Enzymatic spectrophotometric method for aflatoxin B detection based on acetylcholinesterase inhibition

Article abstract of DOI:10.1021/ac061819j

A new method for aflatoxin B (AFB) determination is proposed. The AFB determination is based on acetylcholinesterase (AChE) inhibition, and the AChE residual activity is determined using the colorimetric method (Ellman's method). Cholinesterases (ChEs) from various sources were tested using AFB₁ as reference aflatoxin. AChE from electric eel has shown the highest sensitivity to AFB₁, and it was chosen for the rest of the work. To select and optimize the analytical procedures, an investigation on the type of AChE inhibition by AFB₁ was carried out. The AChE degree of inhibition by AFB₁ was independent of the incubation time and the enzyme concentrations, showing the reversibility of the inhibition. This reversibility of the inhibition permits a rapid analysis of AFB₁, requiring only 3 min. For the development of the AFB₁ assay, the pH, the time of reaction, temperature, and substrate concentration were evaluated and optimized. The linear range of 10-60 ng mL^-1 was determined. To evaluate the selectivity of this method, the cross-reactivity with other aflatoxins such as aflatoxin B₂, aflatoxin G₁, aflatoxin G₂, and aflatoxin M₁ was investigated. Finally, the suitability of the assay for AFB₁ quantification in barley was evaluated. This study shows a new approach to detect anatoxins based on enzyme inhibition and has advantages such as the ease of use, rapidity, and cost effectiveness. Thus, it could find a possible use as a screening method for this type of mycotoxins.

Full text of DOI:10.1021/ac061819j

Anal. Chem. 2007, 79, 3409-3415
Enzymatic Spectrophotometric Method for
Aflatoxin B Detection Based on
Acetylcholinesterase Inhibition
†
†
‡
†
†
Fabiana Arduini, Ilenia Errico, Aziz Amine, Laura Micheli, Giuseppe Palleschi, and
Danila Moscone*,†
Dipartimento di Scienze e Tecnologie Chimiche, Universit a` di Roma Tor Vergata, Via della Ricerca Scientifica,
1-00133 Roma, Italy, and Facult e´ de Sciences et Techniques de Mohammadia, B.P. 146, Mohammadia, Morocco
and proteins,3,4 aflatoxin B
(AFB ) is the most acutely and
1 1
A new method for aflatoxin B (AFB) determination is
proposed. The AFB determination is based on acetylcho-
linesterase (AChE) inhibition, and the AChE residual
activity is determined using the colorimetric method
5,6
chronically toxic member of the aflatoxin family. It is also the
most widely found of the aflatoxins (AFB , AFG , AFG , and the
2
1
2
metabolic product AFM ). The legal limits set for AFB or for total
1
1
(
Ellman’s method). Cholinesterases (ChEs) from various
sources were tested using AFB as reference aflatoxin.
AChE from electric eel has shown the highest sensitivity
to AFB , and it was chosen for the rest of the work. To
select and optimize the analytical procedures, an inves-
aflatoxins vary significantly from country to country (e.g., for total
aflatoxins from 0 to 50 ng/g).7
1
In light of this overall scenario, several important issues require
a response. Most studies of aflatoxins have focused on their
chronic toxic effects as carcinogens, mutagens, and hepatotoxins.
So regulatory limits have been established as a means to protect
human and animal health and the food supply. At the same time,
there are limited analytical methods available for the determination
of aflatoxins within this strict regulatory context. The current
reference methods are primarily chromatographic, relying on
1
tigation on the type of AChE inhibition by AFB
carried out. The AChE degree of inhibition by AFB
independent of the incubation time and the enzyme
concentrations, showing the reversibility of the inhibition.
This reversibility of the inhibition permits a rapid analysis
of AFB
the AFB
and substrate concentration were evaluated and opti-
mized. The linear range of 10-60 ng mL-1 was deter-
mined. To evaluate the selectivity of this method, the
cross-reactivity with other aflatoxins such as aflatoxin B
aflatoxin G , aflatoxin G , and aflatoxin M was investi-
gated. Finally, the suitability of the assay for AFB
quantification in barley was evaluated. This study shows
a new approach to detect aflatoxins based on enzyme
inhibition and has advantages such as the ease of use,
rapidity, and cost effectiveness. Thus, it could find a
possible use as a screening method for this type of
mycotoxins.
1
was
was
1
1
, requiring only 3 min. For the development of
assay, the pH, the time of reaction, temperature,
methods such as high-performance liquid chromatography
1
(
HPLC).8 A promising alternative approach involves the use of
-12
13,14
enzyme-linked immunoassay (ELISA).
While they have often
demonstrated the required sensitivity and acceptable specificity,
these assays need specific antibodies and, indirectly, the use of
animals in order to produce these “receptors”.
2
,
1
2
1
1
(
(
1) IARC. In Mycotoxins; International Agency for Research on Cancer: Lyon,
France, 1993; Vol. 56, pp 249-395.
2) Hussein, H. S.; Brasel, J. M. Toxicology 2001, 167, 101-134.
(3) Iwaki, M.; Kitagawa, T.; Akamatsu, Y.; Aibara, K. Biochim. Biophys. Acta
1
990, 1035, 146-153.
(4) Cole, K. E.; Jones, T. W.; Lipsky, M. M.; Trump, B. F.; Hsu, I. C.
Carcinogenesis 1988, 9, 711-716.
(5) Lakkawar, A. W.; Chattopadhyay, S. K.; Johri, T. S. Slovak Vet. Res. 2004,
1, 73-81.
4
(
6) Wilson, D. M.; Payne, G. A. In The Toxicology of Aflatoxin, Human Health,
Veterinary and Agricultural Significance; Eaton D. L., Groopman J. D., Eds.;
Academic Press: San Diego, 1994; pp 309-325.
7) Delmulle, B. S.; De Saeger, S. M. D. G.; Siba, L.; Barna-Vetro, I.; Van
Peteghem, C. H. J. Agric. Food Chem. 2005, 53, 3364-3368.
Cereals and a wide variety of food products are susceptible to
damage from mold during pre- and postharvest stages of produc-
tion. The family of aflatoxins, which can be produced by several
species of the mold Aspergillus (Aspergillus flavus, Aspergillus
parasitucus and the rare Aspergillus nomius), has by now been
shown to represent a significant class of mycotoxins because of
their documented impact on both human and animal health and
on economic aspects of international trade involving food and
(
(8) Blesa, J.; Soriano, J. M.; Molt o` , J. C.; Marin, R.; Manes, J. J. Chromatogr. A
003, 1011, 49-54.
9) Elizalde-Gonzalez, M. P.; Mattusch, J.; Wennich, R. J. Chromatogr. 1998,
28, 439-444.
(10) Reif, K.; Metzger, W. J. Chromatogr. 1995, 692, 131-136.
2
(
8
(
11) Jaimez, J.; Fente, C. A.; Vazquez, B. I.; Franco, C. M.; Cepeda, A.; Mahuzier,
G.; Prognon, P. J. Chromatogr., A 2000, 882, 1-10.
1
,2
animal feeds. Due to its capacity for covalent binding to DNA
(
12) Ventura, M.; Gomez, A.; Anaya, I.; Diaz, J.; Broto, F.; Montserrat, A.; Luis,
C. J. Chromatogr. 1990, 1084, 25-29.
*
Corresponding author. Tel.: +39 0672594421. Fax: +39 06 72594328.
(13) Ammida, N. H. S.; Micheli, L.; Palleschi, G. Anal. Chim. Acta 2004, 520,
159-164.
(14) Micheli, L.; Greco, R.; Badea, M.; Moscone, D.; Palleschi, G. Biosens.
Bioelectron. 2005, 21, 588-596.
E-mail: danila.moscone@uniroma2.it.
†
Universit a` di Roma Tor Vergata.
Facult e´ de Sciences et Techniques de Mohammadia.
‡
1
0.1021/ac061819j CCC: $37.00 © 2007 American Chemical Society
Analytical Chemistry, Vol. 79, No. 9, May 1, 2007 3409
Published on Web 04/05/2007

Scheme 1. Reaction between 5,5′-Dithiobis-2-nitrobenzoic Acid (DTNB) and Thiocholine to give
2-Nitro-5-thiobenzoic Acid (TNB)
Given the demonstrated toxicity of aflatoxins, their potential
linesterase (AChE) by AFB
of nerve impulses. The AFB
mouse brain was studied and the implications in terms of kinetic
mechanism and toxicity discussed. In this study, it was first shown
1
, a key enzyme in the transmission
1
inhibition on AChE extracted from
widespread presence in the food chain, and the economic costs
of contaminated lots, much attention has also been focused on a
complementary approach, the development of practical screening
assays that could be used for monitoring at all stages of food and
feed production, so that contaminated ingredients can be identified
or rejected before feeding to animals or humans. Such methods
must be rapid, sufficiently sensitive, and applicable in the field in
order to allow the necessary corrective actions to be taken in a
timely fashion. To respond to these various issues, enzymatic
methods have shown promise in some cases, as an alternative to
that AFB
this effect, a method to quantify AFB
in enzyme activity, determined using Ellman’s spectrophotometric
1
inhibits the AChE from electric eel. On the basis of
1
by measuring the decrease
method, after exposure of the enzyme to AFB
optimized.
1
was proposed and
EXPERIMENTAL SECTION
Apparatus and Reagents. All chemicals from commercial
sources were of analytical grade. Acetylcholinesterase from
electric eel (EC 3.1.1.7), butyrylcholinesterase (BChE) from
equine serum (EC 3.1.1.8), S-butyrylthiocholine chloride, acetyl-
thiocholine chloride (ACTh), and 5,5′-dithiobis-(2-nitrobenzoic acid
classical methods, to achieve faster and simpler detection of some
environmental pollutants such as pesticides, heavy metals, etc.15-21
In the case of the aflatoxins, this approach pointed to either an
enzyme that metabolizes AFB
enzyme that can be inhibited by AFB
1
into a detectable compound or an
. It can be noted that, as
1
(DTNB) were obtained from Sigma Chemical Co. (St. Louis, MO).
for biosensors, the use of biological molecules (enzymes or
receptors) in analytical schemes can be particularly advantageous
if the action of the analyte is specifically related to the mechanism
of toxicity toward humans and animals. In this perspective, recent
work has provided insight into the acute effects of the aflatoxins
on the gastrointestinal, respiratory, cardiovascular, and central
The AChEs from Drosophila melanogaster wild type and mutants
were obtained from Marty’s group (University of Perpignan,
France). The AChE from D. melanogaster was dialyzed before use.
Aflatoxins B
1
, B
2
, G
1
, G
2
, and M were purchased from Vinci-
Biochem.
2
2-26
Stock solutions of AFB were preparated in methanol, and their
1
nervous systems of both humans and animals.
In particular,
concentration was confirmed with the HPLC method using the
official AOAC method with some modifications.13 Different con-
centrations of aflatoxin were obtained by diluting the stock solution
with methanol.
For spectrophotometric measurements, a Unicam 8625 UV/
vis spectrophotometer was used.
AFB could be shown to evoke contractile responses in the rumen
1
2
7
28
intestine and on isolated guinea pig ileum. These results
demonstrated that the aflatoxin induces its contractile effect
indirectly through the cholinergic system by stimulating acetyl-
choline release. Moreover, the work of Egbunike and Ikegwuonu29
suggested that AFB
1
changes acetylcholine turnover and hence
Measurement of Cholinesterase Activity. The AChE activity
was evaluated by measuring the product of the enzymatic reaction.
The ACTh was chosen as substrate and the thiocholine, enzymati-
cally produced using AChE, was evaluated using the spectropho-
tometric Ellman’s method. The thiocholine is measured using
Scheme 1: DTNB reacts with thiocholine to give 2-nitro-5-
thiobenzoic acid (TNB), a yellow product with a maximum
cholinergic transmission in rat brain and adenohypophysis. In a
recent study, Cometa et al. analyzed the inhibition of acetylcho-
30
(
(
15) Trojanowicz, M.; Hitchman, M. L. Trends Anal. Chem. 1996, 15, 38-45.
16) Ch aˆ teau, C.; Dzyadevych, S.; Durrieu, C.; Chovelon, J. M. Biosens. Bioelec-
tron. 2005, 21, 273-281.
(17) Ricci, F.; Palleschi, G. Biosens. Bioelectron. 2005, 21, 89-407.
(18) Kulys, J.; Vidziunaite, R. Biosens. Bioelectron. 2003, 18, 319-325.
(19) Vidal, J. C.; Esteban, S.; Gil, J.; Castillo, J. R. Talanta 2006, 68, 791-799.
(20) Verma, N.; Singh, M. Biometals 2005, 18, 121-129.
(21) Tsai, H.; Doong, R. Biosens. Bioelectron. 2005, 20, 1796-1804.
(22) Hussein, H. S.; Brasel, J. M. Toxicology 2001, 167, 101-134.
(23) Hayes, R. B.; Van Nieuwenhuiz, J. P.; Raatgever, J. W.; Ten Kate, F. J. Food
Chem. Toxicol. 1984, 22, 39-43.
31
absorbance peak at 412 nm.
For thiocholine measurement, 900 µL of phosphate buffer
-
1
solution (0.1 M, pH 8), 100 µL of 0.1 M DTNB, 40 mU mL
enzyme (AChE), and 0.4 mM ATCh were put in a spectrophoto-
metric cuvette. Absorbance was measured after 3 min, and the
AChE activity was calculated, using the Lambert-Beer law and
(
(
(
(
24) Abdel-Haq, H.; Palmery, M.; Leone, G. M.; Saso, L.; Silvestrini, B. Toxicol.
in Vitro 2000, 14, 193-197.
25) Abdel-Haq, H.; Palmery, M.; Leone, G. M.; Saso, L.; Silvestrini, B. Toxicol.
Sci. 2000, 55, 162-170.
-1
known molar extinction coefficient of TNB (λ ) 13.600 M
-
1
31
cm ).
Measurement of the Effect of Methanol on Ellman’s
26) Peraica, M.; Radiae, B.; Luciae, A.; Pavloviae, M. Bull. World Health Org.
1
999, 77, 754-766.
27) Cook, W. O.; Richard, J. L.; Osweiler, G. D.; Trampel, D. W. J. Am. Vet. Res.
986, 47, 1817-1825.
1
Reaction. Since methanol is often used to extract the AFB from
1
many contaminated agricultural samples,3
2-34
it was chosen as
(28) Luzi, A.; Cometa, M. F.; Palmery, A. Toxicol. in Vitro 2002, 16, 525-529.
(29) Egbunike, G. N.; Ikegwuonu, F. I. Neurosci. Lett. 1984, 52, 171-174.
(30) Cometa, M. F.; Lorenzini, P.; Fortuna, S.; Volpe, M. T.; Meneguz, A.; Palmery,
A. Toxicology 2005, 206, 125-135.
1
organic solvent to solubilize AFB in our experiments.
(31) Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70-77.
3410 Analytical Chemistry, Vol. 79, No. 9, May 1, 2007

In the experimental procedure, the effect of methanol on
the Ellman’s method was evaluated. In this perspective, the
cysteamine was taken as a representative thiol for this investiga-
tion, since it is commercially available whereas thiocholine has
by centrifugation for 5 min at 4000 rpm. After the separation of
the two layers, the aqueous layer was collected36 and concentrated
in a rotary evaporator to remove the solvent. The dried matrix
mixture was redissolved in 1 mL of methanol and the solution
obtained was filtered on a glass fiber membrane (1 µm). The
filtered solution was diluted 1:15 v/v in buffer and used for the
35
to be enzymatically produced. An aliquot of 900 µL of phosphate
buffer solution (0.1 M, pH 8.0) containing different percentages
of methanol (from 0 to 90% v/v) and cysteamine to a final
AFB
Measurement of AFB
the same procedure described above was used. The inhibition
due the AFB in barley was calculated by using eq 1, where I% is
percent inhibition, A is the absorbance obtained by adding extract
of barley without AFB , and A is absorbance obtained by adding
extract of barley with AFB
1
spectrophotometric assay.
-
5
concentration of 1.9 × 10 M was added to a cuvette. The
absorbance was measured using the procedure described above.
Measurement of the Effect of Methanol on AChE Activity.
The effect of methanol on the enzyme activity was also evaluated.
An aliquot of 900 µL of phosphate buffer solution (0.1 M, pH 8.0)
containing different percentages of methanol (from 0 to 90% v/v)
and a known amount of AChE was added. After the substrate was
added, the residual enzymatic activity was evaluated by using the
procedure described above. Inactivation of enzyme by exposure
to organic solvent was calculated by using eq 1, where I% is
1
1
in Barley. For AFB measurements,
1
0
1
i
1
.
In order to achieve a lower detection limit, the aqueous solution
separated after the centrifugation was concentrated in a rotary
evaporator and redissolved in the minimum amount of methanol.
This amount, chosen in order to provide the best solubility of the
analyte in the least amount of the solvent, corresponded to 1 mL.
Safety Conditions. The stock solutions of aflatoxins (highly
toxic compounds) were prepared under appropriate safety condi-
tions. In fact, in order to avoid contact with the powder or
inhalation of vapor of aflatoxins, the operators were dressed with
lab dresses, gloves, mask, and glasses. Also, a dedicated hood
has been used during sample preparation and analysis.
I% ) (A - A )/A
0
(1)
0
i
percent of enzyme inactivation due to methanol, A
0
is absorbance
i
obtained in aqueous solution, and A is the absorbance obtained
in aqueous solution with methanol.
Measurement of the Effect of Methanol on AChE Inhibi-
tion. The effect of methanol on the AFB inhibition on AChE was
also evaluated. An aliquot of 900 µL of phosphate buffer solution
0.1 M, pH 8.0) containing a known amount of AChE and various
percentages of methanol (from 0 to 50% v/v) in the absence or
presence of different concentrations of AFB was added. Afterward,
the substrate was added and the residual enzymatic activity was
evaluated by using the procedure described above. AFB inhibition
on enzyme was calculated by using eq 1, where I% is percent of
1
RESULTS AND DISCUSSION
Aflatoxin Inhibition on Different ChEs. Since our objective
(
1
is to devise an assay that can measure AFB at the lowest possible
concentration, a survey of cholinesterases (ChEs) from various
sources was carried out to find the enzyme having the highest
1
degree of inhibition by AFB
1
. For this purpose, we have studied
1
AFB inhibition using AChE from electric eel, BChE from equine
1
serum, and AChE from D. melanogaster wild type and mutants.
The AChE from electric eel was the most sensitive: in fact, IC50
AFB
solution with methanol, and A
solution with methanol and AFB
1
inhibition on enzyme, A
0
is absorbance obtained in aqueous
is absorbance obtained in aqueous
i
(IC50 equal to amounts of inhibitor that gave 50% enzyme
1
. To consider the AChE inactiva-
0
was also measured in the presence of
inhibition) was obtained with 60 ng mL AFB
by AFB on BChE from equine serum was also investigated. In
the case of BChE, at a concentration of 60 ng mL AFB
-1
. The inhibition
1
tion by methanol, A
1
methanol.
-1
1
no
Measurement of Aflatoxin Stock Solution. For aflatoxin
measurements, the same procedure described above was used.
The inhibition due the toxin was calculated by using eq 1, where
inhibition was observed, while only 10% inhibition was obtained
-1
using 1.4 mg mL AFB1. This demonstrated the very low affinity
1 1
between AFB and BChE. The AFB inhibition on AChE from D.
I% is percent of inhibition, A
0
is absorbance obtained using
melanogaster wild type and two different mutants (B24 and B394)
was tested. The AChE was engineered in the laboratory by Marty’s
and Fournier’s groups to increase the sensitivity toward the
methanol, and A is absorbance obtained using this analyte in
i
methanol, this being the preferred solvent.
Spiked Barley Sample Preparation and Extraction. Non-
contaminated barley seeds (from the local market) were first
ground in a household blender at high speed for 1 min. Then, 5
37-39
organophosphorous and carbammic pesticides.
The degree
-1
of inhibition obtained with AFB
1
at 60 ng mL using AChE wild
type, B24, and B394 were 20, 17, and 21%, respectively. Overall,
the results obtained demonstrated that the AChE from electric
eel had the highest sensitivity to AFB among the cholinesterases
1
g of ground barley samples was spiked with 200 µL of AFB
solution at several concentrations (in order to have barley at 100,
1
-
1
1
20, and 150 ng g ). The samples were fully mixed using
previously tested and was also considerably more sensitive than
the unpurified AChE from mouse brain tested by Cometa et al.,
autovortexing for 1 min and extracted with 25 mL of extraction
solvent (70% methanol/29 %PBS/1% DMF) by shaking for 15 min,
and then the extract was separated from the insoluble materials
-1 30
which showed IC50 at 10 mg mL
.
(36) Ammida, N. H. S.; Micheli, L.; Moscone, D.; Palleschi, G. Anal. Lett. 2006,
(
(
32) Bourais, I.; Amine, A.; Venanzi, M.; Micheli, L.; Moscone, D.; Palleschi, G.
Microchim. Acta 2005, 539, 195-201.
33) Lin, L.; Zhang, J.; Wang, P.; Wang, Y.; Chen, J. J. Chromatogr., A 1998,
39, 1559-1572.
(37) Bucur, B.; Fournier, D.; Danet, A. F.; Marty, J. M. Anal. Chim. Acta 2006,
562, 115-121.
8
15, 3-20.
(38) Bachmann, T. T.; Leca, B.; Villate, F.; Marty, J. M.; Fournier, D.; Schmid,
(
(
34) AOAC Official Method 991.31 A, 1994.
35) Amine, A.; Mohammadi, H.; Bourais, I.; Palleschi, G. Biosens. Bioelectron.
R. D. Biosens. Bioelectron. 2000, 15, 193-201.
(39) Bucur, B.; Dandoi, M.; Danet, A. F.; Marty, J. M. Anal. Chim. Acta 2005,
539, 195-201.
2006, 21, 1405-1423.
Analytical Chemistry, Vol. 79, No. 9, May 1, 2007 3411

1
In order to better understand the type of AFB inhibition on
AChE, and mainly to select the best analytical procedure, kinetic
studies were carried out.
1
Mechanism of the AChE Inhibition by AFB . Measure-
ment of AChE Activity. The first step was the study of the K
M
of the enzyme (AChE from electric eel) using ACTh as substrate.
Different concentrations of substrate were evaluated in the
dynamic range 0.1-5 mM ACTh. In this range of ACTh, values
of absorbance from 0.050 ( 0.002 up to 0.80 ( 0.02 were observed.
Enzymatic analysis of ATCh hydrolysis using AChE yielded
the following kinetic parameters: K
L
M
0.33 mM and Vmax 0.26 mmol
min using a Lineweaver-Burk plot (data not shown).
Reversibility of the AChE Inhibition by AFB . It was
-
1
-1
1
essential to establish certain aspects of the inhibitory mechanism
in order to determine the suitability of a given enzyme for a
screening assay. Since little is known about the inhibition by
2
2,30
AFB
AFB
1
,
the mechanism of AChE inhibition by the mycotoxin
was first investigated.
1
Figure 1. Lineweaver-Burk plot representing reciprocals of the
initial enzyme velocity vs ATCh concentration without (O) and with
In the first instance, whether the inhibition is of a reversible
or irreversible type is crucial, since for irreversible inhibition, it
becomes important to use a low enzyme concentration in order
to obtain a low limit of detection, as in the case of pesticide
-
1
various concentrations of AFB1(75 (9) and 300 (2) ng mL AFB1).
-
1
AChE ) 40 mU mL , 3 min of reaction, 0.1 M phosphate buffer, pH
. All the values are the average of triplicate measurements.
8
3
5,40
detection.
On the other hand, for reversible inhibition, no
competitive in nature. In fact, pure competitive inhibition is
characterized by an increase of inhibition degree as the substrate
decreases.
To understand if the inhibition is uncompetitive, noncompeti-
tive, or mixed type, the AChE activity was determined with the
change in degree of inhibition should be observed using different
enzyme concentrations.41
1
The degree of inhibition at a fixed concentration of AFB (60
ng mL-1) using various concentrations of AChE was determined.
The enzyme concentrations used for this experiment were 70, 40,
-
1
previous range of ATCh concentration (from values near K
to 4 K ) either in the absence or presence of different AFB
concentrations.
M
/4
and 7 mU mL , and the degrees of inhibition obtained were 45
3, 50 ( 4, and 47 ( 3%, respectively. These results (essentially
no change in degree of inhibition) seem to support the hypothesis
M
1
(
The analysis of the linear transformation of Michaelis-Menten
equation, i.e., the Lineweaver-Burk plot (Figure 1) confirmed that
the lines are not parallel and thus the inhibition is not uncom-
petitive. In this way, the mechanism of inhibition could be
noncompetitive or mixed type. In the case here of the AChE from
electric eel, the lines pass through different points on the ordinate
and intersect at a point slightly displaced from the abscissa in
the second quadrant. Thus, we can suppose that the inhibition
seems a mixed type.
In the case of the mixed-type inhibition, the inhibition can be
regarded conceptually as being composed of a competitive and
noncompetitive inhibition. The dissociation constants for inhibitor
differ from each other, depending on whether they refer to the
that the inhibition of AChE by AFB
nism.
1
follows a reversible mecha-
To confirm this hypothesis, a study of the incubation time with
the inhibitor was performed. While for the case of irreversible
inhibition, an increase in incubation time is required to reach a
low detection limit, for reversible inhibition, a longer incubation
time does not lead to an increase in the degree of inhibition. Then,
the effect of incubation time (the time of reaction between the
1
inhibitor, AFB , and AChE) was also evaluated between 0 and 30
min, obtaining a degree of inhibition equal to 48 ( 3%.
Given that the incubation time has no effect on degree of
inhibition, that is, that the interactions were rapid and reversible,
there was no need for a prior incubation of the enzyme with
inhibitor before substrate was added to start the reaction.
free enzyme or the complex.41 The constants K
dissociation constant for binding to the free enzyme) and RK
inhibitor dissociation constant for binding to the enzyme-
substrate complex) were calculated using the kinetic equations
for mixed inhibition. For K calculation, the equation KMapp/Vmaxapp
(1 + [I]/K ) was used with 75 ng mL AFB
value of K equal to 61 ng mL . The RK
(inhibitor
i
i
1
Study of the Type of AChE Inhibition by AFB . After
(
demonstrating the reversibility of this inhibition, experiments were
performed to evaluate whether the inhibition was competitive,
uncompetitive, noncompetitive, or mixed.
i
-
1
)
K
M
i
1
obtaining a
was calculated equal to
130 ng mL , from the eq 1/Vmaxapp) (1/Vmax + ([I]/ RK
AChE activity was determined using ATCh concentrations over
-
1
i
i
the range from values near K
or presence of fixed AFB
M
/4 up to 4 K
concentrations (60 ng mL ). The
M
either in the absence
-
1
-
1
i max
V )
1
-
1
using 75 ng mL AFB
type seems similar to the hypothesis proposed by Cometa et al.
for the AFB inhibition of unpurified AChE from mouse brain,
1
. Our hypothesis of the mixed-inhibition
degree of inhibition (47 ( 5%) does not change with substrate
concentration. This study indicates that the inhibition is not
3
0
1
(
40) Suprun, E.; Evtugun, G.; Budnikov, H.; Ricci, F.; Moscone, D.; Palleschi, G.
Anal. Bioanal. Chem. 2005, 383, 597-604.
but further investigations are required in order to fully understand
the mechanism of the inhibition under study.
(
41) Meleti, T. In Basic Enzyme Kinetics; Akademia Khado: Budapest, 1986.
42) Arduini, F.; Ricci, F.; Bourais, I.; Amine, A.; Moscone, D.; Palleschi, G. Anal.
Lett. 2005, 38, 1703-1719.
Optimization of the AFB
1
Assay Based on AChE Inhibi-
determination
(
tion. In order to develop an effective assay for AFB
1
3412 Analytical Chemistry, Vol. 79, No. 9, May 1, 2007

-
1
Figure 3. Effect of temperature on AChE storage conditions. AChE
) 40 mU mL-1, 3 min of reaction time, ATCh ) 0.4 mM. The residual
Figure 2. Effect of pH on AChE inhibition by 60 ng mL AFB1.
-
1
AChE ) 40 mU mL , 3 min of reaction, ATCh ) 0.4 mM. At pH 7,
7
.5, and 8, the 0.1 M phophate buffer (b) was used while at pH 8,
activity was evaluated for AChE maintained at room temperature (4)
and at 4 °C (b). All the values are the average of triplicate
measurements.
8.5, and 9 a 0.1 M Tris buffer (]) was chosen. All the values are the
average of triplicate measurements.
based on the decrease in enzyme activity, the spectrophotometric
Ellman’s method for the determination of AChE activity had to
be adapted and optimized, taking into consideration parameters
such as substrate concentration, solvent, pH, etc.
while no decrease of enzymatic activity was observed when the
enzyme was maintained at 4 °C. Thus, AChE was stored at 4 °C.
Study of Methanol Effect. In the previous experiments, the
AFB
AFB
1
inhibition of AChE was carried out in methanol because
is insoluble in completely nonpolar solvents and soluble in
As noted above, the characteristics of the inhibition made it
possible to adjust the enzyme and substrate concentration of the
assay mixture to achieve suitable assay times and levels of
inhibition. An enzyme concentration of 40 mU mL- was chosen
because it permitted the achievement of reasonable optical
densities from Ellman’s reaction in only 3 min. Furthermore, at a
1
1
slightly polar solvents. The AFB , in fact, is normally extracted
using mixtures of organic solvents such as methanol, acetonitrile,
chloroform, or acetone. Methanol has previously been used to
1
3
0-32
extract AFB
and was chosen as the organic solvent to solubilize AFB
experiments. With a goal to adopt the assay for AFB detection
1
from many contaminated agricultural samples
1
in our
-
1
constant concentration of AFB
concentrations of substrate (0.1-1.6 mM), the degree of inhibition
47 ( 5%) was found to be constant. Finally, a substrate concentra-
1
of 60 ng mL , with different
1
in barley, the effect of methanol in the assay was evaluated.
Since Ellman’s method was to be utilized to monitor the
thiocholine produced enzymatically, a first step was to evaluate
the direct methanol effect on thiol determination. Cysteamine was
taken as a representative thiol for this investigation since it is
commercially available whereas thiocholine had to be enzymati-
(
tion of 0.4 mM was chosen for the rest of the work.
Given that the incubation time has no effect on degree of
inhibition, because of the reversible inhibition, there was no need
for a prior incubation of the enzyme with inhibitor before substrate
was added to initiate the reaction. For this reason, no incubation
time was adopted for the rest of work.
Effect of pH. As reported in our previous work,39 the best
buffer and pH value for AChE is phosphate buffer 0.1 M at pH 8.
-
5
cally produced. The reaction between cysteamine (1.9 × 10 M)
and DTNB (Ellman’s reagent) was evaluated in the presence of
different percentages of methanol, in the range from 0 to 90%,
mixed with buffer. Since the same value of absorbance (0.230 (
1
To evaluate AChE inhibition by AFB as a function of pH, the
0
.007) was obtained when the methanol percentage in phosphate
AChE, ATCh, and AFB were added to buffer at different pHs.
1
buffer was 0, 50, 60, 70, and 80%, it was concluded that methanol
had no direct effect on thiol detection using Ellman’s method.
In terms of the enzyme’s sensitivity to solvent effects, it is
known that AChE is inactivated by organic solvents such as
The degrees of inhibition obtained are shown in Figure 2. We
observed the highest degree of inhibition at pH 8 with phosphate
buffer, and a rapid decrease at pH 9 was observed in Tris buffer.
Thus, phosphate buffer at pH 8 was chosen for further experi-
ments.
42,44,45
dimethyl sulfoxide, toluene, dichloromethane, and chloroform.
For this reason, AChE inactivation by methanol was investigated.
Figure 4 shows that the AChE activity decreased in parallel with
an increase in the amount of methanol. At 50% methanol (in
phosphate buffer), the AChE activity decreased by 30%. If the
percentage of methanol was increased to 80%, the inactivation
Effect of Temperature on Enzyme Activity in Storage
Condition. Storage stability, or shelf life, refers to an enzyme’s
maintaining its catalytic properties in the period between manu-
facture and eventual use. A common inactivation model for an
enzyme involves the unfolding of the polypeptide’s native tertiary
structure. The unfolded protein may refold to the native conforma-
tion or undergo some further change leading to permanent
inactivation. Temperature is an important parameter that influ-
(
(
(
43) Rochu, D.; Cedric, G.; Repiton, J.; Vigui e` , N.; Saliou, B.; Bon, C.; Masson,
P. Biochim. Biophys. Acta 2001, 1545, 216-226.
44) Montesinos, T.; Perez-Munguia, S.; Valdez, F.; Marty, J. L. Anal. Chim. Acta
2
001, 431, 231-237.
4
3
ences the enzyme stability. As can be seen in Figure 3, AChE
45) Mionetto, N.; Marty, J. L.; Karube, I. Biosens. Bioelectron. 1994, 9, 463-
maintained at room temperature (25 °C) was gradually inactivated,
470.
Analytical Chemistry, Vol. 79, No. 9, May 1, 2007 3413

Table 1. Concentration of Various Aflatoxins Giving
5
0% Enzyme Inhibition (IC50)a
aflatoxin
IC50 (ng mL-1) ( SD
AFB1
AFB2
AFG1
AFG2
AFM1
60 ( 5
72 ( 4
850 ( 70
310 ( 10
175 ( 8
a
-1
AChE ) 40 mU mL , 3 min of reaction time, ATCh ) 0.4 mM.
The data reported in the table represent the average of 3 measurements.
Calibration Curve. From an analysis of inhibition by AFB
over the concentration range 10-1000 ng mL (data not shown),
1
-1
it was found that the response for the optimized assay was linear
-
1
in the range between 10 and 60 ng mL . Five successive
calibration curves were performed, and the results showed a linear
Figure 4. Effect of methanol (percentage from 0 to 80%) on AChE
activity. AChE ) 40 mU mL- , 3 min of reaction, acetylthiocholine )
1
0.4 mM, 0.1 M phosphate buffer, pH 8. All the values are the average
correlation: y ) (13.4 ( 0.9) + (0.602 ( 0.009)x, where y is degree
of triplicate measurements.
2
of inhibition and x is AFB
1
concentration with r equal to 0.955.
The lower limit of the linear range, defined as the concentration
-
1
giving an inhibition of 20%, was 10 ng mL AFB
1
while the IC50
-
1
was 60 ng mL AFB . The detection limits reached allow the use
this assay for detection in feed at the regulatory limits.
1
4
6
Cross-Reactivity. To evaluate the selectivity of the proposed
assay, IC50 values were determined using the other aflatoxins
(
AFB
in Table 1, both AFB
of AChE, while both AFG
2
, AFG
1
, AFG
2
, AFM
1
) for comparison with AFB
1
. As shown
1
and AFB
2
provoked considerable inhibition
1
and AFG
2
showed limited capacity to
were intermediate with
inhibit the enzyme. The results for AFM
1
an IC 50 of 175 ng mL-1. The structural difference between AFG
and AFB forms is the presence of a cyclohexane in the structure
of AFG, while in AFB there is a cyclopentane. This difference
probably affects the interaction between aflatoxin and its binding
site on the catalytic unit of AChE.
The results obtained showed that the assay can detect AFB
and AFB with almost the same sensitivity. Thus, if AFB and AFB
2
occur simultaneously in the sample, the assay detects both
aflatoxins, because it cannot discriminate between them. To
understand the behavior of the assay in the case of an aflatoxin
1
2
1
Figure 5. Effect of methanol (percentage from 0 to 50%) on AChE
-1
inhibition by AFB1. AChE ) 40 mU mL , 3 min of reaction time, ATCh
)
0.4 mM, AFB1 ) 60 ng mL-1, 0.1 M phosphate buffer, pH 8. All
the values are the average of triplicate measurements.
mixture, AFB
mixture. A concentration of 25 ppb AFB
of inhibition of 29 ( 3% was observed. Then, 50 ppb AFB
also measured, showing a degree of inhibition of 47 ( 2%. The
same experiments were performed using AFB , obtaining a degree
1
and AFB
2
were analyzed individually and as a
was added and a degree
was
1
seemed to level off at 60% of the original activity. Using lower
concentrations of methanol, it was found that in the range 0-5%
methanol in buffer no inactivation was observed. Thus, in setting
up the inhibition assay system, a methanol percentage lower than
1
2
of inhibition equal to 27 ( 4 and 41 ( 3%, respectively. The degree
of inhibition was also measured for a mixture of aflatoxins (25
5
% was always used.
The effect of the methanol on the inhibition of AChE by AFB
1
ppb AFB
to 46 ( 3%, confirming that the system simultaneously detects
AFB and AFB with similar sensitivity.
Among the aflatoxins tested, the AFM
highest degree of interference in the AFB detection, but since
AFM is present only in milk, this seems not to present a problem
for monitoring of cereals.
AFB Measurement in Fortified Barley Samples. The
AChE inhibition assay was then applied for the determination of
AFB using spiked barley samples in order to test its performance
1 2
and 25 ppb AFB ), observing a degree of inhibition equal
was also investigated using the optimized assay conditions (Figure
). This study was carried out by performing the assay in the
presence of different amounts of methanol (from 0 to 50%). The
degree of inhibition by 60 ng mL-1 AFB
was equal to (50 ( 3%)
5
1
2
1
seems to show the
1
in the presence of different amounts of methanol (from 0 to 50%).
These results demonstrate that even the highest percentage of
methanol has no effect on the degree of AChE inhibition by AFB ,
1
though this amount of methanol in buffer decreased the control
level of AChE activity. This result means that it is possible to
1
determine AFB using a percentage of methanol as high as 50%,
1
1
1
in a real matrix. First, the absorbance of the sample in the absence
1
that is diluting the AFB standard solution (prepared in pure
methanol) 2-fold.
(46) European Community Regulation 2003/100, 31/10/2003.
3414 Analytical Chemistry, Vol. 79, No. 9, May 1, 2007

The advantage of this method is that it is much simpler and
faster than reference methods such as HPLC;8 moreover, it uses
Table 2. Recovery Studies of Spiked Barley Samples
Using Assay for AFB1 Based on AChE Inhibition
(
-12
Concentration in Barley)a
less expensive reagents than the specific antibodies adopted in
ELISA.1 The disadvantage is the low selectivity for AFB
3,14
and
AFB1 (ng mL-1
1
)
2
AFB because the degree of inhibition of AChE is almost the same,
added
found
% recovery ( SD
but this does not necessarily compromise its value as a screening
assay. The ease of performance and cost effectiveness of the
system for AFB detection and the good results obtained in terms
of recovery in real samples show the high potentiality of this assay
as a screening method for AFB detection in feed.
150
120
100
152
117
99
101 ( 2
98 ( 3
99 ( 9
a
The data reported in the table represent the average of 3
measurements.
CONCLUSIONS
A new method for AFB detection was developed. The deter-
mination of AFB is based on its capacity to inhibit AChE from
electric eel, and it makes use of Ellman’s spectrophotometric
method to measure the enzyme activity in the absence and
presence of analyte. In fact, within a few minutes, it is possible to
obtain results suitable for rapid screening of the prepared samples.
If it can be demonstrated that the inhibition of a given AChE
by aflatoxins bears a direct relation to their toxic effects (e.g.,
cholinergic ones), then it has an additional advantage relative to
the ultimate objective of effectively detecting the presence of
harmful substances in order to remove contaminated lots from
the food production chains.
of Ellman’s reagent was evaluated. An absorbance equal to 1.760
was observed after 2-fold sample dilution in buffer. Different
dilutions of the sample in buffer were evaluated in order to obtain
the lowest matrix effect. It was necessary to make a dilution (1:
1
5 v/v) of the sample in order to decrease the absorbance of the
sample to a value of 0.100. This dilution factor (15) was adopted
for the recovery study.
The extraction efficiency of the toxin from barley was calcu-
lated using samples spiked before the extraction with known
amounts of the toxin as reported by Ammida et al.3 A 200-µL
6
1
aliquot of AFB standard solution at different concentrations was
added to 5 g of blank barley in order to obtain 100, 120, and 150
A preliminary investigation of the kinetics and inhibitory
-1
1
ng g of barley sample. For each AFB level, three samples were
mechanism using AFB
to note that the binding of AFB
orders of magnitude stronger than that found for the mouse brain
AChE28 and this behavior allowed AFB
to be detected, through
1
indicated a mixed type. It is interesting
independently processed and each sample was analyzed in
triplicate. On the basis of the calibration curves prepared in barley
extract, it was possible to calculate the extraction efficiency (Table
1
to the electric eel enzyme is ∼3
1
2
) of the analyte. The results show that the procedure adopted
an enzyme inhibition based assay, at nanogram per milliliter levels.
Finally, the cross-reactivity results showed that the assay detected
1
for extraction of AFB from barley is quick and shows a recovery
of 99 ( 5%.
AFB
had low capacity to detect AFM
detection (LOD) achieved for AFB
2
with a sensitivity similar to that for AFB
, AFG , and AFG
was 10 ng mL , rendering
1
, while this method
Among the possible interferences in the detection of AFB using
the assay based on AChE inhibition, the organophosphorous and
carbammic pesticides, which also inhibit the AChE, cannot be
considered as interferences because these compounds are ir-
reversible inhibitors. In fact, for the irreversible inhibition, a
certain incubation time and a low concentration of the enzyme
are necessary. On the contrary, the aflatoxins are reversible
inhibitors and the degree of inhibition is independent of the
enzyme loading and of the incubation time. Thus, we can suppose
that the concentration of the enzyme adopted in this work together
with no incubation should avoid the interferences due to the
pesticides eventually present in real samples.
1
1
2
. The limit of
-
1
1
this method useful for the screening of AFB in feed.
Future work will be focused on reaching a lower LOD (2 ng
mL- ) according to EC regulation (Commission Regulation (EC)
1
1
525/1998) for analysis of aflatoxin in human food.
ACKNOWLEDGMENT
This study was carried out in the framework of the AFLARID
project and financed by the MIPAF of Italy. This work was also
supported by the Protocol for Scientific and Technological Col-
laboration between the Republic of Italy and the Kingdom of
Marocco (2004-2006). The authors thank Prof. J.L. Marty and D.
Fournier for the AChE from Drosophila melanogaster wild type
and mutants. F.A. is very grateful to Prof. J.L Marty for the kind
hospitality in his laboratory. The authors also thank N. Downer
for revising the English manuscript.
Regarding other possible interferences such the heavy metals
4
7
(
reversible inhibitors), we can suppose that these inhibitors
cannot interfere with the proposed assay because in the extraction
step the aflatoxins are extracted into an organic phase. This would
eliminate the presence in the sample solution of heavy metals and
other water-soluble enzymatic inhibitors such as fluoride48 given
their low solubility in organic solvents.
Received for review September 27, 2006. Accepted March
(
47) Stoytcheva, M. Electroanalysis 2002, 14, 923-927.
9, 2007.
(
48) Tran-Minh, C.; Pandey, P. C.; Kuraman, S. Biosens. Bioelectron. 1990, 5,
4
61-471.
AC061819J
Analytical Chemistry, Vol. 79, No. 9, May 1, 2007 3415