1Department of Pharmacology and Toxicology School of Pharmacy Ahvaz Jundishapur University of Medical Sciences Ahvaz Iran2Department of Toxicology Faculty of Medical Sciences Tarbiat Modares University Tehran Iran3Protein Technology Research Center Shahid Beheshti University of Medical Sciences Tehran Iran
Correspondence should be addressed to Mohsen Rezaei rezaeimohsengmailcom and Bahram Daraei bdaraeimodaresacir
Received 2 January 2017 Accepted 19 April 2017 Published 14 May 2017
Academic Editor Orish Ebere Orisakwe
Copyright copy 2017 Marzieh Jafari 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
Combination of aptamers with DNAzymes attracted intense attention for development of DNA-based biosensors for detection ofmycotoxins In the present study a combination of aflatoxin B1 specific aptamer and HRP- (horseradish peroxidase-) mimickingDNAzyme was optimized for detecting aflatoxin B1 Detecting approach is based on the binding affinity of aflatoxin B1 to its specificaptamer and conversion of substrate to a detectable colorimetric signal by a linked DNAzyme Compared to conventional methodsfor aflatoxin B1 detection DNA-based assay has the advantages of low cost long-term stability and rapid simple and user-friendlysteps
1 Introduction
Mycotoxins are the well-known secondary metabolites offungi with established adverse health effects in human andanimals [1] Aflatoxins are a group of mycotoxins producedby a variety of Aspergillus species including A flavus and Aparasiticus [2] Human contacts to these toxic metabolitesare inevitable during preharvest storage or processing ofagricultural products [3] AFB1 the most toxic form ofaflatoxins has been linked to various health problems and hasbeen placed in group 1 of human carcinogens by the Inter-national Agency for Research on Cancer (IARC) [4] Foodsafety strategies and imminent contamination of crops withaflatoxins have increased the importance of AFB1 detectionworldwide
Various analytical methods have been developed fordetermination of AFB1 levels in food stuffs [5] Despite theirsensitivity and accuracy chromatography basedmethodsmetsome limitations that can be time consuming and expensivefrom sample preparation to the detection steps Disadvan-tages of protein based assays are stability of antibodies understrict physical and chemical conditions and their long-termproduction processes Consequently it is required to develop
a rapid simple and low cost technique for AFB1 detection infood samples
Numerous DNA sequences with enzymatic functionsbesides carrying of genetic information have been identified[6 7] Single-stranded DNAs because of their folding prop-erties have been enrolled in many analytical procedures fordeveloping of several DNA-based biosensors [8 9]
The term aptamer refers to a single-stranded DNA whichfolds in to three-dimensional structure and interacts with itsspecific target [10] This specific and short single-strandedoligonucleotide is chosen from a random sequence libraryand can bind to a wide variety of small molecule targetsincluding proteins nucleic acids and cell receptors [1112] DNAzyme also known as deoxyribozyme refers to aguanine-rich nucleic acid sequence with catalytic capabilitiessimilar to common enzymes [13] Several single-strandedDNA sequences with enzymatic activity in various colorimet-ric reactions have been identified [14]
DNAzymes and aptamers have remarkable advantagesincluding of low cost preparation and high efficiency [15 16]They are stable in different chemical or physical conditionsIn contrast to proteins they return to their original con-formations when pH and temperature return to the initial
HindawiJournal of ToxicologyVolume 2017 Article ID 2461354 6 pageshttpsdoiorg10115520172461354
2 Journal of Toxicology
Table 1 Sequences of oligonucleotides used in this study ssDNA single stranded DNA B blocker complementary sequence
Scheme 1 Schematic representation of AFB1 detection by conju-gated DNAzyme-aptamer
condition [17 18] and their combination has been recruitedto analyze a wide range of molecules [19ndash22] Based onthese properties many DNA-based biosensors have beenintroduced for detection of mycotoxins [23 24]
In the present study a combination of AFB1 specificaptamer [25] and HRP- (horseradish peroxidase-) mimick-ing DNAzyme [13] was optimized for detecting of AFB1(Scheme 1) Binding of AFB1 to its specific aptamer recogni-tion sequence prevents the blocker from being annealed tothe aptamer and the reaction proceeds to yield a blue colorproduct in a concentration dependent manner
2 Materials and Methods
21 Reagents Aflatoxin B1 (AFB1) TrisHCl sodium chloride(NaCl) magnesium chloride (MgCl2) 3355-tetramethylbe-nzidine (TMB) are purchased from Sigma (USA) Hemin waspurchased from Serva (USA) All the chemical reagents wereof highest grade and used without further purification Allsolutions were prepared with diethyl pyrocarbonate (DEPC)treated deionized water
Oligonucleotides contained a sequence of 49 bp aflatoxinB1 aptamer [25] and 18 bp DNAzyme [13] in two different se-quential orders (51015840-aptamer-DNAzyme-31015840 or 51015840-DNAzyme-aptamer-31015840) and blockers complementary sequences werepurchased from Biolegio (Netherlands) These sequences are
(ii) AFB1 (different concentrations)
Read at 630nm
10 min
10 min
15 min
5min
(v) TMBH2O2 substrate
(iv) Hemin (05휇M)
(iii) Blocker complementary sequence (01휇M)
(i) DNAzyme-aptamer (01휇M)
Scheme 2 Schematic representation for optimized assay procedure
shown in Table 1 Mfold software was used for prediction ofthe secondary structure of used single-stranded nucleic acids[26]
22 Assay Procedure DNA stock solutions (100120583M) wereprepared in DEPC deionized water and stored in smallaliquots DNA working solutions were prepared in incuba-tion buffer (10mM TrisndashHCl pH 8 120mM NaCl 25mMMgCl2 and 5mM KCl) [22] Before starting the experi-ments oligonucleotides were denatured at 95∘C for 5minThen 70 120583L of AFB1 aptamer (final concentration of 01 120583M)incubated with different concentrations of 10120583L of AFB1for 15min 10 120583L of 1120583M blocker complementary sequencewas then added in to 96-well microplate and incubated for10min at room temperature 10 120583L of 05 120583M hemin wasalso added to wells followed by 10min incubation TMBsubstrate containing H
2O2was prepared immediately before
use and 100 120583L of substrate solutionwas added to themixtureFor kinetic assay absorbance of TMB color product wasmeasured at wavelength of 630 nm for 5min (Scheme 2) All
Figure 1 Predicted secondary structure of DNAzyme-aptamer and aptamer-DNAzyme using Mfold tool
absorbances were measured by a BioTek (ELx800) microtiterplate reader (BioTek USA)
3 Results and Discussion
DNAzyme sequence can attach to either 51015840 or 31015840 ends ofaptamer sequence yield DNAzyme-aptamer or aptamer-DNAzyme (Table 1 and Figure 1) In our previous studywe investigated the relationship between the orientationof the DNAzyme and aptamer conjugation and their finalperoxidase activities [27] As seen in Table 2 DNAzyme-aptamer displayed a higher enzymatic activity than 31015840 ori-ented conjugation Aptamer-DNAzyme revealed its priorityfor further evaluations in biosensor design
For achieving of best results several parameters wereoptimized including blocker complementary sequences (B1B2 and B3) selection incubation times reagent concen-trations and their order of addition Optimal annealing toDNAzyme-aptamer and inhibiting of its enzymatic activity
Table 2 Kinetic parameters for DNAzyme-aptamer and aptamer-DNAzyme catalytic activity
were influenced mainly through the blocker complementarysequence B1 B2 and B3 were designed with different num-bers and sequences of nucleotides As shown in Figure 2the blockade of peroxidase activity has considerably beenattained by B3 sequence compared to B1 and B2
At the next step different molar ratios of DNAzyme-aptamer and B3 blocker from 1 1 to 1 5 were tested With aconstant amount of DNAzyme-aptamer (01120583M) increasingthe blocker concentration from 01 to 05120583M resulted ina concentration dependent decline of DNAzyme activity(Figure 3) Given that AFB1 and complementary sequence of
4 Journal of Toxicology
000
005
010
015
020
Abso
rban
ce
Apt + B1Apt Apt + B3Apt + B2
Figure 2 Blockade of peroxidase activity of DNAzyme-aptamerby B1 B2 and B3 sequences Apt DNAzyme-aptamer B blockercomplementary sequence
000
002
004
006
008
010
Abso
rban
ce
02 03 04 0501(휇M)
Figure 3 Blockade of peroxidase activity of DNAzyme-aptamer byincreasing concentration of B3
blocker compete with each other for binding to DNAzyme-aptamer blocker at high concentration decreases the sensi-tivity of assay So a conservative lower molar ratio of 1 1 wasselected for further evaluations
Orders of addition of AFB1 blocker complementarysequence and hemin were evaluated in different settingsResults showed that adding blocker prior to AFB1 resulted inunaffected remaining of blocker at its binding site ThereforeAFB1 was scheduled to incubate with aptamer before theblocker addition Also we observed that enzymatic activ-ity will be temporally influenced when hemin is addedto the reaction containing blocker As hemin preventedthe hybridization of blocker complementary sequence andDNAzyme-aptamer it was added subsequently to the blockersequences addition
The best incubation times for AFB1 blocker complemen-tary sequence and hemin were achieved via incubation of allreagents separately at 10min intervals up to 60 minutes Theshortest and most efficient incubation times were 15 10 and10min for AFB1 blocker and hemin respectively
Figure 4 Peroxidase activity of DNAzyme-aptamer in the presenceof B3 anddifferent concentrations ofAFB1AptDNAzyme-aptamerB blocker complementary sequence and AFB1 aflatoxin B1
In the present work the sensing strategy is based on thebinding affinities of AFB1 to its specific aptamer that producea detectable colorimetric signal by DNAzyme (Scheme 1)In the absence of AFB1 annealing of blocker sequence(complementary sequence to a part of DNAzyme-aptamer)to DNAzyme-aptamer decreases the enzymatic activity Inthe presence of AFB1 the aptamer binds to AFB1 and formsa hairpin structure Consequently blocker complementarysequence was prevented from being bound to DNAzyme-aptamer and following addition of heminDNAzyme displaysa colorimetric signal that is directly associated with AFB1concentrations (Figure 4)
Under optimal conditions the limit of detection of10 ngmL was achieved AFB1 aptamer has been used asa recognition probe in several detection systems based onPCR electrochemical chemiluminescent colorimetric andfluorescent platforms In Guo et alrsquos study AFB1 aptamerwith 31015840-terminal biotin groups has been immobilized onthe surface of PCR tubes for developing of an aptasensor(LOD 25 fgmL) based on RT-qPCR [28] An aptamer-based dipstick assay (LOD 01 ngmL) using biotin-modifiedaptamer has also been reported by Shim et al [29] In anotherwork by Shim et al based on chemiluminescence competitiveassay AFB1-OVA conjugate was coated on the wells (LOD011 ngmL) [30] Castillo et al have developed an aptamer-based biosensor (LOD 040 nM) using immobilization ofamino-modified aptamers and electrochemistry [25]
One of the important goals of this work was designing asimple and cost-effective method for AB1 detection withoutintricate steps and equipment In thementioned publicationsaptamer has been modified with functional groups or immo-bilized on surfaces In our experiment AB1 aptamer has been
Journal of Toxicology 5
employed without any immobilization and modification Allsteps of procedure were done as a simple solution Sinceaptamer interaction with its target depends on folding intounique structures [31] intact aptamers were excepted to havemore appropriate folding
AFB1 aptamer and DNAzyme also have also been usedby Seok et al with some alterations [32] In their studytwo split DNAzyme halves anneal with aptamer that formG-quadruplex The AFB1 aptamer complex prevented theannealing of split DNAzyme and aptamer therefore weakcolor intensity will be observed upon addition of ABTSsubstrate The accuracy of this method is depending onthe correct annealing of two split DNAzymes with aptamerThe amount of this annealing may be variable in eachperformance and cause false negative results In our workto have the stable signal DNAzyme sequence has beenattached to 51015840 ends of aptamer sequenceTherefor its catalyticactivity remained constant in all experiments Also in theirstudy all reagents including split DNAzyme probes aptamerhemin and AB1 were added simultaneously while our resultsshowed that the order of addition of reagents is an importantparameter
4 Conclusions
Advantages of using DNAzymes and aptamers over proteinenzymes and antibodies have been reported in many studiesIn this study we optimized a colorimetric simple assay usingDNAzyme-aptamer conjugate to detect AFB1 Under opti-mized conditions the formation of AFB1 aptamer complexprevents the hybridization of its complementary sequencesHence the catalytic activity of DNAzyme increases corre-sponding to AFB1 concentration To improve the procedurewe will work on the limit of detection and sensitivity of thisaptasensor for a more accurate and sensitive determinationof AFB1
Conflicts of Interest
The authors declare that they have no conflicts of interest
Acknowledgments
This work was supported by a Grant (U-94080) from theAhvaz Jundishapur University of Medical Sciences AhvazIran
References
[1] S Marin A J Ramos G Cano-Sancho and V Sanchis ldquoMyco-toxins occurrence toxicology and exposure assessmentrdquo Foodand Chemical Toxicology vol 60 pp 218ndash237 2013
[2] M A Gacem and A Ould El Hadj-Khelil ldquoToxicology biosyn-thesis bio-control of aflatoxin and new methods of detectionrdquoAsian Pacific Journal of Tropical Biomedicine vol 6 no 9 pp808ndash814 2016
[3] S Z Iqbal M R Asi and A Arino ldquoAflatoxinsrdquo in ReferenceModule in Life Sciences Elsevier 2017
[4] M E Smela S S Currier E A Bailey and J M EssigmannldquoThe chemistry and biology of aflatoxin B
1 from mutational
spectrometry to carcinogenesisrdquo Carcinogenesis vol 22 no 4pp 535ndash545 2001
[5] A P Wacoo D Wendiro P C Vuzi and J F HawumbaldquoMethods for detection of aflatoxins in agricultural food cropsrdquoJournal of Applied Chemistry vol 2014 Article ID 706291 15pages 2014
[6] M Darmostuk S Rimpelova H Gbelcova and T RumlldquoCurrent approaches in SELEX an update to aptamer selectiontechnologyrdquoBiotechnology Advances vol 33 no 6 pp 1141ndash11612014
[7] S Tom H-E Jin and S-W Lee ldquoAptamers as functional bio-nanomaterials for sensor applications a2mdashgrumezescu alexan-dru mihairdquo in Engineering of Nanobiomaterials chapter 6 pp181ndash226 William Andrew Publishing 2016
[8] C Acquah M K Danquah J L S Yon A Sidhu and CM Ongkudon ldquoA review on immobilised aptamers for highthroughput biomolecular detection and screeningrdquo AnalyticaChimica Acta vol 888 pp 10ndash18 2015
[9] Y Seok Kim N H Ahmad Raston andM Bock Gu ldquoAptamer-based nanobiosensorsrdquo Biosensors and Bioelectronics vol 76pp 2ndash19 2016
[10] R Nezlin ldquoUse of aptamers in immunoassaysrdquo MolecularImmunology vol 70 pp 149ndash154 2016
[11] M Tabarzad and M Jafari ldquoTrends in the design and develop-ment of specific aptamers against peptides and proteinsrdquoProteinJournal vol 35 no 2 pp 81ndash99 2016
[12] A Chen and S Yang ldquoReplacing antibodies with aptamers inlateral flow immunoassayrdquo Biosensors and Bioelectronics vol 71pp 230ndash242 2015
[13] J Kosman and B Juskowiak ldquoPeroxidase-mimickingDNAzymes for biosensing applications a reviewrdquo AnalyticaChimica Acta vol 707 no 1-2 pp 7ndash17 2011
[14] X-H Zhao H-M Meng L Gong L Qiu X-B Zhang andW-H Tan ldquoRecent progress of DNAzyme-nanomaterial basedbiosensorsrdquoChinese Journal of Analytical Chemistry vol 43 no11 pp 1611ndash1619 2015
[15] S Mackay D Wishart J Z Xing and J Chen ldquoDevelopingtrends in aptamer-based biosensor devices and their applica-tionsrdquo IEEE Transactions on Biomedical Circuits and Systemsvol 8 no 1 pp 4ndash14 2014
[16] S M Bone N E Lima and A V Todd ldquoDNAzyme switchesformolecular computation and signal amplificationrdquo Biosensorsand Bioelectronics vol 70 pp 330ndash337 2015
[17] MMcKeague andM C Derosa ldquoChallenges and opportunitiesfor small molecule aptamer developmentrdquo Journal of NucleicAcids vol 2012 Article ID 748913 20 pages 2012
[18] M Citartan S C B Gopinath J Tominaga S-C Tan and T-H Tang ldquoAssays for aptamer-based platformsrdquo Biosensors andBioelectronics vol 34 no 1 pp 1ndash11 2012
[19] X Gong J Li W Zhou Y Xiang R Yuan and Y Chai ldquoTargetrecycling amplification for label-free and sensitive colorimetricdetection of adenosine triphosphate based on un-modifiedaptamers andDNAzymesrdquoAnalytica Chimica Acta vol 828 pp80ndash84 2014
[20] H Mun E-J Jo T Li et al ldquoHomogeneous assay of tar-get molecules based on chemiluminescence resonance energytransfer (CRET) using DNAzyme-linked aptamersrdquo Biosensorsand Bioelectronics vol 58 pp 308ndash313 2014
6 Journal of Toxicology
[21] C Yang V Lates B Prieto-Simon J-L Marty and X YangldquoAptamer-DNAzyme hairpins for biosensing of Ochratoxin ArdquoBiosensors and Bioelectronics vol 32 no 1 pp 208ndash212 2012
[22] C Yang V Lates B Prieto-Simon J-L Marty and X YangldquoRapid high-throughput analysis of ochratoxin A by the self-assembly of DNAzyme-aptamer conjugates in winerdquo Talantavol 116 pp 520ndash526 2013
[23] R Chauhan J Singh T Sachdev T Basu and B D MalhotraldquoRecent advances in mycotoxins detectionrdquo Biosensors andBioelectronics vol 81 pp 532ndash545 2016
[24] X-H Yang W-J Kong M-H Yang M Zhao and Z OuyangldquoApplication of aptamer identification technology in rapid anal-ysis ofmycotoxinsrdquoChinese Journal of Analytical Chemistry vol41 no 2 pp 297ndash306 2013
[25] G Castillo K Spinella A Poturnayova M Snejdarkova LMosiello and T Hianik ldquoDetection of aflatoxin B1 by aptamer-based biosensor using PAMAM dendrimers as immobilizationplatformrdquo Food Control vol 52 pp 9ndash18 2015
[26] M Zuker ldquoMfold web server for nucleic acid folding andhybridization predictionrdquoNucleic Acids Research vol 31 no 13pp 3406ndash3415 2003
[27] M Jafari M Rezaei H Kalantari M Tabarzad and BDaraei ldquoDNAzyme-aptamer or aptamer-DNAzyme paradigmbiochemical approach for aflatoxin analysisrdquo Biotechnology andApplied Biochemistry In press
[28] X Guo F Wen N Zheng et al ldquoDevelopment of an ultrasensi-tive aptasensor for the detection of aflatoxin B1rdquo Biosensors andBioelectronics vol 56 pp 340ndash344 2014
[29] W B Shim M J Kim H Mun and M G Kim ldquoAn aptamer-based dipstick assay for the rapid and simple detection ofaflatoxin B1rdquo Biosensors and Bioelectronics vol 62 pp 288ndash2942014
[30] W-B ShimHMunH-A Joung J A Ofori D-H Chung andM-G Kim ldquoChemiluminescence competitive aptamer assayfor the detection of aflatoxin B1 in corn samplesrdquo Food Controlvol 36 no 1 pp 30ndash35 2014
[31] H Hasegawa N Savory K Abe and K Ikebukuro ldquoMethodsfor improving aptamer binding affinityrdquo Molecules vol 21 no4 article 421 2016
[32] Y Seok J-Y Byun W-B Shim and M-G Kim ldquoA structure-switchable aptasensor for aflatoxin B1 detection based onassembly of an aptamersplit DNAzymerdquo Analytica ChimicaActa vol 886 pp 182ndash187 2015
Submit your manuscripts athttpswwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Scheme 1 Schematic representation of AFB1 detection by conju-gated DNAzyme-aptamer
condition [17 18] and their combination has been recruitedto analyze a wide range of molecules [19ndash22] Based onthese properties many DNA-based biosensors have beenintroduced for detection of mycotoxins [23 24]
In the present study a combination of AFB1 specificaptamer [25] and HRP- (horseradish peroxidase-) mimick-ing DNAzyme [13] was optimized for detecting of AFB1(Scheme 1) Binding of AFB1 to its specific aptamer recogni-tion sequence prevents the blocker from being annealed tothe aptamer and the reaction proceeds to yield a blue colorproduct in a concentration dependent manner
2 Materials and Methods
21 Reagents Aflatoxin B1 (AFB1) TrisHCl sodium chloride(NaCl) magnesium chloride (MgCl2) 3355-tetramethylbe-nzidine (TMB) are purchased from Sigma (USA) Hemin waspurchased from Serva (USA) All the chemical reagents wereof highest grade and used without further purification Allsolutions were prepared with diethyl pyrocarbonate (DEPC)treated deionized water
Oligonucleotides contained a sequence of 49 bp aflatoxinB1 aptamer [25] and 18 bp DNAzyme [13] in two different se-quential orders (51015840-aptamer-DNAzyme-31015840 or 51015840-DNAzyme-aptamer-31015840) and blockers complementary sequences werepurchased from Biolegio (Netherlands) These sequences are
(ii) AFB1 (different concentrations)
Read at 630nm
10 min
10 min
15 min
5min
(v) TMBH2O2 substrate
(iv) Hemin (05휇M)
(iii) Blocker complementary sequence (01휇M)
(i) DNAzyme-aptamer (01휇M)
Scheme 2 Schematic representation for optimized assay procedure
shown in Table 1 Mfold software was used for prediction ofthe secondary structure of used single-stranded nucleic acids[26]
22 Assay Procedure DNA stock solutions (100120583M) wereprepared in DEPC deionized water and stored in smallaliquots DNA working solutions were prepared in incuba-tion buffer (10mM TrisndashHCl pH 8 120mM NaCl 25mMMgCl2 and 5mM KCl) [22] Before starting the experi-ments oligonucleotides were denatured at 95∘C for 5minThen 70 120583L of AFB1 aptamer (final concentration of 01 120583M)incubated with different concentrations of 10120583L of AFB1for 15min 10 120583L of 1120583M blocker complementary sequencewas then added in to 96-well microplate and incubated for10min at room temperature 10 120583L of 05 120583M hemin wasalso added to wells followed by 10min incubation TMBsubstrate containing H
2O2was prepared immediately before
use and 100 120583L of substrate solutionwas added to themixtureFor kinetic assay absorbance of TMB color product wasmeasured at wavelength of 630 nm for 5min (Scheme 2) All
Figure 1 Predicted secondary structure of DNAzyme-aptamer and aptamer-DNAzyme using Mfold tool
absorbances were measured by a BioTek (ELx800) microtiterplate reader (BioTek USA)
3 Results and Discussion
DNAzyme sequence can attach to either 51015840 or 31015840 ends ofaptamer sequence yield DNAzyme-aptamer or aptamer-DNAzyme (Table 1 and Figure 1) In our previous studywe investigated the relationship between the orientationof the DNAzyme and aptamer conjugation and their finalperoxidase activities [27] As seen in Table 2 DNAzyme-aptamer displayed a higher enzymatic activity than 31015840 ori-ented conjugation Aptamer-DNAzyme revealed its priorityfor further evaluations in biosensor design
For achieving of best results several parameters wereoptimized including blocker complementary sequences (B1B2 and B3) selection incubation times reagent concen-trations and their order of addition Optimal annealing toDNAzyme-aptamer and inhibiting of its enzymatic activity
Table 2 Kinetic parameters for DNAzyme-aptamer and aptamer-DNAzyme catalytic activity
were influenced mainly through the blocker complementarysequence B1 B2 and B3 were designed with different num-bers and sequences of nucleotides As shown in Figure 2the blockade of peroxidase activity has considerably beenattained by B3 sequence compared to B1 and B2
At the next step different molar ratios of DNAzyme-aptamer and B3 blocker from 1 1 to 1 5 were tested With aconstant amount of DNAzyme-aptamer (01120583M) increasingthe blocker concentration from 01 to 05120583M resulted ina concentration dependent decline of DNAzyme activity(Figure 3) Given that AFB1 and complementary sequence of
4 Journal of Toxicology
000
005
010
015
020
Abso
rban
ce
Apt + B1Apt Apt + B3Apt + B2
Figure 2 Blockade of peroxidase activity of DNAzyme-aptamerby B1 B2 and B3 sequences Apt DNAzyme-aptamer B blockercomplementary sequence
000
002
004
006
008
010
Abso
rban
ce
02 03 04 0501(휇M)
Figure 3 Blockade of peroxidase activity of DNAzyme-aptamer byincreasing concentration of B3
blocker compete with each other for binding to DNAzyme-aptamer blocker at high concentration decreases the sensi-tivity of assay So a conservative lower molar ratio of 1 1 wasselected for further evaluations
Orders of addition of AFB1 blocker complementarysequence and hemin were evaluated in different settingsResults showed that adding blocker prior to AFB1 resulted inunaffected remaining of blocker at its binding site ThereforeAFB1 was scheduled to incubate with aptamer before theblocker addition Also we observed that enzymatic activ-ity will be temporally influenced when hemin is addedto the reaction containing blocker As hemin preventedthe hybridization of blocker complementary sequence andDNAzyme-aptamer it was added subsequently to the blockersequences addition
The best incubation times for AFB1 blocker complemen-tary sequence and hemin were achieved via incubation of allreagents separately at 10min intervals up to 60 minutes Theshortest and most efficient incubation times were 15 10 and10min for AFB1 blocker and hemin respectively
Figure 4 Peroxidase activity of DNAzyme-aptamer in the presenceof B3 anddifferent concentrations ofAFB1AptDNAzyme-aptamerB blocker complementary sequence and AFB1 aflatoxin B1
In the present work the sensing strategy is based on thebinding affinities of AFB1 to its specific aptamer that producea detectable colorimetric signal by DNAzyme (Scheme 1)In the absence of AFB1 annealing of blocker sequence(complementary sequence to a part of DNAzyme-aptamer)to DNAzyme-aptamer decreases the enzymatic activity Inthe presence of AFB1 the aptamer binds to AFB1 and formsa hairpin structure Consequently blocker complementarysequence was prevented from being bound to DNAzyme-aptamer and following addition of heminDNAzyme displaysa colorimetric signal that is directly associated with AFB1concentrations (Figure 4)
Under optimal conditions the limit of detection of10 ngmL was achieved AFB1 aptamer has been used asa recognition probe in several detection systems based onPCR electrochemical chemiluminescent colorimetric andfluorescent platforms In Guo et alrsquos study AFB1 aptamerwith 31015840-terminal biotin groups has been immobilized onthe surface of PCR tubes for developing of an aptasensor(LOD 25 fgmL) based on RT-qPCR [28] An aptamer-based dipstick assay (LOD 01 ngmL) using biotin-modifiedaptamer has also been reported by Shim et al [29] In anotherwork by Shim et al based on chemiluminescence competitiveassay AFB1-OVA conjugate was coated on the wells (LOD011 ngmL) [30] Castillo et al have developed an aptamer-based biosensor (LOD 040 nM) using immobilization ofamino-modified aptamers and electrochemistry [25]
One of the important goals of this work was designing asimple and cost-effective method for AB1 detection withoutintricate steps and equipment In thementioned publicationsaptamer has been modified with functional groups or immo-bilized on surfaces In our experiment AB1 aptamer has been
Journal of Toxicology 5
employed without any immobilization and modification Allsteps of procedure were done as a simple solution Sinceaptamer interaction with its target depends on folding intounique structures [31] intact aptamers were excepted to havemore appropriate folding
AFB1 aptamer and DNAzyme also have also been usedby Seok et al with some alterations [32] In their studytwo split DNAzyme halves anneal with aptamer that formG-quadruplex The AFB1 aptamer complex prevented theannealing of split DNAzyme and aptamer therefore weakcolor intensity will be observed upon addition of ABTSsubstrate The accuracy of this method is depending onthe correct annealing of two split DNAzymes with aptamerThe amount of this annealing may be variable in eachperformance and cause false negative results In our workto have the stable signal DNAzyme sequence has beenattached to 51015840 ends of aptamer sequenceTherefor its catalyticactivity remained constant in all experiments Also in theirstudy all reagents including split DNAzyme probes aptamerhemin and AB1 were added simultaneously while our resultsshowed that the order of addition of reagents is an importantparameter
4 Conclusions
Advantages of using DNAzymes and aptamers over proteinenzymes and antibodies have been reported in many studiesIn this study we optimized a colorimetric simple assay usingDNAzyme-aptamer conjugate to detect AFB1 Under opti-mized conditions the formation of AFB1 aptamer complexprevents the hybridization of its complementary sequencesHence the catalytic activity of DNAzyme increases corre-sponding to AFB1 concentration To improve the procedurewe will work on the limit of detection and sensitivity of thisaptasensor for a more accurate and sensitive determinationof AFB1
Conflicts of Interest
The authors declare that they have no conflicts of interest
Acknowledgments
This work was supported by a Grant (U-94080) from theAhvaz Jundishapur University of Medical Sciences AhvazIran
References
[1] S Marin A J Ramos G Cano-Sancho and V Sanchis ldquoMyco-toxins occurrence toxicology and exposure assessmentrdquo Foodand Chemical Toxicology vol 60 pp 218ndash237 2013
[2] M A Gacem and A Ould El Hadj-Khelil ldquoToxicology biosyn-thesis bio-control of aflatoxin and new methods of detectionrdquoAsian Pacific Journal of Tropical Biomedicine vol 6 no 9 pp808ndash814 2016
[3] S Z Iqbal M R Asi and A Arino ldquoAflatoxinsrdquo in ReferenceModule in Life Sciences Elsevier 2017
[4] M E Smela S S Currier E A Bailey and J M EssigmannldquoThe chemistry and biology of aflatoxin B
1 from mutational
spectrometry to carcinogenesisrdquo Carcinogenesis vol 22 no 4pp 535ndash545 2001
[5] A P Wacoo D Wendiro P C Vuzi and J F HawumbaldquoMethods for detection of aflatoxins in agricultural food cropsrdquoJournal of Applied Chemistry vol 2014 Article ID 706291 15pages 2014
[6] M Darmostuk S Rimpelova H Gbelcova and T RumlldquoCurrent approaches in SELEX an update to aptamer selectiontechnologyrdquoBiotechnology Advances vol 33 no 6 pp 1141ndash11612014
[7] S Tom H-E Jin and S-W Lee ldquoAptamers as functional bio-nanomaterials for sensor applications a2mdashgrumezescu alexan-dru mihairdquo in Engineering of Nanobiomaterials chapter 6 pp181ndash226 William Andrew Publishing 2016
[8] C Acquah M K Danquah J L S Yon A Sidhu and CM Ongkudon ldquoA review on immobilised aptamers for highthroughput biomolecular detection and screeningrdquo AnalyticaChimica Acta vol 888 pp 10ndash18 2015
[9] Y Seok Kim N H Ahmad Raston andM Bock Gu ldquoAptamer-based nanobiosensorsrdquo Biosensors and Bioelectronics vol 76pp 2ndash19 2016
[10] R Nezlin ldquoUse of aptamers in immunoassaysrdquo MolecularImmunology vol 70 pp 149ndash154 2016
[11] M Tabarzad and M Jafari ldquoTrends in the design and develop-ment of specific aptamers against peptides and proteinsrdquoProteinJournal vol 35 no 2 pp 81ndash99 2016
[12] A Chen and S Yang ldquoReplacing antibodies with aptamers inlateral flow immunoassayrdquo Biosensors and Bioelectronics vol 71pp 230ndash242 2015
[13] J Kosman and B Juskowiak ldquoPeroxidase-mimickingDNAzymes for biosensing applications a reviewrdquo AnalyticaChimica Acta vol 707 no 1-2 pp 7ndash17 2011
[14] X-H Zhao H-M Meng L Gong L Qiu X-B Zhang andW-H Tan ldquoRecent progress of DNAzyme-nanomaterial basedbiosensorsrdquoChinese Journal of Analytical Chemistry vol 43 no11 pp 1611ndash1619 2015
[15] S Mackay D Wishart J Z Xing and J Chen ldquoDevelopingtrends in aptamer-based biosensor devices and their applica-tionsrdquo IEEE Transactions on Biomedical Circuits and Systemsvol 8 no 1 pp 4ndash14 2014
[16] S M Bone N E Lima and A V Todd ldquoDNAzyme switchesformolecular computation and signal amplificationrdquo Biosensorsand Bioelectronics vol 70 pp 330ndash337 2015
[17] MMcKeague andM C Derosa ldquoChallenges and opportunitiesfor small molecule aptamer developmentrdquo Journal of NucleicAcids vol 2012 Article ID 748913 20 pages 2012
[18] M Citartan S C B Gopinath J Tominaga S-C Tan and T-H Tang ldquoAssays for aptamer-based platformsrdquo Biosensors andBioelectronics vol 34 no 1 pp 1ndash11 2012
[19] X Gong J Li W Zhou Y Xiang R Yuan and Y Chai ldquoTargetrecycling amplification for label-free and sensitive colorimetricdetection of adenosine triphosphate based on un-modifiedaptamers andDNAzymesrdquoAnalytica Chimica Acta vol 828 pp80ndash84 2014
[20] H Mun E-J Jo T Li et al ldquoHomogeneous assay of tar-get molecules based on chemiluminescence resonance energytransfer (CRET) using DNAzyme-linked aptamersrdquo Biosensorsand Bioelectronics vol 58 pp 308ndash313 2014
6 Journal of Toxicology
[21] C Yang V Lates B Prieto-Simon J-L Marty and X YangldquoAptamer-DNAzyme hairpins for biosensing of Ochratoxin ArdquoBiosensors and Bioelectronics vol 32 no 1 pp 208ndash212 2012
[22] C Yang V Lates B Prieto-Simon J-L Marty and X YangldquoRapid high-throughput analysis of ochratoxin A by the self-assembly of DNAzyme-aptamer conjugates in winerdquo Talantavol 116 pp 520ndash526 2013
[23] R Chauhan J Singh T Sachdev T Basu and B D MalhotraldquoRecent advances in mycotoxins detectionrdquo Biosensors andBioelectronics vol 81 pp 532ndash545 2016
[24] X-H Yang W-J Kong M-H Yang M Zhao and Z OuyangldquoApplication of aptamer identification technology in rapid anal-ysis ofmycotoxinsrdquoChinese Journal of Analytical Chemistry vol41 no 2 pp 297ndash306 2013
[25] G Castillo K Spinella A Poturnayova M Snejdarkova LMosiello and T Hianik ldquoDetection of aflatoxin B1 by aptamer-based biosensor using PAMAM dendrimers as immobilizationplatformrdquo Food Control vol 52 pp 9ndash18 2015
[26] M Zuker ldquoMfold web server for nucleic acid folding andhybridization predictionrdquoNucleic Acids Research vol 31 no 13pp 3406ndash3415 2003
[27] M Jafari M Rezaei H Kalantari M Tabarzad and BDaraei ldquoDNAzyme-aptamer or aptamer-DNAzyme paradigmbiochemical approach for aflatoxin analysisrdquo Biotechnology andApplied Biochemistry In press
[28] X Guo F Wen N Zheng et al ldquoDevelopment of an ultrasensi-tive aptasensor for the detection of aflatoxin B1rdquo Biosensors andBioelectronics vol 56 pp 340ndash344 2014
[29] W B Shim M J Kim H Mun and M G Kim ldquoAn aptamer-based dipstick assay for the rapid and simple detection ofaflatoxin B1rdquo Biosensors and Bioelectronics vol 62 pp 288ndash2942014
[30] W-B ShimHMunH-A Joung J A Ofori D-H Chung andM-G Kim ldquoChemiluminescence competitive aptamer assayfor the detection of aflatoxin B1 in corn samplesrdquo Food Controlvol 36 no 1 pp 30ndash35 2014
[31] H Hasegawa N Savory K Abe and K Ikebukuro ldquoMethodsfor improving aptamer binding affinityrdquo Molecules vol 21 no4 article 421 2016
[32] Y Seok J-Y Byun W-B Shim and M-G Kim ldquoA structure-switchable aptasensor for aflatoxin B1 detection based onassembly of an aptamersplit DNAzymerdquo Analytica ChimicaActa vol 886 pp 182ndash187 2015
Submit your manuscripts athttpswwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Figure 1 Predicted secondary structure of DNAzyme-aptamer and aptamer-DNAzyme using Mfold tool
absorbances were measured by a BioTek (ELx800) microtiterplate reader (BioTek USA)
3 Results and Discussion
DNAzyme sequence can attach to either 51015840 or 31015840 ends ofaptamer sequence yield DNAzyme-aptamer or aptamer-DNAzyme (Table 1 and Figure 1) In our previous studywe investigated the relationship between the orientationof the DNAzyme and aptamer conjugation and their finalperoxidase activities [27] As seen in Table 2 DNAzyme-aptamer displayed a higher enzymatic activity than 31015840 ori-ented conjugation Aptamer-DNAzyme revealed its priorityfor further evaluations in biosensor design
For achieving of best results several parameters wereoptimized including blocker complementary sequences (B1B2 and B3) selection incubation times reagent concen-trations and their order of addition Optimal annealing toDNAzyme-aptamer and inhibiting of its enzymatic activity
Table 2 Kinetic parameters for DNAzyme-aptamer and aptamer-DNAzyme catalytic activity
were influenced mainly through the blocker complementarysequence B1 B2 and B3 were designed with different num-bers and sequences of nucleotides As shown in Figure 2the blockade of peroxidase activity has considerably beenattained by B3 sequence compared to B1 and B2
At the next step different molar ratios of DNAzyme-aptamer and B3 blocker from 1 1 to 1 5 were tested With aconstant amount of DNAzyme-aptamer (01120583M) increasingthe blocker concentration from 01 to 05120583M resulted ina concentration dependent decline of DNAzyme activity(Figure 3) Given that AFB1 and complementary sequence of
4 Journal of Toxicology
000
005
010
015
020
Abso
rban
ce
Apt + B1Apt Apt + B3Apt + B2
Figure 2 Blockade of peroxidase activity of DNAzyme-aptamerby B1 B2 and B3 sequences Apt DNAzyme-aptamer B blockercomplementary sequence
000
002
004
006
008
010
Abso
rban
ce
02 03 04 0501(휇M)
Figure 3 Blockade of peroxidase activity of DNAzyme-aptamer byincreasing concentration of B3
blocker compete with each other for binding to DNAzyme-aptamer blocker at high concentration decreases the sensi-tivity of assay So a conservative lower molar ratio of 1 1 wasselected for further evaluations
Orders of addition of AFB1 blocker complementarysequence and hemin were evaluated in different settingsResults showed that adding blocker prior to AFB1 resulted inunaffected remaining of blocker at its binding site ThereforeAFB1 was scheduled to incubate with aptamer before theblocker addition Also we observed that enzymatic activ-ity will be temporally influenced when hemin is addedto the reaction containing blocker As hemin preventedthe hybridization of blocker complementary sequence andDNAzyme-aptamer it was added subsequently to the blockersequences addition
The best incubation times for AFB1 blocker complemen-tary sequence and hemin were achieved via incubation of allreagents separately at 10min intervals up to 60 minutes Theshortest and most efficient incubation times were 15 10 and10min for AFB1 blocker and hemin respectively
Figure 4 Peroxidase activity of DNAzyme-aptamer in the presenceof B3 anddifferent concentrations ofAFB1AptDNAzyme-aptamerB blocker complementary sequence and AFB1 aflatoxin B1
In the present work the sensing strategy is based on thebinding affinities of AFB1 to its specific aptamer that producea detectable colorimetric signal by DNAzyme (Scheme 1)In the absence of AFB1 annealing of blocker sequence(complementary sequence to a part of DNAzyme-aptamer)to DNAzyme-aptamer decreases the enzymatic activity Inthe presence of AFB1 the aptamer binds to AFB1 and formsa hairpin structure Consequently blocker complementarysequence was prevented from being bound to DNAzyme-aptamer and following addition of heminDNAzyme displaysa colorimetric signal that is directly associated with AFB1concentrations (Figure 4)
Under optimal conditions the limit of detection of10 ngmL was achieved AFB1 aptamer has been used asa recognition probe in several detection systems based onPCR electrochemical chemiluminescent colorimetric andfluorescent platforms In Guo et alrsquos study AFB1 aptamerwith 31015840-terminal biotin groups has been immobilized onthe surface of PCR tubes for developing of an aptasensor(LOD 25 fgmL) based on RT-qPCR [28] An aptamer-based dipstick assay (LOD 01 ngmL) using biotin-modifiedaptamer has also been reported by Shim et al [29] In anotherwork by Shim et al based on chemiluminescence competitiveassay AFB1-OVA conjugate was coated on the wells (LOD011 ngmL) [30] Castillo et al have developed an aptamer-based biosensor (LOD 040 nM) using immobilization ofamino-modified aptamers and electrochemistry [25]
One of the important goals of this work was designing asimple and cost-effective method for AB1 detection withoutintricate steps and equipment In thementioned publicationsaptamer has been modified with functional groups or immo-bilized on surfaces In our experiment AB1 aptamer has been
Journal of Toxicology 5
employed without any immobilization and modification Allsteps of procedure were done as a simple solution Sinceaptamer interaction with its target depends on folding intounique structures [31] intact aptamers were excepted to havemore appropriate folding
AFB1 aptamer and DNAzyme also have also been usedby Seok et al with some alterations [32] In their studytwo split DNAzyme halves anneal with aptamer that formG-quadruplex The AFB1 aptamer complex prevented theannealing of split DNAzyme and aptamer therefore weakcolor intensity will be observed upon addition of ABTSsubstrate The accuracy of this method is depending onthe correct annealing of two split DNAzymes with aptamerThe amount of this annealing may be variable in eachperformance and cause false negative results In our workto have the stable signal DNAzyme sequence has beenattached to 51015840 ends of aptamer sequenceTherefor its catalyticactivity remained constant in all experiments Also in theirstudy all reagents including split DNAzyme probes aptamerhemin and AB1 were added simultaneously while our resultsshowed that the order of addition of reagents is an importantparameter
4 Conclusions
Advantages of using DNAzymes and aptamers over proteinenzymes and antibodies have been reported in many studiesIn this study we optimized a colorimetric simple assay usingDNAzyme-aptamer conjugate to detect AFB1 Under opti-mized conditions the formation of AFB1 aptamer complexprevents the hybridization of its complementary sequencesHence the catalytic activity of DNAzyme increases corre-sponding to AFB1 concentration To improve the procedurewe will work on the limit of detection and sensitivity of thisaptasensor for a more accurate and sensitive determinationof AFB1
Conflicts of Interest
The authors declare that they have no conflicts of interest
Acknowledgments
This work was supported by a Grant (U-94080) from theAhvaz Jundishapur University of Medical Sciences AhvazIran
References
[1] S Marin A J Ramos G Cano-Sancho and V Sanchis ldquoMyco-toxins occurrence toxicology and exposure assessmentrdquo Foodand Chemical Toxicology vol 60 pp 218ndash237 2013
[2] M A Gacem and A Ould El Hadj-Khelil ldquoToxicology biosyn-thesis bio-control of aflatoxin and new methods of detectionrdquoAsian Pacific Journal of Tropical Biomedicine vol 6 no 9 pp808ndash814 2016
[3] S Z Iqbal M R Asi and A Arino ldquoAflatoxinsrdquo in ReferenceModule in Life Sciences Elsevier 2017
[4] M E Smela S S Currier E A Bailey and J M EssigmannldquoThe chemistry and biology of aflatoxin B
1 from mutational
spectrometry to carcinogenesisrdquo Carcinogenesis vol 22 no 4pp 535ndash545 2001
[5] A P Wacoo D Wendiro P C Vuzi and J F HawumbaldquoMethods for detection of aflatoxins in agricultural food cropsrdquoJournal of Applied Chemistry vol 2014 Article ID 706291 15pages 2014
[6] M Darmostuk S Rimpelova H Gbelcova and T RumlldquoCurrent approaches in SELEX an update to aptamer selectiontechnologyrdquoBiotechnology Advances vol 33 no 6 pp 1141ndash11612014
[7] S Tom H-E Jin and S-W Lee ldquoAptamers as functional bio-nanomaterials for sensor applications a2mdashgrumezescu alexan-dru mihairdquo in Engineering of Nanobiomaterials chapter 6 pp181ndash226 William Andrew Publishing 2016
[8] C Acquah M K Danquah J L S Yon A Sidhu and CM Ongkudon ldquoA review on immobilised aptamers for highthroughput biomolecular detection and screeningrdquo AnalyticaChimica Acta vol 888 pp 10ndash18 2015
[9] Y Seok Kim N H Ahmad Raston andM Bock Gu ldquoAptamer-based nanobiosensorsrdquo Biosensors and Bioelectronics vol 76pp 2ndash19 2016
[10] R Nezlin ldquoUse of aptamers in immunoassaysrdquo MolecularImmunology vol 70 pp 149ndash154 2016
[11] M Tabarzad and M Jafari ldquoTrends in the design and develop-ment of specific aptamers against peptides and proteinsrdquoProteinJournal vol 35 no 2 pp 81ndash99 2016
[12] A Chen and S Yang ldquoReplacing antibodies with aptamers inlateral flow immunoassayrdquo Biosensors and Bioelectronics vol 71pp 230ndash242 2015
[13] J Kosman and B Juskowiak ldquoPeroxidase-mimickingDNAzymes for biosensing applications a reviewrdquo AnalyticaChimica Acta vol 707 no 1-2 pp 7ndash17 2011
[14] X-H Zhao H-M Meng L Gong L Qiu X-B Zhang andW-H Tan ldquoRecent progress of DNAzyme-nanomaterial basedbiosensorsrdquoChinese Journal of Analytical Chemistry vol 43 no11 pp 1611ndash1619 2015
[15] S Mackay D Wishart J Z Xing and J Chen ldquoDevelopingtrends in aptamer-based biosensor devices and their applica-tionsrdquo IEEE Transactions on Biomedical Circuits and Systemsvol 8 no 1 pp 4ndash14 2014
[16] S M Bone N E Lima and A V Todd ldquoDNAzyme switchesformolecular computation and signal amplificationrdquo Biosensorsand Bioelectronics vol 70 pp 330ndash337 2015
[17] MMcKeague andM C Derosa ldquoChallenges and opportunitiesfor small molecule aptamer developmentrdquo Journal of NucleicAcids vol 2012 Article ID 748913 20 pages 2012
[18] M Citartan S C B Gopinath J Tominaga S-C Tan and T-H Tang ldquoAssays for aptamer-based platformsrdquo Biosensors andBioelectronics vol 34 no 1 pp 1ndash11 2012
[19] X Gong J Li W Zhou Y Xiang R Yuan and Y Chai ldquoTargetrecycling amplification for label-free and sensitive colorimetricdetection of adenosine triphosphate based on un-modifiedaptamers andDNAzymesrdquoAnalytica Chimica Acta vol 828 pp80ndash84 2014
[20] H Mun E-J Jo T Li et al ldquoHomogeneous assay of tar-get molecules based on chemiluminescence resonance energytransfer (CRET) using DNAzyme-linked aptamersrdquo Biosensorsand Bioelectronics vol 58 pp 308ndash313 2014
6 Journal of Toxicology
[21] C Yang V Lates B Prieto-Simon J-L Marty and X YangldquoAptamer-DNAzyme hairpins for biosensing of Ochratoxin ArdquoBiosensors and Bioelectronics vol 32 no 1 pp 208ndash212 2012
[22] C Yang V Lates B Prieto-Simon J-L Marty and X YangldquoRapid high-throughput analysis of ochratoxin A by the self-assembly of DNAzyme-aptamer conjugates in winerdquo Talantavol 116 pp 520ndash526 2013
[23] R Chauhan J Singh T Sachdev T Basu and B D MalhotraldquoRecent advances in mycotoxins detectionrdquo Biosensors andBioelectronics vol 81 pp 532ndash545 2016
[24] X-H Yang W-J Kong M-H Yang M Zhao and Z OuyangldquoApplication of aptamer identification technology in rapid anal-ysis ofmycotoxinsrdquoChinese Journal of Analytical Chemistry vol41 no 2 pp 297ndash306 2013
[25] G Castillo K Spinella A Poturnayova M Snejdarkova LMosiello and T Hianik ldquoDetection of aflatoxin B1 by aptamer-based biosensor using PAMAM dendrimers as immobilizationplatformrdquo Food Control vol 52 pp 9ndash18 2015
[26] M Zuker ldquoMfold web server for nucleic acid folding andhybridization predictionrdquoNucleic Acids Research vol 31 no 13pp 3406ndash3415 2003
[27] M Jafari M Rezaei H Kalantari M Tabarzad and BDaraei ldquoDNAzyme-aptamer or aptamer-DNAzyme paradigmbiochemical approach for aflatoxin analysisrdquo Biotechnology andApplied Biochemistry In press
[28] X Guo F Wen N Zheng et al ldquoDevelopment of an ultrasensi-tive aptasensor for the detection of aflatoxin B1rdquo Biosensors andBioelectronics vol 56 pp 340ndash344 2014
[29] W B Shim M J Kim H Mun and M G Kim ldquoAn aptamer-based dipstick assay for the rapid and simple detection ofaflatoxin B1rdquo Biosensors and Bioelectronics vol 62 pp 288ndash2942014
[30] W-B ShimHMunH-A Joung J A Ofori D-H Chung andM-G Kim ldquoChemiluminescence competitive aptamer assayfor the detection of aflatoxin B1 in corn samplesrdquo Food Controlvol 36 no 1 pp 30ndash35 2014
[31] H Hasegawa N Savory K Abe and K Ikebukuro ldquoMethodsfor improving aptamer binding affinityrdquo Molecules vol 21 no4 article 421 2016
[32] Y Seok J-Y Byun W-B Shim and M-G Kim ldquoA structure-switchable aptasensor for aflatoxin B1 detection based onassembly of an aptamersplit DNAzymerdquo Analytica ChimicaActa vol 886 pp 182ndash187 2015
Submit your manuscripts athttpswwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Figure 2 Blockade of peroxidase activity of DNAzyme-aptamerby B1 B2 and B3 sequences Apt DNAzyme-aptamer B blockercomplementary sequence
000
002
004
006
008
010
Abso
rban
ce
02 03 04 0501(휇M)
Figure 3 Blockade of peroxidase activity of DNAzyme-aptamer byincreasing concentration of B3
blocker compete with each other for binding to DNAzyme-aptamer blocker at high concentration decreases the sensi-tivity of assay So a conservative lower molar ratio of 1 1 wasselected for further evaluations
Orders of addition of AFB1 blocker complementarysequence and hemin were evaluated in different settingsResults showed that adding blocker prior to AFB1 resulted inunaffected remaining of blocker at its binding site ThereforeAFB1 was scheduled to incubate with aptamer before theblocker addition Also we observed that enzymatic activ-ity will be temporally influenced when hemin is addedto the reaction containing blocker As hemin preventedthe hybridization of blocker complementary sequence andDNAzyme-aptamer it was added subsequently to the blockersequences addition
The best incubation times for AFB1 blocker complemen-tary sequence and hemin were achieved via incubation of allreagents separately at 10min intervals up to 60 minutes Theshortest and most efficient incubation times were 15 10 and10min for AFB1 blocker and hemin respectively
Figure 4 Peroxidase activity of DNAzyme-aptamer in the presenceof B3 anddifferent concentrations ofAFB1AptDNAzyme-aptamerB blocker complementary sequence and AFB1 aflatoxin B1
In the present work the sensing strategy is based on thebinding affinities of AFB1 to its specific aptamer that producea detectable colorimetric signal by DNAzyme (Scheme 1)In the absence of AFB1 annealing of blocker sequence(complementary sequence to a part of DNAzyme-aptamer)to DNAzyme-aptamer decreases the enzymatic activity Inthe presence of AFB1 the aptamer binds to AFB1 and formsa hairpin structure Consequently blocker complementarysequence was prevented from being bound to DNAzyme-aptamer and following addition of heminDNAzyme displaysa colorimetric signal that is directly associated with AFB1concentrations (Figure 4)
Under optimal conditions the limit of detection of10 ngmL was achieved AFB1 aptamer has been used asa recognition probe in several detection systems based onPCR electrochemical chemiluminescent colorimetric andfluorescent platforms In Guo et alrsquos study AFB1 aptamerwith 31015840-terminal biotin groups has been immobilized onthe surface of PCR tubes for developing of an aptasensor(LOD 25 fgmL) based on RT-qPCR [28] An aptamer-based dipstick assay (LOD 01 ngmL) using biotin-modifiedaptamer has also been reported by Shim et al [29] In anotherwork by Shim et al based on chemiluminescence competitiveassay AFB1-OVA conjugate was coated on the wells (LOD011 ngmL) [30] Castillo et al have developed an aptamer-based biosensor (LOD 040 nM) using immobilization ofamino-modified aptamers and electrochemistry [25]
One of the important goals of this work was designing asimple and cost-effective method for AB1 detection withoutintricate steps and equipment In thementioned publicationsaptamer has been modified with functional groups or immo-bilized on surfaces In our experiment AB1 aptamer has been
Journal of Toxicology 5
employed without any immobilization and modification Allsteps of procedure were done as a simple solution Sinceaptamer interaction with its target depends on folding intounique structures [31] intact aptamers were excepted to havemore appropriate folding
AFB1 aptamer and DNAzyme also have also been usedby Seok et al with some alterations [32] In their studytwo split DNAzyme halves anneal with aptamer that formG-quadruplex The AFB1 aptamer complex prevented theannealing of split DNAzyme and aptamer therefore weakcolor intensity will be observed upon addition of ABTSsubstrate The accuracy of this method is depending onthe correct annealing of two split DNAzymes with aptamerThe amount of this annealing may be variable in eachperformance and cause false negative results In our workto have the stable signal DNAzyme sequence has beenattached to 51015840 ends of aptamer sequenceTherefor its catalyticactivity remained constant in all experiments Also in theirstudy all reagents including split DNAzyme probes aptamerhemin and AB1 were added simultaneously while our resultsshowed that the order of addition of reagents is an importantparameter
4 Conclusions
Advantages of using DNAzymes and aptamers over proteinenzymes and antibodies have been reported in many studiesIn this study we optimized a colorimetric simple assay usingDNAzyme-aptamer conjugate to detect AFB1 Under opti-mized conditions the formation of AFB1 aptamer complexprevents the hybridization of its complementary sequencesHence the catalytic activity of DNAzyme increases corre-sponding to AFB1 concentration To improve the procedurewe will work on the limit of detection and sensitivity of thisaptasensor for a more accurate and sensitive determinationof AFB1
Conflicts of Interest
The authors declare that they have no conflicts of interest
Acknowledgments
This work was supported by a Grant (U-94080) from theAhvaz Jundishapur University of Medical Sciences AhvazIran
References
[1] S Marin A J Ramos G Cano-Sancho and V Sanchis ldquoMyco-toxins occurrence toxicology and exposure assessmentrdquo Foodand Chemical Toxicology vol 60 pp 218ndash237 2013
[2] M A Gacem and A Ould El Hadj-Khelil ldquoToxicology biosyn-thesis bio-control of aflatoxin and new methods of detectionrdquoAsian Pacific Journal of Tropical Biomedicine vol 6 no 9 pp808ndash814 2016
[3] S Z Iqbal M R Asi and A Arino ldquoAflatoxinsrdquo in ReferenceModule in Life Sciences Elsevier 2017
[4] M E Smela S S Currier E A Bailey and J M EssigmannldquoThe chemistry and biology of aflatoxin B
1 from mutational
spectrometry to carcinogenesisrdquo Carcinogenesis vol 22 no 4pp 535ndash545 2001
[5] A P Wacoo D Wendiro P C Vuzi and J F HawumbaldquoMethods for detection of aflatoxins in agricultural food cropsrdquoJournal of Applied Chemistry vol 2014 Article ID 706291 15pages 2014
[6] M Darmostuk S Rimpelova H Gbelcova and T RumlldquoCurrent approaches in SELEX an update to aptamer selectiontechnologyrdquoBiotechnology Advances vol 33 no 6 pp 1141ndash11612014
[7] S Tom H-E Jin and S-W Lee ldquoAptamers as functional bio-nanomaterials for sensor applications a2mdashgrumezescu alexan-dru mihairdquo in Engineering of Nanobiomaterials chapter 6 pp181ndash226 William Andrew Publishing 2016
[8] C Acquah M K Danquah J L S Yon A Sidhu and CM Ongkudon ldquoA review on immobilised aptamers for highthroughput biomolecular detection and screeningrdquo AnalyticaChimica Acta vol 888 pp 10ndash18 2015
[9] Y Seok Kim N H Ahmad Raston andM Bock Gu ldquoAptamer-based nanobiosensorsrdquo Biosensors and Bioelectronics vol 76pp 2ndash19 2016
[10] R Nezlin ldquoUse of aptamers in immunoassaysrdquo MolecularImmunology vol 70 pp 149ndash154 2016
[11] M Tabarzad and M Jafari ldquoTrends in the design and develop-ment of specific aptamers against peptides and proteinsrdquoProteinJournal vol 35 no 2 pp 81ndash99 2016
[12] A Chen and S Yang ldquoReplacing antibodies with aptamers inlateral flow immunoassayrdquo Biosensors and Bioelectronics vol 71pp 230ndash242 2015
[13] J Kosman and B Juskowiak ldquoPeroxidase-mimickingDNAzymes for biosensing applications a reviewrdquo AnalyticaChimica Acta vol 707 no 1-2 pp 7ndash17 2011
[14] X-H Zhao H-M Meng L Gong L Qiu X-B Zhang andW-H Tan ldquoRecent progress of DNAzyme-nanomaterial basedbiosensorsrdquoChinese Journal of Analytical Chemistry vol 43 no11 pp 1611ndash1619 2015
[15] S Mackay D Wishart J Z Xing and J Chen ldquoDevelopingtrends in aptamer-based biosensor devices and their applica-tionsrdquo IEEE Transactions on Biomedical Circuits and Systemsvol 8 no 1 pp 4ndash14 2014
[16] S M Bone N E Lima and A V Todd ldquoDNAzyme switchesformolecular computation and signal amplificationrdquo Biosensorsand Bioelectronics vol 70 pp 330ndash337 2015
[17] MMcKeague andM C Derosa ldquoChallenges and opportunitiesfor small molecule aptamer developmentrdquo Journal of NucleicAcids vol 2012 Article ID 748913 20 pages 2012
[18] M Citartan S C B Gopinath J Tominaga S-C Tan and T-H Tang ldquoAssays for aptamer-based platformsrdquo Biosensors andBioelectronics vol 34 no 1 pp 1ndash11 2012
[19] X Gong J Li W Zhou Y Xiang R Yuan and Y Chai ldquoTargetrecycling amplification for label-free and sensitive colorimetricdetection of adenosine triphosphate based on un-modifiedaptamers andDNAzymesrdquoAnalytica Chimica Acta vol 828 pp80ndash84 2014
[20] H Mun E-J Jo T Li et al ldquoHomogeneous assay of tar-get molecules based on chemiluminescence resonance energytransfer (CRET) using DNAzyme-linked aptamersrdquo Biosensorsand Bioelectronics vol 58 pp 308ndash313 2014
6 Journal of Toxicology
[21] C Yang V Lates B Prieto-Simon J-L Marty and X YangldquoAptamer-DNAzyme hairpins for biosensing of Ochratoxin ArdquoBiosensors and Bioelectronics vol 32 no 1 pp 208ndash212 2012
[22] C Yang V Lates B Prieto-Simon J-L Marty and X YangldquoRapid high-throughput analysis of ochratoxin A by the self-assembly of DNAzyme-aptamer conjugates in winerdquo Talantavol 116 pp 520ndash526 2013
[23] R Chauhan J Singh T Sachdev T Basu and B D MalhotraldquoRecent advances in mycotoxins detectionrdquo Biosensors andBioelectronics vol 81 pp 532ndash545 2016
[24] X-H Yang W-J Kong M-H Yang M Zhao and Z OuyangldquoApplication of aptamer identification technology in rapid anal-ysis ofmycotoxinsrdquoChinese Journal of Analytical Chemistry vol41 no 2 pp 297ndash306 2013
[25] G Castillo K Spinella A Poturnayova M Snejdarkova LMosiello and T Hianik ldquoDetection of aflatoxin B1 by aptamer-based biosensor using PAMAM dendrimers as immobilizationplatformrdquo Food Control vol 52 pp 9ndash18 2015
[26] M Zuker ldquoMfold web server for nucleic acid folding andhybridization predictionrdquoNucleic Acids Research vol 31 no 13pp 3406ndash3415 2003
[27] M Jafari M Rezaei H Kalantari M Tabarzad and BDaraei ldquoDNAzyme-aptamer or aptamer-DNAzyme paradigmbiochemical approach for aflatoxin analysisrdquo Biotechnology andApplied Biochemistry In press
[28] X Guo F Wen N Zheng et al ldquoDevelopment of an ultrasensi-tive aptasensor for the detection of aflatoxin B1rdquo Biosensors andBioelectronics vol 56 pp 340ndash344 2014
[29] W B Shim M J Kim H Mun and M G Kim ldquoAn aptamer-based dipstick assay for the rapid and simple detection ofaflatoxin B1rdquo Biosensors and Bioelectronics vol 62 pp 288ndash2942014
[30] W-B ShimHMunH-A Joung J A Ofori D-H Chung andM-G Kim ldquoChemiluminescence competitive aptamer assayfor the detection of aflatoxin B1 in corn samplesrdquo Food Controlvol 36 no 1 pp 30ndash35 2014
[31] H Hasegawa N Savory K Abe and K Ikebukuro ldquoMethodsfor improving aptamer binding affinityrdquo Molecules vol 21 no4 article 421 2016
[32] Y Seok J-Y Byun W-B Shim and M-G Kim ldquoA structure-switchable aptasensor for aflatoxin B1 detection based onassembly of an aptamersplit DNAzymerdquo Analytica ChimicaActa vol 886 pp 182ndash187 2015
Submit your manuscripts athttpswwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
employed without any immobilization and modification Allsteps of procedure were done as a simple solution Sinceaptamer interaction with its target depends on folding intounique structures [31] intact aptamers were excepted to havemore appropriate folding
AFB1 aptamer and DNAzyme also have also been usedby Seok et al with some alterations [32] In their studytwo split DNAzyme halves anneal with aptamer that formG-quadruplex The AFB1 aptamer complex prevented theannealing of split DNAzyme and aptamer therefore weakcolor intensity will be observed upon addition of ABTSsubstrate The accuracy of this method is depending onthe correct annealing of two split DNAzymes with aptamerThe amount of this annealing may be variable in eachperformance and cause false negative results In our workto have the stable signal DNAzyme sequence has beenattached to 51015840 ends of aptamer sequenceTherefor its catalyticactivity remained constant in all experiments Also in theirstudy all reagents including split DNAzyme probes aptamerhemin and AB1 were added simultaneously while our resultsshowed that the order of addition of reagents is an importantparameter
4 Conclusions
Advantages of using DNAzymes and aptamers over proteinenzymes and antibodies have been reported in many studiesIn this study we optimized a colorimetric simple assay usingDNAzyme-aptamer conjugate to detect AFB1 Under opti-mized conditions the formation of AFB1 aptamer complexprevents the hybridization of its complementary sequencesHence the catalytic activity of DNAzyme increases corre-sponding to AFB1 concentration To improve the procedurewe will work on the limit of detection and sensitivity of thisaptasensor for a more accurate and sensitive determinationof AFB1
Conflicts of Interest
The authors declare that they have no conflicts of interest
Acknowledgments
This work was supported by a Grant (U-94080) from theAhvaz Jundishapur University of Medical Sciences AhvazIran
References
[1] S Marin A J Ramos G Cano-Sancho and V Sanchis ldquoMyco-toxins occurrence toxicology and exposure assessmentrdquo Foodand Chemical Toxicology vol 60 pp 218ndash237 2013
[2] M A Gacem and A Ould El Hadj-Khelil ldquoToxicology biosyn-thesis bio-control of aflatoxin and new methods of detectionrdquoAsian Pacific Journal of Tropical Biomedicine vol 6 no 9 pp808ndash814 2016
[3] S Z Iqbal M R Asi and A Arino ldquoAflatoxinsrdquo in ReferenceModule in Life Sciences Elsevier 2017
[4] M E Smela S S Currier E A Bailey and J M EssigmannldquoThe chemistry and biology of aflatoxin B
1 from mutational
spectrometry to carcinogenesisrdquo Carcinogenesis vol 22 no 4pp 535ndash545 2001
[5] A P Wacoo D Wendiro P C Vuzi and J F HawumbaldquoMethods for detection of aflatoxins in agricultural food cropsrdquoJournal of Applied Chemistry vol 2014 Article ID 706291 15pages 2014
[6] M Darmostuk S Rimpelova H Gbelcova and T RumlldquoCurrent approaches in SELEX an update to aptamer selectiontechnologyrdquoBiotechnology Advances vol 33 no 6 pp 1141ndash11612014
[7] S Tom H-E Jin and S-W Lee ldquoAptamers as functional bio-nanomaterials for sensor applications a2mdashgrumezescu alexan-dru mihairdquo in Engineering of Nanobiomaterials chapter 6 pp181ndash226 William Andrew Publishing 2016
[8] C Acquah M K Danquah J L S Yon A Sidhu and CM Ongkudon ldquoA review on immobilised aptamers for highthroughput biomolecular detection and screeningrdquo AnalyticaChimica Acta vol 888 pp 10ndash18 2015
[9] Y Seok Kim N H Ahmad Raston andM Bock Gu ldquoAptamer-based nanobiosensorsrdquo Biosensors and Bioelectronics vol 76pp 2ndash19 2016
[10] R Nezlin ldquoUse of aptamers in immunoassaysrdquo MolecularImmunology vol 70 pp 149ndash154 2016
[11] M Tabarzad and M Jafari ldquoTrends in the design and develop-ment of specific aptamers against peptides and proteinsrdquoProteinJournal vol 35 no 2 pp 81ndash99 2016
[12] A Chen and S Yang ldquoReplacing antibodies with aptamers inlateral flow immunoassayrdquo Biosensors and Bioelectronics vol 71pp 230ndash242 2015
[13] J Kosman and B Juskowiak ldquoPeroxidase-mimickingDNAzymes for biosensing applications a reviewrdquo AnalyticaChimica Acta vol 707 no 1-2 pp 7ndash17 2011
[14] X-H Zhao H-M Meng L Gong L Qiu X-B Zhang andW-H Tan ldquoRecent progress of DNAzyme-nanomaterial basedbiosensorsrdquoChinese Journal of Analytical Chemistry vol 43 no11 pp 1611ndash1619 2015
[15] S Mackay D Wishart J Z Xing and J Chen ldquoDevelopingtrends in aptamer-based biosensor devices and their applica-tionsrdquo IEEE Transactions on Biomedical Circuits and Systemsvol 8 no 1 pp 4ndash14 2014
[16] S M Bone N E Lima and A V Todd ldquoDNAzyme switchesformolecular computation and signal amplificationrdquo Biosensorsand Bioelectronics vol 70 pp 330ndash337 2015
[17] MMcKeague andM C Derosa ldquoChallenges and opportunitiesfor small molecule aptamer developmentrdquo Journal of NucleicAcids vol 2012 Article ID 748913 20 pages 2012
[18] M Citartan S C B Gopinath J Tominaga S-C Tan and T-H Tang ldquoAssays for aptamer-based platformsrdquo Biosensors andBioelectronics vol 34 no 1 pp 1ndash11 2012
[19] X Gong J Li W Zhou Y Xiang R Yuan and Y Chai ldquoTargetrecycling amplification for label-free and sensitive colorimetricdetection of adenosine triphosphate based on un-modifiedaptamers andDNAzymesrdquoAnalytica Chimica Acta vol 828 pp80ndash84 2014
[20] H Mun E-J Jo T Li et al ldquoHomogeneous assay of tar-get molecules based on chemiluminescence resonance energytransfer (CRET) using DNAzyme-linked aptamersrdquo Biosensorsand Bioelectronics vol 58 pp 308ndash313 2014
6 Journal of Toxicology
[21] C Yang V Lates B Prieto-Simon J-L Marty and X YangldquoAptamer-DNAzyme hairpins for biosensing of Ochratoxin ArdquoBiosensors and Bioelectronics vol 32 no 1 pp 208ndash212 2012
[22] C Yang V Lates B Prieto-Simon J-L Marty and X YangldquoRapid high-throughput analysis of ochratoxin A by the self-assembly of DNAzyme-aptamer conjugates in winerdquo Talantavol 116 pp 520ndash526 2013
[23] R Chauhan J Singh T Sachdev T Basu and B D MalhotraldquoRecent advances in mycotoxins detectionrdquo Biosensors andBioelectronics vol 81 pp 532ndash545 2016
[24] X-H Yang W-J Kong M-H Yang M Zhao and Z OuyangldquoApplication of aptamer identification technology in rapid anal-ysis ofmycotoxinsrdquoChinese Journal of Analytical Chemistry vol41 no 2 pp 297ndash306 2013
[25] G Castillo K Spinella A Poturnayova M Snejdarkova LMosiello and T Hianik ldquoDetection of aflatoxin B1 by aptamer-based biosensor using PAMAM dendrimers as immobilizationplatformrdquo Food Control vol 52 pp 9ndash18 2015
[26] M Zuker ldquoMfold web server for nucleic acid folding andhybridization predictionrdquoNucleic Acids Research vol 31 no 13pp 3406ndash3415 2003
[27] M Jafari M Rezaei H Kalantari M Tabarzad and BDaraei ldquoDNAzyme-aptamer or aptamer-DNAzyme paradigmbiochemical approach for aflatoxin analysisrdquo Biotechnology andApplied Biochemistry In press
[28] X Guo F Wen N Zheng et al ldquoDevelopment of an ultrasensi-tive aptasensor for the detection of aflatoxin B1rdquo Biosensors andBioelectronics vol 56 pp 340ndash344 2014
[29] W B Shim M J Kim H Mun and M G Kim ldquoAn aptamer-based dipstick assay for the rapid and simple detection ofaflatoxin B1rdquo Biosensors and Bioelectronics vol 62 pp 288ndash2942014
[30] W-B ShimHMunH-A Joung J A Ofori D-H Chung andM-G Kim ldquoChemiluminescence competitive aptamer assayfor the detection of aflatoxin B1 in corn samplesrdquo Food Controlvol 36 no 1 pp 30ndash35 2014
[31] H Hasegawa N Savory K Abe and K Ikebukuro ldquoMethodsfor improving aptamer binding affinityrdquo Molecules vol 21 no4 article 421 2016
[32] Y Seok J-Y Byun W-B Shim and M-G Kim ldquoA structure-switchable aptasensor for aflatoxin B1 detection based onassembly of an aptamersplit DNAzymerdquo Analytica ChimicaActa vol 886 pp 182ndash187 2015
Submit your manuscripts athttpswwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
[21] C Yang V Lates B Prieto-Simon J-L Marty and X YangldquoAptamer-DNAzyme hairpins for biosensing of Ochratoxin ArdquoBiosensors and Bioelectronics vol 32 no 1 pp 208ndash212 2012
[22] C Yang V Lates B Prieto-Simon J-L Marty and X YangldquoRapid high-throughput analysis of ochratoxin A by the self-assembly of DNAzyme-aptamer conjugates in winerdquo Talantavol 116 pp 520ndash526 2013
[23] R Chauhan J Singh T Sachdev T Basu and B D MalhotraldquoRecent advances in mycotoxins detectionrdquo Biosensors andBioelectronics vol 81 pp 532ndash545 2016
[24] X-H Yang W-J Kong M-H Yang M Zhao and Z OuyangldquoApplication of aptamer identification technology in rapid anal-ysis ofmycotoxinsrdquoChinese Journal of Analytical Chemistry vol41 no 2 pp 297ndash306 2013
[25] G Castillo K Spinella A Poturnayova M Snejdarkova LMosiello and T Hianik ldquoDetection of aflatoxin B1 by aptamer-based biosensor using PAMAM dendrimers as immobilizationplatformrdquo Food Control vol 52 pp 9ndash18 2015
[26] M Zuker ldquoMfold web server for nucleic acid folding andhybridization predictionrdquoNucleic Acids Research vol 31 no 13pp 3406ndash3415 2003
[27] M Jafari M Rezaei H Kalantari M Tabarzad and BDaraei ldquoDNAzyme-aptamer or aptamer-DNAzyme paradigmbiochemical approach for aflatoxin analysisrdquo Biotechnology andApplied Biochemistry In press
[28] X Guo F Wen N Zheng et al ldquoDevelopment of an ultrasensi-tive aptasensor for the detection of aflatoxin B1rdquo Biosensors andBioelectronics vol 56 pp 340ndash344 2014
[29] W B Shim M J Kim H Mun and M G Kim ldquoAn aptamer-based dipstick assay for the rapid and simple detection ofaflatoxin B1rdquo Biosensors and Bioelectronics vol 62 pp 288ndash2942014
[30] W-B ShimHMunH-A Joung J A Ofori D-H Chung andM-G Kim ldquoChemiluminescence competitive aptamer assayfor the detection of aflatoxin B1 in corn samplesrdquo Food Controlvol 36 no 1 pp 30ndash35 2014
[31] H Hasegawa N Savory K Abe and K Ikebukuro ldquoMethodsfor improving aptamer binding affinityrdquo Molecules vol 21 no4 article 421 2016
[32] Y Seok J-Y Byun W-B Shim and M-G Kim ldquoA structure-switchable aptasensor for aflatoxin B1 detection based onassembly of an aptamersplit DNAzymerdquo Analytica ChimicaActa vol 886 pp 182ndash187 2015
Submit your manuscripts athttpswwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
