**5. Miscellaneous methods**

ence of different parameters, such as amplicon concentration, dilution, and PCR specificity

An important point of these kinds of devices is that the crystal only can be used for 25 meas‐ urements without losing sensitivity; this is because the devices that work with mechanical effects are majorly affected with the use. This is the reason for coupling the PCR protocol and the DNA piezoelectric biosensor. After its characterization with synthetic oligonucleoti‐ des, the piezoelectric-DNA biosensor led to the clear identification and quantification of

The principle of the optical waveguide light-mode spectroscopy (OWLS) technique is the measurement of the resonance angle of polarized laser light, diffracted by a grating and in‐ coupled into a thin waveguide. Incoupling resonance occurs at very precise angles depend‐ ing on the optical parameters of the sensor chips and the complex refractive index of the covering sample medium. The intensity of the incoupled light guided within the waveguide layer by multiple internal reflections is measured with a photodiode [31].The refractive in‐ dex is determined from the resonance incoupling angle detected at high precision. Such in‐ dex allows the determination of layer thickness and coverage of the adsorbed or bound material with high sensitivity. This method allows the construction of both chemical and bi‐ osensors. Therefore, it can be applied for direct sensing of various types of biomolecules.

Other optical based biosensor uses a high-tech semiconductor material–silicon for the effi‐ cient accuracy registration of narrow spectral bands or specific wavelengths. This biosensor is used for detecting and quantifying aflatoxins that are commonly found in a variety of ag‐

Based on the above mentioned techniques it was developed a structure with two oppositely directed potential barriers, the total current conditioned by these barriers depended on both, the external voltage and the wavelength of the absorbed radiation. A modification in these parameters resulted in the obtaining of high-accuracy data of aflatoxins contaminants in

Detection and identification of harmful organisms, such as aflatoxins, in a cost and time ef‐ fective way is a challenge for the researchers. Biosensors have proved to be useful tools for detection and quantification of such organisms. These sensors have advantages such as: fast response, relative easiness of use, a huge realm of applications, and flexibility for combina‐ tion of techniques. Such advantages are derived from the involvement of multidisciplinary research activities. But, even though the vast research on biosensors, it is still needed the in‐ jection of economical funds to locate them in the commercial market, and impulse their use

The research on biosensor has the aim to develop, at low cost an analytical approach simpler and faster. Being an alternative improving the classical techniques. It is necessary that the improvement of the processes is focused in autonomous measurement, in order to avoid, as much as possible, the human error. The mostly classical measurements are linked to the lab‐

on the biosensor's response.

302 Aflatoxins - Recent Advances and Future Prospects

**4.9. Optoelectronic**

ricultural products.

in real applications.

contaminated feed samples with aflatoxins [49].

food and provender in natural conditions [51].

### **5.1. Electrochemical methods for aflatoxins determination**

Aflatoxins measurement usually implies complex, expensive and slow methods. However, this determination can be carried out taking into account the response of aflatoxins to deter‐ minate electrical stimulus. These methods are called electrochemical, where immunosensors are applied to determine the presence of aflatoxins in a sample. Usually, these sensors are composed by two screen-printed-electrodes (SPE), the first one is made of graphite, plati‐ num, or gold; and it is known as working, active, or measuring electrode. The second elec‐ trode is the reference and is commonly made of Ag/AgCl. In general, this technique involves two basic steps. In the first, the immunosensor working electrode is coated with an anti‐ body; after an incubated time, the sample that contains the aflatoxins is added to this elec‐ trode, while the left one reacts for a determinate time; finally, a conjugated of aflatoxins and enzymes is added to the electrode, it is then when the competitive reaction begins. In this reaction, free aflatoxins compete to link to antibodies present in the working electrode. After a stabilization time, the measuring electrode is removed from the sample and rinsed with a buffer solution. The second step implies to apply an electrical potential (commonly 100 mV) to the electrode, which changes its electrical conductivity according with the aflatoxins con‐ centration. After sampling the electrode; an increase or reduction in the electric current flow will appear according with the concentration of aflatoxins in the sample. This technique has received improvements; disposable immunosensors have been reached for measurement of aflatoxins M1 (AFM1) directly in milk following a simple centrifugation step but without di‐ lution or other pretreatment steps. Exhibiting a good working range, comparable to the ones obtained in buffer; linearity between 30 and 240 ng/ml making it useful for AFM1 monitor‐ ing in milk (maximum acceptable level of AFM1 in milk is 50 ppt) [52]. It is easy to notice that electrochemical techniques offer some advantages over traditional methods for aflatox‐ ins determination, among which it can be found: reliability, low cost, *in-situ* measurements, fast processes, and easier methodology than common chromatography techniques through a similar performance

Other improvements to this methodology involve the analysis of thermal stability given that the conductivity properties of materials also change with temperature variations and not on‐ ly for the aflatoxins concentration in the electrode. SPEs with platinum as substrate for the working electrode have been used to achieve long-term stability. Probes have shown that this type of electrodes maintain a good biorecognition affinity for antibodies on its layer and a decrease in the detected signal of less than 10% after two weeks inside a refrigerator (5 °C) and less than 22% at laboratory temperature (25 °C), values that allow partial usability for practical assaying [37]. Using this type of electrodes, a voltage of 50 mV, and a stabilization time of 1 minute are suggested to begin current measurements. Limits detection of 2.4 ppb has been reached in real capsicum spice samples, producing good correlations comparing with data from HPLC with fluorescence detector.

0.05 and 2 ng/mL. Aflatoxin AFM1 was also quantified by this method. The suitability of the immunosensor for the direct analysis of the toxin in milk was assessed. AFM1 was correctly measured with a working range of 5-250 pg/ml and a detection limit of 1 pg/ml was ach‐ ieved. For this experiment, the intermittent pulse amperometry parameters were adjusted to

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Variation of electrochemical immunosensors appeared recently to determinate aflatoxins through detection of a specific DNA [56]. The detection technique was optimized applying DNA sequences from *Aspergillus* gene *aflR* that codes a biochemical pathway of aflatoxins B1 production. Then, voltametric detection of the specific *Aspergillus* DNA sequence is based on hybridization of adsorbed target DNA with a biotinylated probe and subsequent binding with streptavidin alkaline phosphatase conjugated. Then, the modified electrode surface of car‐ bon paste electrode is incubated in a buffer solution with an electrochemically inactive sub‐ strate (1-naphthyl phosphate). Alkaline phosphatase converts 1-naphthyl phosphate into 1 naphthol, which is determinate by the selected voltammetric technique. The optimize procedure is capable to distinguish potentially aflatoxigenic fungi from other *Aspergillus* species.

Spectroscopy techniques have been popularized because they present fast, low-cost and non-destructive analytical methods suitable to work with solid and liquid samples. This method involves the study of the interrelationships between the spectral characteristics of objects and their biophysical attributes, specifically, the interaction with radiated energy as a

In the particular case of aflatoxins, different studies have been carried out to determinate the wavelength in which these substances respond to radiant energy. The different spectroscopy systems available in the market have the facility to scan a sample over a determinate wave‐ length range and acquire the spectral data in different modes as reflectance, absorbance, or transmittance. The procedure to detect aflatoxins in a sample is quite similar to the afore‐ mentioned methods. The sample preparation implies extraction and clean up. However, some authors use the sample without any preparation. The samples are scanned with a spectrophotometer commonly over a wavelength range from 250 nm to 2500 nm at different steps (2 nm steps can be reached). Finally the results are shown in a graph of wavelength

Near infrared spectroscopy (NIRS) is an excellent method for a rapid and low cost detection of aflatoxins in cereals [58]. Aflatoxin B1 was successfully measured in maize and barley by applying grating and Fourier transform NIR spectroscopy instruments with multivariable statistical methods on intact, non-milled samples. This technique quantifies aflatoxins in or‐ der of 20 ppb. Variations to this method imply the use of horizontal attenuated total reflec‐ tance technique for determination of aflatoxin B1, B2, G1 and G2 in groundnut. The mid-band infrared attenuated total reflectance (ATR) spectra were obtained with a Fourier transform spectrometer equipped with a horizontal ATR accessory. This variant in the method gener‐ ates rapid and substantial spectra of aflatoxins with a minimum sample size (>2 mL) and chemicals [59]. Other authors have incorporated a bundle reflectance fiber-optic probe to


**5.2. Spectroscopy techniques**

function of its wavelength or frequency [57].

against reflectance or absorbance.

Working range for electrochemical immunnosensors from 0.1 to 10 ng/ml with a detection limit of 0.06 ng/ml has been achieved by using gold electrodes and enzymatic silver deposi‐ tion amplification. In this procedure, an aflatoxin B1-bovine serum albumin (AFB1-SBA) con‐ jugated is immobilized on the measuring electrode (gold electrode). An indirect competitive format between the selected analyte in solution and the AFB1-BSA on the electrode is per‐ formed. After the competition step, monoclonal antibody against AFB1 was bounded to the electrode and then conjugated to a secondary antibody-alkaline phosphatase (ALP) conju‐ gated. The ALP could catalyze the substrate, ascorbic acid 2-phosphate, into ascorbic acid, and the latter could reduce silver ions in solution to metal silver deposited onto de electrode surface. Finally, the metallic silver deposited onto the electrode was determined by linear sweep voltametry (LSV). The peak current for this immunosensor exhibited a negative line‐ ar correlation to AFB1 concentration [53].

As it can be noticed, electrochemical sensors and biosensors have, in some cases, the advant‐ age of rapidity and sensitivity over the traditional techniques. Electrochemical sensors based on acetycholinesterase (AChE) inhibition by aflatoxins have been rapidly applied due to de‐ tection limits of 2 ppb. As reported by [53], the AFB1 determination can be based on AChE inhibition, while the AChE residual activity is determined by using a choline oxidase am‐ perometric biosensor coupled with AChE enzyme in solution. The amperometric detection of AChE activity is based on a second enzyme, cholesterol oxidease (ChOx), providing a consecutive conversion of the native substrate (acetylcholine) to an electrochemically active H2O2. Finally this component is measured at the screen-printed electrode previously modi‐ fied with Prussian Blue (PB) at a potential of -0.05V versus screen-printed internal silver pseudo reference electrode. The linear working range was assessed to be 10-60 ppb.

Single electrode immunosensors have proved to be a reliable alternative to complex meth‐ ods for aflatoxins determination. However, devices with multiple electrodes have been de‐ veloped to offer the possibility to combine the high sensitivity of electrochemical SPE-based immunosensors with the favourable characteristics of high throughput ELISA procedures. An analytical immunosensor array, based on a microtiter plate coupled to a multichannel electrochemical detection system using the intermittent pulse amperometry technique is presented for detection of aflatoxins B1 [54].

The device is composed by 96-well screen-printed microplated, their thick-film carbon sen‐ sors was modified according with a competitive indirect enzyme-linked immunoassay (ELI‐ SA) format for aflatoxins detection. Spectrophotometry and electrochemical procedures were both applied to determinate the reliability of the proposed system. The principal ad‐ vantage of the aforementioned system is the possibility to separately apply the amperomet‐ ric to each of the 96 sensing electrodes. The applied potential is +400 mV with a pulse of 1 ms and a selected frequency of 50 Hz. This immunoassay was applied for analysis of corn samples. AFB1 could be measured at a level of 30 pg/ml and with a working range between 0.05 and 2 ng/mL. Aflatoxin AFM1 was also quantified by this method. The suitability of the immunosensor for the direct analysis of the toxin in milk was assessed. AFM1 was correctly measured with a working range of 5-250 pg/ml and a detection limit of 1 pg/ml was ach‐ ieved. For this experiment, the intermittent pulse amperometry parameters were adjusted to -100mV with a pulse width of 10 ms and a 5 Hz frequency [55].

Variation of electrochemical immunosensors appeared recently to determinate aflatoxins through detection of a specific DNA [56]. The detection technique was optimized applying DNA sequences from *Aspergillus* gene *aflR* that codes a biochemical pathway of aflatoxins B1 production. Then, voltametric detection of the specific *Aspergillus* DNA sequence is based on hybridization of adsorbed target DNA with a biotinylated probe and subsequent binding with streptavidin alkaline phosphatase conjugated. Then, the modified electrode surface of car‐ bon paste electrode is incubated in a buffer solution with an electrochemically inactive sub‐ strate (1-naphthyl phosphate). Alkaline phosphatase converts 1-naphthyl phosphate into 1 naphthol, which is determinate by the selected voltammetric technique. The optimize procedure is capable to distinguish potentially aflatoxigenic fungi from other *Aspergillus* species.

#### **5.2. Spectroscopy techniques**

practical assaying [37]. Using this type of electrodes, a voltage of 50 mV, and a stabilization time of 1 minute are suggested to begin current measurements. Limits detection of 2.4 ppb has been reached in real capsicum spice samples, producing good correlations comparing

Working range for electrochemical immunnosensors from 0.1 to 10 ng/ml with a detection limit of 0.06 ng/ml has been achieved by using gold electrodes and enzymatic silver deposi‐ tion amplification. In this procedure, an aflatoxin B1-bovine serum albumin (AFB1-SBA) con‐ jugated is immobilized on the measuring electrode (gold electrode). An indirect competitive format between the selected analyte in solution and the AFB1-BSA on the electrode is per‐ formed. After the competition step, monoclonal antibody against AFB1 was bounded to the electrode and then conjugated to a secondary antibody-alkaline phosphatase (ALP) conju‐ gated. The ALP could catalyze the substrate, ascorbic acid 2-phosphate, into ascorbic acid, and the latter could reduce silver ions in solution to metal silver deposited onto de electrode surface. Finally, the metallic silver deposited onto the electrode was determined by linear sweep voltametry (LSV). The peak current for this immunosensor exhibited a negative line‐

As it can be noticed, electrochemical sensors and biosensors have, in some cases, the advant‐ age of rapidity and sensitivity over the traditional techniques. Electrochemical sensors based on acetycholinesterase (AChE) inhibition by aflatoxins have been rapidly applied due to de‐ tection limits of 2 ppb. As reported by [53], the AFB1 determination can be based on AChE inhibition, while the AChE residual activity is determined by using a choline oxidase am‐ perometric biosensor coupled with AChE enzyme in solution. The amperometric detection of AChE activity is based on a second enzyme, cholesterol oxidease (ChOx), providing a consecutive conversion of the native substrate (acetylcholine) to an electrochemically active H2O2. Finally this component is measured at the screen-printed electrode previously modi‐ fied with Prussian Blue (PB) at a potential of -0.05V versus screen-printed internal silver

pseudo reference electrode. The linear working range was assessed to be 10-60 ppb.

Single electrode immunosensors have proved to be a reliable alternative to complex meth‐ ods for aflatoxins determination. However, devices with multiple electrodes have been de‐ veloped to offer the possibility to combine the high sensitivity of electrochemical SPE-based immunosensors with the favourable characteristics of high throughput ELISA procedures. An analytical immunosensor array, based on a microtiter plate coupled to a multichannel electrochemical detection system using the intermittent pulse amperometry technique is

The device is composed by 96-well screen-printed microplated, their thick-film carbon sen‐ sors was modified according with a competitive indirect enzyme-linked immunoassay (ELI‐ SA) format for aflatoxins detection. Spectrophotometry and electrochemical procedures were both applied to determinate the reliability of the proposed system. The principal ad‐ vantage of the aforementioned system is the possibility to separately apply the amperomet‐ ric to each of the 96 sensing electrodes. The applied potential is +400 mV with a pulse of 1 ms and a selected frequency of 50 Hz. This immunoassay was applied for analysis of corn samples. AFB1 could be measured at a level of 30 pg/ml and with a working range between

with data from HPLC with fluorescence detector.

304 Aflatoxins - Recent Advances and Future Prospects

ar correlation to AFB1 concentration [53].

presented for detection of aflatoxins B1 [54].

Spectroscopy techniques have been popularized because they present fast, low-cost and non-destructive analytical methods suitable to work with solid and liquid samples. This method involves the study of the interrelationships between the spectral characteristics of objects and their biophysical attributes, specifically, the interaction with radiated energy as a function of its wavelength or frequency [57].

In the particular case of aflatoxins, different studies have been carried out to determinate the wavelength in which these substances respond to radiant energy. The different spectroscopy systems available in the market have the facility to scan a sample over a determinate wave‐ length range and acquire the spectral data in different modes as reflectance, absorbance, or transmittance. The procedure to detect aflatoxins in a sample is quite similar to the afore‐ mentioned methods. The sample preparation implies extraction and clean up. However, some authors use the sample without any preparation. The samples are scanned with a spectrophotometer commonly over a wavelength range from 250 nm to 2500 nm at different steps (2 nm steps can be reached). Finally the results are shown in a graph of wavelength against reflectance or absorbance.

Near infrared spectroscopy (NIRS) is an excellent method for a rapid and low cost detection of aflatoxins in cereals [58]. Aflatoxin B1 was successfully measured in maize and barley by applying grating and Fourier transform NIR spectroscopy instruments with multivariable statistical methods on intact, non-milled samples. This technique quantifies aflatoxins in or‐ der of 20 ppb. Variations to this method imply the use of horizontal attenuated total reflec‐ tance technique for determination of aflatoxin B1, B2, G1 and G2 in groundnut. The mid-band infrared attenuated total reflectance (ATR) spectra were obtained with a Fourier transform spectrometer equipped with a horizontal ATR accessory. This variant in the method gener‐ ates rapid and substantial spectra of aflatoxins with a minimum sample size (>2 mL) and chemicals [59]. Other authors have incorporated a bundle reflectance fiber-optic probe to NIRS system. Here, the fiber-optic probe is immersed in the sample without any previous treatment or manipulation of the samples. Then, NIR spectra are recorded direct from the fiber. This combination of technologies has proved to quantify aflatoxin B1, ocharatoxin A and total aflatoxins in paprika successfully [60].

In the case of immunological methods, there are several research papers reporting advances in the development and improvement of immunological techniques for detection of aflatox‐ ins. Most of them are based on ELISA, although there are other techniques such as ICA and real-time PCR that have been used for this purpose; the objective of these studies is to ach‐

Future aflatoxins detection methods shall be guided by biosensors with mixed techniques, which have already proved their contribution, and utility in sensing and detection technolo‐ gy. Such sensors might be also used in biosecurity brigades along international borders. Bio‐ sensors may play a major role in this field as they provide rapid and specific detection compared to other techniques. A barrier that shall be overcome is the production of biosen‐ sors for harsh environments. Research on materials, techniques and working parameters need to be made to solve such problems. Portability is another obstacle to be defeated. The use of biosensors in small laboratories and the agricultural industry will increase as biosen‐

Tendencies in the development of new methods for quantifying the aflatoxins suggest a con‐ tinuous combination among the different techniques. The combination of different techni‐

Authors give thanks to Consejo Nacional de Ciencia y Tecnología (CONACyT), in Mexico, for its financial support through the scholarships with Registration Numbers: 201401

, Rafael Francisco Muñoz-Huerta1

, Ramon Gerardo Guevara-Gonzalez1

1 Biosystems Engineering CA, Postgraduate Study Division, Engineering Faculty, Autono‐

2 Faculty of Physics and Mathematics, Autonomous University of Sinaloa, Culiacán, Sina‐

, Carlos Duarte-Galvan1

,

and

Characteristics of Mycotoxin Analysis Tools for Tomorrow

http://dx.doi.org/10.5772/51683

307

,

ques allows increasing the sensibility, portability and rapidness of analysis.

(LMCM), 239421 (AEC), 231946 (CDG), 226888 (AAFJ), 209021 (RFMH).

Luis Miguel Contreras-Medina1\*, Alejandro Espinosa-Calderon1

\*Address all correspondence to: mcontreras@hspdigital.org

mous University of Querétaro, Querétaro, Qro., México

ieve the development of rapid, simple, highly sensitive and low-cost techniques.

sors become more portable.

**Acknowledgements**

**Author details**

Arturo Alfonso Fernandez-Jaramillo1

Jesus Roberto Millan-Almaraz2

Irineo Torres-Pacheco1

loa , México

#### **5.3. Fluorescence methods**

Aflatoxins have a native luminescence due to their oxygenated pentaherocyclic structure. Thus, most analytical and microbiological methods for detection and quantification of afla‐ toxins are based on this feature. There are a number of microbiological methods that can be used for the direct visual detection of aflatoxin-producing *Aspergillus* strains*.* The aim of these procedures is to increase the production of aflatoxins and elicit at bright blue or bluegreen fluorescent areas surrounding colonies under UV radiation. Complex agar media con‐ taining different additives to increase the production of aflatoxins have been implemented for this purpose. The addition of a methylated derivative of of β-CD plus sodium deoxycho‐ late (NaDC) to yeast extract agar (YES) was found to be suitable for the identification of afla‐ toxigenic *Aspergillus* strains. This was achieved through the visualization of a beige ring surrounding the colonies. When this ring was examined under UV light, it exhibited blue fluorescence. Furthermore, it was observed that aflatoxigenic colonies grown in such envi‐ ronment also emitted room temperature phosphorescence (RTP), when examined in the dark, following excitation with a UV light lamp [61]. The main problem with this technique is related with the disturbance due to the background emission origination from matrix con‐ stituents, this because the emission maxima depends on the solvent and the pH. This prob‐ lematic was addressed and solved by applying two-photon excitation conditions [62].

#### **6. Conclusions**

More than 300 micotoxins are discovered. They are toxic metabolites of a variety of fungi growing in a wide range of food and animal feedstuffs. Of all micotoxins, the aflatoxins are the major concerns as they are mutagenic, carcinogenic, teratogenic and immunosuppres‐ sive compounds. Consumption even at very low concentration may cause serious health problems. For the aforementioned reasons, it is important to develop new methodologies and systems able to quantify the aflatoxins concentrations that satisfy the restrictions pro‐ posed by the organizations in charge of control this compounds. To do this, several techni‐ ques have been employed such as: chromatography, immunological methods, biosensors and others methods. Through the paper can be noticed that almost all techniques need to combine efforts to accomplish precise quantifications. These combinations have depended greatly of technology development during the last years. In the case of chromatography, if the methods of pre-process, derivatization and detections improve their capabilities to ach‐ ieve their functions, it can be developed new systems with higher sensitivity and portability than the so far developed systems.

In the case of immunological methods, there are several research papers reporting advances in the development and improvement of immunological techniques for detection of aflatox‐ ins. Most of them are based on ELISA, although there are other techniques such as ICA and real-time PCR that have been used for this purpose; the objective of these studies is to ach‐ ieve the development of rapid, simple, highly sensitive and low-cost techniques.

Future aflatoxins detection methods shall be guided by biosensors with mixed techniques, which have already proved their contribution, and utility in sensing and detection technolo‐ gy. Such sensors might be also used in biosecurity brigades along international borders. Bio‐ sensors may play a major role in this field as they provide rapid and specific detection compared to other techniques. A barrier that shall be overcome is the production of biosen‐ sors for harsh environments. Research on materials, techniques and working parameters need to be made to solve such problems. Portability is another obstacle to be defeated. The use of biosensors in small laboratories and the agricultural industry will increase as biosen‐ sors become more portable.

Tendencies in the development of new methods for quantifying the aflatoxins suggest a con‐ tinuous combination among the different techniques. The combination of different techni‐ ques allows increasing the sensibility, portability and rapidness of analysis.
