An electrochemical aptasensor based on functionalized graphene oxide assisted electrocatalytic signal amplification of methylene blue for aflatoxin B1 detection
Graphical abstract
Introduction
Last decade has witnessed rapid increment in a special kind of electrochemical aptasensors (E-Apt) [1], [2], [3], which involved an aptamer containing labeled redox molecule immobilized on the surface of the electrode. In an E-Apt sensor engineering, the aptamer is labeled with redox probe at 3′-end (ferrocene or methylene blue) and with the carboxylic group at 5′-end [4], [5], [6]. When analyte molecule interacts with the specifically labeled aptamer causes conformation changes in immobilized aptamer probe, which leads to change in the efficiency of the electron transfer resistance between the electrocatalytic solution and the electrode active surface. The conformation change of aptamer is measured in faradaic current upon target analyte interaction, these current values resemble the concentration of target analyte molecule. By virtue of their reagent-less nature, simplicity in operational procedure, ease of doing, effortlessness in recovery and extraordinary selectivity, combining all these E-Apt labeled biosensors have developed as an assuring biosensor stage for the various analyte molecules detection from little particles to bigger protein targets. The change in the signaling of E-Apt sensors is due to the effective electron transfer tendency resulting from a conformational turnover of an aptamer with target analyte interaction, these signal changes follow the concentration of the analyte [7], [8], [9], [10], [11]. In consequence of this principle, a few techniques have been proposed in which the typical changes of the aptamer occurred upon interact with target analyte molecule.
Despite the lot of electrochemical aptasensors were developed for detection of numerous target analyte molecules, they need a huge change in the aptamer conformation to regulate the distance between aptamer probe and electrode surface to get the output signal. Background current was the main problem in these type of aptasensors. To overcome these issues and to increase the sensor signal, we described a new simplified methodology utilizing functionalized graphene oxide as a signal enhancing platform [12], [13], [14].
Herein, we have demonstrated the quantification of AFB1 in beer and wine samples. Alcoholic beverages are an important part in present days human social life. Further, the known injurious effects of alcohol can also be considered as a possible source of toxic pollutants which were transfer from raw grains in the preparation process. The main grains used for the alcohol preparation process were wheat [15], barley [16] and maize [17], could be able to contaminate with possible mycotoxins like aflatoxins. There is a need to monitor the alcohol brewing process in every step and in general alcohols to ensure the public safety.
Due to the contamination of all food products with a different type of mycotoxins, lot of countries has put regulation limits for Mycotoxins. Among the all the kind of Mycotoxins aflatoxins are more carcinogenic molecules. The European Commission has set legal regulation limits for AFB1 in a different type of food materials and products [18]. In the literature, a few analytical techniques were developed for the detection of AFB1 like antibody-based ELISA assays [19], [20], electrochemical aptasensors [21], [22], [23] and chromatographic methods. Electrochemical methods have some advantages over traditional analytical techniques like low cost, simple procedure, portable field monitoring and no need of the well-trained person. Additionally, the reported methods limit for the detection of AFB1 sample in a different type of food products. Therefore, cost effective biosensor system was needed to screen the alcohol beverages samples.
In this work, we developed an electrochemical aptasensor by using methylene blue (MB) redox probe labeled aptamer as a signaling fragment and functional graphene oxide (FGO) as the signal-enlarging platform. The FGO was first to drop cast onto screen-printed carbon electrode (SPCE) interface and the aptamer was immobilized by using hexamethylenediamine (HMDA) as a spacer via carbodiimide amide-bonding chemistry. Additionally, the FGO on the SPCE surface not only useful for the making a bridge to link the aptamer but also useful to greatly enhance the conductive and catalytic properties leads to increase the electrochemical signal of the sensor. The proposed FGO based methylene blue aptasensor platform was potentially applied for AFB1 detection in alcoholic beverages with high sensitivity and reliability by using differential pulse voltammetry (DPV) [24], [25]. And the developed principle aptasensor was further used to screen the alcoholic beverage samples for AFB1 detection. This proposed method could be promptly useful for the sensing of further analytes like mycotoxins and drugs.
Section snippets
Chemicals and materials
Methylene blue redox probe labeled anti-AFB1 aptamer was procured from M/s Microsynth (Schutzenstrasse, Balgach, Switzerland). The base pairs of aptamer sequence were showed below.
(5′-COOH modified) 5′-TGGGGTTTTGGTGGCGGGTGGTGTACGGGCGAGGG-3′. (3′-MB modified)
All other chemicals, hexamethylenediamine (HMDA), graphite powder, chloroacetic acid, sodium nitrite (NaNO2), sodium hydroxide (NaOH), ethanolamine, N-hydroxysuccinimide (NHS), 4-aminobenzoic acid,
Principle and framework of the developed aptasensor
The proposed aptasensor mechanism and framework of molecular folding based aptasensor of present work were represented in the Schematic diagram (Fig. 1). The aptamer immobilization was done via functionalized graphene oxide deposition on SPCE and long chain HMDA spacer by using EDC-NHS chemistry. The SPCE/FGO surface first immobilized with HMDA long spacer molecule which is useful for leaving a long distance of MB tagged aptamer with the electrode surface. The FGO layer was useful for the three
Conclusion
In this paper, we report a new electrochemical aptasensor by using redox probe (MB) labeled aptamer as a signaling molecule and FGO has a signal enhancing the platform. The constructed aptasensor has fabricated by using FGO molecule. This FGO molecule has some unique properties like high conductivity, high surface area, and high catalytic properties. So these properties helpful for the enhancement of the electrochemical response and leads to the development of sensitive and selective biosensor.
Acknowledgments
K. Yugender Goud gratefully acknowledges the EUPHRATES Program for ERASMUS Mundus Doctoral Fellowship.
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