer cells. This dye selectively stains non-viable cells and exhibits distinctive blue under the microscope. Briefly, a suspension of cancer cells (HepG2 or MCF-7) in PBS was diluted in Trypan blue solution (0.4%) at a 1:1 ratio. When cell viability was above 85%, the cells were used for further experiments.
2.5. Flow Cytometry Analysis
Flow cytometry was conducted for HepG2 and MCF-7 cancer cells using a Beckman Coulter Elite Xl (Nyon, Switzerland) with OV-6 phycoerythrin monoclonal antibody (R&D Systems). Briefly, both cell lines (1 × 106 cells/mL) were incubated with 10 µL of antibody for 30 min in the dark followed by washing with PBS; the cells were resuspended in fresh PBS and analyzed by flow cytometer immediately. The cells were passed through the laser beam in the flow cytometer at a rate of 10,000 cells/second.
2.6. Electrochemical Measurements
The three-electrode system was printed on ceramic substrates with dimensions: L3.4 × W1.0 × H0.05 cm, and three-electrode configuration was incorporated: counter electrode (CE, carbon), reference electrode (RE, silver), and working electrode (WE, MWCNT, 400 µm diameter). All CV and SWV measurements were performed at least in duplicate using a potentiostat (Zimmer and Peacock, Royston, UK). Cyclic voltammetry measurements were recorded for each functionalized layer of the developed sensor after rinsing with PBS. The modified electrodes were embedded into the 3D-printed flow cell, which then connected to a flow control system (Fluigent, Paris, France) that allows cancer cell injection at different concentrations, and SWV measurements were recorded after rinsing with PBS to remove unbound cells.