Review Article Methods for Detection of Aflatoxins...

Review ArticleMethods for Detection of Aflatoxins in Agricultural Food Crops

Alex P Wacoo12 Deborah Wendiro1 Peter C Vuzi2 and Joseph F Hawumba2

1 Microbiology and Biotechnology Centre Department of Product Development Uganda Industrial Research InstitutePO Box 7086 Kampala Uganda

2Department of Biochemistry and Sports Science School of Biological Sciences College of Natural SciencesMakerere University PO Box 7082 Kampala Uganda

Correspondence should be addressed to Joseph F Hawumba jhawumbacnsmakacug

Received 25 August 2014 Accepted 18 October 2014 Published 13 November 2014

Academic Editor Zhen Cheng

Copyright copy 2014 Alex P Wacoo et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Aflatoxins are toxic carcinogenic secondary metabolites produced predominantly by two fungal species Aspergillus flavus andAspergillus parasiticus These fungal species are contaminants of foodstuff as well as feeds and are responsible for aflatoxincontamination of these agro products The toxicity and potency of aflatoxins make them the primary health hazard as well asresponsible for losses associated with contaminations of processed foods and feeds Determination of aflatoxins concentration infood stuff and feeds is thus very important However due to their low concentration in foods and feedstuff analytical methods fordetection and quantification of aflatoxins have to be specific sensitive and simple to carry out Severalmethods including thin-layerchromatography (TLC) high-performance liquid chromatography (HPLC) mass spectroscopy enzyme-linked immune-sorbentassay (ELISA) and electrochemical immunosensor among others have been described for detecting and quantifying aflatoxinsin foods Each of these methods has advantages and limitations in aflatoxins analysis This review critically examines each of themethods used for detection of aflatoxins in foodstuff highlighting the advantages and limitations of each method Finally a wayforward for overcoming such obstacles is suggested

1 Introduction

11 Aflatoxins and Their Metabolism Aflatoxins are cancer-ous secondary metabolites produced primarily by Aspergillusflavus and Aspergillus parasiticus in agricultural foodstuffsuch as peanuts maize grains cereals and animal feeds Afla-toxins are difuranocoumarin molecules synthesized throughthe polyketide pathway [1] Six out of 18 different types ofaflatoxins that have been identified are considered importantand are designated as B

1 B2 G1 G2 M1 andM

2 respectively

[2] These aflatoxin groups exhibit molecular differencesFor example the B-group aflatoxins (B

1and B

2) have a

cyclopentane ring while the G-group (G1and G

2) contains

the lactone ring [3] Whereas the B-group aflatoxins exhibitblue fluorescence the G-group exhibits yellow-green fluo-rescence under ultraviolet (UV) light thus making the useof fluorescence important in identifying and differentiatingbetween the B and G groups Aflatoxin B

1is the most

common [4] and the most widespread [5 6] in the world and

accounts for 75 of all aflatoxins contamination of food andfeeds [7] AflatoxinsM

1andM

2are hydroxylated products of

aflatoxins B1and B

2 respectively and are associated with cow

milk upon ingestion of B1and B

2aflatoxinsrsquo contaminated

feedMoreover once formed fromB1and B

2forms aflatoxins

M1and M

2remain stable during milk processing [8]

In order to understand the metabolism of aflatoxinsthe biotransformation of aflatoxin B

1 the most abundant

form has been explored [9ndash11] Aflatoxin B1metabolism

takes place in the microsome of the liver and is medi-ated by mixed function monooxygenases belonging to thecytochrome P

450super family of enzymes [12] (Figure 1) In

humans cytochrome P450

enzymes CYP1A2 and CYP3A4[10] catabolize aflatoxin B

1through two separate electron

transfer oxidation reactions [9 11 13] While CYP1A2 breaksdown aflatoxin B

1to exoepoxide endoepoxide and aflatoxin

M1 CYP3A4 breaks down aflatoxin B

1to aflatoxin B

1-exo-

89-epoxide and aflatoxin Q1 Aflatoxins M

1and Q

1are not

broken any further but are excreted in the urine Aflatoxin

Hindawi Publishing CorporationJournal of Applied ChemistryVolume 2014 Article ID 706291 15 pageshttpdxdoiorg1011552014706291

2 Journal of Applied Chemistry

Aflatoxin-exo-89-epoxide

Aflatoxin-endo-89-epoxide

Urine

Aflatoxin-mercapturic acid

Peripheral blood

Aflatoxin-glucuronide

Aflatoxin-dialcoholAflatoxin-albumin

Aflatoxin-dihydrodiol

Aflatoxin-dialdehyde

1A23A4 (3A5)

1A2 and 3A4 (3A5) 1A2

DNA

GST

AFARAflatoxin-DNA

O O

O

O O

O

O O

O O OMe

OO

O

O

O O

O

O

O O

OO

O

O

OO O

O

O O

O

O O

O

O

O

N

O

O O

O

O

O

O

OH

HO

OOH

OH

HO

O

O

O

N

NHN

N

O

O OO

O O

OO

O

O O

OOOH

H

H

HO

Aflatoxin B1

Aflatoxin M1

Aflatoxin Q1

Aflatoxin-G-S

Aflatoxin-N7-guanine

OH

OH

HOHO

HO

HO

HO

S

S

Cys

Cys

Gly

Glu

NH CO CH3

H2NdR DNA

Albumin

OMe

OMe OMe

OMe

OMe

OMe

OMe

OMe

OMe

O

O O

O

O O

N

NHN

N

HO

H2N

OMe

OMe

OMe

Figure 1 Principal metabolism of aflatoxin B1leading to reactive metabolites and biomarkers 1A2 CYP1A2 3A4 CYP3A4 3A5 CYP3A5

GST glutathione S-transferase AFAR aflatoxin aldehyde reductase aflatoxin-S-G aflatoxin-glutathione conjugate (adopted fromWild andTurner 2002) [9]

B1-exo-89-epoxide may be converted either to aflatoxin-

mercapturic acid via the GST conjugate mediated routeor into aflatoxin-glucuronide via the aflatoxin-dihydrodiolroute described as follows The activated form of aflatoxinB1(exoepoxides and endoepoxides) is detoxified through

glutathione S-transferase- (GST-) mediated conjugation byusing reduced glutathione (GSH) to form AFB

1exoepoxide-

GSHand endoepoxide-GSH conjugates respectively [14]Thereactive exoepoxides and endoepoxides also undergo rapidnonenzymatic hydrolysis to aflatoxin B

1-89-dihydrodiol that

slowly transforms into a dialdehyde phenolate ion [9 15]Dialdehyde phenolate ion is subsequently hydrolyzed byaflatoxin aldehyde reductase (AFAR) to a dialcohol in theNADPH-dependent reduction reactionThereafter dialcoholis excreted in urine as aflatoxin-glucuronide [16 17] Afla-toxin B

1dialdehydes also form Schiff bases with primary

amine groups of amino acid residues such as lysine ofsuch a protein as albumin to form aflatoxin B

1-albumin

conjugate [9] This conjugate persists in the systemic bloodas permanent and irreversible aflatoxin B

1-albumin adducts

and is thus considered one of the factors accounting for thelow excretion of aflatoxins and their metabolites in urine [18]

12 Toxicity of Aflatoxins From the foregoing (Figure 1)it can be observed that the primary derivatives of afla-toxin B

1biotransformation comprise (a) aflatoxin M1 and

aflatoxin-exo-89-epoxide (products of CYP1A2 activity) and(b) aflatoxin Q

1and aflatoxin-exo-89-epoxide (products of

CYP3A4 activity) Aflatoxins M1and Q

1 although toxic are

less reactive with other molecules and are easily eliminatedfrom the body in the urine [9] However aflatoxin B

1-

89-exo-epoxide is a known mutagen which is extremelyelectrophilic and covalently reacts with nucleophilic sitesof either deoxyribonucleic acid (DNA) or ribonucleic acid(RNA) or proteins [21] thereby introducing mutations thatmay affect the normal function of cells The formation ofaflatoxin B

1-DNA adducts is extremely associated with the

carcinogenicity of aflatoxin B1 Typically aflatoxin B

1reacts

with DNA (methylation) resulting in GrarrT transversion

Journal of Applied Chemistry 3

AA A A

AT T

T

T T

C

C

CC

C

C

GGG

G G

AA A A

AAT T

TT

T T

C

C

CC

C

G

G

G G

1

2SNP

Figure 2 Point mutation of G rarr T at codon 249 in the p53 generesulting in aflatoxin induced hepatocellular cancer (adopted fromBbosa et al 2013) [13]

mutation (Figure 2) [22] Such amutation has been associatedwith hepatocellular carcinoma a type of cancer wherebyaflatoxin B

1promotesAGGrarrAGT (Argrarr Ser) transversion

point mutation of p53 gene at codon 249 that alters p53 genewhich is responsible for DNA repair [23] Apart from GrarrT transversions GrarrC transversions and GrarrA transitionshave also been reported [22]

Nucleic acids and proteins interact covalently with afla-toxins and this results in alteration in base sequences innucleic acids (bothDNA and RNA) and in protein structuresleading to impairment of their activity The highly reac-tive aflatoxin B

1-89-exo-epoxide and its hydration product

dihydrodiol bind covalently to DNA RNA and proteins toinhibit protein synthesis [24] Typically RNA polymeraseand ribosomal translocase have been demonstrated to beinhibited by aflatoxin B

1-89-exo-epoxide [24] While the

epoxide reacts at the N7 position of guanine of both DNAand RNA the dihydrodiol reacts with the amino groups ofthe bases forming a Schiff base [25] Aflatoxin B

1has also

been reported to negatively impact carbohydratemetabolismwhich results in both the reduction in hepatic glycogen andalso the increased blood glucose levels (Figure 3)Notably thenegative effects of aflatoxin B

1on carbohydrate metabolism

Aflatoxin B1

Aflatoxin B1

Aflatoxin B1Glycogenn

Glucose-1-P

Glucose-6-P

Cyclochlorotine

Cyclochlorotine

Citreoviridin

6-P-gluconolactone

P-enolpyruvate Pyruvate

NADP+

LactateAlanine and otherglucogenic aminoacidsOchratoxin A

Oxaloacetate

Malate

Malate

P-enolpyruvatecarboxykinase

Cytoplasm

MoniliforminPyruvateMonilifrmin

Moniliformin

Patulin

Propionate Succinate

Odd-carbonfatty acids

CO2

CO2

Glutamate

TCA-cycle

Oxalo-acetateAspartate

Acetyl-CoA

Citrate

InhibitionActivation

120572-Ketoglut

NADPH + H+

Figure 3 Inhibition ofoxidative phosphorylation by aflatoxins(adopted from Kiessling 1986) [19]

appear to stem from its inhibitory effects of glycogen syn-thetase and transglycosylase enzymes which in turn bringsto a halt glycogen synthesis [19] Besides aflatoxin B

1also

inhibits phosphoglucomutase an enzyme that reversiblycatalyzes the conversion of glucose-6-phosphate into glucose-1-phosphate leading to a decrease in its activity therebypromoting both the accumulation of glucose 6-phosphateand a decrease in glycogen synthesis Consequently excessglucose cannot be stored as glycogen but either accumulatesin blood or is converted to glucose-6-phosphate for synthesisof more metabolic intermediates via the pentose phosphatepathwayOther effects of aflatoxins B

1 G1andM

1include the

inhibition of electron transport system in the mitochondriaspecifically cytochrome oxidase activity (Figure 4) [19 26]

Besides inhibition of the electron transport chain afla-toxins are also good carcinogens Consequently the Inter-national Agency for Research on Cancer (IARC) of theWorld Health Organization in 1987 classified aflatoxins andin 1993 it classified aflatoxin B

1as Group 1 carcinogen

[26] Since then aflatoxins concentration has become oneof the most critical indicators of food and feed toxicityAccordingly several methods have been developed for thedetection and quantification of aflatoxins in agricultural


NADH NADH dehydrogenase quinone c Cytox

I IIIII

ATP ATPATP

LuteoskyrinEmodin

RotenoneAmytal Antimycin A Rubratoxin B

Electron transport

Phosphory-lation

Uncoupling of oxidative phosphorylationInhibition of electron transport

Cyt b1 Cyt c1

ADP + PiADP + Pi ADP + Pi

Aflatoxin B1G1M1

Aflatoxin B1G1M1

H2O

Aflatoxin B1

2H + 12O2

Cyanide CO H2S

Ub1-

Figure 4 Sites of inhibition of the electron transport chain by aflatoxins (adopted from Kiessling 1986) [19]

produce and processed food products [27ndash29] Basing on theprinciples of detection the methods can broadly be groupedinto chromatographic spectroscopic and immunochemicalmethods This review explores the different methods ofaflatoxins detection and quantification in food and feed stuffhighlighting their strengths and weaknesses hence offeringsuggestions on how some of the current drawbacks of themethods can be addressed

2 Aflatoxins Extraction from Food Samples

The detection and quantification of aflatoxins in food sam-ples require an efficient extraction step Aflatoxins are gen-erally soluble in polar protic solvents such as methanolacetone chloroform and acetonitrile Thus the extractionof aflatoxins involves the use of these organic solventssuch as either methanol or acetonitrile or acetone mixedin different proportion with small amounts of water [4647] Several studies exploring the extraction efficiency ofdifferent organic-aqueous solvents have been carried out onthe commonly contaminated matrices [48ndash50] and differentresults have been reported Aflatoxin determination based onimmunoassay technique requires extraction using mixture ofmethanol-water (8 + 2 vv) [48 51] because methanol hasless negative effect on antibodies compared to other organicsolvents such as acetone and acetonitrile

The extraction of aflatoxins is usually followed by acleanup step The common cleanup technique used isimmunoaffinity column (IAC) chromatography [52] This isconsidered the method of choice for the purification andconcentration of aflatoxins [53] before their determinationusing high-performance liquid chromatography (HPLC)Immunoaffinity column chromatography employs the highspecificity and reversibility of binding between an antibodyand antigen to separate and purify target analytes from

matrices [54] During sample cleanup the crude sampleextract is applied to the immunoaffinity column containingspecific antibodies to aflatoxin immobilized on a solid sup-port such as agarose or silica As the crude sample movesdown the column the aflatoxin binds to the antibody and isretained onto the column Another washing step is normallyrequired to remove impurities and unbound proteins Thisis achieved by using appropriate buffers and ionic strengthsThereafter the aflatoxin is recovered by using such solvents asacetonitrile which breaks the bond between the antibody andthe aflatoxin Table 1 provides a summary of the current ana-lytical methods used in aflatoxin determination A detailedaccount of each method is presented in sections that followbelow

3 Methods for Detection andQuantification of Aflatoxins

31 ChromatographicMethods Chromatographic techniquesare based on the physical interaction between a mobile phaseand a stationary phase The components to be separatedare distributed between the two phases (stationary phaseand mobile phase) [55] The mobile phase is usually a fluidthat penetrates through or along the stationary bed (liquidor solid) Liquid gas and supercritical fluids are currentlyused asmobile phase and chromatographic techniques derivetheir names from the nature of the mobile phase liquidchromatography gas chromatography and supercritical fluidchromatography respectively (Table 1) In practice the sam-ple to be analyzed is dissolved in the mobile phase andapplied as a spot on the stationary phase The analyte orsample is carried along by the mobile phase and partitionsbetween the solid and liquid stationary phase are called thesorbent The various constituents in the analytes travel at


Table 1 Comparison of different methods of aflatoxin analysis

Method Need for a labelNeed for

prior samplepreparation

LOD Multipleanalysis

Need forskilledoperator

Field usage Reference

TLC densitometer SPE 1ndash20 ngKg Yes Yes No [30 31]

HPTLC Extractiononly Pictogram yes yes No [32]

HPLC IAC or SPE Yes Yes No [33]

LC-MSMS Extractiononly 08 120583gKg Yes Yes No [34]

Fluorometer IAC 5ndash5000 120583gKg Yes Yes No [35]FTIR lt10120583gKg Yes Yes No [36]

RIA Radio isotope Extractiononly 1 120583gKg Yes Yes No [37]

ELISA Enzymes Extractiononly Yes Yes No [38]

Immunodipstick Colloidal gold Extractiononly 5120583gKg Yes Yes Yes [39]

QCMs Extractiononly 001ndash10 ngmL Yes Yes No [40 41]

SPR Extractiononly 30ndash98 ngmL Yes Yes No [42]

OLWS Extractiononly 05ndash10 ngmL Yes Yes No [43]

Electrochemical Extractiononly 2 120583gKg Yes Yes No [44]

Electrochemical Extractiononly 1 femtomolar Yes Yes No [45]

different speeds resulting in differential partitioning of theconstituents between the mobile and the stationary phasesThe most commonly used chromatography techniques foranalysis of aflatoxins are thin-layer chromatography (TLC)high-performance liquid chromatography (HPLC) and gaschromatography (GC) Although many of the chromato-graphic techniques are very sensitive they require trainedskilled technician cumbersome pretreatment of sample andexpensive apparatusequipment [56]

311 Thin-Layer Chromatography (TLC) Thin-layer chro-matographywas first used by de Iongh et al [57] and has beenregarded by the Association of Official Analytical Chemist(AOAC) as the method of choice since 1990Thin-layer chro-matography is one of the most widely used separation tech-niques in aflatoxins analysis It consists of a stationary phasemade of either silica or alumina or cellulose immobilized onan inertmaterial such as glass or plastic called thematrixThemobile phase is comprised of methanol acetonitrile watermixture [58] which carries the sample along as it movesthrough the solid stationary phase In TLC the distribution ofaflatoxins between the mobile and stationary phases is basedprimarily on differences in solubility of the analytes in thetwo phases Different analytes depending on their molecularstructures and interaction with the stationary and mobilephases either adhere to the stationary phase more or remain

in the mobile phase thereby allowing for quick and effectiveseparationThin-layer chromatography has been widely usedin the determination of aflatoxins in different foods [59ndash61] and as low as 1ndash20 ppb of aflatoxins has been reported[62] The advantage of using the TLC method is that it candetect several types of mycotoxins in single test sample [3062] Whereas TLC has excellent sensitivities it also requiresskilled technician pretreatment of sample and expensiveequipment [8 63] In addition TLC lacks precision due toaccumulated errors during sample application plate devel-opment and plate interpretation Attempts to improve TLChave led to the development of automated formof TLC calledthe high-performance thin-layer chromatography (HPTLC)The HPTLC has since overcome the problems associatedwith the conventional TLC techniques through automationof sample application development and plate interpretationIt is not surprising that currently HPTLC is one of the mostefficient and precise methods in aflatoxins analysis [64 65]Nevertheless the requirement for skilled operators the costsof the equipment coupledwith its bulkiness and the extensivesample pretreatment limit the HPTLC to the laboratory andthus it is inapplicable in field situations

312 High-Performance Liquid Chromatography (HPLC)High-performance liquid chromatography (HPLC) is themost popular chromatographic technique for separation


and determination of organic compounds About 80 oforganic compounds in the world are determined usingHPLC[66] The HPLC technique makes use of a stationary phaseconfined to either a glass or a plastic tube and a mobile phasecomprising aqueousorganic solvents which flow throughthe solid adsorbent When the sample to be analyzed islayered on top of the column it flows through and distributesbetween both the mobile and the stationary phases Thisis achieved because the components in the samples to beseparated have different affinities for the two phases andthus move through the column at different rates The liquid(mobile) phase emerging from the column yields separatefractions containing individual components in the sampleIn practice the HPLC technique employs a stationary phasesuch as C-18 chromatography column a pump thatmoves themobile phase(s) through the column a detector that displaysthe retention times of each molecule and mobile phases(Figure 5)

The sample to be analyzed is usually injected into thestationary phase and the analytes are carried along throughthe stationary phase by the mobile phase using high pressuredelivered by a pump The analytes are distributed differentlywithin the stationary phase [67] through chemical as well asphysical interactions with the stationary and mobile phases[33]The time at which a specific analyte elutes is recorded bya detector as its retention timeThe retention time depends onthe nature of the analyte and composition of both stationaryand mobile phases [68] Programmable detectors such aseither the fluorescent detector (FLD) or the ultraviolet (UV)detector or the diode array detector (DAD) may be used inthe detection and identification of aflatoxins High pressureliquid chromatography methods used for the determinationof aflatoxins in foods include the normal-phase and reversed-phase high pressure liquid chromatography techniques [33]The reversed phase HPLC method is the most widely usedfor separation and determination of aflatoxins Occasionallychemical derivatization of aflatoxins B

1and G

1may be

required to enhance sensitivity of HPLC during analysis sincethe natural fluorescence of aflatoxins B

1and G

1may not be

high enough to reach the required detection limit [69] Thederivatization reactions of aflatoxin B

1with both the acid and

halogens are presented in Figure 6 Whilein the first reactionstep the second furan ring of aflatoxin B

1is hydrolyzed by

trifluoroacetic acid (TFA) into a highly fluorescent aflatoxinB2a in the second and the third derivatization reaction steps

bromine and iodine are used as reagent respectively Theyreact with aflatoxin B

1to form highly fluorescent aflatoxin B

1

derivatives of these halogens respectivelyPapadopoulou-Bouraoui et al [20] compared two post-

column derivatization methods for the determination ofaflatoxins B

1 B2 G1 and G

2by fluorescence detection after

liquid chromatographic separation (Figure 7) The resultsshowed that both bromination and irradiation by UV lightwere suitable for the determination of aflatoxins in variousfoods and animal feed matrices and both generated compa-rable results for fluorescence amplification and repeatabilityThe fluorescence of aflatoxins B

1and G

1was significantly

enhanced after derivatization reaction either by brominationor by irradiation by UV light

High-performance liquid chromatography provides fastand accurate aflatoxins detection results within a shorttime A sensitivity of detection as low as 01 ngKg usingFLD has been reported [70] However the disadvantage ofusing HPLC for aflatoxins analysis is the requirement ofrigorous sample purification using immunoaffinity columnsIn addition HPLC requires tedious pre- and postcolumnderivatization processes to improve the detection limits ofaflatoxins B

1and G

1[66] Therefore to overcome the chal-

lenges associated with derivatization processes in aflatoxinsanalysis a modification of the HPLC method whereby theHPLC is coupled to mass spectroscopy has been made andis currently employed in the determination of aflatoxin [71]Since the mass spectrometer requires neither use of UVfluorescence nor the absorbance of an analyte the need forchemical derivatization of compounds is eliminated TheHPLC-MSMS uses small amounts of sample to generatestructural information and exhibits low detection limits[33] However HPLC-MSMS is bulky and very expensiveequipment which can only be operated by trained and skilledpersonnel Besides this also limits its use to only laboratoryenvironment and not field conditions

313 Gas Chromatography (GC) In gas chromatographythe mobile phase is a carrier gas and the stationary phaseis a liquid coated onto inert solid particles As with otherchromatographic methods sample analysis by GC is basedprimarily on differential partitioning of analytes between thetwo phases The stationary phase consists of inert particlescoated with a layer of liquid and is normally confined to along stainless steel or glass tube called the column whichis maintained at appropriate temperature The sample tobe analyzed is vaporized into gaseous phase and carriedthrough the stationary phase by a carrier gas The differentchemical constituents in the samplewill distribute themselvesbetween the mobile phase and the stationary phase Thecomponents of the samples mixture with higher affinity forthe stationary phase are retarded in their movement throughthe column while those of low affinity pass through thecolumn less impeded For that matter each component ofthe analyte should have a specific partition coefficient whichin turn will govern its rate of passage through the column[72] Once separation has been achieved the detection ofthe volatile products is carried out using either a flameionization detector (FID) or an electron capture detector(ECD) and mass spectrometer (MS) [73] Owing to theirnonvolatility in nature aflatoxins may need derivatizationin order to be detected [74] Gas chromatography howeveris less common in commercial analysis of aflatoxins dueto the existence of other cheaper chromatographic methods[75] Besides gas chromatography also requires a preliminarycleanup step before analysis and it is therefore limited toanalysis of a few mycotoxins such as A-trichothecenes andB-trichothecenes Even in such analyses the GC has suchdisadvantages as nonlinearity of calibration curves driftingresponses memory effects from previous samples and highvariation in reproducibility and repeatability [76]


Wat

er

Met

hano

l

Acet

onitr

ile

Mobile phase reservoir

Pump Injector for samples Column

Detector

Data display

Figure 5 Schematic diagram of components of high-performance liquid chromatography

Br

Br

I

I

TFA

OCH3

OCH3

OCH3

OCH3

Br2

I2Aflatoxin B1

TFA derived aflatoxin B1

Br2 derived aflatoxin B1

I2 derived aflatoxin B1

30min 50∘C

4 s 20∘C

40 s 60∘C

O

1

OO

O

O O

O

O

O O

O

O O

O

O

OHO

O

O

OO

3

2

Figure 6 Derivatization of aflatoxin B1with trifluoroacetic acid bromine and iodine [6]

32 Spectroscopic Methods

321 Fluorescence Spectrophotometry Absorption in theultraviolet-visible region is very important procedure forunraveling the molecular structures of materials Howeverfor some molecules the process of absorption is followedby emission of light of different wavelength In other wordssuch molecules are said to fluoresce Fluorescence is veryimportant in the characterization and analysis of moleculesthat emit energy at specific wavelengths and has been usedto analyze aflatoxins in grains and raw peanut [77] The fluo-rometric method can quantify aflatoxin from 5 to 5000 ppbwithin less than 5 minutes However for better analysis ofaflatoxins using fluorometry derivatization may be required

to improve on the fluorescence of aflatoxins The limit ofdetection is also slightly higher than the limit of 4 120583gKg setfor European settings and thusmay not be good for analyzingsamples from products to be exported to Europe

322 Frontier Infrared Spectroscopy Another spectroscopicmethod useful in aflatoxin analysis is infrared spectroscopy(IR) Infrared spectroscopy relies on the alteration in molec-ular vibrations upon irradiation with infrared radiationsThe vibrations by the bonds within the molecule can bemeasured Since the atomic size bond length and bondstrength vary greatly from molecule to molecule the rateat which a particular bond absorbs infrared radiation will


00

10

20

30

40

50

60

70

5 10 15Time (min)

(mV

)

AFG2

AFG1

AFB2

AFB1

(a)

(mV

)

010203040506070

0 5 10 15Time (min)

AFG2

AFG1

AFB2

AFB1

(b)

(mV

)

0 5 10 15Time (min)

0

10

20

30

40

50

60

70

AFG2

AFG1

AFB2

AFB1

(c)

Figure 7 Chromatograms obtained for amixed aflatoxin standard (AFB1andAFG

1 each at 20 ngmL andAFB

2andAFG

2 each at 4 ngmL)

by using (a) no derivatization (b) PCDUV2 and (c) PCDEC (adopted from Papadopoulou-Bouraoui et al 2002) [20]

differ from bond to bond and in the mode of vibrationFor instance the various bonds of organic molecules shouldvibrate at different frequencies in tandem with the type ofbond excited So when an infrared spectrometer is used inthe analysis of a compound infrared radiations covering arange of different frequencies are passed through the sampleand the radiant energy absorbed by each type of bonds inthe molecules is measured A spectrum is then producednormally consisting of plot of transmittance against thewave number No two organic compounds have the sameinfrared spectrum and thus individual pure compounds canbe identified by examination of their infrared spectraThe useof Fourier transform infrared spectroscopy which employsattenuated total internal reflectance has been reported foranalysis of aflatoxins in peanuts and peanut cake byMirghaniet al [78] Pearson et al [36] also used transmittance andreflectance spectroscopy to detect aflatoxin in single cornkernelsMore than 95 of the kernels analysedwere correctlycategorized as having either high (gt100 ppb) or low (lt10 ppb)concentrations of aflatoxins

33 Immunochemical Methods Immunochemical techni-ques rely on the specificity of binding between antibodiesand antigens The high affinity and specificity of antibodiesfor antigens have been used in the development of thevarious immunochemical methods Moreover specific bind-ing is not limited to antigens and antibodies but rather

receptors and ligands too exhibit such affinity as well ashigh specificity [79] The formation of either the antibody-antigen or the receptor-ligand complexes can be quantifiedby following the change in the absorbance of photons oflight energy spectrophotometrically In other instances thebinding events as well as the resultant complexes mayrequire amplification for better signal recognition This hasbeen achieved by using various labels comprising enzymesfluorophores and radioisotopes among others Owing tothese advances immunochemistry techniques applicable ina wide array of fieldsdisciplines including such analysesin the foods and drugs industry have been developed Thepopularity of the antibody-antigen based techniques sincetheir development by the late 1970s [77] is due to their highlevel of specificity and sensitivity even in the presence ofcontaminatingmaterials Besides immunochemicalmethodsdo not require skilled and highly trained personnel totroubleshoot in case of any problems during separationthey are less labour intensive and consume less time inwhich respects they are preferable to both chromatographicand spectrophotometric techniques The major immuno-chemical methods used in aflatoxins analysis compriseradioimmunoassay (RIA) enzyme-linked immunosorbentassay (ELISA) immunoaffinity column assay (ICA) andimmunosensors Detailed discussions of the principles aswellas the practical applications of these methods are presentedunder the proceeding subheadings


Control line

Sample well Test line

(a)

Control lineTest line

Absorbent pad

Nitrocellulose membrane

Sample pad

Release pad

(b)

Figure 8 Schematic of a lateral flow device in the dipstick format (a) external details and (b) internal details

331 Radioimmunoassay (RIA) The radioimmunoassaytechnique relies on the principle of competitive bindingbetween a radioactive-labeled antigen and a nonradioactiveantigen The radioactive-labeled antigen competes withunlabelled nonradioactive antigen for a fixed number ofantibody or antigen binding sites on the same antibody [80]A known quantity of labeled antigen and unknown amountof unlabeled antigen from standards competitively react witha known and limiting amount of the antibody The amountsof labeled antigen are inversely proportional to the amountof unlabeled antigen in the sample [81] Radioimmunoassaywas the first immunoassay technique to be developed andwas applied in the detection of insulin in human blood[82] Radioimmunoassay has also been used for analysisof aflatoxins in food samples Langone and van Vunakis[83] reported the use of solid phase radioimmunoassaytechnique in the determination of aflatoxin B

1in peanut

and a detection limit of 1 120583gkg was achieved Similarlyradioimmunoassays have been used for the qualitativeand quantitative determination of aflatoxin B

1levels

[37 84] and aflatoxin M1levels [82] The major advantage

of radioimmunoassay is the ability to perform multipleanalyses simultaneously with high levels of sensitivity andspecificity [85] However RIAs also suffer from a number ofdisadvantages (a) it requires an antigen in a pure state (b) aradioactive isotope is used as a label and is associated withpotential health hazards and (c) it has problems associatedwith the storage and disposing of the low-level radioactivewaste [86] These disadvantages have limited the frequentuse of RIA in the day to day analysis of aflatoxins

332 Enzyme-Linked Immunosorbent Assay (ELISA) Thepotential health hazards related to the use of radioimmunoas-say led to the lookout for a safer alternative and a suitablealternative to radioimmunoassay was to replace a radioactivesignal with a nonradioactive one This was achieved bylabeling either the antigens or the antibodies with enzymesinstead of isotopes The preparation of enzyme-antigen con-jugates and enzyme-antibody conjugates byAvrameas in 1969[87] paved the way for the enzyme-linked immunosorbentassay (ELISA) development Enzyme immunoassay (EIA)and typically the ELISA have become the methods of choicefor medical diagnostic laboratories research institutions and

regulatory bodies for quality assessment and proficiency-testing among others The principle of enzyme immunoas-says is essentially the same as other immunochemical meth-ods that is it relies on the specificity of antibodies forantigens and the sensitivity of the assay is increased bylabeling either the antibodies or the antigens with an enzymethat can be easily assayed by use of specific substrates Hencean antibody immobilized onto a solid support may capturean unlabeled antigen in the analyte which is subsequentlydetected by a labeled antibody [86]The EIAELISA principlehas generated a whole series of test formats [88] For instancecompetitive enzyme immunoassays format not only is simpleto perform but provides a useful measure of either antigen orantibody concentration and is also highly sensitive

The ELISA technique is currently used in the detectionof aflatoxins in agricultural products [89ndash93] and a numberof commercially available ELISA kits based on a competitiveimmunoassay format are widely used [8 94 95] Most ofthe kits use horseradish peroxidase (HRP) and alkalinephosphatase (AP) enzymes as labels in analysis of aflatoxins[96 97] The ELISA method offers a number of advantages(a) it is possible to perform the test on a 96-well assayplatform which means that large number of samples can beanalysed simultaneously [95] (b) ELISA kits are cheap andeasy to use and do not require extensive sample cleanupand (c) there are no inherent health hazards associated withenzyme labels as there are for isotopes However the ELISAtechnique requires multiple washing steps which may attimes prove not only laborious but also time consuming

333 Lateral Flow Devices (Immunodipsticks) Immunodip-sticks are immunochromatographic assays also known aslateral flow devices The principle is based on the use of highsensitivity and specificity of antibody-antigen reactions forthe rapid detection of analytes Lateral flow devices containa porous membrane which ensures the flow an absorbentpad that increases the volume of the flowing liquid a samplepad that ensures contact between the liquid sample andthe membrane and a rigid backing that gives support tothe device (Figure 8) Lateral flow devices use labels suchas colloidal gold and gold coated with the antibody whichcommonly provide red-colored binding zones [95] Theliquid sample added to the sample pad moves towards theextreme end through the membrane by capillary flow to the


Thin metal filmFlow cell

To the waste

Antibody Antigen

Laser

Polarized light

Photodetector

Prism

From the pump

120579

Figure 9 Surface plasmon resonance spectroscopy commonly used for the detection of antigen-antibody interactions in a buffered sample

absorbent pad [26] When the liquid component containingaflatoxins reaches the gold particles the sample suspends thegold particles and the aflatoxins bind to the particles therebycoloring the line red

Delmulle et al [38] developed a lateral flow device fordetecting aflatoxin B

1in pig feed The device would detect

5 120583gKg aflatoxinwithin 10minwhich iswithin the EuropeanCommission (EC) stringent limit fixed for feedstuffs Anotherimmunochromatographic method was developed by Ho andWauchope [98] The assay is based on competition betweenfree AFB

1and AFB

1-tagged dye-containing liposomes for

the corresponding antibody The device can detect 18 ng ofthe aflatoxin in less than 12 minutes Whereas the devicewas designed for direct qualitative visual reading it has alsobeen adapted for use in the optical density scanning modewhich allows for quantitative determination of aflatoxinsLateral flow devices are easy to use and provide quick on-site detection of aflatoxins within few minutes They arecost-effective devices that can be adapted for day to daymonitoring of aflatoxins

334 Immunosensors An immunosensor is a biosensor thatuses an antigen or antibody species as biological recognitioncomponents coupled to a signal transducer such as graphitegold and carbon that help to detect the binding of thecomplementary species [99 100] With respect to type ofsignal transduction in use immunosensors may be groupedinto piezoelectric optical and electrochemical sensors [39]

(1) Piezoelectric Quartz Crystal Microbalances (QCMs) arelabel-free devices used for direct detection of antigens Thepiezoelectric quartz crystal relies on changes in mass on theelectrode surface when an antigen interacts with a cognateantibody immobilized on the quartz crystal surface Sincethe change in mass is proportional to the concentration ofthe antigen-antibody complex the method permits detectionand quantification of the immune complex (Ab-Ag) [101]Piezoelectric quartz crystal microbalance has been reportedfor aflatoxin B

1analysis During development Spinella et al

coated both sides of the QCM sensor with gold electrodesliquid side was in contact with the solution while the contactside of the crystal was dry Piezoelectric immunosensorwas tested for aflatoxin B

1detection by immobilization

of DSP-anti-AFLAB1antibody on gold-coated quartz crys-

tals (AT-cut5MHz) The 331015840-dithiodipropionic-acid-di-N-hydroxysuccinimide ester (DSP) was used for the covalentbinding of the proteins The sensor was capable of detectingaflatoxin B

1concentration in the range of 05ndash10 ppb [102] Jin

et al [103] also developed quartz crystal microbalance basedsensor for detection of aflatoxin B

1and their device could

detect aflatoxin B1in artificially contaminated milk samples

at a concentration range of 001ndash100 ngmL Quartz crystalmicrobalance is a very good label-free technology althoughits use for direct detection of mycotoxins may be a challengedue to the small sizes of most mycotoxins

(2) Optical ImmunosensorsAnumber of optical immunosen-sors have been developed for aflatoxins based on differenttransduction approaches One of these optical immunosen-sors already developed for aflatoxin analysis is surface plas-mon resonance (SPR) (Figure 9) Surface plasmon resonance(SPR) platform relies on measurement of changes in refrac-tive index produced by the binding of analyte to its biospecificpartner immobilized on the sensor surfaceWhen the analyteis flowed over the sensor surface there is a shift in resonantSPR wavelength which is proportional to the refractivechange at the sensor surface and can be calibrated to thesurface concentration of bound analyte [104]The SPR sensorsurface contains a biorecognition layer that selectively bindseither an antigen or antibody which in turn causes parallelincrease in themass on the sensor surface that is proportionalto an increase in refractive index The increase in refractiveindex will be observed as a shift in the resonance angle Themeasurable changes in concentration are those due to bindingand dissociation of antibody to its target antigen [105]

The operationalization of SPR immunosensor technologyfor aflatoxin B

1detection and quantification has already

been attempted by using both monoclonal and polyclonal


antibodies to aflatoxin B1[106] The SPR immunosensor

immobilized with monoclonal antibodies however encoun-tered regeneration problems at the sensor surface due to thehigh-affinity binding of the monoclonal antibodies Yet whenpolyclonal anti-aflatoxin B

1antibodies were immobilized

onto the sensor surface regeneration was achieved usingsolution of 1M ethanolamine with 20 (vv) acetonitrile pH120 Besides the sensor achieved a linear detection range of30ndash980 ngmL with good reproducibility Van der Gaag etal [40] have also used the SPR immunosensor for multipledetection of mycotoxins Therefore SPR immunosensorsshould offer label-free detection of aflatoxins if the currentregeneration problems are overcome

Another form of a label-free biosensor operates on anoptical waveguide platform The optical waveguide platformrelies on the evanescent fluorescence excitation to measurebinding events at the surface of the waveguide [41 107]and one such technique is the optical waveguide light-modespectroscopy (OWLS) Typically the OWLS technique isbased on the precise measurement of the resonance angle ofa polarized laser light diffracted by a grating and incoupledinto a thin waveguide Such incoupling resonance occursat very precise angles depending on the optical parametersof the sensor chips and the complex refractive index ofthe covering sample medium [108] The intensity of theincoupled light guidedwithin thewaveguide layer bymultipleinternal reflections is detected by photodiodes When usedto quantify the adsorption of proteins to waveguide surfacesthe platform is coated with a thin layer of materials thathave higher refractive index [42] Suchmaterials would allowfor polarized light to exit the original waveguide undergototal internal reflection (TIR) at the coating-liquid interfaceand eventually reenter the waveguide However some lighttraverses the waveguide and this is done at a precise anglethat reflects both the properties of the coating layer andsorptiondesorption events at the layer-liquid interface [42]Thus the measurement of the resonance angle of polarizedgrating diffracted light coupled into a thin waveguide canbe used to study the adsorption of macromolecules onto thesurface of sensor chips [105 109] Adanyi et al [43] have usedOWLS to detect aflatoxin and ochratoxin in both competitiveand direct immunoassays The detection range of 05 and10 ngmL of aflatoxins was achieved when barley and wheatflour samples were analysed

(3) Electrochemical Immunosensors An electrochemicalimmunosensor is a device that uses antibodies incorporatedinto a biorecognition layer to produce electroactive signalsdetectable by transducers (amplifiers) which generatemeasurable signals The signal is generated in the form of amembrane potential when ions bind to a sensing membraneThe potential difference is then measured A logarithmicrelationship exists between the potential difference (pd)and concentration [99] The signal measurement can bein the form of differential pulse voltammetry cyclic volta-mmetry chronoamperometry electrochemical impedancespectroscopy or linear sweep voltammetry [110] A numberof electrochemical immunosensors have been reported to

be used in aflatoxins analysis [44 45 111 112] and most ofthem involve immobilization of antibodies onto the surfaceof an electrode Although majority of the electrochemicalimmunosensors [111 112] developed for aflatoxins analysis useenzymes as active biological component to generate signalsMasoomi et al [44] developed a nonenzymatic sandwichform of an electrochemical immunosensor The sensor inthe nonenzymatic sandwich type was developed throughmodification of glassy carbon electrodes using chitosangold nanoparticle anti-aflatoxin B

1 and iron III oxide

(Fe3O4) magnetic core with a gold shell functionalized with

3-((2-mercaptoethylimino)methyl) benzene-12-diol andlabeled with AFB

1 This immunosensor achieved aflatoxin

B1detection range of 06ndash110 ngmL and a detection limit of

02 ngmL Another form of nonenzymatic electrochemicalimmunosensor was developed by Linting et al [45] Thistype of immunosensor was developed by electrodepositing ofgraphene oxide and gold nanoparticles respectively on thesurface of gold electrode Aflatoxin B

1antibody immobilized

on the conducting polymer film and ionic liquid and chitosansolution dropped onto this electrode This immunosensorattained a dynamic range of 32ndash032 picomoles and detectionlimit of one femtomole with excellent long-term stability

4 Conclusions

Various analytical methods employed in analysis of aflatoxinsin agricultural food crops and feeds have been exploredWhile chromatographic methods such as TLC and HPLC areconsidered the gold standard and are thus the most widelyused techniques in aflatoxins analysis they remain largelycumbersome requiring extensive sample preparations letalone very expensive equipmentThismakes their routine usein analysis confined to laboratories It is on the account ofsuch limitations that it was necessary to develop more sensi-tive and better techniques for aflatoxins analyses Analyticalmethods based on spectroscopy and immunochemistry havebeen added to the earlier chromatographicmethods of whichimmunoassays emerged as better alternatives for routine andon-site detection of aflatoxins Improvement in analyticalchemistry and recent advances in immunochemistry have ledto more specific sensitive simple and rapid immunoassayswhich have become the method of choice for on-site androutine analysis of mycotoxins in foods and feeds It worthnoting that although many sensitive methods have beendescribed for analysis of aflatoxins based on immunochem-ical format most of them require labeling as well as skilledand well trained operators Therefore the search for simplelabel-free and more rapid and sensitive tools that are basedon immune-biosensor format appears to offer for the nearfuture versatile portable sensitive and accurate field usedevices for aflatoxin detection

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper


Acknowledgments

The authors thank the Canadian International DevelopmentResearch Council (IDRC) and Uganda Industrial ResearchInstitute for financially supporting this studywhich led to thisreview and EngineerOchengMathew of the InstrumentationUnit Technical Development Centre Uganda IndustrialResearch Institute for his technical support

References

[1] J W Bennett andM Klich ldquoMycotoxinsrdquo Clinical MicrobiologyReviews vol 16 no 3 pp 497ndash516 2003

[2] G C Dors S Caldas V Feddern et al ldquoAflatoxins contam-ination analysis and controlrdquo in Aflatoxins-Biochemistry andMolecular Biology pp 415ndash438 InTech Shanghai China 2011

[3] H Gourama and L B Bullerman ldquoDetection of molds in foodsand feeds potential rapid and selective methodsrdquo Journal ofFood Protection vol 58 no 12 pp 1389ndash1394 1995

[4] H S Hussein and J M Brasel ldquoToxicity metabolism andimpact of mycotoxins on humans and animalsrdquo Toxicology vol167 no 2 pp 101ndash134 2001

[5] J M Cullen P M Newberne D Eaton and J GroopmanldquoAcute hepatotoxicity of aflatoxinsrdquo in The Toxicology of Afla-toxins Human Health Veterinary and Agricultural Significancepp 3ndash26 1993

[6] W T Kok ldquoDerivatization reactions for the determination ofaflatoxins by liquid chromatography with fluorescence detec-tionrdquo Journal of Chromatography B Biomedical Sciences andApplications vol 659 no 1-2 pp 127ndash137 1994

[7] M Ayub and D Sachan ldquoDietary factors affecting aflatoxin bicarcinogenicityrdquoMalaysian Journal of Nutrition vol 3 pp 161ndash197 1997

[8] J Stroka and E Anklam ldquoNew strategies for the screeningand determination of aflatoxins and the detection of aflatoxin-producing moulds in food and feedrdquo TrACmdashTrends in Analyti-cal Chemistry vol 21 no 2 pp 90ndash95 2002

[9] C P Wild and P C Turner ldquoThe toxicology of aflatoxins as abasis for public health decisionsrdquoMutagenesis vol 17 no 6 pp471ndash481 2002

[10] E PGallagher K L Kunze P L Stapleton andD L Eaton ldquoThekinetics of aflatoxin B

1oxidation by human cDNA-expressed

and human liver microsomal cytochromes P450 1A2 and 3A4rdquoToxicology and Applied Pharmacology vol 141 no 2 pp 595ndash606 1996

[11] M Vondracek Z Xi P Larsson et al ldquoCytochrome P450expression and related metabolism in human buccal mucosardquoCarcinogenesis vol 22 no 3 pp 481ndash488 2001

[12] F P Guengerich W W Johnson T Shimada Y-F Ueng HYamazaki and S Langouet ldquoActivation and detoxication ofaflatoxin B

1rdquo Mutation ResearchFundamental and Molecular

Mechanisms of Mutagenesis vol 402 no 1-2 pp 121ndash128 1998[13] G S Bbosa D Kitya J Odda and J Ogwal-Okeng ldquoAflatoxins

metabolism effects on epigenetic mechanisms and their role incarcinogenesisrdquo Health vol 5 no 10 pp 14ndash34 2013

[14] W W Johnson H Yamazaki T Shimada Y-F Ueng and F PGuengerich ldquoAflatoxin B1 89-epoxide hydrolysis in the pres-ence of rat and human epoxide hydrolaserdquo Chemical Researchin Toxicology vol 10 no 6 pp 672ndash676 1997

[15] W W Johnson T M Harris and F P Guengerich ldquoKineticsand mechanism of hydrolysis of aflatoxin B1 exo-89-epoxide

and rearrangement of the dihydrodiolrdquo Journal of the AmericanChemical Society vol 118 no 35 pp 8213ndash8220 1996

[16] J D Hayes D J Judah and G E Neal ldquoResistance toaflatoxin B1 is associated with the expression of a novel aldo-keto reductase which has catalytic activity towards a cytotoxicaldehyde-containing metabolite of the toxinrdquo Cancer Researchvol 53 no 17 pp 3887ndash3894 1993

[17] L P Knight T Primiano J D Groopman T W Kenslerand T R Sutter ldquocDNA cloning expression and activity of asecond human aflatoxin B

1-metabolizing member of the aldo-

keto reductase superfamily AKR7A3rdquo Carcinogenesis vol 20no 7 pp 1215ndash1223 1999

[18] A Y Nassar S E Megalla H M Abd El-Fattah A H Hafezand T S El-Deap ldquoBinding of aflatoxin b1 g1 and M to plasmaalbuminrdquoMycopathologia vol 79 no 1 pp 35ndash38 1982

[19] K-H Kiessling ldquoBiochemical mechanism of action of myco-toxinsrdquo Pure and Applied Chemistry vol 58 pp 327ndash338 1986

[20] A Papadopoulou-Bouraoui J Stroka and E Anklam ldquoCom-parison of two post-column derivatization systems ultravioletirradiation and electrochemical determination for the liquidchromatographic determination of aflatoxins in foodrdquo Journalof AOAC International vol 85 no 2 pp 411ndash416 2002

[21] C de Oliveira and P Germano ldquoAflatoxins current concepts onmechanisms of toxicity and their involvement in the etiology ofhepatocellular carcinomardquo Revista de Saude Publica vol 31 no4 pp 417ndash424 1997

[22] D D Levy J D Groopman S E Lim M M Seidman andK H Kraemer ldquoSequence specificity of aflatoxin B1-inducedmutations in a plasmid replicated in xeroderma pigmentosumand DNA repair proficient human cellsrdquo Cancer Research vol52 no 20 pp 5668ndash5673 1992

[23] F Aguilar S P Hussain and P Cerutti ldquoAflatoxin B1 inducesthe transversion of G rarr T in codon 249 of the p53 tumorsuppressor gene in human hepatocytesrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 90 no 18 pp 8586ndash8590 1993

[24] D L Eaton and E P Gallagher ldquoMechanisms of aflatoxin car-cinogenesisrdquo Annual Review of Pharmacology and Toxicologyvol 34 pp 135ndash172 1994

[25] M McLean and M F Dutton ldquoCellular interactions andmetabolism of aflatoxin an updaterdquo Pharmacology ampTherapeu-tics vol 65 no 2 pp 163ndash192 1995

[26] P Li Q Zhang and W Zhang ldquoImmunoassays for aflatoxinsrdquoTrACmdashTrends in Analytical Chemistry vol 28 no 9 pp 1115ndash1126 2009

[27] M N Pascale ldquoDetection methods for mycotoxins in cerealgrains and cereal productsrdquo Zbornik Matice Srpske za PrirodneNauke vol 117 pp 15ndash25 2009

[28] I Y Goryacheva S de Saeger S A Eremin and C vanPeteghem ldquoImmunochemical methods for rapid mycotoxindetection evolution from single to multiple analyte screeninga reviewrdquo Food Additives and Contaminants vol 24 no 10 pp1169ndash1183 2007

[29] M Z Zheng J L Richard and J Binder ldquoA review of rapidmethods for the analysis of mycotoxinsrdquo Mycopathologia vol161 no 5 pp 261ndash273 2006

[30] I Balzer C Bogdanic and S Pepeljnjak ldquoRapid thin layer chro-matographic method for determining aflatoxin B1 ochratoxinA and zearalenone in cornrdquo Journal of the Association of OfficialAnalytical Chemists vol 61 no 3 pp 584ndash585 1978


[31] D L Park M W Trucksess S Nesheim M Stack and R FNewell ldquoSolvent-efficient thin-layer chromatographic methodfor the determination of aflatoxins B1 B2 G1 and G2 in cornand peanut products collaborative studyrdquo Journal of AOACInternational vol 77 no 3 pp 637ndash646 1994

[32] Y Liang M E Baker B T Yeager and M B DentonldquoQuantitative analysis of aflatoxins by high-performance thin-layer chromatography utilizing a scientifically operated charge-coupled device detectorrdquo Analytical Chemistry vol 68 no 22pp 3885ndash3891 1996

[33] A Rahmani S Jinap and F Soleimany ldquoQualitative andquantitative analysis of mycotoxinsrdquo Comprehensive Reviews inFood Science and Food Safety vol 8 no 3 pp 202ndash251 2009

[34] M Kokkonen M Jestoi and A Rizzo ldquoDetermination ofselected mycotoxins in mould cheeses with liquid chromatog-raphy coupled tandem with mass spectrometryrdquo Food Additivesand Contaminants vol 22 no 5 pp 449ndash456 2005

[35] B R Malone C W Humphrey T R Romer and J L RichardldquoDetermination of aflatoxins in grains and raw peanuts by arapid procedure with fluorometric analysisrdquo Journal of AOACInternational vol 83 no 1 pp 95ndash98 2000

[36] T Pearson D Wicklow E Maghirang F Xie and F DowellldquoDetecting aflatoxin in single corn kernels by transmittance andreflectance spectroscopyrdquo Transactions of the American Societyof Agricultural Engineers vol 44 no 5 pp 1247ndash1254 2001

[37] P Sun and Y Chu ldquoA simple solid-phase radioimmunoassay foraflatoxin b1rdquo Journal of Food Safety vol 1 pp 67ndash75 1979

[38] B S Delmulle S M D G de Saeger L Sibanda I Barna-Vetroand C H van Peteghem ldquoDevelopment of an immunoassay-based lateral flow dipstick for the rapid detection of aflatoxin B1in pig feedrdquo Journal of Agricultural and Food Chemistry vol 53no 9 pp 3364ndash3368 2005

[39] P B Luppa L J Sokoll and D W Chan ldquoImmunosensormdashprinciples and applications to clinical chemistryrdquo Clinica Chim-ica Acta vol 314 no 1-2 pp 1ndash26 2001

[40] B van der Gaag S Spath H Dietrich et al ldquoBiosensors andmultiple mycotoxin analysisrdquo Food Control vol 14 no 4 pp251ndash254 2003

[41] Z Liron L M Tender J P Golden and F S Ligler ldquoVoltage-induced inhibition of antigen-antibody binding at conductingoptical waveguidesrdquo Biosensors and Bioelectronics vol 17 no 6-7 pp 489ndash494 2002

[42] H Yu C M Eggleston J Chen W Wang Q Dai and JTang ldquoOptical waveguide lightmode spectroscopy (OWLS) asa sensor for thin film and quantum dot corrosionrdquo Sensors vol12 no 12 pp 17330ndash17342 2012

[43] N Adanyi I A Levkovets S Rodriguez-Gil A RonaldM Varadi and I Szendro ldquoDevelopment of immunosensorbased on OWLS technique for determining Aflatoxin B1 andOchratoxin Ardquo Biosensors and Bioelectronics vol 22 no 6 pp797ndash802 2007

[44] L Masoomi O Sadeghi M H Banitaba A Shahrjerdi andS S H Davarani ldquoA non-enzymatic nanomagnetic electro-immunosensor for determination of Aflatoxin B

1as a model

antigenrdquo Sensors and Actuators B Chemical vol 177 pp 1122ndash1127 2013

[45] Z Linting L Ruiyi L Zaijun X Qianfang F Yinjun andL Junkang ldquoAn immunosensor for ultrasensitive detection ofaflatoxin B

1with an enhanced electrochemical performance

based on grapheneconducting polymergold nanoparticlestheionic liquid composite film on modified gold electrode with

electrodepositionrdquo Sensors and Actuators B Chemical vol 174pp 359ndash365 2012

[46] T Bertuzzi S Rastelli A Mulazzi and A Pietri ldquoEvaluationand improvement of extraction methods for the analysis ofaflatoxins B

1 B2 G1and G

2from naturally contaminated

maizerdquo Food Analytical Methods vol 5 no 3 pp 512ndash519 2012[47] S L Taylor J W King J L Richard and J I Greer ldquoAnalytical-

scale supercritical fluid extraction of aflatoxin b1 from field-inoculated cornrdquo Journal of Agricultural and Food Chemistryvol 41 no 6 pp 910ndash913 1993

[48] J Stroka M Petz U Joerissen and E Anklam ldquoInvestigationof various extractants for the analysis of aflatoxin B1 in differentfood and feed matricesrdquo Food Additives and Contaminants vol16 no 8 pp 331ndash338 1999

[49] I Arranz E Sizoo H van Egmond et al ldquoDetermination ofaflatoxin B1 in medical herbs interlaboratory studyrdquo Journal ofAOAC International vol 89 no 3 pp 595ndash605 2006

[50] A Gallo F Masoero T Bertuzzi G Piva and A Pietri ldquoEffectof the inclusion of adsorbents on aflatoxin B-1 quantificationin animal feedstuffsrdquo Food Additives and ContaminantsmdashPartA Chemistry Analysis Control Exposure and Risk Assessmentvol 27 no 1 pp 54ndash63 2010

[51] N A Lee S Wang R D Allan and I R Kennedy ldquoA rapidaflatoxin B

1Elisa development and validation with reduced

matrix effects for peanuts corn pistachio and soybeansrdquoJournal of Agricultural and Food Chemistry vol 52 no 10 pp2746ndash2755 2004

[52] F Ma R Chen P Li Q Zhang W Zhang and X HuldquoPreparation of an immunoaffinity column with amino-silicagel microparticles and its application in sample cleanup foraflatoxin detection in agri-productsrdquo Molecules vol 18 no 2pp 2222ndash2235 2013

[53] PM Scott andMWTrucksess ldquoApplication of immunoaffinitycolumns tomycotoxin analysisrdquo Journal of AOAC Internationalvol 80 no 5 pp 941ndash949 1997

[54] W L Shelver G L Larsen and J K Huwe ldquoUse ofan immunoaffinity column for tetrachlorodibenzo-p-dioxinserum sample cleanuprdquo Journal of Chromatography B Biomedi-cal Applications vol 705 no 2 pp 261ndash268 1998

[55] A Braithwaite F J Smith and R Stock ChromatographicMethods Chapman and Hall 1985

[56] K E Sapsford C R Taitt S Fertig et al ldquoIndirect competitiveimmunoassay for detection of aflatoxin B

1in corn andnut prod-

ucts using the array biosensorrdquo Biosensors and Bioelectronicsvol 21 no 12 pp 2298ndash2305 2006

[57] H de Iongh R Vles and P de Vogel ldquoThe occurrence anddetection of aflatoxin in foodrdquo in Proceedings of the Symposiumon Mycotoxins in Foodstuffs G H Wogan Ed p 235 MITPress Cambridge Mass USA 1964

[58] V Betina ldquoThin-layer chromatography of mycotoxinsrdquo Journalof Chromatography vol 334 no 3 pp 211ndash276 1985

[59] H Gulyas ldquoDetermination of aflatoxins b1 b2 g1 g2 andm1 by high pressure thin layer chromatographyrdquo Journal ofChromatography A vol 319 pp 105ndash111 1985

[60] K M Abdel-Gawad and A A Zohri ldquoFungal flora andmycotoxins of six kinds of nut seeds for human consumption inSaudi ArabiardquoMycopathologia vol 124 no 1 pp 55ndash64 1993

[61] Y M H Younis and K M Malik ldquoTlc and hplc analysisof aflatoxin contamination in sudanese peanut and peanutproductsrdquo Kuwait Journal of Science and Engineering vol 30no 1 2003


[62] M TrucksessW Brumley and S Nesheim ldquoRapid quantitationand confirmation of aflatoxins in corn andpeanut butter using adisposable silica gel column thin layer chromatography and gaschromatographymass spectrometyrdquo Journal of the Associationof Official Analytical Chemists vol 67 no 5 pp 973ndash975 1984

[63] E Papp K H-Otta G Zaray and E Mincsovics ldquoLiquidchromatographic determination of aflatoxinsrdquo MicrochemicalJournal vol 73 no 1-2 pp 39ndash46 2002

[64] J Ramesh G Sarathchandra and V Sureshkumar ldquoAnalysis offeed samples for aflatoxin b1 contamination by hptlc-a validatedmethodrdquo International Journal of Current Microbiology andApplied Sciences vol 2 pp 373ndash377 2013

[65] S Nawaz R D Coker and S J Haswell ldquoDevelopment andevaluation of analytical methodology for the determination ofaflatoxins in palm kernelsrdquo Analyst vol 117 no 1 pp 67ndash741992

[66] P Li Q Zhang D Zhang et al ldquoAflatoxin measurement andanalysisrdquo inAflatoxinsmdashDetection Measurement and Control ITorres-Pacheco Ed pp 183ndash208 Intech Shanghai China 2011

[67] R Malviya V Bansal O P Pal and P K Sharma ldquoHighperformance liquid chromatography a short reviewrdquo Journal ofGlobal Pharma Technology vol 2 no 5 pp 22ndash26 2010

[68] Y Xiang Y Liu and M L Lee ldquoUltrahigh pressure liquidchromatography using elevated temperaturerdquo Journal of Chro-matography A vol 1104 no 1-2 pp 198ndash202 2006

[69] W T Kok T C H van Neer W A Traag and L G T TuinstraldquoDetermination of aflatoxins in cattle feed by liquid chromatog-raphy and post-column derivatization with electrochemicallygenerated brominerdquo Journal of Chromatography A vol 367 pp231ndash236 1986

[70] S M Herzallah ldquoDetermination of aflatoxins in eggs milkmeat and meat products using HPLC fluorescent and UVdetectorsrdquo Food Chemistry vol 114 no 3 pp 1141ndash1146 2009

[71] M Takino and T Tanaka Determination of Aflatoxins in Foodby LC-MSMS Agilent Technologies 2008

[72] R F Boyer Modern Experimental Biochemistry BenjaminCu-mmings Publishing Company 1993

[73] M N Pascale ldquoDetection methods for mycotoxins in cerealgrains and cereal productsrdquo Matica Srpska Proceedings forNatural Sciences vol 117 pp 15ndash25 2009

[74] P Scott ldquoMycotoxin methodologyrdquo Food Additives amp Contami-nants vol 12 no 3 pp 395ndash403 1995

[75] Y Liang C Zhang and L Liu ldquoChromatographic analysis ofmycotoxinsrdquo ZhongguoWeisheng Jianyan Zazhi vol 15 p 12732005

[76] H Petterson and W Langseth ldquoIntercomparison of tri-chothecenes analysis and feasibility to produce certified cali-brantsrdquo EU Reports EUR 20285 European Commission BCRInformation Project 2002

[77] D Babu Rapid and Sensitive Detection of Aflatoxin in AnimalFeeds and Food Grains Using Immunomagnetic Bead BasedRecovery and Real-Time Immuno Quantitative PCR (RT-iqPCR)Assay Oklahoma State University 2010

[78] M E S Mirghani Y B C Man S Jinap B S Baharin and JBakar ldquoA new method for determining aflatoxins in ground-nut and groundnut cake using Fourier transform infraredspectroscopy with attenuated total reflectancerdquo Journal of theAmerican Oil Chemistsrsquo Society vol 78 no 10 pp 985ndash9922001

[79] A Sargent and O A Sadik ldquoMonitoring antibody-antigenreactions at conducting polymer-based immunosensors using

impedance spectroscopyrdquo Electrochimica Acta vol 44 no 26pp 4667ndash4675 1999

[80] S A Berson and R S Yalow ldquoGeneral principles of radioim-munoassayrdquo Clinica Chimica Acta vol 22 no 1 pp 51ndash69 1968

[81] D S Skelley L P Brown andPK Besch ldquoRadioimmunoassayrdquoClinical Chemistry vol 19 no 2 pp 146ndash186 1973

[82] P Rauch L Fukal J Prosek P Brezina and J Kas ldquoRadioim-munoassay of aflatoxin M

1rdquo Journal of Radioanalytical and

Nuclear Chemistry vol 117 no 3 pp 163ndash169 1987[83] J J Langone and H van Vunakis ldquoAflatoxin B1 specific

antibodies and their use in radioimmunoassayrdquo Journal of theNational Cancer Institute vol 56 no 3 pp 591ndash595 1976

[84] S Tsuboi T Nakagawa M Tomita et al ldquoDetection ofaflatoxin B1 in serum samples of male Japanese subjects byradioimmunoassay and high-performance liquid chromatogra-phyrdquo Cancer Research vol 44 no 3 pp 1231ndash1234 1984

[85] T C Tseng C Chow and G Waller ldquoImmunoassay inmycotoxin researchrdquo in Phytochemical Ecology AllelochemicalsMycotoxins and Insect Phenomones and Allomones p 363 1989

[86] R Twyman Immunoassays applications 2005[87] S Avrameas ldquoCoupling of enzymes to proteins with glutaralde-

hyde Use of the conjugates for the detection of antigens andantibodiesrdquo Immunochemistry vol 6 no 1 pp 43ndash52 1969

[88] R M Lequin ldquoEnzyme immunoassay (eia)enzyme-linkedimmunosorbent assay (elisa)rdquo Clinical Chemistry vol 51 pp2415ndash2418 2005

[89] V Anjaiah V Mehan S Jayanthi D Reddy and D McDonaldldquoEnzyme-linked immunosorbent assay (elisa) for aflatoxin b1estimation in groundnutsrdquo 1989

[90] K T Devi M Mayo K Reddy et al ldquoProduction and charac-terization of monoclonal antibodies for aflatoxin B1rdquo Letters inApplied Microbiology vol 29 no 5 pp 284ndash288 1999

[91] K Thirumala-Devi M A Mayo A J Hall et al ldquoDevel-opment and application of an indirect competitive enzyme-linked immunoassay for aflatoxin M1 in milk and milk-basedconfectioneryrdquo Journal of Agricultural and Food Chemistry vol50 no 4 pp 933ndash937 2002

[92] O Kawamura S Nagayama S Sato K Ohtani I Uenoand Y Ueno ldquoA monoclonal antibody-based enzyme-linkedimmunosorbent assay of aflatoxin B 1 in peanut productsrdquoMycotoxin Research vol 4 no 2 pp 75ndash88 1988

[93] D Ondieki1 S T Lutta M O Okoth and P KeringldquoRapid assessment of aflatoxin contamination of groundnutsby thin layer chromatography and competitive enzyme linkedimmunosorbent assay from selected divisions of Busia countyin Kenyardquo African Journal of Food Science and Technology vol5 no 1 pp 12ndash20 2014

[94] A Ostadrahimi F Ashrafnejad A Kazemi et al ldquoAflatoxin inraw and salt-roasted nuts (pistachios peanuts andwalnuts) soldin markets of tabriz iranrdquo Jundishapur Journal of Microbiologyvol 7 no 1 Article ID e8674 2014

[95] B Huybrechts Evaluation of Immunoassay Kits for AflatoxinDetermination in Corn amp Rice CODA-CERVA National Ref-erence Laboratory on Mycotoxins 2011

[96] J J Pestka P K Gaur and F S Chu ldquoQuantitation of aflatoxinB1 and aflatoxin B1 antibody by an enzyme-linked immunosor-bent microassayrdquo Applied and Environmental Microbiology vol40 no 6 pp 1027ndash1031 1980

[97] D L Park B M Miller L P Hart et al ldquoEnzyme-linkedimmunosorbent assay for screening aflatoxin B1 in cottonseedproducts and mixed feed collaborative studyrdquo Journal of the


Association of Official Analytical Chemists vol 72 no 2 pp326ndash332 1989

[98] J-A Ho and R Wauchope ldquoA strip liposome immunoassay foraflatoxin B

1rdquo Analytical Chemistry vol 74 no 7 pp 1493ndash1496

2002[99] C L Morgan D J Newman and C P Price ldquoImmunosensors

technology and opportunities in laboratory medicinerdquo ClinicalChemistry vol 42 no 2 pp 193ndash209 1996

[100] F Ricci G Volpe L Micheli and G Palleschi ldquoA review onnovel developments and applications of immunosensors in foodanalysisrdquo Analytica Chimica Acta vol 605 no 2 pp 111ndash1292007

[101] L Zhou X He D He K Wang and D Qin ldquoBiosensing tech-nologies for Mycobacterium tuberculosis detection status andnew developmentsrdquo Clinical and Developmental Immunologyvol 2011 Article ID 193963 8 pages 2011

[102] K Spinella L Mosiello G Palleschi and F Vitali ldquoDevelop-ment of a qcm (quartz crystal microbalance) biosensor to thedetection of aflatoxin b1rdquoOpen Journal of Applied Biosensor vol2 no 4 pp 112ndash119 2013

[103] X Jin X Liu L Chen J Jiang G Shen andR Yu ldquoBiocatalyzeddeposition amplification for detection of aflatoxin B

1based on

quartz crystal microbalancerdquo Analytica Chimica Acta vol 645no 1-2 pp 92ndash97 2009

[104] H Vaisocherova K Mrkvova M Piliarik P Jinoch MSteinbachova and J Homola ldquoSurface plasmon resonancebiosensor for direct detection of antibody against Epstein-Barrvirusrdquo Biosensors and Bioelectronics vol 22 no 6 pp 1020ndash1026 2007

[105] M A Brusatori Y Tie and P R van Tassel ldquoProtein adsorptionkinetics under an applied electric field an optical waveguidelightmode spectroscopy studyrdquo Langmuir vol 19 no 12 pp5089ndash5097 2003

[106] S J Daly G J Keating P P Dillon et al ldquoDevelopment ofsurface plasmon resonance-based immunoassay for aflatoxinB1rdquo Journal of Agricultural and Food Chemistry vol 48 no 11

pp 5097ndash5104 2000[107] C A Rowe-Taitt J W Hazzard K E Hoffman J J Cras

J P Golden and F S Ligler ldquoSimultaneous detection of sixbiohazardous agents using a planar waveguide array biosensorrdquoBiosensors and Bioelectronics vol 15 no 11-12 pp 579ndash5892000

[108] A Szekacs N Adanyi I Szekacs K Majer-Baranyi andI Szendro ldquoOptical waveguide light-mode spectroscopyimmunosensors for environmental monitoringrdquo AppliedOptics vol 48 no 4 pp B151ndashB158 2009

[109] G L Duveneck A P Abel M A Bopp G M Kresbach andM Ehrat ldquoPlanar waveguides for ultra-high sensitivity of theanalysis of nucleic acidsrdquo Analytica Chimica Acta vol 469 no1 pp 49ndash61 2002

[110] A-L Valimaa A T Kivisto P I Leskinen and M T KarpldquoA novel biosensor for the detection of zearalenone familymycotoxins inmilkrdquo Journal ofMicrobiologicalMethods vol 80no 1 pp 44ndash48 2010

[111] JHOwinoOAArotibaNHendricks et al ldquoElectrochemicalimmunosensor based on polythioninegold nanoparticles forthe determination of aflatoxin B

1rdquo Sensors vol 8 no 12 pp

8262ndash8274 2008[112] Y Liu Z Qin X Wu and H Jiang ldquoImmune-biosensor for

aflatoxin B1 based bio-electrocatalytic reaction on micro-combelectroderdquo Biochemical Engineering Journal vol 32 no 3 pp211ndash217 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


Aflatoxin-exo-89-epoxide

Aflatoxin-endo-89-epoxide

Urine

Aflatoxin-mercapturic acid

Peripheral blood

Aflatoxin-glucuronide

Aflatoxin-dialcoholAflatoxin-albumin

Aflatoxin-dihydrodiol

Aflatoxin-dialdehyde

1A23A4 (3A5)

1A2 and 3A4 (3A5) 1A2

DNA

GST

AFARAflatoxin-DNA

O O

O

O O

O

O O

O O OMe

OO

O

O

O O

O

O

O O

OO

O

O

OO O

O

O O

O

O O

O

O

O

N

O

O O

O

O

O

O

OH

HO

OOH

OH

HO

O

O

O

N

NHN

N

O

O OO

O O

OO

O

O O

OOOH

H

H

HO

Aflatoxin B1

Aflatoxin M1

Aflatoxin Q1

Aflatoxin-G-S

Aflatoxin-N7-guanine

OH

OH

HOHO

HO

HO

HO

S

S

Cys

Cys

Gly

Glu

NH CO CH3

H2NdR DNA

Albumin

OMe

OMe OMe

OMe

OMe

OMe

OMe

OMe

OMe

O

O O

O

O O

N

NHN

N

HO

H2N

OMe

OMe

OMe

Figure 1 Principal metabolism of aflatoxin B1leading to reactive metabolites and biomarkers 1A2 CYP1A2 3A4 CYP3A4 3A5 CYP3A5

GST glutathione S-transferase AFAR aflatoxin aldehyde reductase aflatoxin-S-G aflatoxin-glutathione conjugate (adopted fromWild andTurner 2002) [9]

B1-exo-89-epoxide may be converted either to aflatoxin-

mercapturic acid via the GST conjugate mediated routeor into aflatoxin-glucuronide via the aflatoxin-dihydrodiolroute described as follows The activated form of aflatoxinB1(exoepoxides and endoepoxides) is detoxified through

glutathione S-transferase- (GST-) mediated conjugation byusing reduced glutathione (GSH) to form AFB

1exoepoxide-

GSHand endoepoxide-GSH conjugates respectively [14]Thereactive exoepoxides and endoepoxides also undergo rapidnonenzymatic hydrolysis to aflatoxin B

1-89-dihydrodiol that

slowly transforms into a dialdehyde phenolate ion [9 15]Dialdehyde phenolate ion is subsequently hydrolyzed byaflatoxin aldehyde reductase (AFAR) to a dialcohol in theNADPH-dependent reduction reactionThereafter dialcoholis excreted in urine as aflatoxin-glucuronide [16 17] Afla-toxin B

1dialdehydes also form Schiff bases with primary

amine groups of amino acid residues such as lysine ofsuch a protein as albumin to form aflatoxin B

1-albumin

conjugate [9] This conjugate persists in the systemic bloodas permanent and irreversible aflatoxin B

1-albumin adducts

and is thus considered one of the factors accounting for thelow excretion of aflatoxins and their metabolites in urine [18]

12 Toxicity of Aflatoxins From the foregoing (Figure 1)it can be observed that the primary derivatives of afla-toxin B

1biotransformation comprise (a) aflatoxin M1 and

aflatoxin-exo-89-epoxide (products of CYP1A2 activity) and(b) aflatoxin Q

1and aflatoxin-exo-89-epoxide (products of

CYP3A4 activity) Aflatoxins M1and Q

1 although toxic are

less reactive with other molecules and are easily eliminatedfrom the body in the urine [9] However aflatoxin B

1-

89-exo-epoxide is a known mutagen which is extremelyelectrophilic and covalently reacts with nucleophilic sitesof either deoxyribonucleic acid (DNA) or ribonucleic acid(RNA) or proteins [21] thereby introducing mutations thatmay affect the normal function of cells The formation ofaflatoxin B

1-DNA adducts is extremely associated with the

carcinogenicity of aflatoxin B1 Typically aflatoxin B

1reacts

with DNA (methylation) resulting in GrarrT transversion


AA A A

AT T

T

T T

C

C

CC

C

C

GGG

G G

AA A A

AAT T

TT

T T

C

C

CC

C

G

G

G G

1

2SNP










1has also



Aflatoxin B1

Aflatoxin B1


Glucose-1-P

Glucose-6-P

Cyclochlorotine

Cyclochlorotine

Citreoviridin

6-P-gluconolactone


NADP+


Oxaloacetate

Malate

Malate


Cytoplasm


Moniliformin

Patulin



CO2

CO2

Glutamate

TCA-cycle


Acetyl-CoA

Citrate


120572-Ketoglut

NADPH + H+



1also


1 G1andM

1include the







I IIIII

ATP ATPATP

LuteoskyrinEmodin


Electron transport

Phosphory-lation


Cyt b1 Cyt c1


Aflatoxin B1G1M1

Aflatoxin B1G1M1

H2O

Aflatoxin B1

2H + 12O2

Cyanide CO H2S

Ub1-




































1and G

1may be


1and G

1may not be




1is hydrolyzed by




1



1 B2 G1 and G



1and G

1was significantly



1and G





Wat

er

Met

hano

l

Acet

onitr

ile



Detector

Data display


Br

Br

I

I

TFA

OCH3

OCH3

OCH3

OCH3

Br2

I2Aflatoxin B1




30min 50∘C

4 s 20∘C

40 s 60∘C

O

1

OO

O

O O

O

O

O O

O

O O

O

O

OHO

O

O

OO

3

2







00

10

20

30

40

50

60

70

5 10 15Time (min)

(mV

)

AFG2

AFG1

AFB2

AFB1

(a)

(mV

)

010203040506070

0 5 10 15Time (min)

AFG2

AFG1

AFB2

AFB1

(b)

(mV

)

0 5 10 15Time (min)

0

10

20

30

40

50

60

70

AFG2

AFG1

AFB2

AFB1

(c)



2andAFG

2 each at 4 ngmL)






Control line


(a)


Absorbent pad


Sample pad

Release pad

(b)



1in peanut


1levels









To the waste

Antibody Antigen

Laser

Polarized light

Photodetector

Prism

From the pump

120579






1and AFB



































4 Conclusions





Acknowledgments


References





















































1as a model







1 B2 G1and G








































































1based on























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


AA A A

AT T

T

T T

C

C

CC

C

C

GGG

G G

AA A A

AAT T

TT

T T

C

C

CC

C

G

G

G G

1

2SNP










1has also



Aflatoxin B1

Aflatoxin B1


Glucose-1-P

Glucose-6-P

Cyclochlorotine

Cyclochlorotine

Citreoviridin

6-P-gluconolactone


NADP+


Oxaloacetate

Malate

Malate


Cytoplasm


Moniliformin

Patulin



CO2

CO2

Glutamate

TCA-cycle


Acetyl-CoA

Citrate


120572-Ketoglut

NADPH + H+



1also


1 G1andM

1include the







I IIIII

ATP ATPATP

LuteoskyrinEmodin


Electron transport

Phosphory-lation


Cyt b1 Cyt c1


Aflatoxin B1G1M1

Aflatoxin B1G1M1

H2O

Aflatoxin B1

2H + 12O2

Cyanide CO H2S

Ub1-




































1and G

1may be


1and G

1may not be




1is hydrolyzed by




1



1 B2 G1 and G



1and G

1was significantly



1and G





Wat

er

Met

hano

l

Acet

onitr

ile



Detector

Data display


Br

Br

I

I

TFA

OCH3

OCH3

OCH3

OCH3

Br2

I2Aflatoxin B1




30min 50∘C

4 s 20∘C

40 s 60∘C

O

1

OO

O

O O

O

O

O O

O

O O

O

O

OHO

O

O

OO

3

2







00

10

20

30

40

50

60

70

5 10 15Time (min)

(mV

)

AFG2

AFG1

AFB2

AFB1

(a)

(mV

)

010203040506070

0 5 10 15Time (min)

AFG2

AFG1

AFB2

AFB1

(b)

(mV

)

0 5 10 15Time (min)

0

10

20

30

40

50

60

70

AFG2

AFG1

AFB2

AFB1

(c)



2andAFG

2 each at 4 ngmL)






Control line


(a)


Absorbent pad


Sample pad

Release pad

(b)



1in peanut


1levels









To the waste

Antibody Antigen

Laser

Polarized light

Photodetector

Prism

From the pump

120579






1and AFB



































4 Conclusions





Acknowledgments


References





















































1as a model







1 B2 G1and G








































































1based on























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



I IIIII

ATP ATPATP

LuteoskyrinEmodin


Electron transport

Phosphory-lation


Cyt b1 Cyt c1


Aflatoxin B1G1M1

Aflatoxin B1G1M1

H2O

Aflatoxin B1

2H + 12O2

Cyanide CO H2S

Ub1-




































1and G

1may be


1and G

1may not be




1is hydrolyzed by




1



1 B2 G1 and G



1and G

1was significantly



1and G





Wat

er

Met

hano

l

Acet

onitr

ile



Detector

Data display


Br

Br

I

I

TFA

OCH3

OCH3

OCH3

OCH3

Br2

I2Aflatoxin B1




30min 50∘C

4 s 20∘C

40 s 60∘C

O

1

OO

O

O O

O

O

O O

O

O O

O

O

OHO

O

O

OO

3

2







00

10

20

30

40

50

60

70

5 10 15Time (min)

(mV

)

AFG2

AFG1

AFB2

AFB1

(a)

(mV

)

010203040506070

0 5 10 15Time (min)

AFG2

AFG1

AFB2

AFB1

(b)

(mV

)

0 5 10 15Time (min)

0

10

20

30

40

50

60

70

AFG2

AFG1

AFB2

AFB1

(c)



2andAFG

2 each at 4 ngmL)






Control line


(a)


Absorbent pad


Sample pad

Release pad

(b)



1in peanut


1levels









To the waste

Antibody Antigen

Laser

Polarized light

Photodetector

Prism

From the pump

120579






1and AFB



































4 Conclusions





Acknowledgments


References





















































1as a model







1 B2 G1and G








































































1based on























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




























1and G

1may be


1and G

1may not be




1is hydrolyzed by




1



1 B2 G1 and G



1and G

1was significantly



1and G





Wat

er

Met

hano

l

Acet

onitr

ile



Detector

Data display


Br

Br

I

I

TFA

OCH3

OCH3

OCH3

OCH3

Br2

I2Aflatoxin B1




30min 50∘C

4 s 20∘C

40 s 60∘C

O

1

OO

O

O O

O

O

O O

O

O O

O

O

OHO

O

O

OO

3

2







00

10

20

30

40

50

60

70

5 10 15Time (min)

(mV

)

AFG2

AFG1

AFB2

AFB1

(a)

(mV

)

010203040506070

0 5 10 15Time (min)

AFG2

AFG1

AFB2

AFB1

(b)

(mV

)

0 5 10 15Time (min)

0

10

20

30

40

50

60

70

AFG2

AFG1

AFB2

AFB1

(c)



2andAFG

2 each at 4 ngmL)






Control line


(a)


Absorbent pad


Sample pad

Release pad

(b)



1in peanut


1levels









To the waste

Antibody Antigen

Laser

Polarized light

Photodetector

Prism

From the pump

120579






1and AFB



































4 Conclusions





Acknowledgments


References





















































1as a model







1 B2 G1and G








































































1based on























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Wat

er

Met

hano

l

Acet

onitr

ile



Detector

Data display


Br

Br

I

I

TFA

OCH3

OCH3

OCH3

OCH3

Br2

I2Aflatoxin B1




30min 50∘C

4 s 20∘C

40 s 60∘C

O

1

OO

O

O O

O

O

O O

O

O O

O

O

OHO

O

O

OO

3

2







00

10

20

30

40

50

60

70

5 10 15Time (min)

(mV

)

AFG2

AFG1

AFB2

AFB1

(a)

(mV

)

010203040506070

0 5 10 15Time (min)

AFG2

AFG1

AFB2

AFB1

(b)

(mV

)

0 5 10 15Time (min)

0

10

20

30

40

50

60

70

AFG2

AFG1

AFB2

AFB1

(c)



2andAFG

2 each at 4 ngmL)






Control line


(a)


Absorbent pad


Sample pad

Release pad

(b)



1in peanut


1levels









To the waste

Antibody Antigen

Laser

Polarized light

Photodetector

Prism

From the pump

120579






1and AFB



































4 Conclusions





Acknowledgments


References





















































1as a model







1 B2 G1and G








































































1based on























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


00

10

20

30

40

50

60

70

5 10 15Time (min)

(mV

)

AFG2

AFG1

AFB2

AFB1

(a)

(mV

)

010203040506070

0 5 10 15Time (min)

AFG2

AFG1

AFB2

AFB1

(b)

(mV

)

0 5 10 15Time (min)

0

10

20

30

40

50

60

70

AFG2

AFG1

AFB2

AFB1

(c)



2andAFG

2 each at 4 ngmL)






Control line


(a)


Absorbent pad


Sample pad

Release pad

(b)



1in peanut


1levels









To the waste

Antibody Antigen

Laser

Polarized light

Photodetector

Prism

From the pump

120579






1and AFB



































4 Conclusions





Acknowledgments


References





















































1as a model







1 B2 G1and G








































































1based on























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Control line


(a)


Absorbent pad


Sample pad

Release pad

(b)



1in peanut


1levels









To the waste

Antibody Antigen

Laser

Polarized light

Photodetector

Prism

From the pump

120579






1and AFB



































4 Conclusions





Acknowledgments


References





















































1as a model







1 B2 G1and G








































































1based on























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



To the waste

Antibody Antigen

Laser

Polarized light

Photodetector

Prism

From the pump

120579






1and AFB



































4 Conclusions





Acknowledgments


References





















































1as a model







1 B2 G1and G








































































1based on























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

















4 Conclusions





Acknowledgments


References





















































1as a model







1 B2 G1and G








































































1based on























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Acknowledgments


References





















































1as a model







1 B2 G1and G








































































1based on























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of
















1as a model







1 B2 G1and G








































































1based on























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



















































1based on























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

Review Article Methods for Detection of Aflatoxins...

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Transcript of Review Article Methods for Detection of Aflatoxins...