aptamer to make aflatoxin sensors

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Biosensors and Bioelectronics 62 (2014) 288–294
Contents lists available at ScienceDirect
Biosensors and Bioelectronics
journal homepage: www.elsevier.com/locate/bios
An aptamer-based dipstick assay for the rapid and simple detection of
a?atoxin B1
Won-Bo Shim a,1, Min Jin Kim a,1, Hyoyoung Mun a, Min-Gon Kim a,b,n
Department of Chemistry, School of Physics and Chemistry, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea
Advanced Photonics Research Institute, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea
art ic l e i nf o
a b s t r a c t
Article history:
Received 25 March 2014
Received in revised form
10 June 2014
Accepted 30 June 2014
Available online 4 July 2014
A rapid and simple dipstick assay based on an aptamer has been developed for the determination of
a?atoxin B1 (AFB1). The dipstick assay format was based on a competitive reaction of the biotin-modi?ed
aptamer speci?c to AFB1 between target and cy5-modi?ed DNA probes. Streptavidin and anti-cy5
antibody as capture reagents were immobilized at test and control lines on a membrane of the dipstick
assay. After optimization, the limit of detection for the dipstick assay was 0.1 ng/ml AFB1 in buffer. The
method was con?rmed to be speci?c to AFB1, and the entire process of the assay can be completed
within 30 min. Aqueous methanol (20%) provided a good extraction ef?ciency, and the matrix in?uence
from corn extracts was successfully reduced through 2-fold dilution. The results of AFB1 analysis for corn
samples spiked with known concentration of AFB1 by the dipstick assay and ELISA showed good
agreement. The cut-off value of the dipstick assay for corn samples was 0.3 ng/g AFB1. Therefore, the
dipstick assay is ?rst reported and considered as a rapid, simple, on-site and inexpensive screening tool
for AFB1 determination in grains as well as a corn.
& 2014 Elsevier B.V. All rights reserved.
A?atoxin B1
Dipstick assay
On-site detection
1. Introduction
A?atoxins are toxic and carcinogenic secondary metabolites
produced by Aspergillus ?avus and A. parasiticus and the most
predominant and toxic of mycotoxins. Since a?atoxins have been
frequently detected in food and agricultural commodities, they
cause signi?cant health and economic problems in many countries
(Bhatnagar et al., 2002; Blesa et al., 2003). Among the a?atoxins,
a?atoxin B1 (AFB1) possesses the highest toxicity and is listed as
group I carcinogens by the International Agency for Research on
Cancer (IARC, 2002). AFB1 is generally detected in agricultural
commodities, such as grains, peanuts, corn, and feedstuffs. In order
to protect human health from exposure to AFB1, many countries
have set regulation limits. For examples, permissible levels set by
the European Union are 2 mg/kg for AFB1 and 4 mg/kg for total
a?atoxins in groundnuts, nuts, dried fruits, and cereals
(Commission Regulation, 2001), whereas Korean maximum levels
is 10 mg/kg for AFB1 and 15 mg/kg for total a?atoxins in food,
respectively (MFDS, 2014).
Corresponding author at: Department of Chemistry, School of Physics and
Chemistry, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712,
Republic of Korea. Tel.: þ 82 62 715 2874; fax: þ82 62 715 3419.
E-mail address: mkim@gist.ac.kr (M.-G. Kim).
These authors contributed equally to this work.
0956-5663/& 2014 Elsevier B.V. All rights reserved.
Therefore, analytical methods for AFB1 are essential to prevent
exposure to the mycotoxin. Various methods based on chromatographic method and immunoassay have been developed and
practically used for the determination of AFB1 in real samples.
Chromatographic methods including a high performance liquid
chromatography (HPLC) and liquid chromatography–mass spectrometry (LC–MS) have been of?cially accepted for the quanti?cation of AFB1 but are time-consuming, unsuitable for large numbers
of samples, laborious, and not amenable to on-site detection (Shim
et al., 2007). Immunoassays are simpler and easier-to-use methods
compared with chromatographic methods and are being used
increasingly for screening of AFB1 in food and agricultural commodities. Especially, enzyme-linked immunosorbent assay (ELISA)
has signi?cantly grown in the development of methods for AFB1.
However, ELISA often requires long reaction times and involves
multiple incubation and washing steps and its utilization has been
con?ned to laboratories equipped with tools and special devices
for analysis (Paek et al., 2000). Thus, these disadvantages make it
dif?cult for on-site detection.
On-site detection technology has received great attention in
the development of analytical methods for mycotoxins determination as well as diagnosing human diseases and hazards in
environmental and food samples (Wang et al., 2011a; Cella et al.,
2010). Therefore, there are many studies that have been conducted
for the development of rapid, simple, and easy-to-use detection
methods to detect mycotoxins. Immunochromatographic and
W.-B. Shim et al. / Biosensors and Bioelectronics 62 (2014) 288–294
dipstick assays are representative on-site detection technologies
and are based on a membrane containing detector and capture
reagents. Both methods combine several bene?ts such as a userfriendly format, long-term stability, and cost-effectiveness. These
properties make both methods attractive for on-site screening by
untrained personnel (Lattanzio et al., 2012; Wang et al., 2011b).
Immunochromatographic and dipstick assays for various mycotoxins such as AFB1, ochratoxin A, zearalenone, fumonisins, T-2 and
HT-2 toxins, and deoxynivalenol have been well developed. However, the assays reported involve the use of antibodies speci?c to
targets (Wang et al., 2011b) and the utilization of antibodies has
many disadvantages in terms of the high cost of production
(economically, labor-wise, and time involved) and their stability
as they are easily denatured during the storage in a buffer solution
(Huang et al., 2012).
Aptamers have been considered as a good alternative to
antibodies. Aptamers are single-stranded DNA or RNA that can
speci?cally bind to a target molecule and offer signi?cant advantages over antibodies as their production is less expensive and
labor-intensive. Aptamers are easier to label with ?uorescent dyes,
enzymes, biotin, and DNA ligands (Jayasena, 1999), and to regenerate by heating (McKeague et al., 2010). An added advantage of
using aptamers is the option of hybridization with complementary
DNA and the deconstruction of hybridization once aptamers meet
a target molecule (Chen et al., 2012). These advantages make
aptamers attractive in the development of low-cost, reusable, and
robust analytical methods.
Recently, several aptamers which are speci?c to mycotoxins
(McKeague et al., 2010; Cruz-Aguado and Penner, 2008) have been
developed. Especially, ochratoxin A aptamer was ?rstly reported in
2008 and has been well investigated to develop various aptamer
sensors. Membrane based chromatographic strip assay (lateral
?ow strip) with ochratoxin A aptamer was reported for on-site
detection of ochratoxin A (Wang et al., 2011a, 2011b). In the
method, two probes (complementary single strand DNAs) were
immobilized on a membrane to hybridize with residue aptamers
on gold nanoparticles. However, it is often dif?cult to hybridize an
aptamer unbound with a target and a complementary single
strand DNA used as a probe since a sample solution quickly passed
the membrane within 10 min and this period was not enough to
get hybridization form on the membrane. Wei et al. (2005)
reported that 500 s as a minimum time to hybridize DNA on a
microarray using a micro?uidic device was required in liquid
solution. Although lateral ?ow strip is simple and user-friendly
format intended to detect the target analyte without the need for
specialized and costly equipment, we considered that a lateral
?ow strip based on an aptamer may be limited with the hybridization of the aptamer and a complementary single strand DNA. On
the other hand, a dipstick assay usually includes two steps, prereaction with a target molcule and a receptor (antibody and
aptamer) and dipping a dipstick composed with an absorbent
pad and a membrane treated with capture reagents that can bind
the receptor. At the pre-reaction step, suf?cient hybridization of
the aptamer and complementary single strand DNA was formed
for negative samples whereas interaction of the aptamer to the
target analyte for positive samples are performed in liquid solution. When a dipstick is dipped into the mixtures, the complexes
(target–aptamer and hybridized aptamer/complementary DNA)
were trapped by the capture reagents immobilized on the membrane. However, to the best of our knowledge, no dipstick assay
based on an aptamer is reported for mycotoxin and any analyte
analysis. In this study, we ?rst reported novel aptamer-based
dipstick assay for the rapid, easy-to-perform, and on-site detection
of AFB1. For this study, we used a biotin-modi?ed aptamer speci?c
to AFB1 and cyanine 5 (Cy5)-modi?ed a single strand-DNA probe,
which can hybridize with the aptamer, used as the ?uorescent
reporter. Streptavidin and anti-cy5 antibody were treated as test
and control zones, respectively. The aptamer-based dipstick assay
was successfully optimized to detect AFB1 and applied to corn
samples arti?cially contaminated with AFB1.
2. Materials and methods
2.1. Chemicals and reagents
AFB1 and related mycotoxins (AFB2, AFG1, AFG2, ochratoxin A,
zearalenone, citrinin, T-2 toxin, deoxynivalenol, and patulin),
bovine serum albumin (BSA), and sodium chloride were purchased
from Sigma (St. Louis, MO, USA). 96-Microwell plates (?at bottom)
were purchased from Nunc (Roskilde, Denmark). An anti-cy5
antibody was obtained from abcam (Cambridge, UK). A nitrocellulose membrane and an absorbent pad were purchased from
Millipore Co. (Bedford, MA, USA). Semirigid polyethylene sheets
were obtained from a local market. All chemicals and organic
solvents were reagent grade. Water used in all experiments was
puri?ed with a Purelab Option Water Puri?cation System (ELGA,
Marlow, UK). Phosphate buffer saline (0.1 M, pH 7.4), carbonate
buffer (25 mM, pH 9.6), borate buffer (1 mM, pH 7.4), Tris–HCl (0.1
M, pH 7.4 and 8.8), MES buffer (25 mM, pH 5.0), acetate buffer (0.1
M, pH 5.0), and deionized water were tested as working buffers for
the dilution of aptamers and DNA probes. Standards of mycotoxins
were prepared by diluting stock solutions of each mycotoxin (1
mg/ml) in absolute methanol. The ChemiDocTM MP System (BioRad, Hercules, CA, USA) was used for the measurement of
?uorescent intensities on a dipstick assay.
2.2. Aptamer and DNA probes
Biotin-modi?ed aptamer to AFB1 and cy5-modi?ed DNA
probes with different length (14 mer and 23 mer) were purchased
from GenoTech Corp. (Daejeon, Korea). The sequences of the
aptamer and DNA probes were as follows:
Biotin-modi?ed aptamer: 5′-biotin-AAA AAA AAA AGT TGG GCA
Cy5-modi?ed DNA probe 1 (14 mer): 5′-cy5-AAA TGT GGG CCT
Cy5-modi?ed DNA probe 2 (23 mer): 5′-cy5-AAA TGT GGG CCT
The sequences in italic type mean the real sequences of
aptamer speci?c to AFB1 which was presented by NeoVenture
Bitechnologyj Inc. (Canada) in 2012 (Patent:PCT/CA2010/001292).
Ten adenines at 5′ end were used as a linker to modify biotin.
Additionally, appropriate lengths of complementary single strand
DNA to an aptamer are needed to form a swithching structures
from aptamer/complementary DNA (a duplex DNA) to aptamer/
target complex (Nutiu and Li, 2003, 2005; Chen et al., 2012)
Therefore, two DNA probes, DNA probes 1 and 2, were designed
for the development of the dipstick assay. The sequences underlined on the DNA probes were portions which can hybridize with
the aptamer.
2.3. Sample preparation
Corn samples were purchased from local markets and tested by
LC/MS to gain AFB1-free corn samples for the preparation of AFB1positive corn samples. For the AFB1 analysis by the dipstick assay,
1 g of corn samples was extracted with 3 ml of methanol/water
(20:80, v/v) for 15 min at room temperature and then centrifuged
at 3075g for 5 min at 4 °C. The supernatants were ?ltered through
W.-B. Shim et al. / Biosensors and Bioelectronics 62 (2014) 288–294
a disposable syringe ?lter (0.45 µm and diluted 2-fold with 0.1 M
Tris–HCl (pH 7.4) to minimize matrix and methanol in?uence. The
diluted extracts were analyzed by the dipstick assay.
2.4. Development of the dipstick assay for AFB1
The format of the dipstick assay was based on an indirect
competitive assay. AFB1 competes with a cy5-modi?ed DNA
probes to bind a biotin-modi?ed aptamer speci?c to AFB1. Fig. 1
shows a construction scheme and the principle of the dipstick
assay for AFB1. The dipstick assay was consisted of an absorbance
pad and a nitrocellulose (NC) membrane. A nitrocellulose membrane was treated with streptavidin and anti-cy5 antibody for test
and control lines, respectively and soaked into 1% BSA in order to
prevent non-speci?c binding of the cy5-modi?ed DNA probe on a
residue surface of the NC membrane. The treated NC membrane
was dried at 37 °C for 20 min. The pad and NC membrane were
placed on a semirigid polyethylene sheets.
The aptamer (0.1 mM), AFB1 standard solutions, and DNA
probes (0.5 mM) were sequentially added into wells of microplate.
The mixtures were incubated for 20 min at 37 °C, and a dipstick
was placed into the wells containing the mixture. In case of the
presence of AFB1, the biotin-modi?ed aptamer ?rstly reacts to
AFB1, and cy5-modi?ed DNA probes cannot hybridize with the
aptamer. The complex of biotin-modi?ed aptamer and AFB1 was
migrated to the NC membrane and trapped by streptavidin at the
test line, and free cy5-modi?ed DNA probes migrated up to anticy5 antibody on the control line. Therefore, one ?uorescent dot
was observed on the NC membrane by the ChemiDocTM MP
System. Whereas, the biotin-modi?ed aptamer and DNA-cy5
probes could be completely hybridized under the absence of
AFB1 that caused the formation of double stranded DNA (hybridized biotin-aptamer/cy5-DNA probe). The formed double
stranded DNA migrated up to the NC membrane and was captured
by streptavidin at the test line, and residue free cy5-modi?ed DNA
probes was trapped by anti-cy5 antibody immobilized at control
line. Consequently, two ?uorescent dots on the membrane were
2.5. Sensitivity and speci?city of the dipstick assay
The sensitivity and speci?city of the dipstick assay were
conducted by analyzing AFB1 standards (0, 0.1, 0.3, 1, 3, and 10
ng/ml) and other mycotoxin standards such as AFB2, AFG1, AFG2,
ochratoxin A, zearalenone, citrinin, T-2 toxin, deoxynivalenol, and
patulin at the concentration of 50 ng/ml. The performance of the
dipstick assay was as described above.
2.6. Analysis of AFB1 in corn samples
AFB1-positive corn samples to validate the aptamer-based
dipstick assay were prepared by spiking known amounts of AFB1
at 1, 0.1, 0.5, 1, 5, and 10 ng/g to AFB1-free corn samples. The
spiked samples were kept at room temperature for 4 h under dark
condition to evaporate methanol used to prepare AFB1 standards.
The corn samples (1 g) were extracted 3 ml of 20% methanol/water
Fig. 1. Schematic illustration of the dipstick assay for the simple and rapid detection of AFB1. A construction of dipstick assay is shown on Upperpart. The procedures and
results of the dipstick assay for negative (bottom left) and positive tests are presented on bottom of the schematic illustration.
W.-B. Shim et al. / Biosensors and Bioelectronics 62 (2014) 288–294
(v/v) for 15 min at room temperature and then centrifuged at
2906g for 5 min at 4 °C. Further steps (?ltration and dilution) for
the sample preparation were as previously described. The sample
extracts were directly applied to the dipstick assay. The results
were compared with those obtained by the enzyme-linked
immuosorbent assay (ELISA). The ELISA analysis for corn samples
spiked with AFB1 was performed according to that described
previously (Kolosova et al., 2007).
3. Results and disscussion
3.1. Optimization of the dipstick assay for AFB1
The dipstick assay developed in this study was based on the
competition reaction of the biotin-modi?ed aptamer between
AFB1 and cy5-modi?ed DNA probes. In the dipstick assay, the
biotin-modi?ed aptamer and cy5-modi?ed DNA probe were used
as a detector and competitor to mycotoxin replacing the roles of
antibodies and mycotoxin–protein conjugates respectively in traditional dipstick assays based on a competitive reaction for
mycotoxin. In the general competitive immunoassay, appropriate
amount of immunoreagents such as antibody and antigen or target
is required to develop the sensitive detection method. In addition,
reaction mode and incubation period are also key parameter for
the development of sensitive immunoassays. For the optimization
of the dipstick assay to AFB1, several experimental parameters
such as, the length of DNA probe, amount of the biotin-modi?ed
aptamer and cy5-modi?ed DNA probe, working buffer, and incubation step, time and temperature were investigated. One of
aptamer properties is that an aptamer can bind target or can be
hybridized with complementary DNA, and the length of complementary DNA may affect the interaction of aptamer and target
(Nutiu and Li, 2003, 2005; Chen et al., 2012). For example, if the
length of complementary DNA is the same with that of an aptamer,
the aptamer tends to strongly hybridize with complementary DNA
than a target, while too short length of the complementary DNA
may be dif?cult to hybridize. In the present study, we synthesized
two types of complementary DNA modi?ed with cy5, 14 mer (DNA
probe 1) and 23 mer (DNA probe 2) as DNA probes. Both DNA
probes were tested to select optimal length of DNA probe producing expected performance on the dipstick assay explained in
Fig. 1. As shown in Fig. 2, both DNA probes worked well on the
dipstick system, but a decrease of ?uorescence intensity performed with biotin-modi?ed aptamer (1 mM), cy5-modi?ed DNA
probe 1 (1 mM) and AFB1 10 ng/ml was much higher compared to
that with biotin-modi?ed aptamer (1 mM), cy5-modi?ed DNA
Fig. 2. Comparison test with two cy5-modi?ed DNA probes. The lengths of DNA
probe 1 and probe 2 are 14 mer and 23 mer. Both DNA probes were linked with
cy5-AAA at the 5′ end. The sequences of DNA probes 1 and 2 and biotin-modi?ed
aptamer are described in Section Materials and methods.
Fig. 3. Selection of appropriate concentrations of biotin-modi?ed aptamer (A) and
cy5-modi?ed DNA probe 1 (B) for the development of the aptamer-based dipstick
assay for AFB1 determination.
probe 2 (1 mM), and AFB1 10 ng/ml. Thus, cy5-modi?ed DNA probe
1 was chosen for further experiments.
In typical competitive immunoassay, increasing concentration
of immunoreagents decreases the sensitivity of immunoassays for
small molecules as well as mycotoxins. Therefore, the utilization of
appropriate amount of immunoreagent is a key factor to develop a
sensitive assay. In this study, various concentrations of biotinmodi?ed aptamer (0.1. 0.5, 1, and 2 mM) and cy5-modi?ed DNA
probe l (0.1, 0.5, and 1 mM) were tested. For the determination of
optimal amount of the biotin-modi?ed aptamer, the different
concentrations of aptamers were mixed with 0, 10, and 100 ng/
ml AFB1 and cy5-modi?ed DNA probe 1 (1 mM) and incubated for
30 min at room temperature. The bottom of the dipstick assay
treated with 0.5 mg streptavidin and 0.5 mg anti-cy5 antibody was
placed into wells containing the mixture a …
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