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Bohlokoa ba ho beha leihlo lisampole tsa tikoloho bo ananetsoe haholo ho tloha qalong ea seoa sa COVID-19, 'me boiteko bo bong ba ho beha leihlo bo ntse bo etsoa ho sebelisoa maemo a khauta, le hoja mekhoa e thehiloeng ho qPCR e theko e boima. Tharollo bakeng sa ho beha leihlo mehlala ea metsi a tikoloho linaheng tse nang le chelete e tlase le e bohareng. Mosebetsing ona, re bonts'a tlhahlobo ea electrochemical ea li-amplicon tse fumanoeng ho Phi6 phage e arohaneng le lisampole tsa metsi a letša la spiked (moemeli ea tsebahalang oa SARS-CoV-2), a sebelisa ENIG. ho tlatsa li-electrode tsa PCB ntle le phetoho ea bokaholimo.sex.Karabelo ea sensor ea electrochemical e ne e khetholloa ka botlalo bakeng sa likaroloana tse peli tsa DNA tsa bolelele bo fapaneng (\({117}\,\hbox {bp}\) le \({503}\,\hbox {bp}\)), le phello ea letsoai ho PCR master mixes ka methylene blue (MB) -DNA interactions.Liphetho tsa rona li bontša hore bolelele ba likaroloana tsa DNA bo khetholla haholo kutloisiso ea electrochemical le ho bontša mosebetsing ona hore bokhoni ba ho lemoha li-amplicon tse telele ntle le ho hloekisa gel ea lihlahisoa tsa PCR ke bohlokoa bakeng sa ho metha sebakeng sa lisampole tsa metsi.Tharollo e ikemetseng ka botlalo bakeng sa bongata ba kokoana-hloko e bodes hantle.
Phetiso ea kokoana-hloko e tsoang metsing e tsejoa e le kotsi ea bophelo bo botle ba sechaba ho tloha lilemong tsa bo-1940, ka bopaki ba pele ba ho fetisoa ha pholio le lefu la sebete ka metsing E1.Mokhatlo oa Lefatše oa Bophelo (WHO) o khethile likokoana-hloko tse ngata tse tsoang metsing tsa bohlokoa bo itekanetseng ho isa boemong bo phahameng ba bophelo bo botle2.Traditional virus mekhoa ea ho lemoha e itšetlehile ka mekhoa ea khauta e thehiloeng ho qPCR, e hlokolosi haholo le e tobileng, empa e hloka basebetsi ba nang le tsebo ho etsa liteko laboratoring ba sebelisa lisebelisoa tse theko e boima. tlhahlobo ea sampole e kanna ea tla pele ho tlhahlobo ea metsi a tikoloho. Ka hona, mekhoa e meng e theko e tlase e ea hlokahala bakeng sa tlhahlobo e tšoarellang, ea nako ea 'nete ea lisampole tsa metsi le litšila linaheng tse nang le meputso e tlase le e bohareng e le litemoso tsa pele tsa ho qhoma ha mafu a hlahang, ka hona ho ba sireletsa litlamoraong tse matla tsa moruo oa seoa sa kokoana-hloko.Li-biosensors tsa electrochemical tse theko e tlase bakeng sa li-nucleic acid li ka fana ka tharollo e ts'episang ea tlhoko ena e sa finyelloang.Bongata ba li-biosensors tsena tsa DNA li sebetsa ka taba ea hore likhoele tsa DNA tse tlatselletsang ha li tsamaee holim'a electrode. holimo le ho nyarela ha tatellano e nyallanang e le teng ka sampuling.Sena se ka fetoloa lets'oao ka mekhoa e fapaneng ea electrochemical ho sebelisa li-mediator tsa redox joalo ka potassium iron/ferrocyanide.Methylene blue (MB) is one such redox-active molecule, which has e tlalehiloe hore e kena-kenana le DNA e nang le mefuta e 'meli ea DNA (dsDNA) ho phaella ho tlamahano ea eona e sa tobang ho DNA5,6. Sebopeho sa intercalating sa MB ho etsa li-complexes tsa MB-DNA se etsa hore e be khetho e tummeng e le baemeli ba redox ho tse ngata tsa electrochemical DNA. Sensor configurations5,6,7,8,9. Le hoja ho kena-kenana ha MB ho DNA ho sa khethehe, 'me ho khetheha ha sensor ena ea electrochemical e itšetlehile haholo ka bohloeki ba lisebelisoa tse sebelisoang bakeng sa PCR kapa amplification ea isothermal, e loketse hantle bakeng sa ho kenya ts'ebetsong ea sebele. -nako ea electrochemical-based qPCR kapa fluorescence isothermal amplification e le mokhoa o mong oa tekanyo ea mahloriso ea DNA 9. Ts'ebetsong e 'ngoe e joalo, Won et al.Bokaholimo ba li-electrode tsa khauta bo ile ba fetoloa ka 6-mercapto-1-hexanol (MCH) bakeng sa nako ea sebele. tekanyo ea li-amplicon tsa PCR tse nang le MB ka ho sebelisa phapang ea pulse voltammetry (DPV) 9. Maemong a mang, Ramirez et al. Ho fumanoa ha SARS-CoV-2 metsing a litšila ka RT-LAMP reaction sebelisa MB ka li-electrode tse hatisitsoeng skrineng. Li-electrode tsa Platinum le tsona li 'nile tsa etsoa. e sebelisoang e le li-electrode sebakeng sa microfluidic PCR sethaleng se etselitsoeng ho lemoha li-amplicons ka electrochemically nakong ea karabelo 8. Liphuputso tsena kaofela li hloka phetoho ea holim'a li-electrode, e bolelang ho eketseha ha tlhahiso le litšenyehelo tsa ts'ebetso ka lebaka la litlhoko tse khethehileng tsa polokelo bakeng sa botsitso ba li-electrode tsena tse sebetsang hantle.
Sekema sa phallo ea mosebetsi bakeng sa tlhahlobo ea electrochemical ea li-amplicon tse fumanoeng ho tsoa ho likaroloana tse koalehileng tsa vaerase lisampoleng tsa metsi a letša.
Haufinyane tjena re bonts'itse maikutlo a electrochemical ea li-amplicon tsa SARS-CoV-2 tse nang le li-electrode tsa theko e tlase tse hatisitsoeng (PCB) tse thehiloeng ho DPV le cyclic voltammetry (CV) tse susumetsoang ke ho ata ha li-complexes tsa MB-DNA holim'a li-electrode tse sa fetoloang ) ea joale11.Re tlaleha hore likhechana tse telele tsa DNA (N1-N2, \({943}\, \hbox) tse entsoeng ho sebelisoa N1 e khothalelitsoeng ke CDC le li-primer tsa N2 reverse ha li bapisoa le likhechana tse khuts'oane {bp}\)) li bonts'itse taolo e betere ho karabo ea kutlo. ( N1, \(72\,\hbox {bp}\)) e entsoeng ka ho sebelisa N1 forward le N1 reverse primer set -2 li-amplikhone ka lisampole tsa metsi a litšila tse etsisitsoeng (tse fumanoeng ka spiking kakaretso ea lisampole tsa RNA tse nang le SARS-CoV-2 RNA). Kaha RNA e kotsing ea ho kuta nakong ea ho itšehla thajana le ho sebetsa ka tlase ho noka,12,13 ho thata ho holisa likhechana tse telele ka sampole ena e fapaneng. Ka hona, ponts'o ea electrochemical sensing ea SARS-CoV-2 amplicon metsing a litšila e lekanyelitsoe ho sekhechana se sekhutšoane \(72\,\hbox {bp}\) N1.
Mosebetsing ona, re ile ra batlisisa ho khoneha ha ENIG PCB-based electrochemical sensing of phage Phi6 concentrated le ho arohana le lisampole tsa metsi a letša (Fig. 1) .Phi6 phages li bapisoa ka boholo (80-100 nm) ho SARS-CoV-2 le hape e na le membrane ea lipid le protheine ea spike.Ka mabaka ana, bacteriophage Phi6 ke moemeli ea tsebahalang oa SARS-CoV-2 le likokoana-hloko tse ling tse koahetsoeng ke RNA14,15.RNA e arohaneng le likaroloana tsa phage e ne e sebelisoa e le template bakeng sa motsoako oa cDNA e lateloa ke PCR ho fumana likhechana tse peli tsa DNA tsa 117 le 503 base pairs bolelele \,\hbox {bp}\) le \(503 \,\hbox {bp}\)), ho ipapisitsoe le li-primers tse fumanehang.Karabo ea sensor ea electrochemical e ile ea ithutoa ka mokhoa o hlophisehileng ka mefuta e mengata ea mahloriso (\({10}\,{ \hbox {pg}/{\upmu \hbox {l}}}\) ho \({20}\, {\hbox {ng}/{\upmu \hbox {l}}}\)) Bakeng sa likhechana ka bobeli ho boteng ba MB, phello ea letsoai holim'a karabo ea kutlo e 'nile ea khetholloa' me ea tiisoa ka litekanyo tsa spectrophotometric.Menehelo e ka sehloohong ea mosebetsi ona ke e latelang:
Bolelele ba sekhechana sa DNA le boteng ba letsoai ka har'a sampole bo ama haholo kutlo.Liphetho tsa rona li bonts'a hore ts'ebetso ea electrochemical e itšetlehile ka mekhoa e fapaneng ea ho sebelisana ha MB, DNA, le sensor ka karabo ea voltammetric, ho itšetlehile ka mahloriso a DNA le bolelele, ka likaroloana tse telele li bontša kutloisiso e phahameng, le hoja letsoai le na le phello e mpe litšebelisanong tsa electrostatic pakeng tsa. MB le DNA.
DNA concentration e etsa qeto ea mokhoa oa ho sebelisana ha MB-DNA ka li-electrode tse sa fetotsoeng Re bontša hore mekhoa e fapaneng ea ho sebelisana ha MB-DNA e itšetlehile ka DNA concentration. {l}}}\), re hlokometse hore karabelo ea hona joale ea electrochemical e ne e khethoa haholo ke ho ata ha MB-DNA ho electrode, ha e ntse e le ka tlaase ho maemo a phahameng a DNA, karabo ea hona joale ea electrochemical e ne e khethoa ke thibelo ea steric ea redox. ts'ebetso ka lebaka la ho kenngoa ha MB lipakeng tsa lipara tsa motheo tsa DNA.
ENIG PCB-Based Electrochemical Sensing of Viral Nucleic Acids in Lake Water Samples Maikutlo a netefalitsoe ke tlhahlobo ea electrochemical ea Phi6-added \(503\,\hbox {bp}\) likaroloana tsa DNA tse fumanoeng lisampong tsa metsi ho tsoa Powai Lake, IIT Mumbai Campus. Sephetho phage.
Litsenyehelo tse tlase tsa ts'ebetsong le monyetla oa ho kopanngoa le mekhoa ea ho hlahloba ka ho feletseng, li-oligonucleotide kapa li-aptamers ho li-electrode tse nang le nako e telele ea nako.
Phage Phi6 ke kokoana e enveloped dsRNA ea lelapa la Cytoviridae e tšoaetsang Pseudomonas syringae.Genome ea Phi6 phage e teng ka sebopeho sa likaroloana tse 3: S (\(2.95\,\hbox {Kb}\)), M (\(4.07) \,\hbox {Kb}\)) le L (\ (6.37\ ,\hbox{Kb}\))16,17.Kaha Phi6 phage e tšoaetsa mofuta o seng oa pathogenic oa BSL-1 Pseudomonas, ho bolokehile ho sebelisoa. 'me e ka lengoa habonolo laboratoring.Phage Phi6 le moamoheli oa eona Pseudomonas syringae li rekiloe ho Felix d'Herelle Reference Center for Bacterial Viruss, Laval University, Canada (linomoro tsa setsi sa lireferense ke HER-102 le HER-1102, ka ho latellana) .Phi6 phage le moamoheli oa eona ba ile ba tsosolosoa joalokaha ho laetsoe ke setsi sa litšupiso.Phage Phi6 was purified by plate lysis and elution to find final titers with \(\ about 10^{12}\,{\hbox {PFU}/\hbox { ml}}\) (plaque forming units/ milliliters).RNA e ne e arotsoe ho tloha ho hloekisitsoeng phage particles sebelisa GenElute™ Universal Total RNA Purification Kit (Sigma-Aldrich) ho latela litaelo tsa moetsi.Ka bokhutšoanyane, phage e hloekisitsoeng Phi6 e emisitsoe\({{ 100}\,{{\upmu \hbox {l}}}\) e ile ea hlajoa 'me lysate ea laeloa holim'a kholomo e bilikang ho lumella RNA ho tlama kholumong ea resin .RNA e ntan'o hlakoloa ka tharollo ea elution \({ 10 ({-80}\,{^{\circ }\hbox {C}}\) ho fihlela e sebelisoa hape.\({2}\,{\upmu \hbox {g}}\) The iScript cDNA Synthesis Kit (Bio -Rad Laboratories) e ne e sebelisoa e le template bakeng sa motsoako oa cDNA ho latela litaelo tsa moetsi. )\({5}\,{\hbox {min} }\) , phetisetso ea morao ea \({20}\,{\hbox {min}}\) ho \({46}\,{^{\circ }\hbox {C}}\), le ho kgutlela morao Sehatisi se ho \({95}\,{^{\circ }\hbox {C}}\) bakeng sa \({1}\,{\hbox {min }}\).Ha e tsamaisoa ka gel ea agarose ea 1%, cDNA e ile ea bontša lihlopha tse tharo tse tsamaellanang le likhechana tse tharo tsa RNA tse lebelletsoeng (data e sa bonts'itsoeng). Lintlha tse latelang li ile tsa sebelisoa ho holisa likhechana tse peli tsa DNA tsa 117 le 503 bp ka bolelele. ho sebelisa cDNA joalo ka template ea PCR ho miniPCR® mini8 thermal cycler:
Li-primers tsa \(117\,\hbox {bp}\) le \(503\,\hbox {bp}\) li lumellana le 1476-1575 nucleotide ea karolo ea M le 458-943 nucleotide ea karolo ea L, ka ho latellana acid. .Lihlahisoa tsohle tsa PCR tse matlafalitsoeng li ne li entsoe ka electrophores ka li-gel tsa agarose tsa 1%, 'me sepheo se matlafalitsoeng sa DNA se ile sa hloekisoa ka ho sebelisa GeneJET Gel Extraction Kit (Thermo Fisher Scientific).
Letša la khamphaseng ea IIT Mumbai (Powai Lake, Powai, Mumbai) le ne le sebelisetsoa ho eketsa likaroloana tsa phage. likaroloana tse emisitsoeng, 'me Phi6 phage ea eketsoa.Eketsa \({1}\,{\hbox {ml}}\) ea \(10^{6}\,{\hbox {PFU}/\hbox {ml}} \) ho \( {100}\ ,{\hbox {ml}}\) metsi a hloekisitsoeng a letša, ka \({4}\,{^{\ circle}\hbox {C}}\).Aliquot e nyenyane e ne e le Re lekile mekhoa e 'meli e fapaneng ea ho tsepamisa maikutlo ho likaroloana tsa kokoana-hloko ea Phi6: (1) mokhoa oa aluminium hydroxide adsorption-precipitation, 19 o netefalitsoeng bakeng sa ho bokellana ha likokoana-hloko tse ngata tsa RNA tse tsoang lisampoleng tsa tikoloho, le (2) ) Mokhoa oa mahloriso oa kokoana-hloko ea polyethylene glycol (PEG) o ile oa fetoloa ho tloha Moroallo et al.20 .Kaha katleho ea ho hlaphoheloa ha mokhoa o thehiloeng ho PEG e fumanoe e le molemo ho feta mokhoa oa aluminium hydroxide, mokhoa o thehiloeng ho PEG o ne o sebelisetsoa ho tsepamisa mohopolo likaroloana tsa Phi6 ho tloha lisampoleng tsa metsi a letša.
Mokhoa oa PEG o sebelisitsoeng e ne e le o latelang: PEG 8000 le \(\hbox {NaCl}\) li kenyellelitsoe lisampoleng tsa metsi a letša a Phi6-spiked ho fumana 8 % PEG 8000 le \(0.2\,\hbox {M} \) \( \ hbox {NaCl}\).Mehlala e ile ea kengoa ka shaker\({4}\,{^{\circ }\hbox {C}}\)\({4}\,{\hbox {h}}\ ), ebe centrifuged ho \(4700 \,\hbox {g}\) is \({45}\,{\hbox {min}}\).Lahla supernatant ebe o emisa pellet hape ho \({1}\, {\ hbox {ml}}\) ka supernatant e tšoanang. Liteko tsohle tsa spiking le kokoana-hloko ea kokoana-hloko li ile tsa etsoa ka triplicate.After concentration, aliquot e nyenyane e ne e boloketsoe ho lekanya katleho ea ho hlaphoheloa ka plaque assay.RNA e ne e arotsoe joalokaha e hlalositsoe pele le ho hlakoloa ka kit-supplied elution buffer\({40}\,{\upmu \hbox {l}}\). Kaha mahloriso a RNA a tla fapana ho tloha sampole ho isa ho disampole ka triplicate, the \({2}\,{\upmu \\) hbox {l}}\) ea RNA e sebelisoa ho tse tharo kaofela ho sa natsoe hore na cDNA synthesis ea lisampole ke efe. e ne e sebelisoa e le template bakeng sa \({20}\,{\upmu \hbox {l}}\) PCR bakeng sa lipotoloho tse 35 ho holisa \ (117\,\hbox {bp}\) le \(503\,\hbox { bp}\) likhechana.Mehlala ena e emeloa e le “1:1″, ke hore ntle le dilution.A no-template control (NTC) e ile ea thehoa e le taolo e mpe, ha cDNA synthesized sebelisa RNA e arohaneng le phage e hloekisitsoeng. e le template bakeng sa taolo e ntle (PC).Quantitative PCR (qPCR) e entsoe ka sesebelisoa sa Stratagene Mx3000P RT-PCR ho sebelisoa Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies). Liphetoho li ile tsa thehoa ka makhetlo a mararo joaloka pele e hlalositsoe.Moeli oa potoloho (Ct) o ile oa rekotoa bakeng sa disampole tsohle.Ho feta moo, disampole tse hlapolotsoeng li ne li \({1}\,{\upmu \hbox {l}}\) ho sebelisoa cDNA e hlapolotsoeng 1:100 metsing a hloekisitsoeng a letša e le. \({20}\,{\upmu \hbox {l}}\) PCR bakeng sa lipotoloho tse 35. Mehlala ena e emetsoe e le “1:100″.
Li-electrode tsa PCB li entsoe ka mokhoa o sebelisoang khoebong oa theko e tlaase ea Electroless Nickel Immersion Gold (ENIG) ntle le tlhokahalo ea ho khabisa khauta e eketsehileng.Litlhaloso tsa motlakase oa ENIG PCB li hlalositsoe ka ho qaqileng mosebetsing oa rona o fetileng11.Bakeng sa li-electrode tsa ENIG PCB, mekhoa e tloaelehileng ea ho hloekisa li-electrode tse kang tharollo ea piranha kapa sulfuric acid cyclic voltammetry ha e khothalletsoe kaha e ka baka lesela le tšesaane la khauta (botenya \(\ approx\) \(100\,\hbox {nm}\)) le ho pepesa likarolo tse ka tlase tsa koporo tse bonolo. ho corrosion 21, 22, 23, 24, 25. Ka hona, hloekisa li-electrode ka lesela le se nang lint le kolobisitsoeng ka IPA. Mohlala o lokelang ho hlahlojoa o ne o e-na le \({50}\,{\upmu \hbox {M} }\) MB ho \({4}\,{^{\circ }\hbox {C}\)\({ 1}\,{\hbox {h}}\) hore e kenyelle habonolo. Mosebetsing oa rona o fetileng , re hlokometse hore kutloisiso le linearity ea sensor li ile tsa ntlafatsoa ka ho eketsa mohopolo oa MB 11 .Ho itšetlehile ka optimizations e tlalehiloeng mosebetsing oa rona oa pejana, re sebelisitse \({50}\,{\upmu \hbox {M}}\) MB concentrations to embed DNA in this study.Ho lemoha ka electrochemical ea DNA e nang le likhoele tse peli (ds-DNA) e ka finyelloa ka ho sebelisa li-anionic kapa cationic intercalators.Le hoja li-intercalator tsa anionic li lemoha DNA ka khetho e ntle, li hloka ho kenngoa ha bosiu bosiu, ho fella ka nako e telele ea ho lemoha. ka lehlakoreng le leng, li-intercalator tsa cationic tse kang MB li hloka linako tse khutšoanyane tsa ho qhomisa, hoo e ka bang \({1}\,{\hbox {h}}\) bakeng sa ho lemoha electrochemical ea ds-DNA6. electrode \ ({5}\,{{\ upmu \hbox {l}}}\), ebe o hloekisa ka rag e kolobisitsoeng ka IPA, pele o tsoela pele ka sampuli e 'ngoe.tekanyo e le 'ngoe.Sampole e' ngoe le e 'ngoe e ile ea lekoa ka li-electrode tse 5 tse fapaneng ntle le haeba ho boletsoe ka tsela e fapaneng.Litekanyo tsa DPV le CV li ne li etsoa ho sebelisoa PalmSens Sensit Smart potentiostat,' me software ea PSTrace e ne e sebelisoa bakeng sa tlhophiso ea potentiostat le ho fumana lintlha, ho kenyelletsa le lipalo tse phahameng tsa hona joale.Litlhophiso tse latelang li sebelisoa. bakeng sa litekanyo tsa DPV le CV:
DPV: Nako ea Tekano = \(8\,\hbox {s}\), Voltage Step = \(3\,\hbox {mV}\), Pulse Voltage = \(25\,\hbox {mV}\) , nako ea ho otla ha pelo = \(50\,\hbox {ms}\), sekhahla sa scan = \({20}\,\hbox {mV/s}\)
CV: Nako ea Tekano = \(8\,\hbox {s}\), Mothati oa Voltage = \(3\,\hbox {mV}\), Sweep Rate = \({300}\,\hbox {mV/ s }\)
Maqhubu a holimo a fumanoeng ho tsoa ho li-voltammograms tsa DNA tse entsoeng ka \({50}\,{\upmu \hbox {M}}\) MB: (a) \(503\,\hbox {bp}\) DPV , (b) \ (503\,\hbox {bp}\) CV, (c) \(117\,\hbox {bp}\) DPV, (d) \(117\,\hbox {bp}\) CV.
DPV le CV voltammograms li ile tsa fumanoa ka li-electrode tsa ENIG PCB \({50}\,{\upmu \hbox {M}}\) MB tse kopantsoeng le DNA (ka likhakanyo tsa 10–\({20}\,{\ hbox {ng) }/{\upmu \hbox {l}}}\) ke hore 0.13–\({0.26}\,{\upmu \hbox {M}}\) bakeng sa \(117\,\hbox {bp}\ ) le 0.03 –\({0.06}\,{\upmu \hbox {M}}\) bakeng sa \(503\,\hbox {bp}\)). Li-voltammogram tsa moemeli li bontšoa ho Setšoantšo sa S1 ho Boitsebiso ba Tlatsetso.Setšoantšo sa 2 se bontša liphetho ea litekanyo tsa DPV le CV (tlhōrō ea hona joale) ho sebelisa lihlahisoa tsa PCR tse hloekisitsoeng ka gel. Ha li bapisoa le litekanyo tsa CV, litekanyo tsa DPV li bontša kutloisiso e phahameng (hona joale e le ts'ebetso ea DNA concentration) hobane mokokotlo oa capacitive currents litekanyong tsa CV o pata maqhubu a Faradaic 26 .The data bakeng sa lebokose le leng le le leng ka lebokoseng la lebokose le na le litekanyo tse tsoang ho li-electrode tse 5. Litekanyo tsohle li sebelisa sete e tšoanang ea li-electrode ho qoba liphoso tsa tekanyo ka lebaka la phapang ea electrode-to-electrode. , nako e telele (\(503\,\hbox {bp}\)) \,\hbox {bp}\ bapisoa le \(117) ) sekhechana.Sena se lumellana le mokhoa o lebeletsoeng oa adsorption ea electrode e tlalehiloeng mosebetsing oa rona o fetileng.The adsorption ea MB-DNA complex e thusa ho fetisetsa tefiso ho electrode, e leng se tlatsetsang ho eketseha ha tlhōrō ea hona joale. -cytosine (GC) dikahare tsa li-amplicon tse peli (\(117\,\hbox {bp}\) le \(503\,\hbox {bp}\)) e ne e ka ba 50%, e leng se bontšang hore ho shebella Phapano ho fihlela bolelele ba amplicon.Leha ho le joalo, bakeng sa likhakanyo tse phahameng tsa DNA (\(>{2}\,{\hbox {ng}/{\upmu \hbox {l}}}\), bakeng sa \(503\,\hbox {bp} \) le \( >{10}\,{\hbox {ng}/{\upmu \hbox {l}}}\) bakeng sa \(117\,\hbox {bp}\)), re bona likeletso tse peli maqhubu a tlhōrō ea li-subs a fokotsehile ka bobeli ba DPV le litekanyo tsa CV. Sena se bakoa ke hore MB e tletse le ho kenella pakeng tsa lipara tsa motheo tsa DNA, e leng se etsang hore ho be le thibelo ea steric ea mosebetsi oa redox oa sehlopha se fokotsoang 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.
Matsoai a teng ho PCR master mixes a kena-kenana le litšebelisano tsa electrostatic pakeng tsa MB le DNA, kahoo ka ho eketsa \(2\,\hbox {mM}\) \(\hbox {MgCl }_2\) le \({50} \,{\ upmu \hbox {M}}\) MB sehlahiswa se hlwekisitsweng ka gel ho ithuta phello ya letswai tshebedisanong ya MB-DNA. Joalo ka ha ho bontshitswe ho Setšoantšo sa 3, re hlokometse hore bakeng sa DNA e phahameng (\(>{2}\,{\ hbox {ng}/{\upmu \hbox {l}}}\) (503\,\hbox {bp }\) le \(>{10}\,{\hbox {ng}/{\upmu \hbox { l}}}\) bakeng sa \(117\,\hbox {bp} \)), ho DPV le CV Ho eketsoa ha letsoai ha hoa ka ha ama litekanyo haholo (sheba Figure S2 in Supplementary Information bakeng sa li-voltammograms tse emetseng). Leha ho le joalo, ho DNA e fokolang, ho eketsoa ha letsoai haholo ho fokotsa kutloisiso, ho etsa hore ho se ke ha e-ba le phetoho e kholo ea hona joale ka mahloriso a DNA. Liphello tse tšoanang tse mpe tsa letsoai ka ho sebelisana ha MB-DNA le ho kopana ho 'nile ha tlalehoa pele ke bafuputsi ba bang33,34.\(\ hbox { Mg}^{2+}\) li-cations li tlama le mokokotlo oa phosphate oa DNA, kahoo li sitisa ho sebelisana ha electrostatic pakeng tsa MB le DNA. u se ke ua ama karabelo ea kutlo haholo.Ntlha ea bohlokoa ke hore biosensor ena e loketse hamolemo ho bona likarolo tse phahameng tsa DNA (ka seoelo \({\hbox {ng}/{\upmu \hbox {l}}}\) kapa holimo), bakeng sa ts'ebetso e felletseng ea lisampole tsa metsi a tikoloho, moo tlhoekiso ea lijeli ea lihlahisoa tsa PCR e kanna ea se khonehe.
Sebaka se tlas'a mokoloko oa maqhubu a maqhubu a 600–700 \(\hbox {nm}\) bakeng sa likarolo tse fapaneng tsa DNA tse kopantsoeng ka \({50}\,{\upmu \hbox {M}}\) MB: ( a ) \(503\,\hbox {bp}\) ka letsoai le le se nang letsoai (\(2\,\hbox {m}\) \(\hbox {MgCl}_2\)), (b) \( 117\, \hbox {bp}\) e nang le letsoai le le se nang letsoai (\(2\,\hbox {mM}\) \(\hbox {MgCl}_2\)).\({0}\,{\hbox {pg}/) {\upmu \hbox {l}}}\) DNA concentrations tse tsamaellanang le \({50}\,{\upmu \hbox {M}}\) MB disampole Ha ho DNA.
Ho netefatsa liphetho tse kaholimo ho feta, re entse litekanyo tsa mahlo re sebelisa UV/Vis spectrophotometer (Thermo Scientific Multiskan GO), lisampole \({50}\,{{\upmu \hbox {l}}}\) li sebelisitsoe bakeng sa e 'ngoe le e 'ngoe. Tekanyo.Tshaeno ea ho monya e fokotseha ha DNA e ntse e eketseha, joalo ka ha ho ka bonoa ho tsoa mokhoeng oa sebaka se ka tlas'a lekhalo la maqhubu a maqhubu a fapaneng \(600\,\hbox {nm}\) ho \(700\,\hbox { nm}\) , joalo ka ha ho bonts'itsoe setšoantšong sa 4 (matla a ho monya a bonts'itsoeng ho Fig. S3 in Supplementary Information). Bakeng sa disampole tse nang le DNA concentrations ka tlase ho \({1}\,{\hbox {ng}/{\upmu \hbox {l}}}\), ho ne ho se na phapang e kholo ea ho amoheloa lipakeng tsa lisampole tse nang le DNA le MB feela (bakeng sa \(503\,\hbox {bp}\) le \(117\,\hbox {bp}\) ) likhechana tsa bolelele), tse bontšang ho se be teng ha steric inhibition ea redox-active MB. Likarolong tse phahameng tsa DNA, re ile ra bona ho fokotseha butle-butle ha lets'oao la ho monya 'me ra hlokomela ho fokotseha ho fokolang ha letsoai le le teng. interactions and steric inhibition with base stacking in DNA hybrids.Liphetho tsa rona li lumellana le litlaleho tse hlahang libukeng tsa lithuto tsa spectroscopic tsa MB-DNA intercalation tse amahanya hypochromaticity le matla a fokotsehileng a matla ho \(\pi\)–\(\pi ^*\ ) liphetoho tsa elektroniki ka lebaka la ho hokahana Likarolo tsa 36, 37, 38.
Agarose gel electrophoresis of phage Phi6: PCR lihlahisoa tsa bolelele \(117\,\hbox {bp}\) le \(503\,\hbox {bp}\) ho tloha lisampole tsa metsi a letša.M-DNA marker;Taolo ea NTC-no-template, li-primers tse nang le li-amplicon tse lumellanang;Taolo e ntle ea PC;1, 2, 3-undiluted (1:1) spiked lisampole tsa metsi a letša ka triplicate hbox {bp}\) tsela.
Re ile ra lekola ts'ebeliso ea sensaro ka ho sebelisa lisampole tsa metsi a Letša la Powai tse spiked le Phi6 phage. The RNA concentrations tse arohaneng le phage-spiked water samples ranged from 15.8–\({19.4}\,{\upmu \hbox {g}/\hbox { ml}}\), ha ba arohaneng le ho emisoa ha phage e hloekisitsoeng RNA e hakanngoa e le \({1945}\,{\upmu \hbox {g}/\hbox {ml}}\) e nang le ts'ebetso ea ho hlaphoheloa ea hoo e ka bang 1. %RNA was reverse transcribed into cDNA and used as the template for PCR and qPCR.Bogolo ba sehlahiswa bo netefaditswe ke agarose gel electrophoresis (Figure 5) pele ho tekolo ka sensor.Mehlala ena ha e hlwekisitswe ka gel mme ka hona e na le likarolo tsohle tsa PCR joalo ka Theko ea Ct e tlalehiloeng nakong ea qPCR (Letlapa la 1) e bonts'itsoe hore e amana le bongata ba RNA bo arohaneng le lisampole tsa metsi a spiked. feta moeli kapa lets'oao le ka morao.Litekanyetso tse phahameng tsa Ct li bonts'a maemo a tlase a template le ka tsela e fapaneng. Litekanyetso tsa Ct tsa lisampole tsa NTC li ne li le holimo ka moo ho neng ho lebelletsoe. taolo e ntle le sampole ea tlhahlobo e tsoela pele ho bontša hore sampole e 'ngoe le e 'ngoe ea teko e na le template e ka bang 1% ha e bapisoa le taolo e ntle.Re kile ra buisana ka hore li-amplicon tse telele li lebisa tlhokomelong e ntle. ea ho fokotseha ha kokoana-hloko ea kokoana-hloko le ho senyeha ha RNA.Leha ho le joalo, ka ho ntlafatsa kokoana-hloko ea rona le protocol ea amplification ea PCR, re khonne ho atleha ho holisa sekhechana sa \(503\,\hbox {bp}\) bakeng sa ho utloa electrochemical.
Setšoantšo sa 6 se bonts'a liphetho tsa sensor ea electrochemical tsa sekhechana sa amplicon ea \(503\,\hbox {bp}\), ka bobeli li sebelisa cDNA e sa tsitsang joalo ka template (1:1) le cDNA e hlapollotsoeng ka makhetlo a 100 joalo ka template (1:100 ) e entsoeng PCR. , ha e bapisoa le NTC le PC (sheba Setšoantšo sa S4 ho Boitsebiso bo Tlatsetso bakeng sa li-voltammograms tse emetseng) . Lebokose le leng le le leng ka lebokoseng la lebokose le setšoantšong sa 6 le na le litekanyo tse tsoang ho mehlala e meraro ho li-electrode tsa 5. Li-electrode tse tšoanang li ne li sebelisetsoa ho lekanya mehlala eohle ho qoba liphoso ka lebaka la electrode -to-electrode variation.Ha ho bapisoa le litekanyo tsa CV, litekanyo tsa DPV li bontša qeto e ntlafetseng ea ho khetholla lisampole tsa liteko le tsa PC ho tsoa ho NTCs hobane, joalo ka ha ho boletsoe pejana, maqhubu a Faradaic a patiloe ka lebaka la maqhubu a capacitive a morao-rao.Bakeng sa li-amplicon tse telele, re hlokometse hore taolo e mpe (NTC) e hlahisitse maqhubu a phahameng a CV le DPV a amanang le taolo e nepahetseng, athe disampole tse ntle le tse sa tsitsang tsa liteko li bonts'itse bophahamo bo ts'oanang ba maqhubu a tlhoro ea DPV. ) sampole ea tlhahlobo le PC li ka rarolloa ka ho hlaka ho tsoa ho sephetho sa sensor bakeng sa sampole ea NTC, ha qeto ea 1: 100 sampole e hlapollotsoeng e sa tsejoe haholo. (litsela tse sa bonts'itsoeng ho Setšoantšo sa 5), le maqhubu a phahameng a DPV le CV a tšoanang a ne a tšoana le a neng a lebelletsoe bakeng sa NTC. Liphetho tsa sekhechana sa \(117\,\hbox {bp}\) li bonts'itsoe ho Litaba tsa Tlatsetso. The negative ho laola ho entse hore ho be le karabo ea electrochemical ho tloha ho sensor ea PCB ka lebaka la ho phatlalatsoa ha MB mahala ho electrode le ho sebelisana ha MB le oligonucleotide e nang le makhasi a le mong. tlhōrō ea tlhōrō ea sampole ea teko ha e bapisoa le tlhōrō ea tlhōrō e fumanoeng ke taolo e mpe ho finyella tekanyo e fapaneng (e lekanyelitsoeng)39,40 ho khetholla sampole ea teko e le ntle kapa e mpe.
(a) DPV, le (b) CV peak current bakeng sa ho lemoha electrochemical tsa \(503\,\hbox {bp}\) likaroloana tsa lisampole tsa metsi a letša. Mehlala ea liteko e ile ea lekanngoa ka makhetlo a mararo le ho bapisoa le no template controls (NTC) le li-control tse ntle (PC).
Liphuputso tsa rona li bontša mekhoa e fapaneng e amang ts'ebetso ea li-sensor tsa electrochemical bakeng sa li-amplicon tsa bolelele bo fapaneng bakeng sa li-DNA tse fapaneng, tse nang le lintlha tse netefalitsoeng ke litekanyo tsa mahlo ho sebelisoa UV/Vis spectrophotometer. \(500\,\hbox {bp}\) e ka bonoa ka kutlo e phahameng le hore boteng ba letsoai ka har'a sampole ha bo na "Sensitivity DNA concentration" e amang kutlo e phahameng (ka seoelo \({\hbox {ng}/{\upmu) \hbox {l}}}\) le ka holimo).Ho feta moo, re ile ra batlisisa phello ea mefuta e fapaneng ea lisampole, ho kenyeletsoa liamplicon tse hloekisitsoeng ka gel tse nang le letsoai le ntle le ho eketsoa, le ho eketsoa ha lisampole tsa metsi a letša litekanyong tsa DPV le CV.Re hlokometse hore DPV e fana ka tharollo e betere, kaha bokamorao ba capacitive current bo boetse bo ama tekanyo ea CV, e etsa hore e se utloele bohloko.
Ho phahamisa likhechana tse telele ho itšetlehile ka botšepehi ba likokoana-hloko tsa RNA. .Re hlokometse hore mokhoa oa khatello ea kokoana-hloko e thehiloeng ho PEG e ne e atlehile haholo ho tsepamisa mohopolo oa phage Phi-6 spiked ka lisampole tsa metsi a letša ho feta mokhoa oa aluminium hydroxide-based based virus concentration.Bokhoni ba ho lemoha likaroloana tse telele tsa DNA bo ipakile ho hlōla tlhokahalo ea multiplex PCR ho holisa litempele tse ngata tse khuts'oane tse ngata le ho fokotsa monyetla oa ho fapana.
Li-electrode tsa ENIG PCB tse sebelisitsoeng thutong ena li hloka feela \({5}\,{{\upmu \hbox {l}}}\ lisampole bakeng sa liteko ho koahela sebaka se sebetsang sa li-electrode. Ho ekelletsa moo, eleketrode e tšoanang e ka sebelisoa hape ka mor'a ho hloekisa pele ho fana ka sampuli e latelang. le lik'hemik'hale tse sebelisoang hangata.Kaha electrode ka 'ngoe e bitsa chelete e ka bang $0.55 (kapa INR 40) ho e etsa, biosensor ena e ka ba mokhoa o sa sebetseng hantle ho feta mekhoa e teng ea ho lemoha. Likaroloana tsa DNA ka lisampole tse sa tšoaneng.
Ka lebaka la hore liprothokholo tsa tlhahlobo ea electrochemical tse thehiloeng ho MB li itšetlehile ka ho khetheha ha PCR, moeli o moholo oa mokhoa ona ke monyetla oa ho holisa ka mokhoa o sa tobang ka lisampole tse sa tšoaneng tse kang metsi a litšila le metsi a letša kapa ho sebelisa li-primers tse tlaase. mekhoa ea ho lemoha electrochemical bakeng sa ho lemoha DNA ea lihlahisoa tsa PCR tse sa hloekang ka ho sebelisa li-electrode tsa ENIG PCB tse sa fetoloang, hoa hlokahala ho utloisisa hamolemo liphoso tse hlahisoang ke li-dNTP tse sa sebelisoeng le li-primers, le ho ntlafatsa maemo a ho itšoara le mekhoa ea ho hlahloba. tlhokahalo ea oksijene (BOD) ea sampole ea metsi le eona e kanna ea hloka ho lekanyetsoa molemong oa ho ntlafatsa ho nepahala ha tekanyo.
Qetellong, re fana ka mochine o theko e tlaase oa electrochemical ENIG PCB bakeng sa ho fumanoa ha kokoana-hloko tikolohong (metsi a letša).Ho fapana le li-electrode tsa oligonucleotide tsa immobilized kapa li-substrates tse tloaelehileng bakeng sa ho utloa DNA tse hlokang polokelo ea cryogenic ho boloka kutlo,53,54 mokhoa oa rona o sebelisa PCB e sa fetotsoeng. li-electrode tse nang le nako e telele ea nako e telele 'me ha li na litlhoko tse khethehileng tsa polokelo 'me kahoo li loketse bakeng sa nts'etsopele ea litharollo tsa ho lekanya ka mokhoa o ikemetseng oa ho etsa mohlala o sebelisoang ho LMICs e tloaelehileng lisampoleng tsa tikoloho e fokotsa ho khetheha ha mokhoa ona oa ho utloa ka lebaka la ho tlamaha ha MB ho sa khethehe ho oligonucleotides e le 'ngoe le e habeli. le maqhubu a tlhōrō ea DPV a fumanoang lisampoleng tse lekiloeng li lokela ho hlalosoa ho latela likarabo tse fumanoang ho tsoa taolong e mpe (NTC) bakeng sa tlhahlobo e 'ngoe le e' ngoe. Meralo le mekhoa ea sensor ea electrochemical e hlahisoang mosebetsing ona e ka kopanngoa le li-autosamplers ho hlahisa mochine o ikemetseng le o tlaase ka ho feletseng. -cost tharollo e ka bokellang le ho sekaseka lisampole le ho fetisetsa liphetho ka mohala ho khutlela laboratoring.
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Nako ea poso: May-27-2022