Label-Free Electrochemical Sensor for Ochratoxin A Using a Microfabricated Electrode with Immobilized Aptamer

Abstract

Ochratoxin A (OTA) is one of the most abundant food-contaminating mycotoxins that is also a potential carcinogen and responsible for many diseases affecting humans. Consequently, a sensitive, portable device for the detection of OTA is highly desirable. In this study, a miniaturized electrochemical aptamer-based sensor was developed for the label-free, sensitive detection of OTA. For the construction of the sensor, a gold thin-film three-electrode system was fabricated using standard microfabrication techniques on a polystyrene substrate (25 mm × 25 mm). Subsequently, the thiol-modified linker, 6-mercaptohexanol, DNA aptamer, and methylene blue (MB) were sequentially applied to the working electrode to construct a sensing layer. MB served as a redox indicator that interacted with the aptamer via the guanine bases and phosphate backbone to form complexes. The addition of OTA to the sensor induced the folding of the aptamer, which was accompanied by the release of the aptamer–MB–OTA complex from the sensor. Thus, the amount of MB decreased with increasing concentration of OTA. Differential pulse voltammetry was used for monitoring the highly sensitive detection. The standard curve for OTA exhibited a wide linearity ranging from 0.1 to 300 ng mL–1 with a detection limit of 78.3 pg mL–1 (S/N = 3). The selectivity test confirmed that the aptamer had high affinity only for the target. The OTA recoveries with the proposed sensor in commercial samples of coffee and beer were 86.4–107%.

An Electrochemical Sensor Based on Structure Switching of Dithiol-modified Aptamer for Simple Detection of Ochratoxin A

“…The procedure for the fabrication of the integrated electrode is described in our previous report. 11 Briefly, an Au film was evaporated on two pieces of PS substrates (5 × 5 cm) with a vacuum chamber until a thickness of 1.4 kÅ was achieved. In this process, no adhesive layer between the Au film and PS substrate, such as Cr or Ti layer, was needed.…”

“…The procedures for surface treatment prior to the self-assembly of the aptamer monolayer on the gold surface was electrode cleaning, which is very crucial to obtain a smooth surface for immobilization of the aptamer. 11 The entire cleaning process for the microfabricated electrode was conducted in a PDMS well (Figs. S1 and S2, Supporting Information).…”

“…Although many analytical techniques, including chromatography, 1,2 spectroscopy, 3,4 and quartz crystal microbalance 5,6 have been employed for this detection, electrochemical techniques exhibit great promise owing to high sensitivity, low cost, and simplicity in instrumentation and operation. [7][8][9][10][11] For mycotoxins, several electrochemical sensors have been designed using aptamers, which are in an emerging class of biorecognition molecules that can potentially replace antibodies. This type of molecule involves nucleic acid ligands that are isolated from a random sequence of DNA pools by systematic evolution of ligands by the exponential enrichment (SELEX).…”

“…17 Moreover, the ability of MB to interact with the guanine bases of DNA via π-π interactions has been reported. 11,18 Therefore, MB has emerged advantageous as a redox label. [19][20][21][22] Besides redox labels, several strategies have been studied to enhance the sensitivity of detection with metal nanoparticles, carbon nanotubes, and magnetic beads.…”

“…For electrochemical aptamer sensors, there have been several reports on a detection scheme based on the structure switching of aptamers, 11,14,29 which causes the target-induced dissociation (TID) of the aptamer from the sensor surface and produces a decreased signal (switch-off scheme). However, TID-based sensors might provide positive errors because external factors might dissociate the aptamer.…”