Resultsanddiscussion3.1.Fabrication

Resultsanddiscussion
3.1.Fabricationofq-MNP–fluorescent polymercomplex
Tosolvetheissueofrapiddetectionandidentification ofpathogen,
a strategyhasbeendevisedforbacterialsensingusinganarrayofthe
q-MNP conjugated fluorescentpolymersystem. Scheme 1 showsa
representationofthisbacteriadetectionmethodbasedontheq-MNP
conjugated PFBTsystem.Inourexperiments,ananionicPFBTis first
associatedwithquaternizedMNPinaqueoussolutiontooffera
sensing system.Whenbacteriasamplesareaddedintothesystem,
the anionicPFBTwillbereplacedonthesurfaceofthebacterial
membraneduetowhichbothelectrostaticandhydrophobicinterac-
tion playkeyrolesinthecomplexsystemofMNPwithbacterialcells.
Toavoidtheinterferenceofq-MNPandq-MNP–fluorescentpolymer
complex,theseparticlesandcomplexwillberemovedusingmagnetic
separationandthe fluorescenceintensityofthesupernatantwas
measuredforthreeresponsepatterns.Fortheresponsereplacement
strategy,weselectedPFBTandthreenanoparticles(q-MNP1–q-MNP3)
as sensorelements(shownin Fig.S4). Thismethodavoidstheneedto
developcomplicatedantibody-orspecific recognitionelement-
functionalizednanoparticles,thusallowingstraightforwardandcom-
pletesurfaceresponselabelsforbacteriadetectionintheabsenceof
instability recognitionelementswithinashortperiodoftime.
Fluorescencetitrationwasperformedtoassessthebindingaffinity
betweenPFBTandq-MNP.The fluorescenceofPFBTwasdistinctly
decreasedandthenormalizedresponseintensitiesofPFBTat538nm
wereplottedversustheratioofq-MNPandPFBTpolymer(shownin
Fig.S6). Thecomplexstabilityconstants(Ks) andassociationstoichio-
metries(n) wereestimatedthroughamathematicalmodelusing
nonlinear least-squarescurve-fittinganalysis(Youetal.,2005).
As depictedin Fig.S6, the Ks and n aredependentonthesidechains
of thenanoparticles.Complexstabilitiesvarywithinoneorderof
magnitudefrom1.7105M
1 forq-MNP3,4.2105M
1 for
q-MNP1to2.8106M
1 for q-MNP2,andtheassociationstoichio-
metriesrangefrom213forq-MNP3,420forq-MNP1to1204for
q-MNP 2.Theseresultsdemonstratedthatthechemicalstructural
changesofthenanoparticles0 surfaceaffecttheir interactionforPBFT
polymer.SincethedifferentbindingabilitiesofPFBTwithq-MNP1,q-
MNP 2andq-MNP3havebeenestablished,theq-MNP–conjugated
PFBT systemwillbeusedtodetectandsensebacteria.
3.2. ‘Chemical nose’ for bacteriaidentification
As asensitiveandreliabletool,thechemicalnosehasbeenapplied
in sensorstomeasurethechangeofresponseintensitybasedon
biological andchemicalmoleculerecognition.Asillustratedin Fig.1a
and b,additionofbacterialsamples(107 cfu mL
1) resultedina
varietyof fluorescenceresponsesduetowhichthePFBTwasreleased
in thesolutionfromthecompetitivebindingabilityofdifferent
bacterialcells.Foreachbacteriasample,wemeasureditsresponse
changeagainstthethreeq-MNPpolymers.The fluorescenceresponse
matrixwasanalyzedbyLDA,amethodusedinstatisticsandpattern
recognitionto find alinearcombinationoffeatureswhichcharac-
terizestwoormoreclassesofevents(MartinezandKak,2001).
The rawdataofmatrixwereclusteredintoeightgroupsfor
different bacteriausingcanonicalscoresofLDAtransformation
(shown in Fig. 1c). Furthermore,another32bacterialsampleswere
testedinanunknowncondition,wherethemethodoffereda
relativelylowaccuracyof87.5%(TableS1) duetowhichthe
canonical scoresareaof E. tarda, M. luteus, and S. oneidensis show
a partialoverlap(Fig. 1c).
Theresponsepattern(Fig. 2a) andthethree-dimensionalrepre-
sentation (Fig. 2b) ofresponsechangeswherethebacterialconcen-
trationisOD600¼0.2arenotablydifferentfromthoseobservedfrom
107 cfumL
1. Asshownin Fig. 2c, LDAcanaccuratelydistinguish
each bacteriaresponsepatternwithoutoverlapbetweenthe95%
confidenceellipses.Withthecanonicalscoreplotin Fig. 2c andinthe
supportingmaterials(shownin TableS2), 32unknownbacteria
sampleschosenfromtheeightdifferentbacteriawithdifferent
concentrationscanbeidentified anddetected.CombinedwithUV–
vismeasurement,ahighaccuracyof96.8%demonstratedreliability
(shownin TableS2). Thisresultcertainlydemonstratesthatour
‘chemicalnose’ holdsasignificantpromiseforidentificationand
detectionofpathogen.
A seriesofunknownbacteriasamples weresuccessfullyestimated
usingthe ‘chemical nose’ withUV–vismeasurement.Thisresult
unambiguouslydemonstratesthatoursensorholdspotentialpromise
forboththedetectionandidentification ofbacterialtargets.Recently,
the efficient andcost-effectivetechniquesbasedonMNPasarapid
and reliabletoolforpathogendetectionhavealreadybeenwidely
used.Forexample,Bakthavathsalametal.reportedarapidmethodfor
immunomagneticseparationof Salmonella along withtheirrealtime
detectionviaPCR.Itenablesdifferentiationof Salmonellatyphi and
Salmonellaparatyphi usingasetoffourspecific primerswithcost-
intensivenessandtechnicalcomplexity(Bakthavathsalametal.,2013).
Kwonetal.useantibodyconjugatedgold-coatedmagneticnanopar-
ticle clustersandmagnetophoreticchromatographywithaprecision
pipettefordetectionof Salmonella (Kwonetal.,2013). Thismethod
showsarelativelyunstableconditionduetowhichtheantibodyis
easilyinactivated.Comparedtootheranalyticaltechniquesbasedon
MNP,itadequatelyservestherequirementsoflarge-scalemonitoring
of environmentalissues.The ‘chemicalnose’ techniqueoffersagood
alternativeforthebiomoleculessensing.

ผลลัพธ์ (ไทย) 1:

Resultsanddiscussion3.1.Fabricationofq-MNP–fluorescent polymercomplexTosolvetheissueofrapiddetectionandidentification ofpathogenstrategyhasbeendevisedforbacterialsensingusinganarrayofthe การfluorescentpolymersystem q MNP กลวง แผน 1 showsarepresentationofthisbacteriadetectionmethodbasedontheq-MNPกลวง PFBTsystem.Inourexperiments,ananionicPFBTis แรกassociatedwithquaternizedMNPinaqueoussolutiontoofferaระบบไร้สาย WhenbacteriasamplesareaddedintothesystemanionicPFBTwillbereplacedonthesurfaceofthebacterialmembraneduetowhichbothelectrostaticandhydrophobicinterac-playkeyrolesinthecomplexsystemofMNPwithbacterialcells สเตรชันToavoidtheinterferenceofq-MNPandq-MNP – fluorescentpolymerซับซ้อน theseparticlesandcomplexwillberemovedusingmagneticseparationandthe fluorescenceintensityofthesupernatantwasmeasuredforthreeresponsepatterns Fortheresponsereplacementstrategy,weselectedPFBTandthreenanoparticles(q-MNP1–q-MNP3)เป็น sensorelements (shownin Fig.S4) Thismethodavoidstheneedtodevelopcomplicatedantibody-orspecific recognitionelement-functionalizednanoparticles, thusallowingstraightforwardandcom-pletesurfaceresponselabelsforbacteriadetectionintheabsenceofความไม่แน่นอน recognitionelementswithinashortperiodoftimeFluorescencetitrationwasperformedtoassessthebindingaffinityfluorescenceofPFBTwasdistinctly betweenPFBTandq-MNP.ThedecreasedandthenormalizedresponseintensitiesofPFBTat538nmwereplottedversustheratioofq-MNPandPFBTpolymer (showninFig.S6) . Thecomplexstabilityconstants(Ks) andassociationstoichio -metries(n) wereestimatedthroughamathematicalmodelusingleast-squarescurve-fittinganalysis(Youetal.,2005) ไม่เชิงเส้นเป็น depictedin Fig.S6, Ks และ n aredependentonthesidechainsของ thenanoparticles.Complexstabilitiesvarywithinoneorderofmagnitudefrom1.7 105M 1 forq MNP3, 105 4.2 M 1 สำหรับq MNP1to2.8 M 106 1 สำหรับ q-MNP2, andtheassociationstoichio -metriesrangefrom213forq-MNP3, 420forq-MNP1to1204for2. q MNP Theseresultsdemonstratedthatthechemicalstructuralchangesofthenanoparticles0 surfaceaffecttheir interactionforPBFTพอลิเมอร์ SincethedifferentbindingabilitiesofPFBTwithq-MNP1, q-Theq-MNP – กลวง 2andq MNP-MNP3havebeenestablishedPFBT systemwillbeusedtodetectandsensebacteria3.2. 'จมูกเคมี' สำหรับ bacteriaidentificationเป็น asensitiveandreliabletool, thechemicalnosehasbeenappliedใน sensorstomeasurethechangeofresponseintensitybasedonandchemicalmoleculerecognition ชีวภาพ Asillustratedin Fig.1aแอนด์ บี resultedina additionofbacterialsamples (107 cfu mL 1)varietyof fluorescenceresponsesduetowhichthePFBTwasreleasedใน thesolutionfromthecompetitivebindingabilityofdifferentbacterialcells Foreachbacteriasample, wemeasureditsresponsefluorescenceresponse changeagainstthethreeq-MNPpolymers.ThematrixwasanalyzedbyLDA, amethodusedinstatisticsandpatternค้นหา recognitionto alinearcombinationoffeatureswhichcharac-terizestwoormoreclassesofevents(MartinezandKak,2001)RawdataofmatrixwereclusteredintoeightgroupsforbacteriausingcanonicalscoresofLDAtransformation แตกต่างกัน(แสดงใน Fig. 1 c) นอกจากนี้ another32bacterialsamplesweretestedinanunknowncondition, wherethemethodofferedarelativelylowaccuracyof87.5%(TableS1) duetowhichtheมาตรฐาน scoresareaof E. tarda, M. luteus และ S. oneidensis ดูpartialoverlap ที่การกิน 1c)Theresponsepattern (Fig. 2a) andthethree-dimensionalrepre -ofresponsechangeswherethebacterialconcen sentation (Fig. 2b) -trationisOD600¼0.2arenotablydifferentfromthoseobservedfrom107 cfumL 1 Asshownin Fig. 2c, LDAcanaccuratelydistinguishแต่ละ bacteriaresponsepatternwithoutoverlapbetweenthe95%confidenceellipses Andinthe Withthecanonicalscoreplotin Fig. 2csupportingmaterials (shownin TableS2), 32unknownbacteriasampleschosenfromtheeightdifferentbacteriawithdifferentconcentrationscanbeidentified ที่ anddetected.CombinedwithUV–vismeasurement,ahighaccuracyof96.8%demonstratedreliability(shownin TableS2) Thisresultcertainlydemonstratesthatourholdsasignificantpromiseforidentificationand 'chemicalnose'detectionofpathogenSeriesofunknownbacteriasamples weresuccessfullyestimatedusingthe 'จมูกเคมี' withUV – vismeasurement Thisresultunambiguouslydemonstratesthatoursensorholdspotentialpromiseforboththedetectionandidentification ofbacterialtargets ล่าสุดeffectivetechniquesbasedonMNPasarapid andcost ที่มีประสิทธิภาพและ reliabletoolforpathogendetectionhavealreadybeenwidelyใช้ Forexample,Bakthavathsalametal.reportedarapidmethodforimmunomagneticseparationof ซัลตาม withtheirrealtimedetectionviaPCR.Itenablesdifferentiationof Salmonellatyphi และSalmonellaparatyphi usingasetoffourspecific primerswithcost-intensivenessandtechnicalcomplexity(Bakthavathsalametal.,2013)Kwonetal.useantibodyconjugatedgold-coatedmagneticnanopar -ticle clustersandmagnetophoreticchromatographywithaprecisionpipettefordetectionof ซัล (Kwonetal. 2013) Thismethodshowsarelativelyunstableconditionduetowhichtheantibodyiseasilyinactivated.ComparedtootheranalyticaltechniquesbasedonMNP, itadequatelyservestherequirementsoflarge-scalemonitoringของ environmentalissues Techniqueoffersagood 'chemicalnose'alternativeforthebiomoleculessensing

ผลลัพธ์ (ไทย) 2:

Resultsanddiscussion
3.1.Fabricationofq-MNP เรืองแสง polymercomplex
Tosolvetheissueofrapiddetectionandidentification ofpathogen,
strategyhasbeendevisedforbacterialsensingusinganarrayofthe
Q-MNP ผัน fluorescentpolymersystem โครงการ 1 แสดง
'representationofthisbacteriadetectionmethodbasedontheq-MNP
PFBTsystem.Inourexperiments ผัน ananionicPFBTis แรก
associatedwithquaternizedMNPinaqueoussolutiontooffera
ตรวจจับ system.Whenbacteriasamplesareaddedintothesystem, sensorelements (shownin Fig.S4) Thismethodavoidstheneedto developcomplicatedantibody-orspecific Thecomplexstabilityconstants (Ks) andassociationstoichio- metries (n) wereestimatedthroughamathematicalmodelusing ไม่เชิงเส้นอย่างน้อย squarescurve-fittinganalysis (Youetal., 2005). ในฐานะที่เป็น depictedin Fig.S6 ที่ Ks และ n aredependentonthesidechains ของ thenanoparticles.Complexstabilitiesvarywithinoneorderof magnitudefrom1.7? 105m 1 forq-MNP3, 4.2? 105m? 1 สำหรับQ-MNP1to2.8? 106m 1 สำหรับ q-MNP2, andtheassociationstoichio- metriesrangefrom213forq-MNP3,420forq MNP1to1204for-Q-MNP 2.Theseresultsdemonstratedthatthechemicalstructural changesofthenanoparticles0 surfaceaffecttheir interactionforPBFT polymer.SincethedifferentbindingabilitiesofPFBTwithq-MNP1, Q- MNP 2andq-MNP3havebeenestablished , theq-MNP-ผันsystemwillbeusedtodetectandsensebacteria PFBT. 3.2 'เคมีจมูกสำหรับ bacteriaidentification เป็น asensitiveandreliabletool, thechemicalnosehasbeenapplied ใน sensorstomeasurethechangeofresponseintensitybasedon ชีวภาพ andchemicalmoleculerecognition.Asillustratedin Fig.1a และ b additionofbacterialsamples (107 cfu mL? 1) resultedina varietyof fluorescenceresponsesduetowhichthePFBTwasreleased ใน fluorescenceresponse matrixwasanalyzedbyLDA, amethodusedinstatisticsandpattern recognitionto หา alinearcombinationoffeatureswhichcharac- terizestwoormoreclassesofevents (MartinezandKak, 2001). rawdataofmatrixwereclusteredintoeightgroupsfor bacteriausingcanonicalscoresofLDAtransformation ที่แตกต่างกัน(แสดงในรูป. 1 c) duetowhichthe ยอมรับ scoresareaof tarda อีเอ็ม luteus และเอส oneidensis แสดงpartialoverlap (รูป. 1 c). Theresponsepattern (รูป. 2a) andthethree-dimensionalrepre- sentation (รูป. 2b) cfumL 1 Asshownin รูป 2c, LDAcanaccuratelydistinguish แต่ละ bacteriaresponsepatternwithoutoverlapbetweenthe95% confidenceellipses.Withthecanonicalscoreplotin รูป 2c andinthe supportingmaterials (shownin TableS2) anddetected.CombinedwithUV- vismeasurement, ahighaccuracyof96.8% demonstratedreliability (shownin TableS2) Thisresultcertainlydemonstratesthatour 'chemicalnose' holdsasignificantpromiseforidentificationand detectionofpathogen. seriesofunknownbacteriasamples weresuccessfullyestimated usingthe 'เคมีจมูก' ofbacterialtargets.Recently, มีประสิทธิภาพ andcost-effectivetechniquesbasedonMNPasarapid และ Salmonella พร้อม withtheirrealtime detectionviaPCR.Itenablesdifferentiationof Salmonellatyphi และSalmonellaparatyphi usingasetoffourspecific clustersandmagnetophoreticchromatographywithaprecision pipettefordetectionof Salmonella (Kwonetal. 2013) environmentalissues.The 'chemicalnose' techniqueoffersagood alternativeforthebiomoleculessensing

ผลลัพธ์ (ไทย) 3:

resultsanddiscussion
3.1 fabricationofq MNP –เรืองแสง polymercomplex

เป็น ofpathogen tosolvetheissueofrapiddetectionandidentification , strategyhasbeendevisedforbacterialsensingusinganarrayofthe
q-mnp conjugated fluorescentpolymersystem . โครงการ 1 showsa

representationofthisbacteriadetectionmethodbasedontheq MNP conjugated pfbtsystem . inourexperiments
ananionicpfbtis , แรกระบบตรวจจับ whenbacteriasamplesareaddedintothesystem associatedwithquaternizedmnpinaqueoussolutiontooffera
. ,
-

anionicpfbtwillbereplacedonthesurfaceofthebacterial membraneduetowhichbothelectrostaticandhydrophobicinterac tion playkeyrolesinthecomplexsystemofmnpwithbacterialcells .
toavoidtheinterferenceofq mnpandq MNP ) fluorescentpolymer
ซับซ้อนtheseparticlesandcomplexwillberemovedusingmagnetic

separationandthe fluorescenceintensityofthesupernatantwas measuredforthreeresponsepatterns กลยุทธ์ fortheresponsereplacement
, weselectedpfbtandthreenanoparticles ( q-mnp1 – q-mnp3 )
เป็น sensorelements ( shownin fig.s4 ) thismethodavoidstheneedto
-
functionalizednanoparticles developcomplicatedantibody orspecific recognitionelement ,thusallowingstraightforwardandcom -

pletesurfaceresponselabelsforbacteriadetectionintheabsenceof ความไม่แน่นอน recognitionelementswithinashortperiodoftime .



fluorescencetitrationwasperformedtoassessthebindingaffinity betweenpfbtandq-mnp.the fluorescenceofpfbtwasdistinctly decreasedandthenormalizedresponseintensitiesofpfbtat538nm wereplottedversustheratioofq mnpandpfbtpolymer ( shownin
fig.s6 )thecomplexstabilityconstants ( KS ) andassociationstoichio -
metries ( N ) wereestimatedthroughamathematicalmodelusing
ไม่เชิงเส้นน้อย squarescurve fittinganalysis ( youetal . , 2005 ) .
เป็น depictedin fig.s6 , KS และ aredependentonthesidechains
ของ thenanoparticles . complexstabilitiesvarywithinoneorderof
magnitudefrom1.7  105m
1 forq-mnp3,4.2  105m
1
q-mnp1to2.8  106m
q-mnp2 1 ,andtheassociationstoichio metriesrangefrom213forq-mnp3420forq-mnp1to1204for
-
q-mnp 2 . theseresultsdemonstratedthatthechemicalstructural

changesofthenanoparticles0 surfaceaffecttheir interactionforpbft พอลิเมอร์ sincethedifferentbindingabilitiesofpfbtwithq-mnp1 , Q -
MNP 2andq-mnp3havebeenestablished TheQ , MNP ) pfbt systemwillbeusedtodetectandsensebacteria conjugated
.
2 .' จมูก ' เคมี bacteriaidentification
เป็น asensitiveandreliabletool thechemicalnosehasbeenapplied
, ใน andchemicalmoleculerecognition ชีวภาพ sensorstomeasurethechangeofresponseintensitybasedon fig.1a

asillustratedin บี additionofbacterialsamples ( 107 CFU ml
1 ) resultedina fluorescenceresponsesduetowhichthepfbtwasreleased

varietyofใน thesolutionfromthecompetitivebindingabilityofdifferent
bacterialcells . foreachbacteriasample wemeasureditsresponse

matrixwasanalyzedbylda , changeagainstthethreeq-mnppolymers.the fluorescenceresponse , recognitionto หา alinearcombinationoffeatureswhichcharac amethodusedinstatisticsandpattern
-
terizestwoormoreclassesofevents ( martinezandkak , 2001 ) rawdataofmatrixwereclusteredintoeightgroupsfor

ที่แตกต่างกัน bacteriausingcanonicalscoresofldatransformation
( แสดงในรูปที่ 1C ) นอกจากนี้ another32bacterialsampleswere testedinanunknowncondition wherethemethodoffereda

, relativelylowaccuracyof87.5 % ( tables1 ) duetowhichthe
Canonical scoresareaof E . tarda M luteus , และ oneidensis แสดง : partialoverlap ( ภาพที่ 1c )
theresponsepattern ( รูปที่ 2A ) andthethree dimensionalrepre -
sentation ( ฟิค2B ) ofresponsechangeswherethebacterialconcen -
trationisod600 ¼ 0.2arenotablydifferentfromthoseobservedfrom
107 cfuml
1 asshownin รูปที่ 2 C , ldacanaccuratelydistinguish
แต่ละ bacteriaresponsepatternwithoutoverlapbetweenthe95 %
confidenceellipses.withthecanonicalscoreplotin รูปที่ 2 andinthe
supportingmaterials ( shownin tables2 32unknownbacteria sampleschosenfromtheeightdifferentbacteriawithdifferent

)concentrationscanbeidentified anddetected . combinedwithuv – vismeasurement ahighaccuracyof96.8 % demonstratedreliability
,
( shownin tables2 ) thisresultcertainlydemonstratesthatour
'chemicalnose ' holdsasignificantpromiseforidentificationand
detectionofpathogen .

ใช้สารเคมีเป็น seriesofunknownbacteriasamples weresuccessfullyestimated ' จมูก ' withuv – vismeasurement thisresult
.unambiguouslydemonstratesthatoursensorholdspotentialpromise
forboththedetectionandidentification ofbacterialtargets เมื่อเร็ว ๆ นี้


และมีประสิทธิภาพที่ใช้ effectivetechniquesbasedonmnpasarapid reliabletoolforpathogendetectionhavealreadybeenwidely ใช้ เช่น bakthavathsalametal . Salmonella immunomagneticseparationof reportedarapidmethodfor

ตาม withtheirrealtime detectionviapcr .itenablesdifferentiationof salmonellatyphi และ
-
salmonellaparatyphi usingasetoffourspecific primerswithcost intensivenessandtechnicalcomplexity ( bakthavathsalametal , 2013 )
-
kwonetal . useantibodyconjugatedgold coatedmagneticnanopar ticle clustersandmagnetophoreticchromatographywithaprecision
pipettefordetectionof Salmonella ( kwonetal . 2556 ) วิธีนี้
showsarelativelyunstableconditionduetowhichtheantibodyis

http itadequatelyservestherequirementsoflarge easilyinactivated . comparedtootheranalyticaltechniquesbasedon , scalemonitoring
ของ environmentalissues . ' chemicalnose ' techniqueoffersagood
alternativeforthebiomoleculessensing .