Electrochemical Biosensors for Detecting Microbial Toxins by Graphene-Based Nanocomposites
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It is important to develop methods to determine microbial toxins at trace levels since these toxins are ubiquitous commonly found in water and foods, and pose potential threats to both human health and ecosystem safety. Taking the advantages of ultrahigh electron-transfer capability, extra-large surface area and easily functionalized ability, the graphene-based nanocomposites have been employed to fabricate electrochemical biosensors including immunosensors and aptasensors for detecting microbial toxins with high sensitivity. The specificity and selectivity of the electrochemical biosensors for targeting toxins can be achieved by combining graphene nanocomposites with antibodies and/or aptamers. The graphene nanocomposite-based electrochemical biosensors could become a promising technique in the detection of microbial toxins for public and environmental health protection.
KeywordsGraphene nanocomposites Electrochemical immunosensors Electrochemical aptasensors Microbial toxins
The microbial toxins are generally catalogued into endotoxins (lipopolysaccharides, LPS) produced by Gram-negative bacteria, exotoxins (peptides and proteins) produced by Gram-negative or Gram-positive bacterial pathogens, and mycotoxins [e.g., deoxynivalenol (DON), aflatoxin, ochratoxin A (OTA), fumonisin B1 (FB1) and zearalenone] produced by certain fungi [1, 2, 3, 4]. With the molecular weight covering a broad range from less than 1 kDa to more than 100 kDa, these toxins exhibit different physico-chemical properties and are one of major threats to the life and health of humans and live stocks. Microbial toxins cause a broad variety of diseases, ranging from mild emesis and diarrhea to severe and fatal cancers and neurological disorders. For instance, Staphylococcus aureus enterotoxins (SEs) can cause gastroenteritis in the gastrointestinal tract and act as a superantigen on the immune system. Aflatoxin B1 (AFB1) has been listed as Group I carcinogens by the International Agency for Research on Cancer (IARC), a body of the World Health Organization. The poisonous effects of some of these molecules can be acute even at very low doses, and the cooccurrence of microbial toxins in nature may cause additive and/or synergistic effects.
Various methods/assays such as polymerase chain reaction (PCR), high-performance liquid chromatography (HPLC), high-performance liquid chromatography–tandem mass spectrometry (HPLC/MS/MS), surface plasmon resonance (SPR), electrochemical biosensors and immunoassays [e.g., competitive enzyme-linked immunosorbent assay (ELISAs) and microfluidic immunoassay] have been developed for detection of microbial toxins in different sources including foods, water and feeds [1, 2, 5, 6, 7, 8]. Among of these methods/assays, electrochemical biosensors are attractive since they have several advantages including high sensitivity, operational simplicity, low cost, and suitable rapid on-site analysis.
Due to its remarkable electrocatalytic activity and conductivity, graphene is an ideal material for electrochemical sensors and biosensors with the structure of the two-dimensional sheet of sp2-conjugated atomic carbon [9, 10, 11, 12, 13]. Researchers have demonstrated that incorporating graphenes and other materials (e.g., polypyrrole and gold nanoparticles) together in sensor platform provide biocompatibility, large surface area, ease of functionalization, and significantly improve the reproducibility, sensitivity, and stability. The graphene-based electrochemical biosensors are widely used in the detection of various analysts including bioactive small molecules, peptides, nucleic acids, proteins, enzymes and living cells [9, 10, 11, 12, 13]. In this chapter, the graphene nanocomposite-based electrochemical biosensors for sensing microbial toxins have been discussed and highlighted by the linear ranges, limits of detection (LODs), reproducibilities, and stabilities of these reported biosensors.
2 Sensing Strategies
Based on the sensing strategies, the electrochemical sensors can be mainly divided into three categories including amperometric sensor, voltammetric sensor and impedimetric sensors . Amperometric sensors are detection of analytes based on the electric current or the change of electric current under a specific electrode potential which can be adjusted to maximize the response for the analyte of interest while minimizing the response for the interfering substances. Amperometric immunosensors are considered as a suitable method for the detection of contaminants in food since the sensors exhibit advantage of rapid, sensitive and selective quantification . Voltammetric biosensors are based on electroanalytical chemistry, in which the current is measured by changing the potential and measuring the generated current when the electrochemical reaction occurs between the analyte and the working electrode surface. Differential-pulse voltammetry (DPV) and cyclic voltammetry (CV) are commonly used voltammetric sensing techniques which can be used to detect analytes with high sensitivities and low LODs and quantitatively analyze/fastly characterize reaction processes that take place on the surface of the sensing electrode. For example, based on exonuclease-catalyzed target recycling, Chen and coauthors have developed a voltammetric aptasensor of OTA with LOD of 1.0 pg mL−1 . Electrochemical impedance spectroscopy (EIS) is a sensitive technology for analyzing the interfacial characteristics related to biometric identification, such as biomolecular recognition events, reactions catalyzed by enzymes occurring at the modified surface. In impedimetric biosensors, the binding of biomolecules can form a blocking layer on the electrode surface, resulting in resistance increases. Impedimetric biosensors allow direct detection of biomolecular recognition events without using labels. Chiriaco and coauthors have developed a flow-injection impedimetric immunosensor for direct and label-free detection of cholera toxin with a LOD smaller than 10 pmol L−1 in the buffer solution .
3 The Roles of Graphene Nanocomposites in the Electrochemical Biosenors
Based on their inherent natures, the graphene nanocomposites can be used as electrochemical labels for generating electrochemical signal, efficient units for immobilizing biomolecules on electrode surface, and strong enhancers for amplifying detecting signal [19, 20, 21, 22]. For instance, reduced graphene oxide-doped polypyrrole/pyrrole propylic acid nanocomposite (rGO-PPy/PPa) has been used to fabricate impedimetric immunosensor, in which rGO greatly improves the conductivity and stability, PPa provides covalent linkers for probe immobilization and PPy endows the film electroactivity from its inherent electrochemical doping/dedoping property for impedance measurements . Graphene-oxide nanoplatelets (GONPs) are directly used as electroactive labels for aptasensing mycotoxin . After treated with HNO3, the cadmium telluride quantum dots (CdTe QDs)-modified graphene/gold nanoparticle (AuNPs) nanocomposites (GAu/CdTe) can be served as electrochemical probe for ultrasensitive detection of OTA through electroredox of releasing Cd2+ from GAu/CdTe . A novel aptasensor is designed by with the dual amplification of gold nanoparticles (AuNPs) and graphene/thionine nanocomposites (GSTH) for sensitive determination of FB1 . In this case, AuNPs is modified to increase the electrical conductivity at the electrode surface and to produce a FB1-specific recognition interface by hybridization with capture DNA and its aptamer. A great quantity of TH molecules loaded on the graphene surface is served as electroactivating probe to increase its electrochemical signal due to the large surface area and excellent conductivity of graphene sheet.
4 Graphene Nanocomposite-Based Electrochemical Immunosensor
In 2001, the International Union of Pure and Applied Chemistry (IUPAC) defined an electrochemical immunosensor as an integrated device based on an antibody/antigen reaction, which can transform their concentration signals or certain chemical substances into a corresponding electric signal (e.g., current, potential, conductance and impedance) through the sensor element, and realize a specific semi-quantitative or quantitative analysis . Up to date, various types of electrochemical immunosensors based on the graphene nanocomposites, including reduced graphene oxide-conductive polymer nanocomposites, amine-terminated dendrimer (PAMAM)-modified graphene oxide nanosheets, have also been proposed to detect microbial toxins in diverse matrices including foods, freshwater and feeds [18, 23, 24].
4.1 Detection Bacterial Toxins
4.2 Detection of Mycotoxins
5 Graphene Nanocomposite-Based Electrochemical Aptasensor
Nucleic acid aptamers are nucleic acid species with appropriate secondary structures containing 10–50 variable bases that have been engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as nucleic acids, small molecules, proteins, and even cells, tissues and organisms [29, 30, 31]. Due to their low cost production, ease to be labelled with different reporter molecules and coupling with different transduction systems, nucleic acid aptamers are ideal reagents for the development of biosensors. As earlier as 2002, Bruno and Kiel selected ssDNA aptamer against cholera toxin . In 2008, Penner and Cruz-aguado evaluated the aptamer sequence (5′-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3′) for OTA using in vitro process SELEX [30, 31]. During past decade, various aptasensors with different sensing strategies including fluorescence and electrochemistry have been developed for detection of multiple microbial toxins [1, 2, 32, 33, 34].
5.1 Detection Bacterial Toxins
Yuan and coauthors have developed a signal-on voltammetric aptasensor for the hypersensitive detection of Endotoxin, also known as LPS by combining the three-way DNA hybridization process and electroactive toluidine blue–graphene–gold nanoparticles (termed as, Tb–Gra–AuNPs) nanocomposite-based amplification . With the cascade signal amplification, the proposed voltammetric aptasensor provides an ultrasensitive electrochemical detection of LPS down to the femtogram level (8.7 fg mL−1) with a linear range of six orders of magnitude (from 10 fg mL−1 to 50 ng mL−1). Zhang and coauthors reported a voltammetric aptasensor which is based on a competitive reaction between free FB1 in the sample and GS-TH nanocomposite, for an immobilized FB1 DNA aptamer (S2)/capture DNA/AuNPs at electrode surface . The constructed aptasensor was applied successfully employed to detect FB1 in feed samples.
5.2 Detection of Mycotoxins
6 Summary and Outlook
The graphene-based nanocomposites have already demonstrated great successes in the development of electrochemical biosensors for sensing microbial toxins. These biosensors exhibit outstanding performance which leads to develop miniaturized electronic and electrochemical devices for the practical or commercialized applications including real-time monitoring of food/feed quality and analysis of clinical samples. Due to the lack of facile methods for controllable, scalable, and reproducible, preparation of graphene materials with defined structures and properties, the development and widespread application of grapheme nanocomposite-based sensors are largely hindered. For example, extensively used graphene synthesis method (Hummers method) will produce acid wastes, and it is still a challenge for the preparation of pure single-layer graphene without residual oxygen groups on the surface. The electrical/chemical properties of reduced graphene-based nanocomposites may differ significantly since the morphology and chemical structure of reduced graphene are very sensitive to the method used for exfoliation and reduction. To move forward, it is, therefore, necessary to collaborate between different disciplines and technologies.
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