A direct detection of Escherichia coligenomic DNA using gold nanoprobes

Jaffar, Padmavathy, Vinoth Kumar, Jaffar Ali

Description

In situation like diagnosis of clinical and forensic samples there exists a need for highly sensitive, rapid and specific DNA detection methods. Though conventional DNA amplification using PCR can provide fast results, it is not widely practised in diagnostic laboratories partially because it requires skilled personnel and expensive equipment. To overcome these limitations nanoparticles have been explored as signalling probes for ultrasensitive DNA detection that can be used in field applications. Among the nanomaterials, gold nanoparticles (AuNPs) have been extensively used mainly because of its optical property and ability to get functionalized with a variety of biomolecules. Results We report a protocol for the use of gold nanoparticles functionalized with single stranded oligonucleotide (AuNP- oligo probe) as visual detection probes for rapid and specific detection of Escherichia coli . The AuNP- oligo probe on hybridization with target DNA containing complementary sequences remains red whereas test samples without complementary DNA sequences to the probe turns purple due to acid induced aggregation of AuNP- oligo probes. The color change of the solution is observed visually by naked eye demonstrating direct and rapid detection of the pathogenic Escherichia coli from its genomic DNA without the need for PCR amplification. The limit of detection was ~54 ng for unamplified genomic DNA. The method requires less than 30 minutes to complete after genomic DNA extraction. However, by using unamplified enzymatic digested genomic DNA, the detection limit of 11.4 ng was attained. Results of UV-Vis spectroscopic measurement and AFM imaging further support the hypothesis of aggregation based visual discrimination. To elucidate its utility in medical diagnostic, the assay was validated on clinical strains of pathogenic Escherichia coli obtained from local hospitals and spiked urine samples. It was found to be 100% sensitive and proves to be highly specific without any cross reaction with non- Escherichia coli strains. Conclusion This work gives entry into a new class of DNA/gold nanoparticles hybrid materials which might have optical property that can be controlled for application in diagnostics. We note that it should be possible to extend this strategy easily for developing new types of DNA biosensor for point of care detection. The salient feature of this approach includes low-cost, robust reagents and simple colorimetric detection of pathogen.

Sujets

• Colloidal gold
• Escherichia coli

Informations

• Publié par: biomed
• Publié le: 01 janvier 2012
• Nombre de lectures: 26
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

etal
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JournalofNanobiotechnology
2012,
10
:8
http://www.jnanobiotechnology.com/content/10/1/8

RESEARCH

OpenAccess

Adirectdetectionof
Escherichiacoli
genomic
DNAusinggoldnanoprobes
PadmavathyBakthavathsalam
1
,VinothKumarRajendran
1
andJaffarAliBaquirMohammed
2*

Abstract
Background:
Insituationlikediagnosisofclinicalandforensicsamplesthereexistsaneedforhighlysensitive,
rapidandspecificDNAdetectionmethods.ThoughconventionalDNAamplificationusingPCRcanprovidefast
results,itisnotwidelypractisedindiagnosticlaboratoriespartiallybecauseitrequiresskilledpersonneland
expensiveequipment.Toovercometheselimitationsnanoparticleshavebeenexploredassignallingprobesfor
ultrasensitiveDNAdetectionthatcanbeusedinfieldapplications.Amongthenanomaterials,goldnanoparticles
(AuNPs)havebeenextensivelyusedmainlybecauseofitsopticalpropertyandabilitytogetfunctionalizedwitha
varietyofbiomolecules.
Results:
Wereportaprotocolfortheuseofgoldnanoparticlesfunctionalizedwithsinglestrandedoligonucleotide
(AuNP-oligoprobe)asvisualdetectionprobesforrapidandspecificdetectionof
Escherichiacoli
.TheAuNP-oligo
probeonhybridizationwithtargetDNAcontainingcomplementarysequencesremainsredwhereastestsamples
withoutcomplementaryDNAsequencestotheprobeturnspurpleduetoacidinducedaggregationofAuNP-
oligoprobes.Thecolorchangeofthesolutionisobservedvisuallybynakedeyedemonstratingdirectandrapid
detectionofthepathogenic
Escherichiacoli
fromitsgenomicDNAwithouttheneedforPCRamplification.The
limitofdetectionwas~54ngforunamplifiedgenomicDNA.Themethodrequireslessthan30minutesto
completeaftergenomicDNAextraction.However,byusingunamplifiedenzymaticdigestedgenomicDNA,the
detectionlimitof11.4ngwasattained.ResultsofUV-VisspectroscopicmeasurementandAFMimagingfurther
supportthehypothesisofaggregationbasedvisualdiscrimination.Toelucidateitsutilityinmedicaldiagnostic,the
assaywasvalidatedonclinicalstrainsofpathogenic
Escherichiacoli
obtainedfromlocalhospitalsandspikedurine
samples.Itwasfoundtobe100%sensitiveandprovestobehighlyspecificwithoutanycrossreactionwithnon-
Escherichiacoli
strains.
Conclusion:
ThisworkgivesentryintoanewclassofDNA/goldnanoparticleshybridmaterialswhichmighthave
opticalpropertythatcanbecontrolledforapplicationindiagnostics.Wenotethatitshouldbepossibletoextend
thisstrategyeasilyfordevelopingnewtypesofDNAbiosensorforpointofcaredetection.Thesalientfeatureof
thisapproachincludeslow-cost,robustreagentsandsimplecolorimetricdetectionofpathogen.
Keywords:
Goldnanoparticles,DNAdetection,colorimetricdetection,clinicaldiagnosis,
Escherichiacoli

Background
inthediagnosticlaboratoriesofdevelopingcountries
ThedevelopmentofhighlysensitiveandselectiveDNApartiallybecauseitrequiresconsiderableskilland
detectionmethodsisextremelyimportantinclinicalexpensiveequipment.Toovercometheseproblems
diagnosis,forensicinvestigationsandgenetherapynanoparticleshavebeenexploredassignallingprobes
becausetheDNAisusuallypresentatverylowconcen-forultrasensitiveDNAdetectionthatcanbeusedin
trations[1-3].AlthoughconventionalDNAamplificationfieldapplications.Amongthenanomaterials,goldnano-
usingPCRcanprovidefastresults,itisnotwidelyusedparticles(AuNPs)havebeenextensivelyusedforbiomo-
leculedetectionbymanyresearchgroupsmainly
*
2
Correspondence:[e-mail]
becauseofitsopticalpropertyandabilitytofunctiona-
CentreforGreenEnergyTechnology,PondicherryUniversity,Puducherry,
lizewithavarietyofbiomolecules.TheAuNPshave
diaInFulllistofauthorinformationisavailableattheendofthearticle
beenintegratedinresearchandroutinediagnostic
©2012Padmavathyetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.

etal
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JournalofNanobiotechnology
2012,
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http://www.jnanobiotechnology.com/content/10/1/8

applicationsandhavebeenshowntohavethegreat
potential[4,5].ThecolloidalAuNPsareusedinthe
developmentofseveralbiodetectionschemes[6-10].
Althoughprotein-coatedgoldcolloidshavebeenused
extensivelyinlateralflowimmunoassaybasedanalytical
techniquestheirapplicationtowardsDNAdetectionhas
beenintroducedbyMirkinandco-workers[11].The
functionalizationofcolloidalAuNPswithalkylthiol
modifiedDNAisthemostcommonones.Such
approachwasusedforpreparingstableoligonucleotide
conjugateswithAu[12],Au-coatedAg[13]andZnS-
coatedCdSenanoparticles[14].Considerableworkwas
carriedoutthatexplorethebasicpropertiesofgold
nanoparticles[15-17].DNA-functionalizedAuNPsare
usedinavarietyofmoleculardiagnosticapplications
suchashighsensitivityDNAdetectioninhomogenous
solution[18]andmicroarray-basedDNAdetection
[19],lab-on-chipbasedsubstrates[20-22],atomicforce
microscopybaseddetection[23],detectionofpolymer-
asechainreaction(PCR)amplicons[24,25].Theoptical
propertyofcolloidalAuNPsformsthebasisofitsappli-
cationinrapidandspecifichybridizationbasedDNA
detections.Thispropertyhasalreadybeenappliedfor
detectionofeukaryoticgeneexpression[26],
Mycobac-
teriumtuberculosis
detection[27,28]andforRNAquan-
tificationtodetectchronicmyeloidleukaemia[29].
Usingaparallelapproachweextendtheapplicationof
AuNPsconjugatedoligonucleotideprobes(AuNP-oligo
probes)asatoolforrapiddetectionofbacterialpatho-
genfromclinicalisolates.Inthiscontext,wedemon-
stratedtheutilityoftheassayondiarrhogenicand
uropathogenic
Escherichiacoli
strainsobtainedfrom
patientsoflocalhospitals.
Diagnosisof
Escherichiacoli
infectionsrequiresbac-
terialculturethatneedsonetotwodaysofincubation,
andsubsequentconfirmatorytesting.Rapiddetection
methodslikeenzymeimmunoassayrequireahighpopu-
lationofthetargetpathogen[30,31].Incontrast,mole-
culardiagnosticassayslikePCRbasedapproaches
requirefeweramountsofsamplebutdedicatedequip-
mentsandtrainedtechnicalpersonnel.Hencethere
existsalwaysaneedforrapidclinicaldiagnosisofpatho-
gensatlowcost.Ourapproachtousinghybridizationof
oligonucleotidesforthecontrolledassemblyofgold
nanoparticlesunderacidicenvironmentfordetectionof
Escherichiacoli
isoutlinedinFigure1.Inbrief,the
hybridizationoftheAuNP-oligoprobewiththecom-
plementary
Escherichiacoli
genomicDNAremainsred.
Howeverthesolutionturnspurplewithoutcomplemen-
taryDNA.Themethodreliesonavisualcomparisonof
solutionsforcolorchangebeforeandafteracidinduced
aggregationofAuNP-oligoprobe.Notably,theconco-
mitantcolorchangeofthesolutioncanbeobservedby
nakedeye.TheassaywasfurtherevaluatedbyUV-Vis

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spectroscopiccharacterization,whichdemonstrates
aggregationinducedred-shiftinAuNP-oligoprobes.
Additionallydispersionandaggregationpatternof
AuNP-oligoprobesrespectivetothehybridizationwas
monitoredatnanoscaleusingAFM.Theapplicationof
thedevelopedassaytomedicaldiagnosticswasshowna)
clinicalisolatesandb)urine-spikedclinicalsamples.
ThisstudydemonstratestheapplicationofAuNP-oligo
probesforvisualdetectionofpathogenatgenomic
DNAlevelwhichcanbeadaptedasaroutinescreening
toolinclinicallaboratories.
Methods
Bacterialstrainsandreagents
Theuropathogenic
Escherichiacoli
(UPEC)were
obtainedfromSundaramMedicalFoundation(SMF)
hospitalandGovernmentGeneralHospital(GH),Chen-
nai.Shiga-toxinproducingdiarrhogenic
Escherichiacoli
O157wasobtainedfromChristianMedicalCollege
(CMC),Vellore.Referencestrainsof
Escherichiacoli
and
non-
Escherichiacoli
organismswereobtainedfrom
MicrobialTypeCultureCollection(MTCC),Instituteof
MicrobialTechnology,Chandigarh.Alltheorganisms
wereculturedinLuria-Bertoni(LB)brothat37°Cfor16-
18hoursat195rpm.Allclinicalisolateswerecharacter-
izedbiochemicallyfollowedbyPCRusingprimerpairs
targeting
fimH
geneof
Escherichiacoli
[32]priortovali-
dationbyAuNP-oligoprobeassay.ThedetailsofPCR
cyclingconditionsweredescribedinadditionalfile1.
Thecolloidalgoldnanoparticlesofsize20nmwerepur-
chasedfromSigmaandstoredat4°Cuntiluse.Thesilica
spincolumnforDNAextractionwaspurchasedfrom
Qiagen.Allotherchemicalswereofanalyticalgradeand
usedaspurchasedwithoutfurthermodifications.
Probedesign
Probesequencewasdesignedbasedonthe
malB
gene
regionthathasthehighhomologyamongthe
Escheri-
chiacoli
strains.A20baseoligonucleotidewasgener-
atedasprobesequenceusingtheNCBIBLAST
nucleotidesearchtool[33].Careistakenindesigning
thesequencesothatitdoesnotshareanysequence
homologywithnon-
Escherichiacoli
familymembers.
Additionally,themeltingtemperatureoftheprobeswas
ensuredtobewithinanarrowrange.Furtherthe
sequenceischeckedforpotentialself-complementarities
andalsoformationofsecondarystructureswasverified
usingmfoldsoftware[34]whichmayotherwisehinder
theassay.Thefinal20baseprobesequenceobtained
was5
’
TACAAAGGGAGAAGGGCATG3
’
.Itcontains
athiolmodifierat5
’
endtoenableconjugationwiththe
colloidalgoldnanoparticles.This5
’
alkylthiolmodified
oligonucleotidewithHPLCpurificationwaspurchased
fromSigma.

etal
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JournalofNanobiotechnology
2012,
10
:8
http://www.jnanobiotechnology.com/content