Research Article Preparation of a Specific ssDNA Aptamer for Brevetoxin …downloads.hindawi.com/journals/jamc/2016/9241860.pdf · 2019-07-30 · Research Article Preparation of a

Research ArticlePreparation of a Specific ssDNA Aptamer forBrevetoxin-2 Using SELEX

Rui-Yun Tian1 Chao Lin12 Shi-Yu Yu3 Sheng Gong1 Pan Hu1

Yan-Song Li1 Zong-ChengWu1 Yang Gao1 Yu Zhou1 Zeng-Shan Liu1

Hong-Lin Ren1 and Shi-Ying Lu1

1Key Laboratory of Zoonosis Research Ministry of Education Institute of Zoonosis College of Veterinary MedicineJilin University Changchun 130062 China2Emergency Department The Eastern Division The First Hospital of Jilin University Changchun 130062 China3Fuqing Entry-Exit Inspection and Quarantine Bureau Port District Qingrong Road Fuqing Fujian 350300 China

Correspondence should be addressed to Hong-Lin Ren renhljlueducn and Shi-Ying Lu lushiying1129163com

Received 10 August 2016 Revised 20 October 2016 Accepted 30 October 2016

Academic Editor Beate Strehlitz

Copyright copy 2016 Rui-Yun Tian 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

The existing assays for detecting brevetoxin (BTX) depend on expensive equipment with a professional operator or on an antibodywith limited stability which requires complex processes a high cost and a considerable amount of time The development of analternative detection probe is another promising research direction This paper reports the use of aptamers binding to BTX-2 inan analytical assay using the systematic evolution of ligands by exponential enrichment (SELEX) After 12 rounds of selectionthe secondary structures of 25 sequences were predicted Compared to other aptamers Bap5 has relatively high affinity with thelowest dissociation constant of 483120583M and IC

50is 7381 ngmLminus1 A good linear regression formula of 119910 = 30688119909 minus 7329 with

a coefficient correlation of 1198772 = 09798 was obtained using a biotin-avidin ELISA Moreover there is no cross-reaction with thedetected marine toxins except for BTX-2 Thus Bap5 has potential to detect BTX-2 in shellfish in the future as a substitute for therecognition probe

1 Introduction

Red tides have bloomed more frequently due to the increasein marine pollution followed by the sprouting of a sig-nificant quantity of harmful algae [1] and the productionof metabolic products from marine biotoxins includingdiarrhetic shellfish poison (DSP) paralytic shellfish poison(PSP) neurotoxic shellfish poison (NSP) and amnesic shell-fish poison (ASP) [2 3] Brevetoxins well-known as typicalNSP toxins are initially located intracellularly in Kareniabrevis but the toxins become more widely available in theenvironment after the cells lyse or die [4] The toxins arealso transferred through the food chain and accumulatedin shellfish fish and other species The persistence of thetoxins in seawater sediments and seagrass epiphytes resultsin chronic or sustained exposures that can damage the health

of marinemammals and other aquatic invertebrates and evencause massive fish killing [5ndash8] NSP toxins activate voltage-sensitive sodium channels causing health issues in humansand animals When humans or animals digest contaminatedfilter-feeding animals or swallow the seawater during bloomsof Karenia brevis they will experience the characteristicsymptoms of NSP such as paresthesia (tingling) reversalof hot-cold temperature sensation myalgia (muscle pain)vertigo ataxia (loss of coordination) abdominal pain nauseadiarrhea headache bradycardia (slow heart rate) dilatedpupils and respiratory distress [5 9]

Based on their individual structures NSP toxins consistof at least 13 different components and have more complexstructures including brevetoxin A (BTX-A) and brevetoxin B(BTX-B) [10] The primary component of BTX-B Ptychodis-cus brevetoxin-2 (BTX-2) is the most common neurotoxic

Hindawi Publishing CorporationJournal of Analytical Methods in ChemistryVolume 2016 Article ID 9241860 8 pageshttpdxdoiorg10115520169241860

2 Journal of Analytical Methods in Chemistry

shellfish poison and has attracted more attention becauseof the red tide event in Florida that resulted in widespreadfish death [7] BTX-2 is a fat-soluble polyether compound[11] The minimum lethal dose in mice is 025mg kgminus1 andLD50

(lethal dose 50) is 200mg kgminus1 Therefore manyresearchers have continued to explore sensitive and conve-nient methods for detecting the toxic substance Currentlythe available detection methods include solid-phase extrac-tion (SPE) for qualitative analysis [12] high-performanceliquid chromatography (HPLC) [13] liquid chromatography-mass spectrometry (LC-MS) [14] radioimmunoassay [4]electrochemiluminescence-based immunoassay [15] thin-layer chromatography [16] and immunological methodssuch as enzyme-linked immunosorbent assay (ELISA) whichutilizes antibodies to quantify the BTX-2 levels [17] Thedisadvantages of these assays are the expensive equipmentwhich require professional staff the high cost of antibodieswith limited stability and the special storage conditionsrequired for the immunological assay Therefore the devel-opment of an alternative probe for the detection methodsthat rapidly cost-effectively and sensitively monitors BTX inshellfish and seawater is a pressing need

Recently aptamers oligonucleotides such as single-stranded DNA (ssDNA) or RNA [18 19] have attractedincreasing attention as the sensing elements in biosensorsused to detect many types of molecules such as proteinsmetals polypeptides and small molecules [20] Aptamershave all of the advantages of antibodies along with the uniqueadvantages of higher specificities and affinities for the targetand can be synthesized chemically modified easily storeddenatured and renatured [21] Aptamers are usually obtainedvia an in vitro process called systematic evolution of ligandsby exponential enrichment (SELEX) whichwas first reportedin 1990 [22] Since then many articles have described theuse of SELEX to select aptamers targeting many types ofsubstances particularly the aptamers targeting and detectingtoxins in recent years such as saxitoxin [23 24] okadaicacid [25 26] ochratoxin A [27ndash30] and the recent reportof BTX-2 in 2015 [31] Based on these advantages aptamersare emerging as novel capturing agents and recognitionreceptors in biosensor applications In this study we reportthe development of DNA aptamers that are able to bind BTX-2 with an dissociation constant of 483 120583M an IC

50value

of 7381 ngmLminus1 and selectivity for BTX only The resultspresented in this study report a novel alternative analyticalprobe for the development of an NSP toxin immunologicalassay These results will contribute to the establishment ofan experimental protocol for a relatively simple affordableand sensitive laboratory method for the detection of BTX inshellfish products and seawater

2 Materials and Methods

21 Apparatus and Reagents TheBTX-2 and saxitoxin (STX)standards (purity ge 98) were purchased from ZEN-UBiotechnology Co Ltd (Tai Wan China) The okadaic acid(OA) and domoic acid (DA) standards were purchased fromALEXIS Biochemicals The monoclonal antibody against

BTX-2 was prepared in our lab (119870119886 value is 082 times 109Mminus1)and stored at minus80∘C [32] Bovine serum albumin (BSA) andovalbumin (OVA) were purchased from the Beijing Ding-guo Biotechnology Development Center (Beijing China)The target BTX-2-BSA and BTX-2-OVA conjugates wereprepared using a previously described method [32] TheDNA mate Taq DNA polymerase PMD-18T and DL2000marker were purchased from Takara Biotechnology Co Ltd(Dalian China) The streptavidin-peroxidase complex waspurchased from Beijing Biosynthesis Biotechnology Co Ltd(Beijing China) Sterile ultrapure water was obtained froma Milli-Q water purification system (Millipore USA) Allof the polymerase chain reactions (PCRs) were conductedin a GeneAmp PCR system 9600 (Applied Perkin-ElmerUSA) The standard 96-well ELISA plates (U-bottom) werepurchased fromCostar (NY USA) Absorbance was recordedusing an Epoch Microvolume Spectrophotometer system(BioTek Instruments Inc USA) The other reagents used inthis study were of analytical grade

22 The ssDNA Library and Primer Synthesis The initialssDNA library used for SELEX and the homologous primerswere synthesized by the Shanghai Sangon Biological Engi-neering Technology amp Services Company (Shanghai China)The sequences included in the ssDNA library were 85nucleotides in length with a central region of 40 randomnucleotides flanked by two primer-binding sites for PCR andcloning as follows 51015840-GAGGCAGCACTTCACACGATC-TG-N40-CTGCGTAATGACTGTAGTGATG-31015840 Primer1 (51015840-GAGGCAGCACTTCACACGATCTG-31015840) and primer2 (51015840-CATCACTACAGTCATTACGCAG-31015840) were used foramplification and cloning The biotin-modified primer (51015840-biotin-GAGGCAGCACTTCACACGAT-31015840) was also synthe-sized for use in the binding assays All of the oligonucleotideswere dissolved in TE buffer (30mMTris-Cl 1mMEDTA pH80) and stored at minus20∘C until further use

23 Preparation of the Target Conjugates The target BTX-2-BSA and BTX-2-OVA conjugates were prepared using apreviously described method [32] The basic procedure isdescribed below A 10-foldmolar excess of succinic anhydridesolubilized in 25mL of anhydrous pyridine was added to2mg of crystalline BTX-2 After a 6 h incubation at 65∘Cthe solvent was evaporated under a stream of nitrogenand the residue was reacted with a 10-fold molar excess oftributylamine and isobutyl chlorocarbonate as 110 dilutionsin dry peroxide-free dioxin for 30min at room temperatureThen the carrier proteins BSA and OVA (molar ratio ofhaptencarrier 50 1) were added and incubated for 30minat room temperature these carrier proteins were used for thetarget (BTX-2-BSA) of SELEX and detection antigen (BTX-2-OVA) respectively The complex was recovered by acetoneprecipitation resuspended in 05mL of distilled water filter-sterilized (022120583m) dispensed into sterile tubes freeze-driedovernight and stored at minus20∘C until use

24 Selection of the Aptamer The typical SELEX procedureis an iterative process in which the sequences that specificallybind to the target are selected from highly diverse synthetic

Journal of Analytical Methods in Chemistry 3

BTX-protein

For next round selection

Second ssDNA pool

PCR amplification

Bound ssDNA Elution

Bound ssDNA

ssDNA pool

Unbound ssDNA

Wash

Scheme 1 Schematic representation of the SELEX process used to select the aptamer

nucleic acid libraries in several rounds The principle forSELEX is shown in Scheme 1 In the selection process theBTX-2-BSA precoated microwell plate was used to screenfor specific aptamers that bound to the target toxin BTX-2 The random ssDNA pool was incubated with the targetand then the separated sequences were amplified to generatean enriched secondary ssDNA pool for the next roundof screening After many rounds of screening the ssDNAsequences with a high affinity for the target were obtained

After each selection round the general ssDNA librarywas amplified using the following two types of PCR thesymmetric PCR method for producing dsDNA and theasymmetric PCR method for preparing ssDNA The randomssDNA library and the amplified ssDNA pool obtained ineach round of selection were dissolved in TE buffer andthen diluted with the SHCMK binding buffer (20mMHepes120mM sodium chloride 5mM potassium chloride 1mMcalcium chloride and 1mMmagnesium chloride) In the firstround 12 120583L of ssDNA (100 nM) was added to 800120583L ofSHCMK binding buffer followed by denaturation at 96∘C for10min and cooling on ice for 10min to prevent misfolding ofthe ssDNA before it bound to the toxin The preconditioningssDNA library (100 120583L per well) was added to 8 wells ofa microtiter plate on which 1 120583gmLminus1 BTX-2-BSA wasimmobilized (100 120583L per well) The mixture was incubatedfor 5 h at 37∘C in the dark Then the liquids were decantedand themicrowell plate was washed three timeswith SHCMKwashing buffer (SHCMK binding buffer containing 005Tween-20 220120583L per well) for 1min with gentle shaking toseparate the unbound sequences from the bound sequencesThe free ssDNA was discarded and the desired ssDNAremained bound to the immobilized toxin conjugates Elutionbuffer (20mM Tris-HCl 4mM guanidine thiocyanate and1mM DL-dithiothreitol pH 83) was added to the wells(100 120583L per well) and incubated at 80∘C for 20min to elute the

ssDNA Phenol chloroform and isoamyl alcohol (25 24 1)were used to purify the ssDNA The ssDNA was precipitatedby adding dehydrated alcohol that had been precooled tominus20∘C and then centrifuging at 12000g for 5min to obtainthe ssDNA targeting BTX-2-BSA Finally the precipitatedssDNA was dissolved in 20120583L of TE buffer and stored atminus20∘Cuntil the next roundThe secondary ssDNA librarywasprepared using the asymmetric PCR method Eight percenturea-polyacrylamide gel electrophoresis and silver stainingwere used to identify the ssDNA silver stainingwas employedafter electrophoresis because of its high sensitivity comparedwith ethidium bromide (EB) or Coomassie Brilliant Bluestaining

25 Amplification and Purification of the ssDNA The elutedand concentrated ssDNA molecules were amplified usingsymmetric and asymmetric PCR The symmetric PCR reac-tion is designed to obtain double-stranded DNA as the tem-plate for asymmetric PCRThe reaction system for symmetricPCR contained 20 120583M upstream primer 20120583M downstreamprimer 10mM dNTP mixture 5 120583L of 10x PCR buffer(100mM Tris-HCl pH 83 500mM KCl and 15mMMgCl

2)

25 ng of ssDNA template the concentration of which wasdetected using BioTek Instruments in the following screen125U of Taq polymerase and sterile distilled water in atotal volume of 50120583L The asymmetric PCR reaction was thesame as the symmetric PCR reaction except that the ratiosof the upstream and downstream primers were increased100-fold The PCR cycling conditions consisted of an initialdenaturation at 96∘C for 2min 25 cycles of 96∘C for 30 s64∘C for 30 s and 72∘C for 30 s and a final extension at 72∘Cfor 5min The ssDNA was amplified from asymmetric PCRusing the same conditions butwith an annealing temperatureof 59∘C for 20 cycles The 51015840-biotin-labeled products of theasymmetric PCR were used for an affinity analysis in each


round The dsDNA obtained from the round with the bestaffinity was used for cloning after it was purified accordingto the instructions of the Poly-Gel DNA Extraction Kit(OMEGA) [33]

26 Cloning and Sequencing Analysis The purified andhighly concentrated oligonucleotide from the best round ofselection was cloned into a pMD18-T vector and transformedinto competent Escherichia coli DH5120572 cells Positive colonieswere verified by PCR Complete sequencing of the sampleswas performed by Shanghai Sangon Biotechnology Aftercloning and sequencing the lowest free-energy shapes andsecondary structures were predicted and the sequences wereanalyzed using the free-energyminimization algorithm in theDNAMAN and Mfold programs

27 Identification of Specificity and Affinity An indirectELISA was performed after each round of selection to testthe process of aptamer selection and evaluate the bindingaffinity of the selected aptamers For this purpose 100 120583Lof 05 120583gmLminus1 BTX-2-OVA (1 05 and 025 120583gmLminus1 BTX-2-OVA were coated to determine the 119870119886 value) dilutedwith coating buffer (005M sodium carbonate pH 96) wasimmobilized on the surface of the microwells and incubatedat 4∘C overnight The plate was washed three times withthe SHCMK cleaning solution (220120583L per well) followedby incubation with 100 120583L of 5 evaporated skim milk at37∘C for 2 h Increasing concentrations (0ndash20 nM) of the 51015840-biotin-labeled aptamer (100 120583L per well) were added to thewashed plate and incubated at 37∘C for 1 h After anotherwashing step with the SHCMK cleaning solution 100120583Lof diluted horseradish peroxidase-conjugated streptavidin(1 1000) in PBS containing 01 BSA was added to eachwell and incubated for 1 h at 37∘C After four gentle washes100 120583L aliquots of the o-phenylenediamineH

2O2solution

in 005M citratendashphosphate buffer (pH 50) were addedto each well The reaction was stopped by the addition of50 120583L of a 2mM H

2SO4solution to each well and a 10min

incubation at room temperature The optical density wasmeasured at 492 nm (OD

492) on an Epoch Microvolume

Spectrophotometer system The 119870119889 value was calculatedusing the equation 119870119889 = 1119870119886 = (119899[Ap1015840]119905 minus [Ap]119905)(119899 minus 1)In this equation 119899 is the concentration ratio of plates coatedwith two different concentrations of BTX-2-OVA in onegroup and [Ap1015840] and [Ap] are the concentrations (mol Lminus1)of the aptamer corresponding to 50 of the maximumabsorbance values of the plates coated with the two differentconcentrations of BTX-2-OVA The average is the affinityconstant The available marine biotoxins BTX STX OA andDA were tested for cross-reactivity using the same affinitymeasurement methods to evaluate the specificity of theaptamer

28 Competitive Inhibitory Activity An indirect competitiveELISA was performed to determine the inhibitory activityof the selected aptamer Bap5 The BTX-2-OVA conjugatewas diluted with 005M bicarbonate buffer (pH 96) addedto a microtiter plate at a concentration of 05 120583gmLminus1 andincubated at 4∘C overnight The plate was washed three

times with the SHCMK cleaning solution (220120583L per well)followed by incubation with 100 120583L of 5 evaporated skimmilk at 37∘C for 2 h After 3 washes equal volumes (50120583Lper well) of solutions of the BTX-2 standard (200 100 5025 625 3125 and 0 ngmLminus1) diluted in 10mM PBS (pH 74)were mixed with 5 nM biotin-labeled ssDNA diluted in theSHCMK binding buffer in each well and incubated at 37∘Cfor 1 h The subsequent steps were performed in the samemanner as those described in Identification of Specificity andAffinityThe calibration curve was obtained with the percentinhibition [(119873minus119878)119873times100] on the 119910-axis and the log BTX-2 concentration on the 119909-axis119873 is the OD

492value when the

toxin standard is not present in the detected sample (control)and 119878 is the OD

492value observed when there is a difference

in the concentration of the standard in the detected sample

3 Results and Discussion

31 Selection of the Aptamer Aptamers against BTX-2 wereobtained using the SELEX method in vitro The specificityand quantity of the ssDNA and the dsDNA were significantlyaffected by the PCR reaction system and the thermocyclerconditions The PCR conditions were optimized to obtaina high volume and specific ssDNA Gradient temperaturePCR was chosen as the optimization method to avoidnonspecific amplification Using this method 64∘C and 25cycles were the optimal settings for the production of ahigh content of dsDNA with sufficient purity (data notshown) The generation of high purity ssDNA is a key factorin obtaining aptamers with high affinity and selectivity Inthis study asymmetric PCR was used to generate ssDNAwith a 1 10 ratio of forward primer and reverse primerThen 2 sepharose gel electrophoresis was used to iden-tify the amplified dsDNA (Figure 1(a)) whereas 8 urea-polyacrylamide gel electrophoresis was used to analyze thessDNA (Figure 1(b)) As shown in Figures 1(a) and 1(b)the target bands indicated the correct dsDNA and ssDNAproducts without dimers and nonspecific DNA The highlyspecific ssDNA provided an excellent secondary library forthe subsequent screen

The counterselection process was performed after every 4rounds of selection using the immobilized BTX-2-OVAas thealternative conjugate to enhance the selectivity and specificityof the aptamer The negative selection steps can remove thesequences that bind to BSA and the plates or moleculesthat may be bound nonspecifically After the 12 rounds ofselection an indirect biotin ELISA was used to identify thebinding capacity of selected aptamers As shown in Figure 2the absorbance values recorded at 492 nmgradually increasedfrom 0108 in the first round to 0537 in round 4 0907 inround 9 and 1256 in round 12 Thus the binding capacitybetween the aptamer and BTX-2 increased Therefore thespecific aptamer library was gradually enriched In contrastthe absorbance values obtained in the 5th and 9th roundswere slightly decreased Thus the nonspecific aptamerstargeting BSA the plates and the other materials hadbeen removed during the negative selection However theabsorbance increased and reached its maximum at the 12thround and then stabilized Therefore as the binding reaches


1 2

20001000750500

250

10085bp

(a)

2000

1000750

500

250

100

1 2

85nt

(b)

Figure 1 Identification of dsDNA and ssDNA using electrophoresis (a) 2 sepharose gel electrophoresis of the 85-bp dsDNA amplified bysymmetric PCR Lane 1 is the 85-bp dsDNA and lane 2 is the DL2000 DNA Marker (b) 8 denaturing urea gel electrophoresis of ssDNALane 1 is the DL2000 DNAMarker and lane 2 is the 85-nt ssDNA amplified by asymmetric PCR

00

02

04

06

08

10

12

14

Abso

rban

ces (

492

nm)

1 2 3 4 5 6 7 8 9 10 11 120

Selection rounds

Figure 2 Binding of the ssDNA library to BTX-2-BSA during the12 SELEX rounds

a plateau the affinity reaches saturation and the enrichedhigh affinity aptamers account for the majority of the ssDNAlibrary After selection we amplified the dsDNA from the12th round using the symmetric PCRprocess described aboveand purified the DNA using an AxyPrep DNAGel ExtractionKit The purified and highly concentrated oligonucleotideswere cloned into the pMD18-T vector and transformed intocompetent Escherichia coliDH5120572 cells and their inserts wereconfirmed by PCR and sequenced prior to the subsequentexperiments

32 Secondary Structure Predictions After cloning andsequencing the secondary structures of the 25 sequenceswere further predicted and analyzed using the DNAMANand Mfold programs The lengths of most of the sequenceswere the same as those in the anticipated library Tworepeated sequences were enriched in guanine and the othersequences reached greater than 70 identity Based on thisresult the ssDNA library was enriched after 12 rounds of

SELEX Generally oligonucleotides fold into various sec-ondary structures such as stem-loop pseudoknot hairpinpocket and even G-quadruplexes which act as the bindingmotifs or the region responsible for biological activity Themost likely minimum energy secondary structures of theselected aptamers were analyzed with the Mfold programto predict the structure or the aptamer that bound to thetarget toxin [33] In this study the secondary structures ofmost of the obtained aptamers were typical stem-loop (a)and hairpin-loop (b) structures Figure 3 shows some of theaptamers The analysis of the secondary structures clearlyshowed that the aptamers formed the loop structure usingthe random region and the G-rich sequence likely forms aG-quartet structure We predict that the structures of all theaptamers might play particularly crucial roles in binding tothe target and are the predicted target-binding site

The letters (a) and (b) indicate the stem-loop and thehairpin-loop which is the possible region in the aptamer thatbinds to the BTX-2 toxin

33 Binding Affinity and Specificity An indirect biotin ELISAwas performed using concentrations of BTX-2-OVA from025ndash1120583gmLminus1as the coated substance to evaluate the disso-ciation constant and specificity of the binding of the candidateaptamers to BTX-2 In addition the aptamer concentrationwas diluted in a gradient from 0625 nM to 20 nM The 119870119889value was calculated using the equation 119870119889 = 1119870119886 =(119899[Ap1015840]119905 minus [Ap]119905)(119899 minus 1) The curve for the Bap5 aptamer isshown in Figure 4(a) The 119870119889 value for the binding betweenthe Bap5 aptamer and BTX-2 was 483 120583M whereas thevalues for Bap 8 Bap 23 Bap 36 and Bap 38 were 980 120583M725 120583M 1047 120583M and 1055 120583M respectively Among thefive selected aptamers Bap5 has the lowest 119870119889 value anda high binding affinity for BTX-2 similar to a previousreport [28] The 119870119889 values for Bap 8 Bap 23 Bap 36and Bap 38 were relatively high and were thus consideredto have weak binding capacity and were discarded fromfurther experiments Cross-reactivity was assessed using theavailable marine toxins DA STX and OA under the sameconditions to further investigate the specificity of the Bap 5


ba

b

a

a

a

b

a

b

b

a

a

Bap5 Bap 8 Bap23

Bap36Bap38

Figure 3 Secondary structures of the candidate aptamers predicted by Mfold

35

25

15

10

20

30

40

05

00

Abso

rban

ces (

492

nm)

ssDNA concentrations (nM)20151050

050120583g mLminus1

025 120583g mLminus1


(a)

100

90

80

70

60

50

40

30

20

10

0

Brevetoxin-2Domoic acid

SaxitoxinOkadaic acid

(NminusS)N

times10

0()

Toxin concentration (ng mLminus1)200180160140120100806040200

(b)

Figure 4 Binding activity and cross-reactivity of Bap5 (a) The 119870119889 value of the Bap5 aptamer was measured using different BTX-2-OVAcoating concentrations (1 05 and 025 120583gmLminus1) to estimate the dissociation constant (119870119889 = 1119870119886 = (119899[Ap1015840]119905 minus [Ap]119905)(119899 minus 1)) The 119909-axis represents the aptamer concentration (ngmLminus1) whereas the 119910-axis represents the absorbance at 492 nm (b) Plots from the ELISAexperiments used to investigate the specificity of the Bap5 aptamer for BTX-2 and other toxins The calibration curve was obtained with theinhibition percent [(119873 minus 119878)119873 times 100] shown on the 119910-axis and the concentration of toxins on the 119909-axis 119873 is the OD

492value when the

toxin standard is not present in the detected sample (control) and 119878 is the OD492

value when there is a difference in the concentration of thestandard in the detected sample All the data represent the means plusmn SD (119899 = 3) of three replicates

aptamer for BTX-2 As shown in Figure 4(b) Bap 5 had anoverwhelmingly positive response to BTX-2 compared withthe weak responses to the other toxins Because the otherBTXs are not available the cross-reactivity with these toxinsmust be assayed in a future study

34 Competitive Inhibitory Activity An indirect competitiveELISA based on the Bap5 aptamer was performed to assay theinhibitory activity of this aptamer A good linear regressionformula of 119910 = 30688119909minus7329 with a coefficient correlationof 1198772 = 09798 was obtained (Figure 5) The standard curve


y = 30688x minus 7329

R2 = 09798

0

10

20

30

40

50

60

70

80

90

100

Inhi

bitio

n pe

rcen

t (

)

05 10 15 20 2500

Log BTX-2 concentration (ng mLminus1)

Figure 5 Plot of the indirect competitive ELISA of the BTX-2 aptamer Bap5 The calibration curve was obtained with theinhibition percent [(119873 minus 119878)119873 times 100] on the 119910-axis and thelog concentration of BTX-2 on the 119909-axis The 119873 and 119878 valueswere the OD

492of the control and the sample respectively As the

concentration of the BTX-2 standard increased (ranging from 3125to 200 ngmLminus1) the inhibition ratio ranged from 1167 to 673

was linear over the range of 3125ndash200 ngmLminus1 with anIC50(half maximal inhibitory concentration of BTX-2) value

of 7381 ngmLminus1 and a detection limit of 3125 ngmLminus1which was greater than the sensitivity of the NSP ELISA kit(Abraxis America Product No PN520026) and the valuereported in our previous study (IC

50is 53 ngmLminus1) using the

monoclonal antibody [32] but less than the current EuropeanUnion andCodexAlimentarius Commission regulatory limit(800 120583gkg shellfish meat) Notably the use of aptamers asa substitute for the antibody has many specific advantagesbecause the aptamers can be synthesized chemically mod-ified easily stored denatured and renatured Therefore weanticipate that the aptamer screened in this study will beapplied as another recognition probe in an aptamer-baseddetection assay for assessing the BTX levels in contaminatedseafood

4 Conclusion

In this study aptamers targeting BTX-2 were successfullyobtained via 12 rounds of SELEX selection Compared toother aptamers Bap5 showed relatively high affinity witha low dissociation constant of 483 120583M and the best IC

50

value of 7381 ngmLminus1The Bap5 aptamer did not show cross-reactivity with the other detectedmarine toxins Based on thehigh sensitivity and specificity of Bap5 it may be used as analternative analytical probe for the development of a detec-tion assay for BTX-2 Future studies should aim to developan assay based on the obtained aptamer for the detection ofBTX-2 in marine food samples to protect consumer safetyand promote the development of aquaculture

Competing Interests

All authors have no conflict of interests to declare

Authorsrsquo Contributions

Rui-Yun Tian Chao Lin and Shi-Yu Yu contributed equallyto this work Shi-Ying Lu and Hong-Lin Ren are correspond-ing authors

Acknowledgments

This work was supported by the National Nature ScienceFoundation of China under Grant no 31071539 the Coop-eration Program from Yunnan Here Biotechnology Co Ltdunder Grant 2015220101000658 and the Central UniversityBasic Scientific Research Business Expenses Special Funds(451160306025)

References

[1] J A Bean L E Fleming B Kirkpatrick et al ldquoFlorida redtide toxins (brevetoxins) and longitudinal respiratory effects inasthmaticsrdquo Harmful Algae vol 10 no 6 pp 744ndash748 2011

[2] M Fraga N Vilarino M C Louzao et al ldquoMultidetection ofparalytic diarrheic and amnesic shellfish toxins by an inhi-bition immunoassay using a microsphere-flow cytometry sys-temrdquo Analytical Chemistry vol 85 no 16 pp 7794ndash7802 2013

[3] S E McNamee C T Elliott P Delahaut and K CampbellldquoMultiplex biotoxin surface plasmon resonance method formarine biotoxins in algal and seawater samplesrdquo EnvironmentalScience and Pollution Research vol 20 no 10 pp 6794ndash68072013

[4] M J Twiner M-Y B Dechraoui Z H Wang et al ldquoExtractionand analysis of lipophilic brevetoxins from the red tide dinoflag-ellateKarenia brevisrdquoAnalytical Biochemistry vol 369 no 1 pp128ndash135 2007

[5] M van Deventer K Atwood G A Vargo et al ldquoKarenia brevisred tides and brevetoxin-contaminated fish a high risk factorfor Floridarsquos scavenging shorebirdsrdquo Botanica Marina vol 55no 1 pp 31ndash37 2012

[6] J H Landsberg ldquoThe effects of harmful algal blooms on aquaticorganismsrdquo Reviews in Fisheries Science vol 10 no 2 pp 113ndash390 2002

[7] J H Landsberg L J Flewelling and J Naar ldquoKarenia brevisred tides brevetoxins in the food web and impacts on naturalresources decadal advancementsrdquo Harmful Algae vol 8 no 4pp 598ndash607 2009

[8] G L Hitchcock J W Fourqurean J L Drake R N Mead andC A Heil ldquoBrevetoxin persistence in sediments and seagrassepiphytes of east Florida coastal watersrdquoHarmful Algae vol 13pp 89ndash94 2012

[9] GMHallegraeffDMAnderson andAD CembellaManualonHarmfulMarineMicroalgae UNESCO Paris France pp 25ndash49 2003

[10] D G Baden A J Bourdelais H Jacocks S Michelliza andJ Naar ldquoNatural and derivative brevetoxins historical back-groundmultiplicity and effectsrdquoEnvironmentalHealth Perspec-tives vol 113 no 5 pp 621ndash625 2005

[11] D Tang J Tang B Su and G Chen ldquoGold nanoparticles-decorated amine-terminated poly(amidoamine) dendrimer forsensitive electrochemical immunoassay of brevetoxins in foodsamplesrdquo Biosensors and Bioelectronics vol 26 no 5 pp 2090ndash2096 2011


[12] M-Y D Bottein J M Fuquay R Munday et al ldquoBioassaymethods for detection ofN-palmitoylbrevetoxin-B2 (BTX-B4)rdquoToxicon vol 55 no 2-3 pp 497ndash506 2010

[13] S M Plakas E L E Jester K R El Said et al ldquoMonitoring ofbrevetoxins in theKarenia brevis bloom-exposed Eastern oyster(Crassostrea virginica)rdquo Toxicon vol 52 no 1 pp 32ndash38 2008

[14] Z H Wang and J S Ramsdell ldquoAnalysis of interactions ofbrevetoxin-B and human serum albumin by liquid chromatog-raphymass spectrometryrdquoChemical Research in Toxicology vol24 no 1 pp 54ndash64 2011

[15] M A Poli V R Rivera D D Neal et al ldquoAn electrochem-iluminescence-based competitive displacement immunoassayfor the type-2 brevetoxins in oyster extractsrdquo Journal of AOACInternational vol 90 no 1 pp 173ndash178 2007

[16] M A Poli K S Rein andD G Baden ldquoRadioimmunoassay forPbTx-2-type brevetoxins epitope specificity of two anti-PbTxserardquo Journal of AOAC International vol 78 no 2 pp 538ndash5421995

[17] S M Plakas and R W Dickey ldquoAdvances in monitoring andtoxicity assessment of brevetoxins in molluscan shellfishrdquo Toxi-con vol 56 no 2 pp 137ndash149 2010

[18] S Marton B Berzal-Herranz E Garmendia F J Cueto andA Berzal-Herranz ldquoAnti-HCV RNA aptamers targeting thegenomic cis-acting replication elementrdquo Pharmaceuticals vol5 no 1 pp 49ndash60 2012

[19] Y Nonaka K Sode andK Ikebukuro ldquoScreening and improve-ment of an anti-VEGF DNA aptamerrdquo Molecules vol 15 no 1pp 215ndash225 2010

[20] K-M Song S Lee and C Ban ldquoAptamers and their biologicalapplicationsrdquo Sensors vol 12 no 1 pp 612ndash631 2012

[21] D T Tran K P F Janssen J Pollet et al ldquoSelection andcharacterization of DNA aptamers for egg white lysozymerdquoMolecules vol 15 no 3 pp 1127ndash1140 2010

[22] C Tuerk and L Gold ldquoSystematic evolution of ligands byexponential enrichment RNA ligands to bacteriophage T4DNApolymeraserdquo Science vol 249 no 4968 pp 505ndash510 1990

[23] X Zheng B Hu S X Gao et al ldquoA saxitoxin-binding aptamerwith higher affinity and inhibitory activity optimized by rationalsite-directed mutagenesis and truncationrdquo Toxicon vol 101 pp41ndash47 2015

[24] S M Handy B J Yakes J A DeGrasse et al ldquoFirst report of theuse of a saxitoxin-protein conjugate to develop a DNA aptamerto a small molecule toxinrdquoToxicon vol 61 no 1 pp 30ndash37 2013

[25] C Lin Z-S Liu D-X Wang et al ldquoGeneration of internal-image functional aptamers of okadaic acid via magnetic-beadSELEXrdquoMarine Drugs vol 13 no 12 pp 7433ndash7445 2015

[26] S Eissa A NgM Siaj A C Tavares andM Zourob ldquoSelectionand identification of DNA aptamers against okadaic acid forbiosensing applicationrdquoAnalytical Chemistry vol 85 no 24 pp11794ndash11801 2013

[27] M McKeague R Velu K Hill V Bardoczy T Meszaros andM C DeRosa ldquoSelection and characterization of a novel DNAaptamer for label-free fluorescence biosensing of ochratoxin ArdquoToxins vol 6 no 8 pp 2435ndash2452 2014

[28] L Barthelmebs J Jonca A Hayat B Prieto-Simon and J-LMarty ldquoEnzyme-Linked Aptamer Assays (ELAAs) based ona competition format for a rapid and sensitive detection ofOchratoxin A in winerdquo Food Control vol 22 no 5 pp 737ndash7432011

[29] J A Cruz-Aguado and G Penner ldquoDetermination of ochra-toxin A with a DNA aptamerrdquo Journal of Agricultural and FoodChemistry vol 56 no 22 pp 10456ndash10461 2008

[30] A De Girolamo M McKeague J D Miller M C DeRosaand A Visconti ldquoDetermination of ochratoxin A in wheat afterclean-up through a DNA aptamer-based solid phase extractioncolumnrdquo Food Chemistry vol 127 no 3 pp 1378ndash1384 2011

[31] S Eissa M Siaj and M Zourob ldquoAptamer-based competitiveelectrochemical biosensor for brevetoxin-2rdquo Biosensors andBioelectronics vol 69 pp 148ndash154 2015

[32] Y Zhou Y-S Li F-G Pan et al ldquoDevelopment of a newmonoclonal antibody based direct competitive enzyme-linkedimmunosorbent assay for detection of brevetoxins in foodsamplesrdquo Food Chemistry vol 118 no 2 pp 467ndash471 2010

[33] S Gong H-L Ren R-Y Tian et al ldquoA novel analyticalprobe binding to a potential carcinogenic factor of N-glycol-ylneuraminic acid by SELEXrdquoBiosensors and Bioelectronics vol49 pp 547ndash554 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


shellfish poison and has attracted more attention becauseof the red tide event in Florida that resulted in widespreadfish death [7] BTX-2 is a fat-soluble polyether compound[11] The minimum lethal dose in mice is 025mg kgminus1 andLD50

(lethal dose 50) is 200mg kgminus1 Therefore manyresearchers have continued to explore sensitive and conve-nient methods for detecting the toxic substance Currentlythe available detection methods include solid-phase extrac-tion (SPE) for qualitative analysis [12] high-performanceliquid chromatography (HPLC) [13] liquid chromatography-mass spectrometry (LC-MS) [14] radioimmunoassay [4]electrochemiluminescence-based immunoassay [15] thin-layer chromatography [16] and immunological methodssuch as enzyme-linked immunosorbent assay (ELISA) whichutilizes antibodies to quantify the BTX-2 levels [17] Thedisadvantages of these assays are the expensive equipmentwhich require professional staff the high cost of antibodieswith limited stability and the special storage conditionsrequired for the immunological assay Therefore the devel-opment of an alternative probe for the detection methodsthat rapidly cost-effectively and sensitively monitors BTX inshellfish and seawater is a pressing need

Recently aptamers oligonucleotides such as single-stranded DNA (ssDNA) or RNA [18 19] have attractedincreasing attention as the sensing elements in biosensorsused to detect many types of molecules such as proteinsmetals polypeptides and small molecules [20] Aptamershave all of the advantages of antibodies along with the uniqueadvantages of higher specificities and affinities for the targetand can be synthesized chemically modified easily storeddenatured and renatured [21] Aptamers are usually obtainedvia an in vitro process called systematic evolution of ligandsby exponential enrichment (SELEX) whichwas first reportedin 1990 [22] Since then many articles have described theuse of SELEX to select aptamers targeting many types ofsubstances particularly the aptamers targeting and detectingtoxins in recent years such as saxitoxin [23 24] okadaicacid [25 26] ochratoxin A [27ndash30] and the recent reportof BTX-2 in 2015 [31] Based on these advantages aptamersare emerging as novel capturing agents and recognitionreceptors in biosensor applications In this study we reportthe development of DNA aptamers that are able to bind BTX-2 with an dissociation constant of 483 120583M an IC

50value

of 7381 ngmLminus1 and selectivity for BTX only The resultspresented in this study report a novel alternative analyticalprobe for the development of an NSP toxin immunologicalassay These results will contribute to the establishment ofan experimental protocol for a relatively simple affordableand sensitive laboratory method for the detection of BTX inshellfish products and seawater

2 Materials and Methods

21 Apparatus and Reagents TheBTX-2 and saxitoxin (STX)standards (purity ge 98) were purchased from ZEN-UBiotechnology Co Ltd (Tai Wan China) The okadaic acid(OA) and domoic acid (DA) standards were purchased fromALEXIS Biochemicals The monoclonal antibody against

BTX-2 was prepared in our lab (119870119886 value is 082 times 109Mminus1)and stored at minus80∘C [32] Bovine serum albumin (BSA) andovalbumin (OVA) were purchased from the Beijing Ding-guo Biotechnology Development Center (Beijing China)The target BTX-2-BSA and BTX-2-OVA conjugates wereprepared using a previously described method [32] TheDNA mate Taq DNA polymerase PMD-18T and DL2000marker were purchased from Takara Biotechnology Co Ltd(Dalian China) The streptavidin-peroxidase complex waspurchased from Beijing Biosynthesis Biotechnology Co Ltd(Beijing China) Sterile ultrapure water was obtained froma Milli-Q water purification system (Millipore USA) Allof the polymerase chain reactions (PCRs) were conductedin a GeneAmp PCR system 9600 (Applied Perkin-ElmerUSA) The standard 96-well ELISA plates (U-bottom) werepurchased fromCostar (NY USA) Absorbance was recordedusing an Epoch Microvolume Spectrophotometer system(BioTek Instruments Inc USA) The other reagents used inthis study were of analytical grade

22 The ssDNA Library and Primer Synthesis The initialssDNA library used for SELEX and the homologous primerswere synthesized by the Shanghai Sangon Biological Engi-neering Technology amp Services Company (Shanghai China)The sequences included in the ssDNA library were 85nucleotides in length with a central region of 40 randomnucleotides flanked by two primer-binding sites for PCR andcloning as follows 51015840-GAGGCAGCACTTCACACGATC-TG-N40-CTGCGTAATGACTGTAGTGATG-31015840 Primer1 (51015840-GAGGCAGCACTTCACACGATCTG-31015840) and primer2 (51015840-CATCACTACAGTCATTACGCAG-31015840) were used foramplification and cloning The biotin-modified primer (51015840-biotin-GAGGCAGCACTTCACACGAT-31015840) was also synthe-sized for use in the binding assays All of the oligonucleotideswere dissolved in TE buffer (30mMTris-Cl 1mMEDTA pH80) and stored at minus20∘C until further use

23 Preparation of the Target Conjugates The target BTX-2-BSA and BTX-2-OVA conjugates were prepared using apreviously described method [32] The basic procedure isdescribed below A 10-foldmolar excess of succinic anhydridesolubilized in 25mL of anhydrous pyridine was added to2mg of crystalline BTX-2 After a 6 h incubation at 65∘Cthe solvent was evaporated under a stream of nitrogenand the residue was reacted with a 10-fold molar excess oftributylamine and isobutyl chlorocarbonate as 110 dilutionsin dry peroxide-free dioxin for 30min at room temperatureThen the carrier proteins BSA and OVA (molar ratio ofhaptencarrier 50 1) were added and incubated for 30minat room temperature these carrier proteins were used for thetarget (BTX-2-BSA) of SELEX and detection antigen (BTX-2-OVA) respectively The complex was recovered by acetoneprecipitation resuspended in 05mL of distilled water filter-sterilized (022120583m) dispensed into sterile tubes freeze-driedovernight and stored at minus20∘C until use

24 Selection of the Aptamer The typical SELEX procedureis an iterative process in which the sequences that specificallybind to the target are selected from highly diverse synthetic


BTX-protein


Second ssDNA pool

PCR amplification

Bound ssDNA Elution

Bound ssDNA

ssDNA pool

Unbound ssDNA

Wash






2)






2O2solution








492value when the








1 2

20001000750500

250

10085bp

(a)

2000

1000750

500

250

100

1 2

85nt

(b)


00

02

04

06

08

10

12

14

Abso

rban

ces (

492

nm)

1 2 3 4 5 6 7 8 9 10 11 120

Selection rounds








ba

b

a

a

a

b

a

b

b

a

a

Bap5 Bap 8 Bap23

Bap36Bap38


35

25

15

10

20

30

40

05

00

Abso

rban

ces (

492

nm)


050120583g mLminus1



(a)

100

90

80

70

60

50

40

30

20

10

0



(NminusS)N

times10

0()


(b)


492value when the






y = 30688x minus 7329

R2 = 09798

0

10

20

30

40

50

60

70

80

90

100

Inhi

bitio

n pe

rcen

t (

)

05 10 15 20 2500









4 Conclusion


50


Competing Interests




Acknowledgments


References












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


BTX-protein


Second ssDNA pool

PCR amplification

Bound ssDNA Elution

Bound ssDNA

ssDNA pool

Unbound ssDNA

Wash






2)






2O2solution








492value when the








1 2

20001000750500

250

10085bp

(a)

2000

1000750

500

250

100

1 2

85nt

(b)


00

02

04

06

08

10

12

14

Abso

rban

ces (

492

nm)

1 2 3 4 5 6 7 8 9 10 11 120

Selection rounds








ba

b

a

a

a

b

a

b

b

a

a

Bap5 Bap 8 Bap23

Bap36Bap38


35

25

15

10

20

30

40

05

00

Abso

rban

ces (

492

nm)


050120583g mLminus1



(a)

100

90

80

70

60

50

40

30

20

10

0



(NminusS)N

times10

0()


(b)


492value when the






y = 30688x minus 7329

R2 = 09798

0

10

20

30

40

50

60

70

80

90

100

Inhi

bitio

n pe

rcen

t (

)

05 10 15 20 2500









4 Conclusion


50


Competing Interests




Acknowledgments


References












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





2O2solution








492value when the








1 2

20001000750500

250

10085bp

(a)

2000

1000750

500

250

100

1 2

85nt

(b)


00

02

04

06

08

10

12

14

Abso

rban

ces (

492

nm)

1 2 3 4 5 6 7 8 9 10 11 120

Selection rounds








ba

b

a

a

a

b

a

b

b

a

a

Bap5 Bap 8 Bap23

Bap36Bap38


35

25

15

10

20

30

40

05

00

Abso

rban

ces (

492

nm)


050120583g mLminus1



(a)

100

90

80

70

60

50

40

30

20

10

0



(NminusS)N

times10

0()


(b)


492value when the






y = 30688x minus 7329

R2 = 09798

0

10

20

30

40

50

60

70

80

90

100

Inhi

bitio

n pe

rcen

t (

)

05 10 15 20 2500









4 Conclusion


50


Competing Interests




Acknowledgments


References












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


1 2

20001000750500

250

10085bp

(a)

2000

1000750

500

250

100

1 2

85nt

(b)


00

02

04

06

08

10

12

14

Abso

rban

ces (

492

nm)

1 2 3 4 5 6 7 8 9 10 11 120

Selection rounds








ba

b

a

a

a

b

a

b

b

a

a

Bap5 Bap 8 Bap23

Bap36Bap38


35

25

15

10

20

30

40

05

00

Abso

rban

ces (

492

nm)


050120583g mLminus1



(a)

100

90

80

70

60

50

40

30

20

10

0



(NminusS)N

times10

0()


(b)


492value when the






y = 30688x minus 7329

R2 = 09798

0

10

20

30

40

50

60

70

80

90

100

Inhi

bitio

n pe

rcen

t (

)

05 10 15 20 2500









4 Conclusion


50


Competing Interests




Acknowledgments


References












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


ba

b

a

a

a

b

a

b

b

a

a

Bap5 Bap 8 Bap23

Bap36Bap38


35

25

15

10

20

30

40

05

00

Abso

rban

ces (

492

nm)


050120583g mLminus1



(a)

100

90

80

70

60

50

40

30

20

10

0



(NminusS)N

times10

0()


(b)


492value when the






y = 30688x minus 7329

R2 = 09798

0

10

20

30

40

50

60

70

80

90

100

Inhi

bitio

n pe

rcen

t (

)

05 10 15 20 2500









4 Conclusion


50


Competing Interests




Acknowledgments


References












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


y = 30688x minus 7329

R2 = 09798

0

10

20

30

40

50

60

70

80

90

100

Inhi

bitio

n pe

rcen

t (

)

05 10 15 20 2500









4 Conclusion


50


Competing Interests




Acknowledgments


References












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

Documents

Research Article Preparation of a Specific ssDNA Aptamer for Brevetoxin …downloads.hindawi.com/journals/jamc/2016/9241860.pdf · 2019-07-30 · Research Article Preparation of a