A Label-Free Microelectrode Array Based on One-Step Synthesis of Chitosan-Multi-Walled Carbon Nanotube-Thionine for Ultrasensitive Detection of Carcinoembryonic Antigen

Abstract

Carcinoembryonic antigen (CEA) has been an extensively used tumor marker responsible for clinical early diagnosis of cervical carcinomas, and pancreatic, colorectal, gastric and lung cancer. Combined with micro-electro mechanical system (MEMS) technology, it is important to develop a novel immune microelectrode array (MEA) not only for rapid analysis of serum samples, but also for cell detection in vitro and in vivo. In this work, we depict a simple approach to modify chitosan-multi-walled carbon nanotubes-thionine (CS-MWCNTs-THI) hybrid film through one-step electrochemical deposition and the CS-MWCNTs-THI hybrid films are successfully employed to immobilize anti-CEA for fabricating simple, label-free, and highly sensitive electro-chemical immune MEAs. The detection principle of immune MEA was based on the fact that the increasing formation of the antigen-antibody immunocomplex resulted in the decreased response currents and the relationship between the current reductions with the corresponding CEA concentrations was directly proportional. Experimental results indicated that the label-free MEA had good selectivity and the limit of detection for CEA is 0.5 pg/mL signal to noise ratio (SNR) = 3. A linear calibration plot for the detection of CEA was obtained in a wide concentration range from 1 pg/mL to 100 ng/mL (r = 0.996). This novel MEA has potential applications for detecting CEA for the research on cancer cells and cancer tissue slices as well as for effective early diagnosis.

Keywords: carcinoembryonic antigen; label-free; microelectrode array; multi-walled carbon nanotube; one-step synthesis.

Scheme 1

The fabrication procedure of the immune microelectrode array.

Figure 1

Characteristics of chitosan-multi-walled carbon nanotubes-thionine (CS-MWCNTs-THI) hybrid film. (A) Scanning electron microscope (SEM) and energy-dispersive X-ray spectroscopy (EDS) image of bare Au microelectrode; (B) SEM-EDS image of CS-MWCNTs-THI hybrid film; (C) SEM image of the CS-MWCNTs-THI hybrid film.

Figure 2

Cyclic voltammograms (CV) of microelectrode modified with CS-MWCNTs-THI/Au (a); anti-CEA/CS-MWCNTs-THI/Au (b); BSA/CS-MWCNTs-THI/Au (c); BSA/anti-CEA/CS-MWCNTs-THI/Au (d). Scan rate: 100 mV/s, solution: pH 7.4 phosphate buffer. BSA: bovine serum albumin; CEA: carcinoembryonic antigen.

Figure 3

(A) Differential pulse voltammetry (DPV) responses of the proposed immune microelectrode arrays (MEAs) after incubation with various concentrations of carcinoembryonic antigen (CEA); (B) Calibration curves of the immune MEAs with regard to CEA. Solution: pH 7.4 phosphate buffer.

Figure 4

Amperometric response of the immune MEA to 1 ng/mL CEA, 1 ng/mL CEA + 1 ng/mL IgG, 1 ng/mL CEA + 1 ng/mL BSA, 1 ng/mL CEA + 1 ng/mL glucose, 1 ng/mL CEA + 1 ng/mL AA, 1 ng/mL CEA +1 ng/mL DA, 1 ng/mL CEA + 1 ng/mL UA. DA: dopamine; UA: uric acid; AA: ascorbic acid.

Figure 5

Amperometric responses of five immune microelectrodes on the MEA to 1 ng/mL CEA.

Figure 6

The CEA concentrations of five serum samples detected by enzyme-linked immune sorbent assay (ELISA) and immune MEA.

Scheme 2

Microelectrode array fabrication process schematic. (a) The first photolithography; (b) the development of the photoresist; (c) magnetron sputtering; (d) lift-off process; (e) chemical vapor deposition; (f) the second photolithography; (g) SF₆ deep reactive ion etcher; (h) display the microelectrode sites and pads; (i) cleaning step.