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Kukosha kwekutarisisa masampula ezvakatipoteredza kwave kufarirwa zvikuru kubva pakatanga denda reCCIDID-19, uye kumwe kuedza kwekutarisa kuri kuitwa pachishandiswa mwero wegoridhe, kunyangwe nzira dzeQPCR-based dzichidhura. Mushandiro uyu, tinoratidza kuongororwa kwe electrochemical yeamplicon inowanikwa kubva kuPhi6 phage yakaparadzaniswa kubva kune spiked dziva samples (yakakurumbira surrogate yeSARS-CoV-2), vachishandisa ENIG. kupedzisa PCB electrodes pasina kuchinjwa kwepamusoro.sex.Mapinduriro emagetsi emagetsi akanyatsotaridzwa pazvidimbu zviviri zveDNA zvehurefu hwakasiyana (\({117}\,\hbox {bp}\) uye \({503}\,\hbox {bp}\)), uye Mhedzisiro yemunyu muPCR master mixes pamethylene blue (MB) -DNA interactions.Zvigumisiro zvedu zvinoratidza kuti kureba kwezvidimbu zveDNA kunonyanya kugadzirisa kunzwisiswa kwe electrochemical uye kuratidza mubasa iri kuti kukwanisa kuona amplicon refu pasina gel kucheneswa kwePCR zvigadzirwa. yakakosha pakuyerwa kwemasamples emvura in situ.Iyo yakazara automated solution yeviral load bodes zvakanaka.
Kutapurirwa kwehutachiona hwemvura kunozivikanwa senjodzi yehutano hweveruzhinji kubva kuma1940, paine humbowo hwekutanga hwekutapurirana nemvura kweporiyo uye hepatitis E1.World Health Organisation (WHO) yakaronga utachiona huzhinji hunobva mumvura hune mwero kusvika pahutano hunokosha2.Traditional virus nzira dzekuona dzinotsamira pahunyanzvi hwegoridhe-standard qPCR-based, ihwo hune hunyanzvi uye hwakananga, asi hunoda vashandi vane hunyanzvi kuti vaongorore murabhoritari vachishandisa zviridzwa zvinodhura.Zvisinei, munyika dzine mari yepasi nepakati (LMICs) ine zviwanikwa zvishoma, vanhu. Sample testing ingangove inotungamira pane yezvakatipoteredza mvura sample monitoring.Naizvozvo, dzimwe nzira dzakachipa-mutengo dzinodiwa kuti dzirambe dzichitariswa, chaiyo-nguva yekutarisisa mvura nemvura yetsvina munyika dzine mari yakaderera nepakati seyambiro dzekutanga dzekubuda kwechirwere, zvichivadzivirira kubva kumatambudziko akanyanya ehupfumi hwehutachiona hwehutachiona.Ma electrochemical biosensors akaderera-mutengo wakaderera we nucleic acids anogona kupa mhinduro inovimbisa kune izvi zvisingaite zvinodiwa.Mazhinji emabiosensors eDNA aya anoshanda nekuti tambo dzeDNA dzinopindirana hadzina kufamba pa electrode. pamusoro uye hybridize kana kuenzanisa kutevedzana kuripo mumuenzaniso.Izvi zvinogona kuchinjwa kuva chiratidzo nemaitiro akasiyana-siyana e electrochemical achishandisa redox mediators se potassium iron/ferrocyanide.Methylene blue (MB) imwe yakadaro redox-active molecule, ine yakashumwa kuti inopindirana muDNA ine-double-stranded DNA (dsDNA) mukuwedzera kune iyo isingatarisirwi inosunga kune imwechete-stranded DNA5,6.Kupindirana kwechimiro cheMBs kuumba MB-DNA complexes inoita kuti ive sarudzo yakakurumbira se redox mediators mune dzakawanda electrochemical DNA. sensor configurations5,6,7,8,9.Kunyange zvazvo kupindirana kweMB kuDNA kusingatarisirwi, uye iyo chaiyo yeiyi electrochemical sensor inoenderana zvakanyanya nekuchena kweprimers inoshandiswa kuPCR kana isothermal amplification, inonyatsokodzera kushandiswa kwechokwadi. -nguva electrochemical-based qPCR kana fluorescence isothermal amplification seimwe nzira yeDNA concentration ye9 .Mumwe kushandiswa kwakadaro, Won et al.Kumusoro kwegoridhe electrodes yakagadziridzwa ne6-mercapto-1-hexanol (MCH) kwenguva chaiyo. kuyerwa kwePCR amplicon ine MB uchishandisa differential pulse voltammetry (DPV)9.Mune zvimwe zviitiko, Ramirez et al.Kuonekwa kweSARS-CoV-2 mumvura yetsvina neRT-LAMP reaction uchishandisa MB ine screen-yakadhindwa maelectrode.Platinum electrodes yakavewo inoshandiswa se in situ electrodes mu microfluidic PCR platform yakagadzirirwa electrochemically kuona amplicon panguva yekuita 8. Zvose izvi zvidzidzo zvinoda kuchinjwa kwepamusoro pema electrode, zvichireva kuwedzera kwekugadzirwa uye kushandiswa kwemari nekuda kwekukosha kwekuchengetedza zvinodiwa pakugadzikana kwemagetsi aya anoshanda.
Schematic of the workflow for electrochemical kuonekwa kwemaamplicon anowanikwa kubva kune concentrated viral particles mumasamples emvura yegungwa.
Isu nguva pfupi yadarika takaratidza kunzwa kwemagetsi eSARS-CoV-2 amplicon ane mutengo wakaderera akadhindwa wedunhu bhodhi (PCB) maelectrodes akavakirwa paDPV uye cyclic voltammetry (CV) inokonzereswa neadsorption yeMB-DNA complexes pamusoro pema electrode asina kuchinjwa ) ikozvino11.Isu tinoshuma kuti zvimedu zveDNA zvakareba (N1-N2, \({943}\, \hbox) zvakaumbwa uchishandisa CDC-inokurudzirwa N1 kumberi uye N2 reverse primers zvichienzaniswa nezvimedu zvipfupi {bp}\)) zvakaratidza mutsara uri nani mumhinduro ye sensor. ( N1, \(72\,\hbox {bp}\)) yakaumbwa uchishandisa N1 mberi uye N1 reverse primer sets.Zvidzidzo izvi zvinonzi zvichishandisa DNA dilutions yakagadzirwa mumvura isina nuclease.Chikuva chakashandiswawo kuona SARS-CoV -2 maamplicon mumasampuli emvura yetsvina akateedzeredzwa (akawanikwa nespiking yakazara RNA samples neSARS-CoV-2 RNA) .Sezvo RNA ichibatwa nekuveura panguva yekuzviparadzanisa nevamwe uye nekudzika kwemvura,12,13 zvinonetsa kukwidziridza zvimedu zvirefu neiyi sampuli yakasiyana. Naizvozvo, kuratidzwa kwe electrochemical sensing yeSARS-CoV-2 amplicon mumvura yetsvina inogumira kune ipfupi \(72\,\hbox {bp}\) N1 chidimbu.
Mubasa iri, takaongorora kugona kwe ENIG PCB-based electrochemical sensing yephage Phi6 yakanangidzirwa uye yakaparadzaniswa kubva kumadziva emvura samples (Fig. 1) . zvakare iine lipid membrane uye spike protein.Nezvikonzero izvi, bacteriophage Phi6 inozivikanwa surrogate yeSARS-CoV-2 uye mamwe akafukidzwa pathogenic RNA mavhairasi14,15.RNA yakaparadzaniswa kubva kune phage particles yakashandiswa setemplate ye cDNA synthesis inoteverwa ne PCR kuwana zvidimbu zviviri zveDNA zve117 uye 503 base pairs pakureba. Tichifunga nezvedambudziko rekukudza \(943\,\hbox {bp}\) N1-N2 zvidimbu mubasa redu rekare, tinonangisa zvimedu zvehurefu hwepakati (\(117) \,\hbox {bp}\) uye \(503 \,\hbox {bp}\)), zvichibva pane zvinotangira zviripo.Mapinduriro emagetsi emagetsi akadzidzwa zvine hungwaru pamusoro pehupamhi hwekuisa pfungwa (\({10}\,{ \hbox {pg}/{\upmu \hbox {l}}}\) kusvika \({20}\, {\hbox {ng}/{\upmu \hbox {l}}}\)) Pazvimedu zviri zviviri kuvapo kweMB, mhedzisiro yemunyu pamhinduro ye sensor yakave yakaratidza uye yakachinjika-yakagadziriswa ne spectrophotometric zviyero.Mipiro mikuru yebasa iri ndeyotevera:
DNA chidimbu kureba uye kuvapo kwemunyu mumuenzaniso kunobata zvakanyanya kunzwa.Mhedzisiro yedu inoratidza kuti electrochemical kuita kunoenderana nemaitiro akasiyana ekudyidzana kweMB, DNA, uye sensor mumhinduro yevoltammetric, zvichienderana nekusangana kweDNA uye kureba, neakareba Zvimedu zvinoratidza kunzwisiswa kwepamusoro, kunyangwe munyu uchikanganisa kupindirana kwemagetsi pakati. MB uye DNA.
DNA concentration inosarudza maitiro eMB-DNA interaction in unmodified electrodes Isu tinoratidza kuti maitiro akasiyana eMB-DNA interaction anoenderana neDNA concentration.Pa DNA concentrations pazasi zvishoma \({\hbox {ng}/{\upmu \hbox {l}}}\), takaona kuti mhinduro ye electrochemical ikozvino yainyanya kutsanangurwa neadsorption yeMB-DNA pa electrode, nepo pakadzika pasi Pahuwandu hweDNA yakawanda, mhinduro ye electrochemical ikozvino yakagadziriswa ne steric inhibition ye redox. chiitiko nekuda kwekuiswa kweMB pakati peDNA base pairs.
ENIG PCB-Yakavakirwa Electrochemical Sensing yeViral Nucleic Acids muLake Water Samples Izvo zvakaonekwa zvakasimbiswa nekuonekwa kwe electrochemical yePhi6-added \(503\,\hbox {bp}\) DNA zvidimbu zvakatorwa kubva kumvura samples kubva kuPowai Lake, IIT Mumbai Campus. Result phage.
Mutengo wakaderera wekushandisa uye mukana wekubatanidzwa mune yakazara otomatiki yekutarisisa masisitimu, oligonucleotides kana maaptamers pama electrode ane hurefu hwehupenyu hwesherufu.
Phage Phi6 chirwere chakaputirwa chedsRNA chemhuri yeCytoviridae chinotapurira Pseudomonas syringae.Genome yePhi6 phage iripo muchimiro chezvikamu zvitatu: S (\(2.95\,\hbox {Kb}\)), M (\(4.07) \,\hbox {Kb}\)) uye L (\ (6.37\ ,\hbox{Kb}\))16,17.Sezvo Phi6 phage inobata chirwere chisiri chepathogenic BSL-1 Pseudomonas, yakachengeteka kushandisa. uye inokwanisa kurimwa murabhoritari zviri nyore.Phage Phi6 nemutambi wayo Pseudomonas syringae yakatengwa kubva kuFelix d'Herelle Reference Center yeBacterial Viruses, Laval University, Canada (nhamba dzekataroji dzereference iHER-102 neHER-1102, zvichiteerana) .Phi6 phage nemukuru wayo akamutsiridzwa sekurairwa kwayakaitwa nereference centre.Phage Phi6 yakacheneswa neplate lysis uye elution kuti iwane final titers ne \(\ about 10^{12}\,{\hbox {PFU}/\hbox { ml}}\) (plaque forming units/milliliters) 100}\,{{\upmu \hbox {l}}}\) yakaiswa lysed uye lysate yaiiswa pane spin column kuti RNA isungirire kune resin column .RNA inobva yaburitswa mumhinduro ye elution \({ 50}\,{{\upmu \hbox {l}}}\) yakapihwa nekiti. Estimate the concentration of RNA by absorbance at \(260\,\hbox {nm}\).RNA was kept in aliquots mu\ ({-80}\,{^{\circ }\hbox {C}}\) kusvikira imwe kushandiswa.\({2}\,{\upmu \hbox {g}}\) The iScript cDNA Synthesis Kit (Bio -Rad Laboratories) yakashandiswa se template ye cDNA synthesis ichitevera mirairo yemugadziri.Muchidimbu, cDNA synthesis reaction ine matanho matatu: priming pa \({25}\,{^{\circ }\hbox {C}}\ )\({5}\,{\hbox {min} }\) , kudzokorora zvinyorwa zve \({20}\,{\hbox {min}}\) pa \({46}\,{^{\circ }\hbhokisi {C}}\), wodzokera kumashure Chirekodha chiri mu \({95}\,{^{\circ }\hbox {C}}\) ye \({1}\,{\hbox {min }}\).Paishandiswa 1% agarose gel, cDNA yakaratidza mabhandi matatu anoenderana nezvinotarisirwa zvitatu zveRNA zvidimbu (data isina kuratidzwa).Maprimers anotevera akashandiswa kukudza zvidimbu zviviri zveDNA zve117 uye 503 bp pakureba, uchishandisa cDNA setemplate yePCR mune miniPCR® mini8 thermal cycler:
Maprimers e \(117\,\hbox {bp}\) uye \(503\,\hbox {bp}\) anoenderana ne1476-1575 nucleotides yeM segment uye 458-943 nucleotides yechikamu cheL, zvakateerana acid. .All amplified PCR products were electrophoresed on 1% agarose gels, and amplified target DNA yakacheneswa pachishandiswa GeneJET Gel Extraction Kit (Thermo Fisher Scientific).
Dziva repaIIT Mumbai campus (Powai Lake, Powai, Mumbai) rakashandiswa kuwedzera phage particles.Mvura yemudhamu yakasefa nekamera kuti ibvise \({5}\,{\upmu \hbox {m}}\) zvidimbu zvakaturikwa, uyezve Phi6 phage yakawedzerwa. Wedzera \({1}\,{\hbox {ml}}\) ye\(10^{6}\,{\hbox {PFU}/\hbox {ml}}} \) ku \( {100}\ ,{\hbox {ml}}\) yakasefa mvura yemudhamu, mu \({4}\,{^{\ circle}\hbox {C}}\).Kariquot kadiki kaive Takaedza nzira mbiri dzakasiyana dzekutarisa spiked Phi6 virus particles: (1) iyo aluminium hydroxide adsorption-precipitation nzira, 19 iyo yakave yakasimbiswa kuunganidzwa kwe akati wandei akafukidzwa mavhairasi eRNA kubva kune zvakatipoteredza samples, uye. (2) ) Iyo polyethylene glycol (PEG)-yakavakirwa hutachiona hwehutachiona nzira yakagadziriswa kubva kuMafashamo et al.20 .Sezvo kushandiswa kwekugadzirisa kwePEG-based method yakawanikwa iri nani pane iyo yealuminium hydroxide nzira, nzira yePEG-based yakashandiswa kuisa pfungwa dzePhi6 particles kubva mumvura yegungwa.
Nzira yePEG yakashandiswa yakaita seinotevera: PEG 8000 uye \(\ hbox {NaCl}\) yakawedzerwa kuPhi6-spiked mvura yemudhamu kuti iwane 8 % PEG 8000 uye \(0.2\,\hbox {M} \) \( \ hbox {NaCl}\). Mienzaniso yakaiswa pane shaker\({4}\,{^{\circ }\hbox {C}}\)\({4}\,{\hbox {h}}\ ), yobva yaiswa centrifuged pa \(4700 \,\hbox {g}\) iri \({45}\,{\hbox {min}}\). Rasa supernatant womisazve pellet mukati \({1}\, {\ hbox {ml}}\) mune imwechete supernatant. Yese spiking uye virus concentration yekuedza yakaitwa in triplicate.After concentration, aliquot diki yakachengeterwa kuyerwa kwekupora kwehunyanzvi neplaque assay.RNA yaive yakaparadzaniswa sezvakatsanangurwa uye yakatsanangurwa kare. mu kit-supplied elution buffer\({40}\,{\upmu \hbox {l}}\).Sezvo kuwandisa kweRNA kuchisiyana kubva kusampuli kuenda kumuenzaniso mune zvakapetwa katatu, \({2}\,{\upmu \ hbox {l}}\) yeRNA inoshandiswa kune ese ari matatu zvisinei neconcentration yawo cDNA synthesis yemasamples.cDNA synthesis yakaitwa sezvakatsanangurwa kare.\({1}\,{\upmu \hbox {l}}\) cDNA yakashandiswa se template ye \({20}\,{\upmu \hbox {l}}\) PCR ye 35 cycles kukudza \ (117\,\hbox {bp}\) uye \(503\,\hbox { bp}\) zvimedu. Aya masampuli anomiririrwa se "1: 1", kureva pasina dilution.A no-template control (NTC) yakaiswa seyakaipa control, ukuwo cDNA yakaitwa ichishandisa RNA yakaparadzaniswa kubva pakacheneswa phage yakaiswa. sechiratidziro chekutonga kwakanaka (PC).Quantitative PCR (qPCR) yakaitwa muStratagene Mx3000P RT-PCR chiridzwa uchishandisa Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies) . zvinotsanangurwa.The cycle threshold (Ct) yakarekodhwa kune ese samples.Pamusoro pezvo, the diluted samples dzaive \({1}\,{\upmu \hbox {l}}\) vachishandisa cDNA yakadirwa 1:100 mumvura yakasefa yedhamu se. \({20}\,{\upmu \hbox {l}}\) PCR yezvikamu makumi matatu neshanu. Masampuli aya anomiririrwa se"1:100".
MaPCB electrode anogadzirwa achishandisa inotengeswa inodhura yakaderera Electroless Nickel Immersion Gold (ENIG) maitiro pasina kudiwa kwekuwedzera kwegoridhe. piranha solution kana sulfuric acid cyclic voltammetry haikurudzirwe sezvo inogona kukonzera kupeperukwa kweiyo goridhe dhata (ukobvu \(\ approx\) \(100\,\hbox {nm }\)) uye kufumura pasi pasi pemhangura zvidimbu zvinowanzoitika. kusvika kune corrosion 21, 22, 23, 24, 25. Nokudaro, chenesa ma electrode nemucheka usina lint-free wakanyoroveswa neIPA. Sample inofanira kuongororwa yakavharwa ne \({50}\,{\upmu \hbox {M}) }\) MB mu \({4}\,{^{\circ }\hbox {C}}\)\({ 1}\,{\hbox {h}}\) kuti inyore zviri nyore.Mubasa redu rekare , takaona kuti kunzwisiswa uye mutsara we sensor zvakagadziridzwa nekuwedzera MB concentration 11 .Kubva pane zvakagadziriswa zvakashumwa mubasa redu rekutanga, takashandisa \({50}\,{\upmu \hbox {M}}\) MB kuisa pfungwa pakuisa DNA muchidzidzo ichi.Kuonekwa kweElectrochemical yeDNA yakapetwa kaviri (ds-DNA) inogona kuwanikwa uchishandisa anionic kana cationic intercalators.Kunyange zvazvo anionic intercalators inoona DNA nekusarudza zviri nani, inoda kuiswa kweusiku humwe, zvichiita kuti nguva yakareba yekuona. nerumwe rutivi, cationic intercalators seMB inoda nguva diki dzekukuchidzira, dzinenge \({1}\,{\hbox {h}}\) yekuongorora kwe electrochemical ye ds-DNA6.Chiyero chega chega chinosanganisira kugovera sampuli kuti iongororwe pa electrode\({5}\,{{\upmu \hbox {l}}}\), wozochenesa ne IPA-yakanyoroveswa rag, usati waenderera neimwe sampuli.chiyero chimwe chete.Sample imwe neimwe yakaedzwa pama electrode mashanu akasiyana kunze kwekunge zvakataurwa.DPV uye CV zviyero zvakaitwa pachishandiswa PalmSens Sensit Smart potentiostat, uye PSTrace software yakashandiswa pakugadzirisa potentiostat uye kutora data, kusanganisira peak ikozvino macalculation.Izvi zvirongwa zvinotevera zvinoshandiswa. yeDPV uye CV kuyerwa:
DPV: Equilibrium Time = \(8\,\hbox {s}\), Voltage Step = \(3\,\hbox {mV}\), Pulse Voltage = \(25\,\hbox {mV}\) , pulse duration = \(50\,\hbox {ms}\), scan rate = \({20}\,\hbox {mV/s}\)
CV: Equilibrium Time = \(8\,\hbox {s}\), Voltage Danho = \(3\,\hbox {mV}\), Sweep Rate = \({300}\,\hbox {mV/ s }\)
Peak currents akawanikwa kubva kuvoltammograms eDNA complexed with \({50}\,{\upmu \hbox {M}}\) MB: (a) \(503\,\hbox {bp}\) DPV , (b) \ (503\,\hbox {bp}\) CV, (c) \(117\,\hbox {bp}\) DPV, (d) \(117\,\hbox {bp}\) CV.
DPV neCV voltammograms zvakawanikwa pa ENIG PCB electrodes \({50}\,{\upmu \hbox {M}}\) MB yakasanganiswa neDNA (pamwero we10–\({20}\,{\ hbox {ng) }/{\upmu \hbox {l}}}\) kureva 0.13–\({0.26}\,{\upmu \hbox {M}}\) ye\(117\,\hbox {bp}\ ) uye 0.03 –\({0.06}\,{\upmu \hbox {M}}\) ye\(503\,\hbox {bp}\)) yeDPV uye CV zviyero (peak current) uchishandisa gel-purified PCR products.Kuenzaniswa neCV zviyero, zviyero zveDPV zvinoratidza kunzwisisika kwepamusoro (ikozvino sekushanda kweDNA concentration) nokuti kumashure capacitive currents muCV zviyero zvinovanza Faradaic currents 26.The data pabhokisi rimwe nerimwe mubhokisi rine zviyero kubva kumagetsi 5. Zvose zviyero zvinoshandisa seti imwe chete ye electrode kudzivisa zvikanganiso zvekuyera nekuda kwekusiyana kwe electrode-to-electrode.Takaona kuwedzera kwemaitiro muDPV uye CV yakayera peak currents yezvikamu zvakaderera zveDNA. , kureba (\(503\,\hbox {bp}\)) \,\hbox {bp}\ zvichienzaniswa ne\(117) ) fragment.Izvi zvinopindirana nemaitiro anotarisirwa e electrode adsorption yakashumwa mubasa redu rekare.The adsorption yeMB-DNA complex inobatsira kutengesa kutengesa pa electrode, iyo inobatsira pakuwedzera kwepamusoro-soro.Zvimwe zvidzidzo zvakaratidza kushanda kweoligonucleotide size uye kutevedzana paMB-DNA intercalation27,28,29,30.Guanine -cytosine (GC) zviri mukati memaamplicon maviri (\(117\,\hbox {bp}\) uye \(503\,\hbox {bp}\)) angangoita 50%, zvichiratidza kuti kucherechedzwa. Kureba kweamplicon.Zvisinei, kune yakakwirira DNA concentrations (\(>{2}\,{\hbox {ng}/{\upmu \hbox {l}}}\), ye \(503\,\hbox {bp} \) uye \( >{10}\,{\hbox {ng}/{\upmu \hbox {l}}}\) ye \(117\,\hbox {bp}\)), tinoona maamplifications maviri. peak currents of the subs yakaderedzwa mune zvose DPV uye CV zviyero.Izvi imhaka yekuti MB inozadza uye inopindirana pakati pezviviri zveDNA, zvichiita kuti steric inhibition yebasa redox reboka reducible muMB31,32.
在存在 \(2\,\hbox {mM}\) \({\hbox {MgCl }_2}\): (a) \(503\,\hbox {bp}\) DPV, (b) \(503 \,\hbhokisi {bp}\) CV, (c) \(117\,\hbox {bp}\) DPV,(d) \(117\,\hbox {bp}\) CV.
Minyu iripo muPCR master misanganiswa inovhiringa kupindirana kwemagetsi pakati peMB neDNA, saka nekuwedzera \(2\,\hbox {mM}\) \(\hbox {MgCl }_2\) ne\({50} \,{\ upmu \hbox {M}}\) MB gel-purified product kuti tidzidze kushanda kwemunyu paMB-DNA kudyidzana.Sezvinoratidzwa mumufananidzo 3, takaona kuti kune yakakwira DNA concentration (\(>{2}\,{\ hbox {ng}/{\upmu \hbox {l}}}\) (503\,\hbox {bp}\) uye \(>{10}\,{\hbox {ng}/{\upmu \hbox { l}}}\) ye \(117\,\hbox {bp} \)), muDPV neCV Kuwedzerwa kwemunyu hakuna kukanganisa zvakanyanya kuyerwa (ona Mufananidzo S2 muSupplementary Information yevanomiririra voltammograms).Zvisinei, pa kuderera kweDNA, kuwedzera kwemunyu kunoderedza zvakanyanya kunzwisisika, zvichiita kuti pasave nekuchinja kwakakosha mune ikozvino neDNA concentration.Similar negative effects of salt on MB-DNA interaction and intercalation have been predified by other researchers33,34.\(\ hbox { Mg}^{2+}\) cations inosunga kune negative phosphate musana weDNA, nokudaro zvichitadzisa kupindirana kwe electrostatic pakati peMB neDNA.Pakukwirisa DNA, steric inhibition ye redox-active MBs inoguma yadzikira peak currents, saka electrostatic interactions. usanyanya kukanganisa mhinduro ye sensor.Chinokosha ndechekuti iyi biosensor inonyatsokodzera kuona yakakwirira DNA concentrations (kashoma \({\ hbox {ng}/{\ upmu \hbox {l}}}\) kana kupfuura), zvizere- Kugadzirisa otomatiki kwemasampuli emvura ezvakatipoteredza, uko kucheneswa kwegel kwePCR zvigadzirwa kungave kusingagoneke.
Nzvimbo iri pasi pekunyudza curve yewavelength renji 600–700 \(\ hbox {nm}\) yezvakasiyana siyana zveDNA zvakasanganiswa ne \({50}\,{\upmu \hbox {M}}\) MB: ( a ) \(503\,\hbox {bp}\) ine uye isina munyu (\(2\,\hbox {m}\) \(\hbox {MgCl}_2\)), (b) \( 117\, \hbhokisi {bp}\) ine uye isina munyu (\(2\,\hbox {mM}\) \(\hbox {MgCl}_2\)).\({0}\,{\hbox {pg}/) {\ upmu \hbox {l}}}\) DNA concentrations inoenderana \({50}\,{\upmu \hbox {M}}\) MB samples Hapana DNA.
Kuenderera mberi nekuona zvawanikwa pamusoro, takaita zviyero zveOptical tichishandisa UV/Vis spectrophotometer (Thermo Scientific Multiskan GO), masampuli \({50}\,{{\upmu \hbox {l}}}\) akashandiswa pane imwe neimwe. Measurement.Siginicha yekunyura inodzikira nekuwedzera DNA concentration, sezvingaonekwa kubva pamaitiro enzvimbo iri pasi peabsorption curve yewavelength range \(600\,\hbox {nm}\) kusvika \(700\,\hbox { nm}\) , sezvinoratidzwa muFig. 4 (absorption spectrum inoratidzwa muFig. S3 muSupplementary Information) .Kumasampuli ane DNA concentrations isingasviki \({1}\,{\hbox {ng}/{\upmu \hbox {l}}}\), pakanga pasina musiyano wakakura mukutora pakati peDNA-ine uye MB-chete samples (ye \(503\,\hbox {bp}\) uye \(117\,\hbox {bp}\) ) kureba zvimedu), zvichiratidza kusavapo kwe steric inhibition ye redox-active MB.Pamusoro weDNA concentrations, takaona kuderera zvishoma nezvishoma kwechiratidzo chekunwa uye takacherechedza kuderera kuduku kwekutora muhupo hwemunyu. kudyidzana uye steric inhibition with base stacking in DNA hybrids.Migumisiro yedu inoenderana nemishumo iri muzvinyorwa zve spectroscopic zvidzidzo zveMB-DNA intercalation inobatanidza hypochromaticity nekudzikiswa kwesimba mu \(\pi\)–\(\pi ^*\ ) shanduko yemagetsi nekuda kwekupindirana Matanho 36, 37, 38.
Agarose gel electrophoresis of phage Phi6: PCR zvigadzirwa zvehurefu \(117\,\hbox {bp}\) uye \(503\,\hbox {bp}\) kubva mumasampuli emvura yegungwa.M-DNA marker;NTC-no-template control, primers ine anowirirana amplicon;PC positive control;1, 2, 3-undiluted (1:1) spiked masamples emvura yemudziva mune triplicate hbox {bp}\) nzira.
Takaongorora kushandiswa kwesensor tichishandisa Powai Lake mvura samples spiked nePhi6 phage. The RNA concentrations yakaparadzaniswa kubva kune phage-spiked mvura samples ranged from 15.8–\({19.4}\,{\upmu \hbox {g}/\hbox { ml}}\), nepo avo vakaparadzaniswa nekumiswa kwephage kwakacheneswa Iyo RNA yaifungidzirwa kuva \({1945}\,{\upmu \hbox {g}/\hbox {ml}}\) nekugona kudzoreredza kungangoita 1. %.RNA was reverse transcribed into cDNA and used as template for PCR and qPCR.Ukuru hwechigadzirwa hwakasimbiswa neagarose gel electrophoresis (Mufananidzo 5) isati yaongororwa nesensor.Masampuli aya haana kucheneswa gel uye naizvozvo ane zvikamu zvose zvePCR se pamwe chete nemaamplicon ekufarira.Iyo Ct tsika dzakarekodhwa panguva yeqPCR (Table 1) dzakaratidzwa kuti dzinoenderana nekusangana kweRNA yakaparadzaniswa kubva kune inoenderana spiked masamples emvura.Kukosha kweCt kunoratidza huwandu hwema cycles anodiwa kuti chiratidzo chefluorescent kudarika chikumbaridzo kana chiratidzo chekumashure. Higher Ct values inoratidza yakaderera template concentrations and versa.The Ct values dzeNTC samples dzaive dzakakwirira sezvaitarisirwa.Musiyano mu \(\ angangoita 3\) Ct values pakati Kudzora kwakanaka uye muenzaniso webvunzo unoratidza zvakare kuti bvunzo imwe neimwe template ine ingangoita 1% template kana ichienzaniswa neiyo yakanaka control.Takambokurukura kuti maamplicon akareba anounza kunzwisiswa kuri nani. yehuwandu hwehutachiona hwehutachiona uye kuparara kweRNA.Zvisinei, nehutachiona hwedu hunowedzera uye PCR amplification protocol, takakwanisa kubudirira kubudirira \(503\,\hbox {bp}\) fragment ye electrochemical sensing.
Figure 6 inoratidza electrochemical sensor results ye \(503\,\hbox {bp}\) fragment amplicon, zvose zvichishandisa undiluted cDNA se template (1:1) uye 100-fold diluted cDNA se template (1:100 ) yakaitwa PCR. , zvichienzaniswa neNTC nePC (ona Mufananidzo S4 muSupplementary Information yevanomiririra voltammograms) .Bhokisi rimwe nerimwe riri mubhokisi rebhokisi riri Mufananidzo 6 rine zviyero kubva pamatatu matatu pamagetsi e 5. Ma electrode akafanana akashandiswa kuyera sampuli dzose kuti dzirege kukanganisa nekuda kwe electrode -ku-electrode variation.Kuenzaniswa neCV zviyero, zviyero zveDPV zvinoratidza kugadzirisa zviri nani kusiyanisa test uye PC samples kubva kuNTCs nokuti, sezvakambotaurwa, Faradaic currents yakavanzwa nekuda kwemashure capacitive currents mune yekupedzisira.Nekuda kweamplicon yakareba, takaona kuti iyo negative control (NTC) yakaguma nepamusoro CV uye DPV peak currents inoenderana neiyo yakanaka control, nepo yakanaka uye isina kunyorwa bvunzo samples yairatidza yakafanana peak urefu hweDPV peak currents. ) test sample uye PC inogona kugadziriswa zvakajeka kubva kune sensor yakabuda yeNTC sampuli, nepo sarudzo ye1: 100 diluted sampuli isinganyanyi kutaurwa.Kuti 100-fold dilution ye cDNA, hatina kucherechedza chero mabhandi panguva yegel electrophoresis. (mikoto isina kuratidzwa muFigure 5), uye inofambirana neDPV neCV peak currents dzakafanana nedzaitarisirwa kuNTC. Mibairo ye \(117\,\hbox {bp}\) fragment inoratidzwa muSupplementary Information.The negative. kudzora kwakakonzera mhinduro ye electrochemical kubva kuPCB sensor nekuda kwekushambadza kwemahara MB pane electrode uye kusangana kweMB neiyo single-stranded primer oligonucleotide.Naizvozvo, nguva imwe neimwe sampu inoedzwa, kutonga kwakashata kunofanirwa kumhanyisa uye iyo peak ikozvino yemuyedzo sampuli kana ichienzaniswa neyepamusoro ikozvino yakawanikwa neakaipa kutonga kuti iwane mutsauko (hama) kuyerwa39,40 kurongedza bvunzo sampu seyakanaka kana yakaipa.
(a) DPV, uye (b) CV peak current yekuonekwa neelectrochemical ye \(503\,\hbox {bp}\) zvimedu mumasampuli emvura yemudhamu. Masampuli ebvunzo akayerwa nekatatu uye akaenzaniswa neasina template controls (NTC) uye positive controls (PC).
Zvatinowana zvinoratidza nzira dzakasiyana-siyana dzinokanganisa kushanda kwemagetsi emagetsi emagetsi emaamplicon ehurefu hwakasiyana-siyana kune maDNA akasiyana-siyana, ane mazinga akasimbiswa nechiyero chemaziso tichishandisa UV / Vis spectrophotometer. \(500\,\hbox {bp}\) inogona kuonekwa nekunzwa kwepamusoro uye kuti kuvapo kwemunyu mumuenzaniso hakuiti Sensitivity DNA concentration inokanganisa kunzwisiswa kwepamusoro (kashoma \({\ hbox {ng}/{\upmu) \hbox {l}}}\) nepamusoro).Pamusoro pezvo, takaongorora maitiro emhando dzakasiyana dzemasamples, kusanganisira maamplicon akacheneswa gel ane uye asina kuwedzera munyu, uye kuwedzera masample emvura yemudhamu muDPV neCV kuyerwa.Isu takaona kuti DPV yakapa zvirinani kugadzirisa, sezvo iyo yekumashure capacitive ikozvino inokanganisawo kuyerwa kweCV, ichiita kuti isanzwe.
Kukwidziridzwa kwezvimedu zvenguva refu kunoenderana nekutendeseka kwehutachiona genomic RNA.Zvidzidzo zvakawanda zvakaratidza kuti kukwidziridzwa kwezvidimbu zvenguva refu hakusi kushanda nguva dzose nekuda kwekuora kweRNA munharaunda uye mukana wekuparadzanisa panguva yekuzviparadzanisa11,41,42,43,44 .Takaona kuti PEG-based virus concentration method yakange yakanyatsoshanda pakuisa phage Phi-6 spiked mumadziva emvura samples pane aluminium hydroxide-based virus concentration method.Kukwanisa kuona zvimedu zveDNA refu kwakaratidza kukunda zvinodiwa multiplex PCR. kukwidziridza akawanda mapfupi mapfupi matemplate uye kuderedza mukana wekuyambuka-chaiyo.
Biological samples shoma, saka panodiwa kugadzira biosensor inoda mashoma mashoma ekuongororwa.Maelectrodes eENIG PCB akashandiswa muchidzidzo chino anongoda chete \({5}\,{{\upmu \hbox {l}}}\ ) masampuli ekuedzwa kuvhara nzvimbo inoshanda ye electrode. Pamusoro pezvo, electrode imwe chete inogona kushandiswazve mushure mekuchenesa isati yapa sampu inotevera. Masampuli akasimudzirwa haadi kuwedzerwa chero makemikari kunze kwemethylene blue, iyo isingadhuri. uye kemikari inowanzoshandiswa.Sezvo electrode imwe neimwe inodhura madhora 0.55 (kana INR 40) kugadzira, iyi biosensor inogona kuva imwe nzira inodhura kune unyanzvi huripo hwekuona.Tafura 2 inoratidza kuenzanisa kwebasa iri nemamwe ma sensor akataurwa mumabhuku kwenguva refu. DNA zvimedu mune heterogeneous samples.
Tichifunga kuti MB-based electrochemical monitors protocol inotsamira pane chaiyo yePCR, muganhu mukuru weiyi nzira ndeyekugona kwekusanyanya kukwidziridzwa mumasampuli akasiyana-siyana akadai semvura yetsvina nemvura yemugungwa kana kushandisa yakaderera-kuchena primers.Kuvandudza kunzwisiswa kwe Electrochemical monitoring nzira dzeDNA kuona zvigadzirwa zvePCR zvisina kucheneswa uchishandisa unmodified ENIG PCB electrodes, zvinodikanwa kuti unzwisise zviri nani kukanganisa kwakaunzwa neasina kushandiswa dNTPs uye maprimers, uye kugadzirisa maitiro ekuita uye assay protocol.Yakawedzera physicochemical paramita senge pH, tembiricha, uye biological. kudiwa kweokisijeni (BOD) yemuyero wemvura ingangoda kuyerwa kuitira kuvandudza kurongeka kwechiyero.
Mukupedzisa, isu tinokurudzira inodhura-yakaderera electrochemical ENIG PCB sensor yekuonekwa kwehutachiona munzvimbo yezvakatipoteredza (mvura yegungwa) samples.Kusiyana neimmobilized oligonucleotide electrodes kana tsika substrates yeDNA sensing inoda cryogenic kuchengetedza kuchengetedza sensitivity,53,54 maitiro edu anoshandisa unmodified PCB. ma electrode ane hurefu hwemasherufu ehupenyu uye pasina chaiwo anodiwa ekuchengetedza uye saka akakodzera kukudziridzwa kwekuyera mhinduro ine otomatiki sampuli yekugadzirisa yakaiswa muLMICs.The biosensor inoshandisa isingadhure DNA-intercalating redox dhayi (MB) kuti ione nekukurumidza maamplicon.The nonspecific amplification. zvakajairika mumasampuli ezvakatipoteredza zvinoderedza kujeka kweiyi nzira yekunzwa nekuda kwekusungwa kusingatarisirwi kweMBs kune imwe- uye kaviri-stranded oligonucleotides.Naizvozvo, kujeka kwemuedzo uyu kunoenderana nekugadzirisa kwekutanga uye PCR maitiro ekuita.Pakuwedzera, iyo CV uye DPV peak currents inowanikwa kubva kune yakaedzwa samples inofanirwa kududzirwa maererano nemhinduro dzakawanikwa kubva kune iyo yakaipa control (NTC) kune yega yega bvunzo.Magadzirirwo emagetsi emagetsi emagetsi uye nzira dzakapihwa mubasa iri dzinogona kubatanidzwa neautosamplers kuti dzigadzire zvizere otomatiki uye zvakaderera. -cost solution iyo inogona kuunganidza nekuongorora samples uye kuendesa zviwanikwa zvisina waya kudzoka murabhoritari.
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Nguva yekutumira: May-27-2022