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O le taua o le mataʻituina o faʻataʻitaʻiga o le siosiomaga ua matua talisapaia talu mai le amataga o le faʻamaʻi COVID-19, ma o nisi o taumafaiga mataʻituina o loʻo faʻatinoina i le faʻaogaina o le auro, e ui lava e taugata auala faʻavae qPCR. E mafai e le eletise DNA biosensors ona maua ai se tau taugofie. fofo mo le mataʻituina o vai faʻataʻitaʻiga i totonu o atunuʻu maualalo ma vaeluagalemu. I lenei galuega, matou te faʻaalia le suʻesuʻeina o le eletise o amplicons na maua mai le Phi6 phage faʻaesea mai faʻataʻitaʻiga vaituloto faʻafefete (o se sui lauiloa mo SARS-CoV-2), faʻaaoga ENIG e faʻamaeʻaina PCB electrodes e aunoa ma suiga luga.feusua'iga.O le tali fa'aeletise fa'amalama sa fa'avasega lelei mo ni vaega DNA se lua e eseese umi (\({117}\,\hbox {bp}\) ma le \({503}\,\hbox {bp}\)), ma le aafiaga o masima i PCR master mixes i luga o le methylene blue (MB) -DNA fegalegaleaiga.O matou taunuuga e faaalia ai o le umi o vaega DNA e matua iloa ai le maaleale eletise ma faʻaalia i lenei galuega o le mafai ona iloa amplicons umi e aunoa ma le faʻamamaina o gel o oloa PCR. e taua mo le fuaina i totonu o nofoaga o vai faataitai.O se vaifofo fa'aautomate atoatoa mo viral load e fa'ailoga lelei.
O fa'ama'i pipisi mai vai ua lauiloa ose lamatiaga o le soifua maloloina lautele talu mai le vaitau o le 1940, fa'atasi ai ma fa'amaoniga muamua o fa'ama'i pipisi mai le vai o le polio ma le hepatitis E1. Ua fa'avasegaina e le Fa'alapotopotoga o le Soifua Maloloina a le Lalolagi (WHO) le tele o fa'ama'i fa'ama'i pipisi i vai e feololo ma maualuga le taua2. Fa'ama'i masani. auala e iloa ai e faʻalagolago i faiga faʻavae qPCR auro, e sili ona maaleale ma faʻapitoa, ae manaʻomia tagata tomai faʻapitoa e suʻe i totonu o le fale suesue e faʻaaoga ai meafaigaluega taugata. faʻataʻitaʻiga faʻataʻitaʻiga e foliga mai e sili atu nai lo le faʻataʻitaʻiina o suʻesuʻega faʻataʻitaʻiga o le siosiomaga. e puipuia ai i latou mai le ogaoga o a'afiaga fa'ale-tamaoaiga o le fa'ama'i. luga ma hybridize pe a i ai se faasologa tutusa i le sample.E mafai ona liua lea i se faailo e ala electrochemical auala eseese e faaaoga redox mediators pei o le potassium iron/ferrocyanide. Ua lipotia mai e faʻafesoʻotaʻi i totonu o le DNA faʻaluaina (dsDNA) faʻaopoopo i lona faʻapipiʻiina e le faʻapitoa i le DNA5, 6. O le natura faʻafesoʻotaʻi o MB e fausia ai MB-DNA faʻalavelave e avea ai i latou ma filifiliga lauiloa e avea ma faufautua redox i le tele o DNA eletise. configurations sensor5,6,7,8,9. E ui lava o le intercalation o le MB i DNA e nonspecific, ma o le faapitoa o lenei masini electrochemical e faalagolago tele i le mama o primers faaaogaina mo PCR po o le isothermal amplification, e fetaui lelei mo le faatinoina moni. -taimi electrochemical-faavae qPCR po fluorescence isothermal amplification e fai ma sui i le fuaina o le DNA 9 . I se tasi o faatinoga faapena, Won et al.O luga o le auro electrodes na suia i le 6-mercapto-1-hexanol (MCH) mo le taimi moni. fuaina o PCR amplicons ma MB e faaaoga ai le eseesega pulse voltammetry (DPV) 9. I isi tulaga, Ramirez et al.Su'esu'eina o le SARS-CoV-2 i le vai otaota e ala i le RT-LAMP fa'aogaina le MB fa'atasi ai ma le fa'aogaina o masini eletise. faʻaaogaina e pei o in situ electrodes i se microfluidic PCR platform fuafuaina e electrochemically iloa amplicons i le taimi o tali 8. O nei suʻesuʻega uma e manaʻomia ai suiga i luga o le eletise, e faʻaalia ai le faʻatupulaia o le gaosiga ma le faʻaogaina o tau ona o manaʻoga faʻapitoa e teu ai mo le mautu o nei electrodes functionalized.
Fuafuaga o le galuega mo su'esu'ega fa'aeletise o amplicons e maua mai fa'ama'i viral fa'ato'ato'a i fa'ata'ita'iga vai vaituloto.
Na matou faʻaalia talu ai nei le faʻaogaina o le faʻaogaina o le SARS-CoV-2 amplicons faʻatasi ai ma le tau maualalo o lomitusi laupapa (PCB) eletise e faʻavae i luga o le DPV ma le cyclic voltammetry (CV) faʻaosoina e ala i le adsorption o MB-DNA complexes i luga o le faʻaogaina o electrodes) suiga i le tumutumu. nei11.Matou te lipoti atu o vaega uumi DNA (N1-N2, \({943}\, \hbox) na fausia e faʻaaoga ai le N1 faʻatonuina e le CDC i luma ma N2 faʻasolosolo faʻataʻitaʻiga faʻatusatusa i vaega pupuu {bp}\)) na faʻaalia ai le sili atu o le laina i le tali atu. (N1, \(72\,\hbox {bp}\)) na faia e faʻaaoga ai le N1 i luma ma le N1 reverse primer sets. O nei suʻesuʻega o loʻo lipotia mai i le faʻaogaina o le DNA faʻafefeteina na saunia i le vai e leai ni faʻamaʻi. -2 amplicons i faʻataʻitaʻiga faʻataʻitaʻiga vai otaota (maua e ala i le spiking atoa RNA samples ma SARS-CoV-2 RNA). Talu ai ona o le RNA e faigofie ona sele i le taimi o le vavae ese ma lalo o le gaosiga, 12,13 e faigata ona faʻalauteleina ni vaega umi ma lenei faʻataʻitaʻiga eseese. O le mea lea, o le faʻaalia o le faʻaogaina o le eletise o le SARS-CoV-2 amplicon i vai otaota e faʻatapulaʻa i le vaega puʻupuʻu \(72\,\hbox {bp}\) N1.
I lenei galuega, sa matou suʻesuʻeina le faʻaogaina o le ENIG PCB faʻavae eletise eletise o le phage Phi6 faʻapitoa ma vavae ese mai vaituloto faʻataʻitaʻiga (Fig. 1). Phi6 phages e faʻatusatusa i le tele (80-100 nm) i le SARS-CoV-2 ma e iai fo'i le lipid membrane ma se polotini spike.Mo nei mafua'aga, o le bacteriophage Phi6 o se sui ta'uta'ua mo le SARS-CoV-2 ma isi fa'ama'i RNA pathogenic14,15.RNA fa'aesea mai vaega phage sa fa'aaogaina e fai ma fa'ata'ita'iga mo le fa'asologa o le cDNA sosoo ai ma le. PCR e maua ai ni vaega se lua o le DNA o le 117 ma le 503 base pairs i le umi. Ona o le luʻitau o le faʻalauteleina o le \(943\,\hbox {bp}\) N1-N2 fragments i la matou galuega talu ai, matou te faʻamoemoeina vaega o le umi o le vaeluagalemu (\(117). \,\hbox {bp}\) ma le \(503 \,\hbox {bp}\)), e fa'avae i luga o fa'amuamua avanoa. O le tali fa'aeletise fa'ainisinia na su'esu'eina fa'apitoa i luga o le lautele o le fa'atonuga (\({10}\,{ \hbox {pg}/{\upmu \hbox {l}}}\) i le \({20}\, {\hbox {ng}/{\upmu \hbox {l}}}\)) Mo vaega uma e lua i totonu. o le i ai o le MB, o le aafiaga o le masima i luga o le tali faʻalogo ua faʻaalia ma faʻamaonia e ala i fuataga spectrophotometric. O sao taua o lenei galuega e pei ona taua i lalo:
Ole umi ole vaega ole DNA ma le iai ole masima ile fa'ata'ita'iga e matua a'afia ai le ma'ale'ale.O a matou taunuʻuga o loʻo faʻaalia ai o le gaioiga eletise e faʻalagolago i auala eseese o le fegalegaleaiga a le MB, DNA, ma le masini i le tali voltammetric, e faʻatatau i le faʻaogaina o le DNA ma le umi, faʻatasi ai ma le umi o Fragments o loʻo faʻaalia ai le maualuga o le lagona, e ui lava o le masima e i ai se aafiaga le lelei i fegalegaleaiga eletise i le va. MB ma DNA.
O le fa'atonuga o le DNA e fa'atatau i le faiga o feso'ota'iga MB-DNA i fa'aeletise e le'i fa'aleleia. {l}}}\), na matou matauina o le tali atu i le electrochemical i le taimi nei e masani lava ona fuafuaina e le adsorption o le MB-DNA i luga o le electrode, ae i le maualalo concentrations I le maualuga DNA concentrations, o le tali electrochemical i le taimi nei na fuafuaina e le steric inhibition o redox. gaioiga ona o le fa'aofiina o le MB i le va o pa'aga fa'avae DNA.
ENIG PCB-Based Electrochemical Sensing of Viral Nucleic Acids in Lake Water Samples O suʻesuʻega na faʻamaonia e ala ile suʻesuʻeina eletise ole Phi6-faʻaopoopo \(503\,\hbox {bp}\) vaega DNA na maua mai vai faʻataʻitaʻiga mai le Vaituloto o Powai, IIT Mumbai Campus I'uga phage.
Tau maualalo o le faʻatinoga ma le ono mafai ona tuʻufaʻatasia i faiga faʻapitoa e mataʻituina ai, oligonucleotides poʻo aptamers i luga o eletise ma umi atu le ola.
O le Phage Phi6 o se faʻamaʻi dsRNA faʻapipiʻiina o le aiga o Cytoviridae e aʻafia ai le Pseudomonas syringae. O le genome o le Phi6 phage o loʻo i ai i le tulaga o 3 vaega: S (\ (2.95 \, \ hbox {Kb} \)), M (\ (4.07) \,\hbox {Kb}\)) ma le L (\ (6.37\ ,\hbox{Kb}\)) 16,17. Talu ai ona o le Phi6 phage e afaina ai le BSL-1 Pseudomonas strain e le faʻamaʻi, e saogalemu le faʻaaogaina. ma e mafai ona faigofie ona tupu i totonu o le falesuesue. Phage Phi6 ma lona talimalo Pseudomonas syringae na faʻatau mai le Felix d'Herelle Reference Center for Bacterial Viruses, Laval University, Kanata (numera faʻamaufaʻailoga nofoaga autu o HER-102 ma HER-1102, faasologa) .Phi6 phage ma lona 'au na toe faʻaolaina e pei ona faʻatonuina e le nofoaga autu. Phage Phi6 na faʻamamaina e le lysis plate ma le elution e maua ai titers mulimuli ma \(\ e tusa ma le 10^{12}\, {\hbox {PFU}/\hbox { ml}}\) (iunite fai ma'a/milliliters).Na vavae'eseina le RNA mai vaega o le phage fa'amamaina e fa'aaoga ai le GenElute™ Universal Total RNA Purification Kit (Sigma-Aldrich) e tusa ai ma faatonuga a le gaosi oloa. 100}\,{{\upmu \hbox {l}}}\) na fa'amama ma fa'aulu le lysate i luga o se koluma vili e fa'ataga ai le RNA e fusifusia i le koluma resin . Ona fa'aulu lea o le RNA i le vaifofo elution \({ 50}\,{{\upmu \hbox {l}}}\) na saunia e le pusa. Fa'atatau le fa'atonuga o le RNA e ala i le mitiia i \(260\,\hbox {nm}\).RNA sa teuina i aliquots i \ ({-80}\,{^{\circ }\hbox {C}}\) se'ia toe fa'aoga.\({2}\,{\upmu \hbox {g}}\) Le iScript cDNA Synthesis Kit (Bio -Rad Laboratories) na faʻaaogaina e fai ma faʻataʻitaʻiga mo le faʻasologa o le cDNA e mulimuli i faʻatonuga a le gaosiga. I se faapuupuuga, o le cDNA synthesis reaction e aofia ai laasaga e 3: priming i \({25}\,{^{\circ }\hbox {C}}\ )\({5}\,{\hbox {min} }\) , toe fa'aliliuina o le \({20}\,{\hbox {min}}\) i \({46}\,{^{\circ }\hbox {C}}\), ma toe fa'afo'i O lo'o i le \({95}\,{^{\circ }\hbox {C}}\) mo \({1}\,{\hbox {min. }}\) .Pe a tamoʻe i luga o le 1% agarose gel, na faʻaalia e le cDNA ni fusi se tolu e fetaui ma le faʻamoemoeina o vaega RNA e tolu (faʻamatalaga e leʻo faʻaalia). fa'aogaina le cDNA e fai ma fa'ata'ita'iga mo le PCR i totonu o le miniPCR® mini8 uila uila:
O mea muamua mo le \(117\,\hbox {bp}\) ma le \(503\,\hbox {bp}\) e fetaui ma le 1476-1575 nucleotides o le vaega M ma le 458-943 nucleotides o le vaega L, fa'asologa o le acid. .O oloa uma a le PCR ua fa'amalosia na fa'aogaina i luga ole 1% agarose gels, ma fa'amamāina le DNA taula'i fa'aogaina le GeneJET Gel Extraction Kit (Thermo Fisher Scientific).
O se vaituloto i le lotoa o le IIT Mumbai (Powai Lake, Powai, Mumbai) na fa'aaogaina e fa'aopoopo ai vaega o le phage. O le vaituloto na fa'amama e ala i se membrane \({5}\,{\upmu \hbox {m}}\) e aveese. o mea ninii ua taofia, ona faaopoopo lea o le Phi6 phage. \) i \( {100}\ ,{\hbox {ml}}\) vai vaitu'i fa'amama, i le \({4}\,{^{\circle}\hbox {C}}\).O se aliquot itiiti sa fa'aagaaga mo le fuaina o viral load e ala ile plaque assay.Sa matou fa'ata'ita'iina ni auala 'ese'ese se lua e fa'atumauina ai vaega fa'ama'i pipisi o le Phi6: (1) o le alumini hydroxide adsorption-timu metotia,19 lea ua fa'amaonia mo le fa'aogaina o le tele o fa'ama'i RNA teutusi mai fa'ata'ita'iga o le si'osi'omaga, ma (2) ) O le polyethylene glycol (PEG)-faʻavaeina faʻamaʻi siama na faʻaogaina mai le Flood et al.20 . Talu ai o le toe faʻaleleia o le faʻaogaina o le PEG na maua ai le sili atu nai lo le alumini hydroxide metotia, o le PEG-faʻaaogaina auala e faʻaogaina ai Phi6 vaega mai faʻataʻitaʻiga vai vaituloto.
Ole auala ole PEG na fa'aogaina e fa'apea: PEG 8000 ma le \(\hbox {NaCl}\) na fa'aopoopo i fa'ata'ita'iga vaituloto Phi6-spiked e maua ai le 8 % PEG 8000 ma le \(0.2\,\hbox {M} \) \( \ hbox {NaCl}\).O fa'ata'ita'iga na tu'u i luga o se lulu\({4}\,{^{\circ }\hbox {C}}\)\({4}\,{\hbox {h}}\ ), ona centrifuge i le \(4700 \,\hbox {g}\) o le \({45}\,{\hbox {min}}\).Tu'u ese le supernatant ma toe fa'anofo le pellet i \({1}\, {\hbox {ml}}\) i le supernatant lava e tasi. O su'esu'ega uma spiking ma siama fa'atonuga na faia i fa'atolu. Ina ua mae'a fa'atonuga, na fa'aagaga se aliquot la'ititi mo le fuaina o le toe fa'aleleia lelei e ala ile plaque assay.RNA na fa'aesea e pei ona fa'amatalaina muamua ma fa'amama. i pusa elution buffer\({40}\,{\upmu \hbox {l}}\).Talu ai o le a eseese le fa'aogaina o le RNA mai lea fa'ata'ita'iga i lea fa'ata'ita'iga i fa'atolu, o le \({2}\,{\upmu \ hbox {l}}\) o le RNA o lo'o fa'aogaina mo uma e tolu e tusa lava po'o le a le fa'aogaina o le cDNA synthesis o samples.cDNA synthesis sa faia e pei ona fa'amatalaina muamua.\({1}\,{\upmu \hbox {l}}\) cDNA sa fa'aaogaina e fai ma fa'ata'ita'iga mo \({20}\,{\upmu \hbox {l}}\) PCR mo ta'amilosaga e 35 e fa'alautele ai le \ (117\,\hbox {bp}\) ma le \(503\,\hbox { bp}\) fragments.O nei faʻataʻitaʻiga o loʻo faʻatusalia e pei o le "1: 1", e aunoa ma se faʻafefeteina. O se pule e leai se faʻataʻitaʻiga (NTC) na faʻatulagaina e avea o se faʻatonuga le lelei, ae o se cDNA faʻapipiʻiina e faʻaaoga ai le RNA vavae ese mai le faʻamamaina phage na faʻatutuina. e fai ma fa'ata'ita'iga mo se fa'atonuga lelei (PC). Quantitative PCR (qPCR) na fa'atinoina i se meafaigaluega Stratagene Mx3000P RT-PCR e fa'aaoga ai le Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies). O tali na fa'atutuina i fa'atolu e pei o le taimi muamua. faʻamatalaina.O le taamilosaga taʻamilosaga (Ct) na faʻamauina mo faʻataʻitaʻiga uma. E le gata i lea, o faʻataʻitaʻiga faʻafefeteina o \({1}\,{\upmu \hbox {l}}\) faʻaaogaina le cDNA faʻafefeteina 1: 100 i vai vaituloto faʻamamaina pei \({20}\,{\upmu \hbox {l}}\) PCR mo ta'amilosaga 35. O nei fa'ata'ita'iga o lo'o fa'atusalia o le “1:100″.
O le PCB electrodes ua fabricated e faaaoga ai se faiga faapisinisi taugofie Electroless Nickel Immersion Gold (ENIG) faagasologa e aunoa ma le manaomia mo isi auro plating.ENIG PCB electrode specifications o loo auiliiliina i la tatou galuega muamua11.Mo ENIG PCB electrodes, auala masani faamamaina electrode pei o e le fautuaina le vaifofo o le piranha po'o le sulfuric acid cyclic voltammetry ona e ono pa'u'u ai le vaega auro manifinifi (mafiafia \(\approx\) \(100\,\hbox {nm }\)) ma fa'aalia ai le 'apa apamemea pito i lalo e faigofie. i le corrosion 21, 22, 23, 24, 25. O le mea lea, faʻamama le eletise i se ie e leai se lint ua faʻafefeteina i le IPA. O le faʻataʻitaʻiga e faʻataʻitaʻiina na faʻaofuina i le \({50}\,{\upmu \hbox {M} }\) MB i le \({4}\,{^{\circ }\hbox {C}}\)\({ 1}\,{\hbox {h}}\) mo le fa'aofi faigofie. I la matou galuega muamua , na matou matauina na faʻaleleia atili le faʻaogaina ma le laina o le masini e ala i le faʻateleina o le MB concentration 11 . Faʻavae i luga o faʻataʻitaʻiga na lipotia i la matou galuega muamua, na matou faʻaaogaina \({50}\,{\upmu \hbox {M}}\) MB concentrations e faʻapipiʻi DNA i totonu o lenei suʻesuʻega.E mafai ona ausia le suʻesuʻeina o le DNA faʻalua-lua (ds-DNA) i le faʻaaogaina o le anionic poʻo le cationic intercalators. i le isi itu, cationic intercalators e pei o le MB e manaʻomia ai taimi faʻapuupuu, e tusa o \({1}\,{\hbox {h}}\) mo suʻesuʻega eletise o le ds-DNA6. O fua taʻitasi e aofia ai le tuʻuina atu o le faʻataʻitaʻiga e faʻataʻitaʻiina i luga o le electrode\({5}\,{{\upmu \hbox {l}}}\), ona fa'amama lea i se ieie susu IPA, a'o le'i faia se isi fa'ata'ita'iga.tasi le fua. Sa faʻataʻitaʻiina faʻataʻitaʻiga taʻitasi i luga o le 5 electrodes eseese seʻi vagana ua taʻua.DPV ma CV fua na faia e faʻaaoga ai le PalmSens Sensit Smart potentiostat, ma PSTrace software na faʻaogaina mo le faʻaogaina o le potentiostat ma le mauaina o faʻamaumauga, e aofia ai faʻatusatusaga o loʻo iai nei. mo fua DPV ma CV:
DPV: Taimi Talafeagai = \(8\,\hbox {s}\), Laasaga Malosi = \(3\,\hbox {mV}\), Malolo Malosi = \(25\,\hbox {mV}\) , umi o fatu = \(50\,\hbox {ms}\), fua fa'ata = \({20}\,\hbox {mV/s}\)
CV: Taimi Talafeagai = \(8\,\hbox {s}\), Laasaga Malosi = \(3\,\hbox {mV}\), Salu fua = \({300}\,\hbox {mV/ s }\)
O le maualuga o galu e maua mai i voltammograms o DNA ua fa'apipi'iina i le \({50}\,{\upmu \hbox {M}}\) MB: (a) \(503\,\hbox {bp}\) DPV , (b) \ (503\,\hbox {bp}\) CV, (c) \(117\,\hbox {bp}\) DPV, (d) \(117\,\hbox {bp}\) CV.
DPV ma CV voltammograms na maua i luga ole ENIG PCB electrodes \({50}\,{\upmu \hbox {M}}\) MB faʻapipiʻiina ma DNA (i faʻamaualuga o 10–\({20}\,{\ hbox {ng). }/{\upmu \hbox {l}}}\) ie 0.13–\({0.26}\,{\upmu \hbox {M}}\) mo \(117\,\hbox {bp}\ ) ma le 0.03 –\({0.06}\,{\upmu \hbox {M}}\) mo \(503\,\hbox {bp}\)).O sui voltammograms o lo'o fa'aalia i le Ata S1 i Fa'amatalaga Fa'aopoopo. Ata 2 o lo'o fa'aalia ai taunu'uga. o le DPV ma le CV fua (peak current) faʻaaogaina gel-faʻamamaina PCR oloa. Faʻatusatusa i fua CV, DPV fua faʻaalia le maaleale maualuga (o loʻo i ai nei o se galuega o le faʻaogaina o le DNA) ona o le faʻasologa capacitive o loʻo i tua i fua CV natia Faradaic au 26 . O faʻamaumauga mo atigipusa taʻitasi i totonu o le boxplot o loʻo i ai fua mai le 5 electrodes. O fua uma e faʻaaogaina le seti tutusa o eletise e aloese ai mai le fuaina o mea sese ona o le fesuiaiga o le electrode-to-electrode. Matou te matauina le faʻatupulaia o le DPV ma le CV fuaina o galu mo le maualalo o le DNA. , umi (\(503\,\hbox {bp}\)) \,\hbox {bp}\ pe a faatusatusa i le \(117) ) fragment.E ogatasi lea ma le tulaga faamoemoeina o le electrode adsorption lipotia i la tatou galuega muamua.The adsorption o le MB-DNA complex e faafaigofieina ai le fesiitaiga o le tau i luga o le eletise, lea e fesoasoani i le faateleina o le maualuga o le taimi nei.O isi suʻesuʻega ua faʻaalia ai le aʻafiaga o le tele o le oligonucleotide ma le faʻasologa i luga ole MB-DNA intercalation27,28,29,30. Le guanine -cytosine (GC) anotusi o amplicons e lua (\ (117 \, \ hbox {bp} \) ma \ (503 \, \ hbox {bp} \)) e tusa ma le 50%, e faʻaalia ai o le matauina O le eseesega e tatau. i le amplicon umi.Ae ui i lea, mo le maualuga o le DNA concentrations (\(>{2}\,{\hbox {ng}/{\upmu \hbox {l}}}\), mo \(503\,\hbox {bp} \) ma le \( >{10}\,{\hbox {ng}/{\upmu \hbox {l}}}\) mo \(117\,\hbox {bp}\)), matou te matauina le fa'aopoopoga e lua. O le maualuga o galu o le subs ua faʻaitiitia i le DPV ma le CV measurements. E mafua ona o le MB saturates ma intercalates i le va o paʻu faavae o le DNA, e mafua ai le taofia o le redox gaioiga o le vaega faʻaitiitia i le MB31,32.
在存在 \(2\,\hbox {mM}\) \({\hbox {MgCl }_2}\): (a) \(503\,\hbox {bp}\) DPV, (b) \(503 \,\hbox {bp}\) CV, (c) \(117\,\hbox {bp}\) DPV，(d) \(117\,\hbox {bp}\) CV.
O masima o loʻo iai i le PCR master mixes e faʻalavelave i fesoʻotaʻiga eletise i le va o le MB ma le DNA, e ala i le faʻaopoopoina o le \(2\,\hbox {mM}\) \(\hbox {MgCl }_2\) ma le \({50} \,{\ upmu \hbox {M}}\) MB gel-fa'amamaina oloa e su'esu'e ai le a'afiaga o le masima i fefa'ataua'iga MB-DNA. E pei ona fa'aalia i le Ata 3, na matou va'aia e mo le maualuga o le DNA (\(>{2}\,{\ hbox {ng}/{\upmu \hbox {l}}}\) (503\,\hbox {bp }\) ma \(>{10}\,{\hbox {ng}/{\upmu \hbox { l}}}\) mo \(117\,\hbox {bp} \)), i le DPV ma le CV O le fa'aopoopoina o le masima e le'i afaina tele ai fua (silasila i le Ata S2 i Fa'amatalaga Faaopoopo mo voltammograms sui). lalo ifo o le DNA concentrations, o le faʻaopoopoga o le masima e matua faʻaitiitia ai le lagona, e leai se suiga tele i le taimi nei ma le faʻaogaina o DNA.O aʻafiaga leaga tutusa o le masima i luga o fegalegaleaiga MB-DNA ma intercalation na lipotia muamua e isi tagata suʻesuʻe33,34.\(\hbox { Mg}^{2+}\) cations e fusifusia i le ivi phosphate le lelei o le DNA, ma faʻalavelaveina ai le fesoʻotaʻiga eletise i le va o le MB ma le DNA. I le maualuga o le DNA concentrations, o le faʻafefeina o le redox-active MBs e maua ai i lalo ifo o galu, o le mea lea o fegalegaleaiga eletise. 'aua ne'i matua a'afia ai le tali fa'alogo.O le manatu autu o lenei biosensor e sili atu ona fetaui e su'esu'e ai fa'aputuga DNA maualuga (seasea \({\hbox {ng}/{\upmu \hbox {l}}}\) pe maualuga atu), mo le atoatoa- Fa'agaoioiga otometi o vai fa'ata'ita'iga o le si'osi'omaga, lea e le mafai ai ona fa'amama gel o oloa PCR.
Vaega o lo'o i lalo o le fa'a'ai'au mo le umi o galu 600-700 \(\hbox {nm}\) mo fa'aputuga eseese o DNA fa'apipi'iina i le \({50}\,{\upmu \hbox {M}}\) MB: ( a ) \(503\,\hbox {bp}\) fa'atasi ma le masima (\(2\,\hbox {mM}\) \(\hbox {MgCl}_2\)), (b) \( 117\, \hbox {bp}\) fa'atasi ma le masima (\(2\,\hbox {mM}\) \(\hbox {MgCl}_2\)).\({0}\,{\hbox {pg}/ {\upmu \hbox {l}}}\) DNA fa'atatau i le \({50}\,{\upmu \hbox {M}}\) MB samples Leai DNA.
Ina ia faʻamaonia atili iʻuga o loʻo taʻua i luga, na matou faia fua faʻapitoa e faʻaaoga ai le UV/Vis spectrophotometer (Thermo Scientific Multiskan GO), o faʻataʻitaʻiga \({50}\, {{\upmu \hbox {l}}}\) sa faʻaaogaina mo taʻitasi. Fua. O le saini absorption e faʻaitiitia i le faʻateleina o le faʻaogaina o le DNA, e pei ona mafai ona vaʻaia mai le tulaga o le vaega i lalo o le absorption curve mo le galu umi \(600\,\hbox {nm}\) i le \(700\,\hbox { nm}\) , e pei ona faaalia i le Ata 4 (absorption spectrum o loʻo faʻaalia i le Ata S3 i Faʻamatalaga Faʻaopoopo). {l}}}\), e leai se eseesega tele i le fa'aogaina i le va o fa'ata'ita'iga o lo'o i ai le DNA ma na'o MB (mo \(503\,\hbox {bp}\) ma le \(117\,\hbox {bp}\ ) vaevaega umi), e faʻaalia ai le leai o se faʻalavelave faʻafefe o le redox-active MB. I le maualuga o le DNA concentrations, na matou matauina ai le faʻaitiitia malie o le faʻailoga o le absorbance ma matauina ai le faʻaitiitia o le faʻaitiitia o le faʻafefe i le i ai o le masima. fegalegaleai ma steric inhibition ma faavae faaputuputu i DNA hybrids.O matou taunuuga e ogatasi ma lipoti i tusitusiga i spectroscopic suesuega o le MB-DNA intercalation e fesootai hypochromaticity ma faaitiitia le malosi maualuga i \(\pi\)-\(\pi ^*\ ) suiga fa'aeletoroni ona o feso'ota'iga Lay 36, 37, 38.
Agarose gel electrophoresis o le phage Phi6: PCR oloa o le umi \(117\,\hbox {bp}\) ma \(503\,\hbox {bp}\) mai vaituloto samples.M-DNA faailoga;NTC-leai-fa'ata'ita'iga pulea, primers o lo'o i ai amplicons tutusa;PC pulea lelei;1, 2, 3-e le'i fa'afefeteina (1:1) fa'ata'ita'iga vaituloto fa'apipi'i i fa'atolu. E iloa atu se fusi i \(\pe tusa o le 50\,\hbox {bp}\) ona o le oligonucleotides e le'i fa'aaogaina i le \(503\,\ hbox {bp}\) laina.
Na matou iloiloina le aoga o le masini e faʻaaoga ai vai faʻataʻitaʻiga o le Vaituloto o Powai na faʻapipiʻiina i le Phi6 phage. O faʻaputuga o le RNA na vavae ese mai faʻataʻitaʻiga vai faʻasolosolo mai le 15.8-\({19.4}\,{\upmu \hbox {g}/\hbox { ml}}\), a'o fa'aesea mai fa'amama fa'amama fa'amalologa O le RNA na fa'atatauina o le \({1945}\,{\upmu \hbox {g}/\hbox {ml}}\) fa'atasi ai ma le toe fa'aleleia lelei e tusa ma le 1. %.RNA na toe fa'aliliuina i le cDNA ma fa'aaogaina e fai ma fa'ata'ita'iga mo le PCR ma le qPCR. O le tele o le oloa na fa'amaonia e le agarose gel electrophoresis (Ata 5) a'o le'i fa'ata'ita'iina ma le masini. fa'apea fo'i ma fa'aputuga o tului. O fa'amaumauga Ct na fa'amauina i le taimi o le qPCR (Table 1) na fa'aalia e feso'ota'i ma le fa'atonuga o le RNA fa'aesea mai fa'ata'ita'iga vai fa'apipi'i tutusa. sili atu i le fa'ailoga po'o le fa'ailoga. o le pulea lelei ma le faʻataʻitaʻiga faʻataʻitaʻiga o loʻo faʻaalia atili ai o faʻataʻitaʻiga faʻataʻitaʻiga taʻitasi e tusa ma le 1% faʻataʻitaʻiga pe a faʻatusatusa i le faʻataʻitaʻiga lelei. Ua matou talanoaina muamua o amplicons umi atu e oʻo atu ai i le sili atu le lagona. o le maualalo o viral concentration ma le RNA degradation. Ae ui i lea, faatasi ai ma la matou siama faʻamaoaigaina ma le PCR faʻalauteleina protocol, na mafai ai ona matou faʻalauteleina le vaega \(503\,\hbox {bp}\) mo le faʻalogoina eletise.
Ata 6 o lo'o fa'aalia ai fa'ai'uga o le masini fa'aeletise o le \(503\,\hbox {bp}\) fragment amplicon, o lo'o fa'aogaina uma le cDNA e le'i fa'aaogaina e fai ma fa'ata'ita'iga (1:1) ma le 100-saili fa'afefeteina cDNA e fai ma fa'ata'ita'iga (1:100) faia PCR. , faʻatusatusa i le NTC ma le PC (silasila i le Ata S4 i Faʻamatalaga Faʻaopoopo mo voltammograms sui). -to-electrode variation.Faatusatusa i fua CV, DPV fua faʻaalia sili atu iugafono e iloa ai suʻega ma PC faʻataʻitaʻiga mai NTCs aua, e pei ona taʻua muamua, o loʻo natia Faradaic au ona o le tafega capacitive pito i tua.Mo amplicons umi, matou matauina lena o le pulega le lelei (NTC) na mafua ai le maualuga o le CV ma le DPV peak currents e faatatau i le pulea lelei, ae o faʻataʻitaʻiga lelei ma faʻataʻitaʻiga faʻataʻitaʻiga na faʻaalia ai le maualuga maualuga o le DPV peak currents. ) faʻataʻitaʻiga faʻataʻitaʻiga ma PC e mafai ona faʻamaonia manino mai le gaosiga o le masini mo le faʻataʻitaʻiga NTC, ae o le iugafono mo le 1: 100 faʻataʻitaʻiga faʻataʻitaʻiga e itiiti ifo faʻaaliga. (ala e le o faʻaalia i le Ata 5), ma o le DPV ma le CV pito i luga o galu e tutusa ma mea na faʻamoemoeina mo le NTC. pulea faʻaosoina se tali eletise mai le PCB sensor ona o le adsorption o free MB i luga o le electrode ma le fegalegaleaiga a le MB ma le tasi-stranded primer oligonucleotide. O lea, o taimi uma e tofotofoina ai se faataitaiga, e tatau ona tamoe se pulea lelei ma le maualuga o le taimi nei o le faʻataʻitaʻiga faʻataʻitaʻiga faʻatusatusa i le maualuga o le taimi nei na maua e le pule le lelei e ausia ai se fua faʻatatau (relative)39,40 e faʻavasega ai le suʻega faʻataʻitaʻiga e lelei pe leaga.
(a) DPV, ma le (b) CV pito maualuga o loʻo iai mo le suʻeina o le eletise o le \(503\,\hbox {bp}\) vaega i vaituloto faʻataʻitaʻiga. Sa fuaina faʻataʻitaʻiga faʻataʻitaʻiga i faʻatolu ma faʻatusatusa i le leai o ni faʻataʻitaʻiga (NTC) ma pulea lelei (PC).
O a matou suʻesuʻega o loʻo faʻaalia ai auala eseese e aʻafia ai le faʻatinoina o masini eletise mo faʻamaufaʻailoga eseese umi mo DNA eseese, faʻatasi ai ma faʻasalalauga faʻamaonia e ala i fua faʻapitoa e faʻaaoga ai le UV / Vis spectrophotometer. \(500\,\hbox {bp}\) e mafai ona iloa i le ma'ale'ale maualuga ma o le iai o le masima i totonu o le fa'ata'ita'iga e le'o Fa'asalaina le fa'aputuga DNA e a'afia ai le ma'ale'ale maualuga (seasea \({\hbox {ng}/{\upmu \hbox {l}}}\) ma luga). E le gata i lea, na matou suʻesuʻeina le aʻafiaga o ituaiga eseese o faʻataʻitaʻiga, e aofia ai amplicons faʻamamaina gel ma ma e aunoa ma se masima faaopoopo, ma le faʻaopoopoina o faʻataʻitaʻiga vai vaituloto i DPV ma fua CV.Na matou matauina o le DPV na maua ai se fofo sili atu, talu ai o le capacitive current e aafia ai foi le fua o le CV, ma faʻaitiitia ai le maaleale.
O le faʻalauteleina o vaega uumi e faʻalagolago i le faʻamaoni o le viral genomic RNA. E tele suʻesuʻega ua faʻaalia ai o le faʻalauteleina o fasi vaega umi e le o taimi uma e lelei ona o le faʻaleagaina o le RNA i le siosiomaga ma le mafai ona faʻapipiʻiina i le taimi o le vavaeeseina11,41,42,43,44 .Sa matou matauina o le PEG-based virus concentration method sa sili atu ona aoga i le faʻaogaina o le phage Phi-6 spiked i vaituloto faʻataʻitaʻiga nai lo le alumini hydroxide-based virus concentration method.O le mafai ona iloa vaevaega DNA umi na faʻamaonia e foia ai le manaʻoga mo PCR multiplex e fa'ateleina le tele o fa'ata'ita'iga pupu'u umi ma fa'aitiitia ai le avanoa e fa'a'ese'ese ai.
E tau le maua ni fa'ata'ita'iga fa'aolaola, o lea e mana'omia ai le mamanuina o se biosensor e mana'omia ai ni fa'ata'ita'iga la'ititi mo su'ega. O le ENIG PCB electrodes na fa'aaogaina i lenei su'esu'ega e na'o le mana'omia \({5}\,{{\upmu \hbox {l}}}\ ) faʻataʻitaʻiga mo suʻega e ufiufi ai le vaega aoga o le eletise. E le gata i lea, e mafai ona toe faʻaaogaina le eletise tutusa pe a uma ona faʻamamāina aʻo leʻi tuʻuina atu le isi faʻataʻitaʻiga. ma e masani ona faʻaaogaina vailaʻau. Talu ai o tau eletise taʻitasi e tusa ma le $ 0.55 (poʻo le INR 40) e gaosia, o lenei biosensor e mafai ona avea ma se faʻaoga taugofie i tekinolosi suʻesuʻe o loʻo i ai nei. Laulau 2 o loʻo faʻaalia ai se faʻatusatusaga o lenei galuega ma isi masini e lipotia i tusitusiga mo le umi. vaevaega DNA i faʻataʻitaʻiga eseese.
Tuuina atu e faʻalagolago le MB-based electrochemical detection protocols i le faʻapitoa o le PCR, o se tapulaʻa tele o lenei metotia o le mafai lea mo le faʻalauteleina o le le faʻapitoa i faʻataʻitaʻiga eseese e pei o le otaota vai ma vaituloto poʻo le faʻaaogaina o primers mama maualalo. auala e su'esu'e ai le eletise mo le su'esu'eina o DNA o oloa PCR e le'i fa'amamāina e fa'aaoga ai le eletise ENIG PCB e le'i suia, e tatau ona malamalama atili i mea sese na fa'aofi mai e le dNTPs e le'i fa'aaogaina ma primers, ma ia fa'amalieina tulaga fa'aalia ma fa'ata'ita'iga fa'ata'ita'iga. Ole mana'oga ole okesene (BOD) ole fa'ata'ita'iga vai e ono mana'omia fo'i ona fuaina ina ia fa'aleleia atili le sa'o o le fua.
I le faaiuga, matou te tuʻuina atu se eletise eletise ENIG PCB eletise taugofie mo le suʻesuʻeina o siama i le siosiomaga (vaituloto vai) faʻataʻitaʻiga. E le pei o le immobilized oligonucleotide electrodes poʻo mea faʻapitoa mo le DNA sensing e manaʻomia ai le teuina o le cryogenic e tausia ai le maaleale, 53,54 o loʻo faʻaogaina e le matou metotia PCB e leʻi suia. electrodes ma se fata ola umi ma e leai ni manaoga faapitoa teuina ma o lea e talafeagai mo le atinae o fua fofo ma faʻataʻitaʻiga faʻataʻitaʻiga faʻapipiʻiina i le LMICs.O le biosensor faʻaaogaina le taugofie DNA-intercalating redox dyes (MB) mo le vave suʻesuʻeina o amplicons sini. masani i faʻataʻitaʻiga o le siosiomaga e faʻaitiitia ai le faʻapitoa o lenei auala faʻalogo ona o le faʻaogaina o le MB i le tasi-ma le lua-stranded oligonucleotides. ma le DPV pito maualuga o loʻo maua mai faʻataʻitaʻiga faʻataʻitaʻiga e tatau ona faʻamatalaina e fesoʻotaʻi ma tali na maua mai le pulega le lelei (NTC) mo suʻega taʻitasi. -totogi fofo e mafai ona aoina ma au'ili'ili fa'ata'ita'iga ma fa'asalalau atu fa'ai'uga i le falesu'esu'e.
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Taimi meli: Me-27-2022