http://informahealthcare.com/ijf ISSN: 0963-7486 (print), 1465-3478 (electronic) Int J Food Sci Nutr, Early Online: 1–8 ! 2015 Informa UK Ltd. DOI: 10.3109/09637486.2015.1034251

COMPREHENSIVE REVIEW

Emerging nanotechnology for detection of mycotoxins in food and feed Mahendra Rai, Priti S. Jogee, and Avinash P. Ingle

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Nanobiotechnology Laboratory, Department of Biotechnology, SGB Amravati University, Amravati, Maharashtra, India

Abstract

Keywords

The term mycotoxin was coined for toxic metabolites secreted by some fungi in food, food products and feed. The most prominent mycotoxins include aflatoxins (AFs), deoxynivalenol, zearalenone, ochratoxin, fumonisin and patulin. Among these some are proved to be strong carcinogenic agents such as AFs B1 while others are under suspicion to have carcinogenic effects. Ingestion of such mycotoxin-contaminated food and feed pose to a threat, mycotoxicoses. Various conventional techniques are available for the detection of mycotoxins, but unfortunately as a consequence of their constraint, the development of new and rapid techniques is the need of the hour. The use of nanotechnology for the development of nanobiosensors would be the alternative sensitive methods for the rapid detection of mycotoxins. Implementation of nanomaterials in the fabrication of nanobiosensors and their use for the detection of the mycotoxins in food and feed is the centre of interest of this review. We have inventoried nanomaterials applied for weaving nanobiosensors, which includes carbon nanotubes, nanowires, nanoparticles, quantum dots, nanorods and nanofibers. In addition, we have extensively reviewed available nanobiosensors specific for different mycotoxins, their advantages and challenges.

Mycotoxicoses, mycotoxins, nanobiosensors, nanomaterial, nanotechnology

Introduction Fungi can grow in any habitat on a wide range of nutrients at different temperature and pH (Boucher & Mannan, 2002). They play dual role in nature, some are beneficial and others are hazardous. Some of the fungal species are responsible to impart an off flavour to the long-termed stored food grains, resulting into the loss of fresh harvest and make these grains unsafe for the consumption. Report suggested that secondary metabolites particularly mycotoxins secreted by the fungi contaminating food were the causative agent for death of thousands of people all over the world (Pitt & Hocking, 2006). There are different types of mycotoxins identified till date which includes aflatoxins (AFs), zearalenone (ZEA), fumonisins (FMs), cyclopiazonic acid (CPA), ochratoxin A (OTA), citrinin (CIT), deoxynivalenol (DON), patuline, vomitoxins, etc. (Bullerman & Bianchini, 2007; Rodriguez-Carrasco et al., 2014). The fungi which secrete these toxins mainly belong to genera Aspergillus, Fusarium, Rhizopus, Alternaria, Penicillium, etc. (Joshaghani et al., 2013). Recent studies have generated hope that nanotechnology will pave the way to nanosensor development, which would be useful for the detection of such mycotoxins. Nanotechnology deals with the nanomaterials (e.g. nanoparticles) in the range of 1–100 nm. Various chemical, physical and biological methods have been successfully used for the synthesis of metal nanoparticles (Dar et al., 2014; Sathiyanarayanan et al., 2014) like gold, silver, palladium, iron, platinum, rhodium (Borodko et al., 2010; Gade et al., 2010; Rai et al., 2008; Stowell & Korgel 2005; Correspondence: Professor Mahendra Rai, Nanobiotechnology Laboratory, Department of Biotechnology, SGB Amravati University, Amravati-444 602, Maharashtra, India. E-mail: [email protected]

History Received 12 November 2014 Revised 7 February 2015 Accepted 10 March 2015 Published online 22 May 2015

Zhu et al., 2005), etc. Ingle et al. (2008) have shown the mycosynthesis of AgNPs from fungus Fusarium acuminatum and examined its activity against some human pathogenic bacteria. Advancement in nanotechnology leads to the discovery of many methods useful for the detection and sensing of mycotoxin level in livestock (Li et al., 2012a,b; Shim et al., 2009). Moreover, the researchers are engaged in development of most sensitive techniques for the detection and management of mycotoxins and mycotoxigenic fungi (Jogee et al., 2012). Recently, much research has been focused on the use of nanoparticles for various applications, which results in the development of different biosensor for the detection of different mycotoxins and to measure their level in the food commodity. Device developed by the combination of nanoscale devices and biological component resulted in the new generation of biosensors known as nanobiosensors. Many reports are available for the development of specific biosensor for the target organism as specific mycotoxins. These reports include different biosensors with portability for onsite detection of mycotoxins in the fields directly where the food is harvested, during storage and transport. In this context, Moon et al. (2013) have developed sensor in the form of strips. These sensitive strips are more economical with respect to the size and price, and hence can be used for on-site analysis of mycotoxins at various stages from field to shop. Sensing ability of developed nanobiosensors is important as it should be more specific and sensitive to mycotoxin even at low concentration, so that one can use it for early detection of mycotoxins contamination in food. In addition, Gan et al. (2013) have developed a nanocomposite for AFs detection with the sensitivity at very low concentration of this mycotoxin, i.e. 0.3 pg/mL. Similarly, a nanosensor was designed by Yotova et al. (2013) for the detection of tyrosinase enzyme in toxic fungi, which detects its target molecule at

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minimal concentration. This acute sensitive mechanism of available nanobiosensors possess advantage that it can be used for early detection of mycotoxins before it get served to humans or animals as they can sense mycotoxins at low concentration than the permissible level by international standards, which is 4 mg/kg set by European Union for total AFs present in food (EU: Commission Regulation, 2006).

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Biosensors for detection of mycotoxins Detection of mycotoxins level and its contamination in food and feed is a vast area of research. Many methods have been described for the detection of mycotoxins including ELISA, GC, TLC, HPLC and some others (Rai et al., 2012a). Among these HPLC is preferable, but the process is very sensitive, tedious, timeconsuming and costly (Pascale, 2009). Most of these conventional techniques require sample extraction and clean-up procedures (pre-treatment) for each assay of analysis of mycotoxins, in some cases, post-concentration is also required. To overcome these problems, much attention has been paid to the toxin sensing technology and the instrument development by many other means. Output of this research is the development of biosensor with the advancement of technology. Biosensors are described as the combination of biological component (whatever secreted by the organisms, e.g. secondary metabolite) with a physicochemical detector or transducer (may vary from optical, electrochemical, piezoelectric elements, etc.). The transducer transforms the signal received from interaction between analyte and biological component into quantified, easily measurable signals. These signals then are displayed by signal processor into user-friendly manner (digital output signals). Use of electrochemical affinity biosensors for the detection of different mycotoxins have been reviewed by Vidal et al. (2013). There are different types of biosensors, such as immunochemical biosensor (in which monoclonal and polyclonal antibodies are used), optical biosensors, electrochemical biosensors, enzyme-based biosensors (Baeumner, 2003), electrodebased biosensors (Tudorache & Bala, 2007), whole cell-based biosensors (Baeumner, 2003), whole organism-based biosensors, amperometric biosensors, acoustic biosensor, potentiommetric biosensors, optical biosensors and colorimetric biosensors (Rana et al., 2010), etc. have been yet developed. Gaag et al. (2003) developed biosensor on the principle of surface plasmon resonance (SPR) for the analysis of four types of mycotoxins. Four types of mycotoxins specific antibodies were immobilised on the sensor surface to which four serially connected flow cells were attached, hence the analysis of four different mycotoxins at a time is possible. Otles & Yalcin (2012) described the use of carbon nanotubes (CNT) in a biosensor. The use of nanotechnology in food and feed was studied by Chaudhary & Castle (2011). They reported the potential use of nanobiosensor in the food labelling. The main purpose behind this was to monitor the conditions of packaged food during storage and transport with the help of nanosized sensor incorporated into them. These sensors are meant for the release of food preservatives when microbial activity was initiated during transport or by rough handling of packages should get abuse. Electronic nose with metal oxide semiconductor was used as sensor for the detection of DON from naturally contaminated wheat. These sensors were used in combination of trap and tenax TA as an adsorbent material to distinguish contaminated samples from those non-contaminated wheat grains (Campagnoli et al., 2011). For detection of AFs, biosensor was developed by the use of SPR which has been proved as the most reliable and promising biosensor. The principle of SPR biosensor development is based on the binding of biological molecules on the thin metal film, which acts as a sensor. This results into changes in the mass concentrations, which are measured by the

Figure 1. Components of biosensor.

detector element. The free electron of sensor metal plate absorbs light of specific wavelength at a specific angle of incidence, as a result, intensity of the reflected light decreases. This phenomenon is dependent on the angle of incidence change because of changes in the mass concentration on the surface of the sensor. The activity of the sensor thus depends on the binding and dissociation of interacting molecules at the metal surface responsible for the mass concentration changes. Mirasoli et al. (2012) developed the chemi-luminescence based biosensor for the detection of FMB1 and FMB2. The developed sensor was very sensitive and rapid, which can detect very low concentration of FMs, i.e. upto 2.5 mg/ L within 25 min (including sample preparation). However, instinct of many researchers to develop advanced biosensor by eliminating the drawbacks of existed biosensors makes them to move towards nanotechnology. Therefore, to enhance the activity of these biosensors, nanotechnology has been proved as promising technology, on account of this, researchers are deliberately moved towards nanotechnology implementation in designing biosensors. Schematic representations of the different component of biosensors are given in Figure 1.

Nanotechnology and nanobiosensors The advancement in the nanotechnology has been a great help for the development of nanobiosensors from existing biosensors. The nanobiosensors are supposed to be the second-generation biosensors, which are ultrasensitive due to involvement of nanomaterial in it. Traditionally used transducers in biosensors are now replaced by the use of nanoscale devices. Katz & Willner (2004) demonstrated the use of CNT in biosensors. The hybrid of semiconductor nanoparticles and biomolecules leads to the formation of nanowires for its use as nanobiosensor. These phenomena of nanoparticles integration into biomolecules are helpful for the fabrication of bio-electronic devices and lead to the way of research for bio-nanomaterials (Tothill, 2009). Different kinds of nanomaterials like CNT, dendrimers, superparamgnetic nanoparticles, quantum dots, metallic nanoparticles including gold, silica and other noble metal nanoparticles, etc. found to have their crucial role in the development of nanobiosensors (Otles & Yalcin, 2010). Enhancement of signal response generated by electrochemical biosensors due to the use of nano-sized material was observed by Soloducho & Cabaj (2013). Singh et al. (2013) fabricated nanobiosensor for sensing the total cholesterol of the body. In this study, they used nickel ferrite and chitosan

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DOI: 10.3109/09637486.2015.1034251

nanocomposite as a platform for the nanocomposite structure used to immobilise enzymes specific for cholesterol. Copious nanobiosensors were inventoried by Rai et al. (2012b) in biomedical applications. For instance, nano-robot for sensing glucose level in diabetic patient, detection of antigen–antibody interaction, nanobiosensor developed by magnetic nanoparticles for sensing activity of telomerase enzyme in cancer patients, detection of pathogenic bacteria by the application of superparamagnetic iron oxide nanoparticles, these and some others. Campas et al. (2012) reviewed the application of nanotechnology in the development of electrochemical biosensors for toxin analysis and reported some novel bio-recognition element for biosensor, use of nanomaterial as transducers and for the immobilisation purpose. Malhotra et al. (2014) reviewed the recent advancements in the synthesis of sensors based on nano-biosystems for mycotoxins detection. Shim et al. (2009) developed biosensors for ZEA mycotoxins detection based on gold nanoparticles immunochromatographic (ICG) assay with the help of monoclonal antibodies. These ICG strips were very accurate, sensitive and rapid for on-site screening test of ZEA with detection limit 30 mg/kg of corn. For the sake of nanobiosensor development, various nanomaterials are used at different stages of nanobiosensor fabrication.

Principle of operational manual of nanomaterial in nanobiosensors Above mentioned properties make nanobiosensors valuable. These qualities of nanobiosensors belong to the nanomaterial applied in weaving nanobiosensors. These nanomaterials act differently to enhance sensing purpose of nanobiosensors by supporting immobilisation process, creating signal probe, amplification of signals, etc. Nanomaterial enhances activity of nanobiosensors at the level of two elements of biosensors: (a) at bioreceptor level and, (b) at transducer level Because of the high ratio of nanomaterial for their surface area to volume, applied nanomaterial can act better at bioreceptor level by providing more surface area for biomolecules attachment and hence, pose an ability to detect target biomolecules even at low concentration in samples. Gan et al. (2013) in their study have used magnetic nanoparticles for immobilisation on graphene oxide and formed magnetic nanocomposites. These nanocomposites were then used as bioreceptor for adsorption of AFs M1 biomolecules. Their results had proven the excellence of nanomaterials at bioreceptor level. Nanomaterials when applied at transducer level, they may increases the electron transfer by behaving as an active quencher of electron and improves the signal amplification. Thus, enhances transducers activity. Holzinger et al. (2014) have given extensive review on, how nanomaterials help to improve transducers functioning in nanobiosensors.

Versatility of nanomaterial used in nanobiosensors Different types of nanomaterials can be used for various means in the nanobiosensors applications such as in the immobilisation of biomolecules on the bio-receptors surface, electrode design, for the enhancement of signal transduction to the detectors, etc. Khalid et al. (2010) reviewed the applications of nanomaterials in the synthesis of DNA biosensors. Otles & Yalcin (2010) developed nanobiosensors to detect microbial contamination in food in which they have used nanoparticles as basic material. Huang & Choi (2007) reviewed the role of nanomaterial in the development of chemical sensors. A group of South Korean scientists under the leadership of Lee in 2011 have developed highly sensitive nanobiosensor for clinical immunoassay by the

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use of elliptical plasmonic gold nano-discarray. Further, Zhang et al. (2009) reviewed the use of nanomaterial for the development of nano-biosensors. According to their study nanoparticles, quantum dots and CNT are excellent nanomaterial for biosensors preparation.

Nanoparticles Use of metallic nanoparticles is now a major area of interest as they possess numerous possible applications in the development of nanobiosensor because of their small size. The chemical, physical or electronic properties of nanoparticles make them different from the bulk metal properties. Hernandez-Santos et al. (2002) investigated the use of nanoparticles in the electroanalysis. Nanoparticles were also used in the immunosensor assay developed by Paniel et al. (2010) for the detection of AFs M1 in milk. They used superparamagnetic nanoparticles, which provides the surface for binding the conjugate of AFs M1-horseradish peroxidase (AFM1-HRP) and antibody anti-AFM1. This complex has an ability to attach the magnet because of superparamagnetic nanoparticles. Thus helps to separate AFs present in milk and gives their concentration on amperometer. Many researchers have developed DNA sensors by the use of metallic nanoparticles as labels in electrochemical DNA sensing, gold nanoparticles-based DNA chips (Im et al., 2006). Fernandez-Baldo et al. (2011) used magnetic nanoparticles in preparation of nanobiosensors, which were used for the detection of OTA. Here, the nanoparticles were used as a platform to enhance the immobilisation of biologically active constituents available in microfluidic system for the fast and accurate sensation of OTA in apples. Zhang et al. (2009) studied application of different nanomaterials in nanobiosensor development. According to them, gold nanoparticles have immense value in development of variable nanobiosensors for quantitative detection of polyionic drugs (protamine and heparin). Liu et al. (2008) used gold nanoparticles for the synthesis of gold immunochromatographic strips to detect OTA from coffee.

Carbon nanotubes Nanotubes are discovered in 1991 by Iijima, a Japanese scientist followed by the development of laboratory synthesis of fullerenes. For the synthesis of the CNT, pyrolysis of hydrocarbons on metal surfaces is done. The research showed the four-step mechanism of the CNT synthesis (Rao et al., 2001). Nanotubes peculiarly carry the structure-dependent electronic and mechanical characteristics, which make them to develop potential for various field applications of biomedical engineering, bioanalysis, nano-electronics to biosensing purpose (Cui et al., 2008). There may be single-walled (SWCNT) or multi- walled (MWCNT) structure of the CNT. Subramanian & Jerzy (2008) studied the multiple use of CNT in the development of superior quality of electrochemical nanobiosensor for the mycotoxins detection in food. Gan et al. (2013) applied cadmium telluride-CNT (CdTe-CNT) for the detection ofAFM1 in milk. The CdTe-CNT were used to form signal tag in sensor development by attaching AFs M1 antibodies on their surface. Mycotoxin STE can be detected by electrochemical biosensors, which are designed with the use of CNT (Yao et al., 2006). By the use of SWCNT, Guo et al. (2011) developed fluorescent aptasensor, which is selective and sensitive for OTA, where SWCNT act as a quencher of fluorescence made by carboxy-fluorescein attached aptamer specific to unfolded free molecules of toxins. MWCNTs, with some modification of platinum nanoparticles, to enhance the sensitivity of electrochemical DNA biosensors were used by Zhu et al. (2005). MWCNT have been used in amperometric nanobiosensor development by Zachetti et al. (2013) for the detection of CIT toxin contamination in rice samples. They used MWCNT containing redox mediators

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Figure 2. Distinguishing features of SWCNT and MWCNT.

for carbon paste electrode formation, which again coated by dialysis membrane were used for CIT detection. Figure 2 represents the distinguishing features of SWCNT and MWCNT.

Nanorods The electrocatalytic activity of brochantite nanoparticles was explored for the fabrication of nanorod. These nanorods were used as nanosensor for the sensing purpose of L-ascorbic acid, purpose behind this to use an electrocatalytic activity of brochantite nanoparticles which oxidises the target molecule L-ascorbic acid. This bio-electrochemical ascorbic acid sensor is excellent with very low response time, i.e. 8 s (Xia & Ning, 2011). Xu et al. (2012) used gold nanorod (GNR) for the fabrication of optical biosensors. These GNR were applied at bioreceptor level. The sensor is developed for the purpose of AFB1 detection with a high sensitivity up to 0.04 ppb in the sample, such a nanobiosensor with low detection value is the greatest achievement in the field of nanobiosensor development. Another report of Xu et al. (2013) showed the label-free construction of optical biosensor requiring minimum time with a single-step assay for AFB1 by GNR competitive dispersion. GNR were used as a platform for sensing purpose because of their high ionic strength and stability and, does not require any stabilising agents. Wang et al. (2010) have synthesised electrochemiluminescent aptamer biosensor by some modification on gold electrode (modified by gold nanoparticles) using a label N-(aminobutyl)-N-ethylisoluminol for the detection of OTA. All the above mentioned nanomaterials proved their applicability in sensor development for betterment of traditional biosensors. Comparative account of Zhang et al. (2009) has given more importance to the GNR use for sensor development than GNP (gold nanoparticles). According to them, GNR with modification in combination of electrode layer exhibit analytical response in a better manner than GNP. Immunosensors with GNR modification are simple, reusable, require low sample volume, no requirement of label and suitable for the device designing in lab-on-chip than the GNP.

Ideal properties of nanomaterials that make them suitable for development of nanobiosensors The nanomaterial used for the development of nanobiosensors include nanoparticles of different metal, noble metal

nanoparticles, nanowires, nanoshells, quantum dots, polymeric nanoparticles, nanotubes, etc. (Katz & Willner, 2004; Malhotra et al., 2014). Some important characteristic properties required by nanomaterial, which make them suitable for their use in nanobiosensors are as below:  High sensitivity for mycotoxins even at low concentration, 0.5 nM concentration of tyrosinase enzyme for tyrosinase biosensor (Yotova et al., 2013)  Result reproducibility, by detecting AFM1 at 5 pg/ml concentration for six times by same nanobiosensor (Gan et al., 2013)  Detection of one or more multitoxins at a time (Gaag et al., 2003)  Minimum assay time, time required for mycotoxins assay is low, about 4–8 s for constructed nanobiosensors (Xia & Ning, 2011)  Cost-effective (Xu et al., 2012)  Portability, nanobiosensor are portable hence they can be used for on-site mycotoxins detection in fields or in storage or at retailers shop (Moon et al., 2013)  Specificity in action (Shim et al., 2009) All these characteristics exhibited by nanobiosensors make them beneficial, with high safety in ensuring quality of food, its management and for risk assessment of mycotoxins contamination. These advances put forth nanobiosensors as a choice of interest among the researchers.

Nanobiosensor for the detection of different mycotoxins Dynamic properties of nanomaterial have attracted the interest of researchers in the development of nanobiosensors. They accompany the reproducibility, short response time and high demonstrable stability with sensitivity and wider linear range as compared to the traditional analytical methods. The advanced nanobiosensors are gaining much popularity because of their enhanced parameters viz. biocompatibility, mobility and the application time (Gan et al., 2013; Moon et al., 2013). Yotova et al. (2013) studied the effect of immobilisation of enzyme tyrosinase on the signal detector in the development of nanobiosensors. According to them, the immobilisation process was essential because immobilised materials increase the contact between the biometrics and the signal detector element of sensor. So, it is helpful in the improvement of analytical sensitivity

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Table 1. Application of nanomaterials in sensor development for analysis of mycotoxins. MTXs

Biosensor principle

Applied nanomaterial

Reference

Aflatoxins

Optical

Amperometer Electrochemiluminescence

GNRs CdTe-CNT MNPs CNT Cadmium telluride quantum dot AuNPs modified gold rod, SWCNT

Lateral flow immunoassay

MNPs

Gan et al., 2013 Paniel et al., 2010 Xu et al., 2012 Xu et al., 2013 Zhang et al., 2014 Fernandez-Baldo et al., 2010; Fernandez-Baldo et al., 2011 Guo et al., 2011; Liu et al., 2008; Moon et al., 2013; Wang et al., 2010

Electrochemical Ochratoxins

Electrochemical Immunosensor SPR aptasensor

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Zearalenone Sterigmatocystin Fumonisin Citrinin Deoxynivalenol

Immunochromatographic assay Electrochemical SPR Electrochemiluminescence Amperometric Biolayer interferometry

GNR Manganese dioxide nanosheets silver nanocluster AuNPs CNT Gold film MWCNT Gold colloid

and selectivity of designed biosensors. For this purpose, gold nanoparticles have been extensively studied and biomimetic nanobiosensors are developed for the detection of mycotoxins in food. Bioconjugates of enzyme and NPs improve the electron transfer among the electrode and catalytic site of immobilised enzyme. This leads to enhance the biological stability of sensor surface and increases its analytical sensitivity. The sensors developed by the use of above mentioned nanomaterial for major mycotoxins are listed below. Table 1 shows the possible application of nanomaterials in sensor and Figure 3 represents the general scheme for nanobiosensors. Rashidi & Darani (2011) reviewed the application of nanobiosensors in food industries and listed many nanobiosensors for the detection of AFs contamination in food or any gases released due to microbial growth within the packaged food. Detection of AFM1 in milk was described by Gan et al. (2013). They developed the nano-biosensor by the use of CdTe-CNT. The study showed the development of sandwich immunoassay for the detection of AFM1 in milk. The magnetic nanocomposite was fabricated by the immobilisation of Fe3O4 nanoparticles on graphene oxides. This nanocomposite was used as absorbent for AFM1. To form the signal tag (ATM1Ab1/CdTe-CNT), the primary antibodies of AFM1 (ATM1Ab1) was attached on the surface of CdTe quantum dots, CNT and nanocomposites. By the use of ATM1Ab1/CdTe-CNT, primary antibodies ofAFM1 and magnetic nanocomposites, i.e. graphene oxides, sandwich immunoassay was structured. This was an ultrasensitive electro-chemiluminescent immunoassay. Paniel et al. (2010) developed electrochemical biosensor using magnetic nanoparticles coated with antibodies specific for AFM1 for the detection of AFM1 from food. Horseradhish peroxidase (HRP) was used as a tag for the competitive immunoassay and it gets linked to AFM1 to form a conjugate, which was used as a tracer. This sensor works on the basis of competition for antibodies between tracer and antigen (AFM1). Then the equilibrated mixture was deposited on carbon electrode to which mediator MPMS (5-methylphenazinium methyl sulphate) is added. The enzymatic reactions taking place at the electrode are detected by amperometer. Detection limit of such sensor is 0.01 ppb having good reproducibility. Conventional optical biosensors were modified by the use of nanomaterial like GNR and proved better in the sensation purpose forAFB1 at very low level of mycotoxins existence (0.04 ppb) in food commodity

Park et al., 2014; Evtugyn et al., 2013 Yuan et al., 2014; Chen et al., 2014 Shim et al., 2009 Chen et al., 2009; Yao et al., 2006 Mullet et al., 1998 Mirasoli et al., 2012; Li et al., 2012b Zachetti et al., 2013 Maragos, 2012

Figure 3. General schematic representation of nanobiosensors.

(Xu et al., 2012). Xu et al. (2013) constructed an optical biosensor by using GNR dispersion on a competitive basis for detection of AFB1. Accordingly, as the concentration of AFB1 increases in solution, GNR dispersion also increases and absorption intensity of UV–visible spectra varies. The changes in the spectra and hydrodynamic size of GNR are recognised as indicator of sensors. Recently, by using quantum dots as fluorescent label, Zhang et al. (2014) have developed fluoroimmuno assay for AFs B1 detection by conjugation of monoclonal antibodies and CdTe quantum dots. This nanobiosensor was developed for the detection of contaminants and their secretion in peanuts. Sterigmatocystin (STE) is the second most important mycotoxin among all mycotoxins. Yao et al. (2006) developed a novel nanobiosensor for the detection of STE using CNT. The authors used modified CNT by the binding of AF-detoxifizyme. The AF-detoxifizyme deposits directly on the electrode of MWCNT and gives the notable signals, which later helps in the detection of mycotoxin. For the detection of STE, enzyme AF oxidase gets immobilised on chitosan-SWCNT, which was attached to poly-ophenylenediamine electrode modified by gold. This electrochemical nanobiosensor has an ability to detect the mycotoxin at very low concentration up to 3 ng/mL (Chen et al., 2009). Wang et al. (2010) reported the procedure for the synthesis of nanobiosensors to detect OTA. They used gold electrode modified by GNP and N-(aminobutyl)-N-ethylisoluminol as label for specific OTA detection from contaminated wheat grains. Guo

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et al. (2011) constructed a nanobiosensor for OTA detection, in which they used SWCNT. The fluorescent aptasensor was constructed on the basis of quenching principle, in which SWCNT are used as quencher. These SWCNT are able to quench the fluorescence of aptamer, which are toxin-specific in a freeunfolded manner attached to carboxy fluorescein FAM. Moon et al. (2013) developed strip assays with nanoparticles used for conjugation to detect OTA. The strips were designed on the basis of competitive assay between OTA in food and OTA-BSA conjugated on the surface of gold nanoparticles with the immobilised OTA antibodies. Fernandez-Baldo et al. (2011) fabricated immunosensor based on microfluidic competitive assay, in which platform for immobilisation of biomolecules used was magnetic nanoparticles (MNPs) for OTA quantification. GNP immunochromatographic strips were constructed by Liu et al. (2008) for the detection of OTA in coffee samples. Electrochemical biosensor for OTA was developed by FernandezBaldo et al. (2010) by the use of MNPs in combination with square wave voltammetry. Park et al. (2014) fabricated aptasensor based on SPR principle for OTA by the use of GNR, which is selective in mechanism and can be reused for seven times by giving the heat treatment at 70  C in methanol each time. Evtugyn et al. (2013) have developed a nanobiosensor by the conjugation of gold electrode coated with modifier. This modifier was developed by electro-polymerised neutral red and suspension of dendrimeric polymer Botlorn H30Õ with gold nanoparticles. This apparatus works on the basis that OTA-specific aptamer form bonding between gold and sulphur from gold nanoparticles and thiolated aptamer, respectively. Their interaction leads to the conformational changes and makes aptamer to detect OTA in the range of 0.02 nM concentration. Hayat et al. (2013) have reviewed the applicability of different nanomaterial for OTA detection. They reviewed on quantum dots, gold and magnetic nanoparticles and, carbon-based nanomaterials for the fabrication of aptasensor, specific for OTA. Manganese dioxide nanosheets have been developed by Yuan et al. (2014) for OTA biosensing purpose by the use of ssDNA probe. Chen et al. (2014) have developed silver nanocluster in combination with fluorescent DNA scaffolds for the detection of OTA at 2 pg/ml concentration. Mullet et al. (1998) developed SPR nanobiosensor for fumonisin by using polyclonal antibodies adsorbed on the gold film substrate coupled with glass prism in the Kretschmann configuration. Immunochromatographic biosensors were found to be the favourite among the scientists due to the use of monoclonal antibodies in its design, which makes them function specific. Shim et al. (2009) developed immunochromatographic nanobiosensor (strips) by the use of monoclonal antibodies functionalised with GNP (ICG). These ICG strips were found to be useful tool for onsite and rapid screening of ZEA in corn samples (direct competitive immunosorbent assay and HPLC assay). Because of the small size and minimum assay time, these ICG strips are applicable for onsite detection of mycotoxins. Maragos (2012) developed a biosensor based on the biolayer interferometry principle, in which colloidal gold was used for the amplification of signals received from transducers for the DON in wheat. Zachetti et al. (2013) developed biosensor for the detection of mycotoxin in rice samples. Contamination of rice by CIT was detected by them with amperometric nanobiosensor based on the use of MWCNT and some other reagents. This nanobiosensor has shown good reproducibility, repeatability and good correlations in electrochemical and spectrophotometric methods. In spite of all the characteristics and advantages of nanobiosensors, one has to think upon the demerits in the use of nanobiosensors. Till date, there are no reports on the controversy for the use of nanobiosensors in detecting mycotoxins presence in food. Still,

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there is a need to focus on its controversial part such as stability and nano-wastes from fabricators.

Advantages and limitations/challenges of using nanobiosensors in mycotoxin detection Analysis for mycotoxin in foods is a very important practice to ensure food quality and safety and to eliminate and control the risk of consuming contaminated foods. Hence, being able to analyse and detect mycotoxins in foods and drinks is a priority to comply with the legislative limits set by food authorities worldwide. Most analysis of these toxins is still conducted using conventional methods, but these methods have some disadvantages: (i) methods are time consuming, (ii) involves more manpower and hence cost is very high, (iii) these methods are less sensitive and cannot detect very low concentration of mycotoxins, (iv) need to send samples to laboratory for analysis and are less specific. However, increase in the number of mycotoxins contamination, required keen monitoring and its control, therefore, new option like multiarray sensors based on micro and nano systems (nanobiosensors) as diagnostics and risk assessment tools have become an attractive option for the researchers (Tothill, 2011). It has been already proved that nanotechnology has the potential to improve food quality and safety significantly through the use of advanced nanobiosensors and tracking system. Moreover, nanobiosensors have been found to be very important for the detection of mycotoxins. They also possess excellent advantages over the conventional methods and biosensors: (i) nanobiosensors are able to provide rapid, robust and cost-effective quantitative methods for on-site detection of mycotoxins, (ii) due to nanobiosensors the development of ultrasensitive devices becomes easy, (iii) nanobiosensors are now used as promising diagnostic tools, (iv) required no laboratory and specific instrument for the analysis, as the onsite analysis can be possible, (v) these methods are highly sensitive and have ability to detect ultra-low concentration of mycotoxins in food, (vi) nanobiosensors are highly specific, (vii) required comparative less manpower, etc. (Kerman et al., 2008; Otles & Yalcin, 2012; Tothill, 2009). Apart from the enormous advantages of nanobiosensors, it has some limitations also, rather we can say the technology has some challenges which needs to be overcome to develop successful commercial products that can compete with traditional methods of mycotoxin analysis. The complex molecular structures of the toxins present in wide range of concentrations need to be detected. There are some bio-analytical challenges facing the application of nanobiosensors for food analysis. Similarly, still there is need to increase the stability of some nanomaterials like quantum dots, which reduce the aggregation in use conditions and also reduce their cost. Moreover, most important concerns regarding the uncontrolled and extensive use of nanobiosensors for the detection of mycotoxins in food is the issue of toxicity risks for humans and the environment, after the disposing of these devices and materials. However, these problems can be easily resolved by ensuring the control and limited use of nanobiosensor and also focus needs to be given on its safe disposal after use.

Conclusions and future prospects Dealing with the application of nanomaterial in biosensor development is the sophisticated way of synthesising nanobiosensors for mycotoxins detection in food and feed. Such type of nanobiosensors acquiring their unique mechanical, magnetic, physical, chemical and optical properties makes them target specific with enhanced sensitivity. The available literature shows the functional enhancement of nanobiosensors by the use of nanomaterial in their construction. These nanobiosensors are fast,

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DOI: 10.3109/09637486.2015.1034251

sensitive, specific, reliable with good reproducibility and cheap. Such type of nanobiosensors will help to improve the quality of food and feed and finally minimise health hazards due to consumption of mycotoxins contaminated food. Developing novel sensors by the application of nanomaterials is the frontier area of research. A large number of applications and versatility in nanobiosensors can make them choice of research. Nanomaterials possessing properties make them different from the conventional biosensors. Applied nanomaterial may improve optical, mechanical, electrochemical or magnetic properties of biosensors. However, regardless of the merits of nanobiosensors, there is a need of extensive studies on the improvement of existing nanobiosensors because of their some limitations in their use and applicability. In addition, extra efforts are needed for the development of nanobiosensors in order to analyse multiple mycotoxins at a time. All these limitations should be considered for the improvement of existing techniques. Modification of biosensors by nanomaterial may enable them to detect mycotoxins at a very low concentration. Use of nanomaterials for the immobilisation of biomolecules and recognition of mycotoxins warrants further research in construction of nanobiosensors with more stability and durability. This will bring a promising resource towards the public health concern for mycotoxin-free food.

Declaration of interest The authors declare no conflicts of interest. The authors alone are responsible for the content and writing of this article.

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