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Ukubaluleka kokubeka iliso kwiisampulu zokusingqongileyo kuye kwaxatyiswa kakhulu ukususela ekuqaleni kobhubhani we-COVID-19, kwaye ezinye iinzame zokubeka esweni ziyenziwa kusetyenziswa umgangatho wegolide, nangona ubuchule obusekwe kwi-qPCR buyaduru. Isisombululo sokuhlola iisampulu zamanzi okusingqongileyo kumazwe anengeniso ephantsi kunye naphakathi. Kulo msebenzi, sibonisa ukufunyanwa kwe-electrochemical yeamplicon efunyenwe kwi-Phi6 phage ekwanti kwiisampulu zamanzi echibi e-spiked (i-surrogate ethandwayo ye-SARS-CoV-2), isebenzisa i-ENIG ukugqiba iielectrode zePCB ngaphandle kokuguqulwa komphezulu.I-sex.Impendulo yesivamvo se-electrochemical ibonakaliswe ngokucokisekileyo kumaqhekeza amabini e-DNA ubude obahlukeneyo (\({117}\,\hbox {bp}\) kunye \({503}\,\hbox {bp}\)), kunye Umphumo weetyuwa kwi-PCR master mixes kwi-methylene blue (MB) -i-DNA interactions.Iziphumo zethu zibonisa ukuba ubude be-DNA fragments ngokuphawulekayo bumisela uvakalelo lwe-electrochemical kwaye lubonise kulo msebenzi ukuba ukukwazi ukubona i-amplicons ezinde ngaphandle kokuhlanjululwa kwe-gel yeemveliso ze-PCR. kubalulekile kwi-situ yokulinganisa iisampulu zamanzi.Isisombululo esizenzekelayo ngokupheleleyo somthamo wentsholongwane egazini bode kakuhle.
Usulelo lwentsholongwane emanzini luyaziwa njengengozi yempilo yoluntu ukususela ngo-1940s, kunye nobungqina bokuqala bokudluliselwa kwamanzi kwepoliyo kunye ne-hepatitis E1.I-World Health Organization (WHO) ihlele iintsholongwane ezininzi eziphuma emanzini eziphakathi ukuya kwi-high health significance2.Traditional virus iindlela zokubhaqa zixhomekeke kubuchule obusekwe kumgangatho wegolide we-qPCR, ezibuthathaka kakhulu kwaye zithe ngqo, kodwa zifuna abasebenzi abanezakhono ukuba bavavanye elabhoratri besebenzisa izixhobo ezibiza imali eninzi.Nangona kunjalo, kumazwe anengeniso ephantsi naphakathi (LMICs) anezibonelelo ezinyiniweyo, abantu Uvavanyo lwesampulu lunokuthi luthathe indawo ephambili ngaphezulu kovavanyo lwesampulu yamanzi yokusingqongileyo. Ke ngoko, ezinye iindlela ezinexabiso eliphantsi ziyafuneka ukuze kugcinwe, ukujonga ixesha lokwenyani kwamanzi kunye neesampulu zamanzi amdaka kumazwe anengeniso ephantsi kunye nephakathi njengezilumkiso zakwangoko zokuqhambuka kwezifo ezikhulayo, ngaloo ndlela ibakhusela kwiimpembelelo zentlalo noqoqosho ezimandundu zobhubhane wentsholongwane.Ixabiso eliphantsi lee-electrochemical biosensors ze-nucleic acids zinokubonelela ngesisombululo esithembisayo kule mfuno ingafezekiyo.Uninzi lwezi biosensors zeDNA zisebenza ngento yokuba imisonto ye-DNA ehambelanayo ayishukumi kwi-electrode. umphezulu kunye ne-hybridize xa ulandelelwano oluhambelanayo lukhona kwisampulu.Oku ke kunokuguqulwa kube ngumqondiso ngeendlela ezahlukeneyo ze-electrochemical usebenzisa abalamli be-redox njenge-potassium yentsimbi / i-ferrocyanide.I-Methylene blue (MB) yenye i-molecule esebenzayo ye-redox, eye kuye kwaxelwa ukuba idibanise kwi-DNA ephindwe kabini (dsDNA) ngaphezu kokubophelela kwayo okungaqhelekanga kwi-DNA ene-stranded enye5,6. Ubume be-intercalating ye-MBs ukwenza i-MB-DNA complexes ibenza ukuba babe lukhetho oludumileyo njengabalamli be-redox kwii-electrochemical DNA ezininzi. I-sensor configurations5,6,7,8,9.Nangona i-intercalation ye-MB kwi-DNA ingabonakaliyo, kwaye inkcazo yale sensor ye-electrochemical ixhomekeke kakhulu ekuhlambulukeni kwee-primers ezisetyenziselwa i-PCR okanye i-isothermal amplification, ifaneleka kakuhle ukuphumeza ngokwenene -ixesha le-electrochemical-based qPCR okanye i-fluorescence isothermal amplification njengenye indlela yokulinganisa i-DNA yoxinaniso 9 .Kolunye uphunyezo olunjalo, Won et al.Umphezulu we-electrode wegolide watshintshwa nge-6-mercapto-1-hexanol (MCH) ngexesha langempela. umlinganiselo we-PCR amplicon kunye ne-MB usebenzisa i-difaultial pulse voltammetry (DPV)9.Kwezinye iimeko, uRamirez et al.Ukufunyanwa kwe-SARS-CoV-2 kumanzi amdaka nge-RT-LAMP reaction usebenzisa i-MB ene-electrode eprintiweyo kwiscreen. isetyenziswe njenge-electrodes ye-situ kwi-PCR ye-microfluidic platform eyenzelwe ukukhangela i-electrochemically amplicon ngexesha lokuphendula 8.Zonke ezi zifundo zifuna ukuguqulwa komphezulu we-electrodes, oku kuthetha ukunyuka kwemveliso kunye neendleko zokusebenza ngenxa yeemfuno ezikhethekileyo zokugcina ukuzinza kwezi electrode ezisebenzayo.
Isicwangciso sokuhamba komsebenzi sokufunyanwa kwe-electrochemical ye-amplicon efunyenwe kumasuntswana entsholongwane exinzeleleyo kwiisampuli zamanzi echibi.
Kutshanje sibonise i-electrochemical sensing ye-SARS-CoV-2 amplicon ene-electrode yebhodi yesekethe eshicilelwe ngexabiso eliphantsi (PCB) esekwe kwi-DPV kunye ne-cyclic voltammetry (CV) ebangelwa kukufakwa kwe-MB-DNA complexes kumphezulu wee-electrode ezingalungiswanga ) utshintsho kwincopho ngoku11.Sinika ingxelo yokuba amaqhekeza amade e-DNA (N1-N2, \({943}\, \hbox) eyenziwe kusetyenziswa i-CDC-ecetyiswayo ye-N1 phambili kunye ne-N2 reverse primers xa kuthelekiswa namaqhekeza amafutshane {bp}\)) ibonise umgca ongcono kwimpendulo yenzwa ( N1, \(72\,\hbox {bp}\)) eyenziwe kusetyenziswa i-N1 phambili kunye ne-N1 reverse primer sets.Ezi zifundo zixelwe kusetyenziswa udilulo lwe-DNA olulungiselelwe kumanzi angena-nuclease.Iqonga likwasetyenziselwa ukubona i-SARS-CoV -2 iamplicon kwiisampulu zamanzi amdaka ezilingisiweyo (ezifunyenwe ngokutshiza iisampulu zizonke ze-RNA nge-SARS-CoV-2 RNA). Kuba i-RNA isesichengeni sokucheba ngexesha lokuhlukaniswa kunye nokusetyenzwa ezantsi komlambo, 12,13 kunzima ukukhulisa amaqhekeza amade ngale sampuli yahlukileyo. Ke ngoko, ukubonakaliswa kwe-electrochemical sensing ye-SARS-CoV-2 amplicon kumanzi amdaka inqunyelwe kwimfutshane \(72\,\hbox {bp}\) N1 fragment.
Kulo msebenzi, siye saphanda ukuba nokwenzeka kwe-ENIG PCB-based electrochemical sensing of phage Phi6 igxininiswe kwaye ihlukaniswe kwiisampuli zamanzi echibi (Fig. 1) . kwakhona abe lipid inwebu kunye spike protein.Ngenxa yezi zizathu, bacteriophage Phi6 i surrogate ethandwayo SARS-CoV-2 kunye nezinye enveloped pathogenic RNA viruses14,15.RNA bebodwa amasuntswana phage yasetyenziswa njenge template for cDNA synthesis ilandelwa I-PCR ukufumana amaqhekeza amabini e-DNA ye-117 kunye ne-503 i-base pairs ubude. Ukunika umngeni wokukhulisa \(943\,\hbox {bp}\) amaqhekeza e-N1-N2 kumsebenzi wethu wangaphambili, sijolise amaqhekeza obude obuphakathi (\(117) \,\hbox {bp}\) kunye \(503 \,\hbox {bp}\)), ngokusekelwe kwiiprimers ezikhoyo.Impendulo yesivamvo se-electrochemical yaphononongwa ngokucwangcisiweyo kuluhlu olubanzi loxinaniso (\({10}\,{ \hbox {pg}/{\upmu \hbox {l}}}\) ukuya \({20}\, {\hbox {ng}/{\upmu \hbox {l}}}\)) Kuwo omabini amaqhekeza kwi ubukho be-MB, umphumo wetyuwa kwi-sensor impendulo ibonakaliswe kwaye i-cross-valided by spectrophotometric measurements.Igalelo eliphambili lalo msebenzi lilandelayo:
Ubude beqhekeza le-DNA kunye nobukho betyuwa kwisampulu buchaphazela kakhulu ubuntununtunu.Iziphumo zethu zibonisa ukuba umsebenzi we-electrochemical uxhomekeke kwiindlela ezahlukeneyo zokusebenzisana kwe-MB, i-DNA, kunye nesivamvo kwimpendulo ye-voltammetric, ngokuxhomekeke kugxininiso lwe-DNA kunye nobude, kunye namaqhekeza amade abonisa ubuntununtunu obuphezulu, nangona ityuwa inefuthe elibi kwintsebenziswano ye-electrostatic phakathi. MB kunye neDNA.
Ugxininiso lwe-DNA lumisela indlela yokusebenzisana kwe-MB-DNA kwii-electrodes ezingaguqukiyo Sibonisa ukuba iindlela ezahlukeneyo zokusebenzisana kwe-MB-DNA zixhomekeke kwi-DNA yoxinaniso. {l}}}\), siye sabona ukuba impendulo yangoku ye-electrochemical yagqitywa kakhulu yi-adsorption ye-MB-DNA kwi-electrode, ngelixa i-concentrations ephantsi Kwiindawo eziphezulu ze-DNA, impendulo yangoku ye-electrochemical inqunywe yi-steric inhibition ye-redox. umsebenzi ngenxa yokufakwa kwe-MB phakathi kweeperi zesiseko se-DNA.
I-ENIG PCB-Isekelwe kwi-Electrochemical Sensing yeViral Nucleic Acids kwiiSampuli zaManzi eLake Ukuqwalaselwa kwaqinisekiswa ngokufunyanwa kwe-electrochemical ye-Phi6-eyongeziweyo \ (503\,\hbox {bp}\) amaqhekeza e-DNA afunyenwe kwiisampuli zamanzi kwi-Powai Lake, IIT Mumbai Campus. Phage isiphumo.
Iindleko eziphantsi zokuphunyezwa kunye namandla okudibanisa kwiinkqubo zokubeka iliso ezizenzekelayo ngokupheleleyo , i-oligonucleotides okanye i-aptamers kwii-electrodes ezinobomi obude beshelufu.
I-Phage Phi6 yintsholongwane egqunyiweyo ye-dsRNA yosapho lwe-Cytoviridae eyosulela i-Pseudomonas syringae. \,\hbox {Kb}\)) kunye L (\ (6.37\ ,\hbox{Kb}\))16,17. Ekubeni i-Phi6 phage isosulela uhlobo lwe-Pseudomonas olungeyo-pathogenic lwe-BSL-1, kukhuselekile ukusebenzisa kwaye ingakhuliswa lula elabhoratri.I-Phage Phi6 kunye ne-host Pseudomonas syringae yathengwa kwi-Felix d'Herelle Reference Centre ye-Bacterial Viruses, iYunivesithi yaseLaval, eKhanada (iinombolo zeziko lokukhangela ikhathalogu yi-HER-102 kunye ne-HER-1102, ngokulandelelanayo) .I-Phi6 phage kunye nomkhosi wayo wavuselelwa njengoko kuyalelwe liziko lokubhekisela.I-Phage Phi6 yahlanjululwa ngokucocwa kwepleyiti kunye ne-elution ukufumana iziqu zokugqibela nge \(\malunga ne-10^{12}\,{\hbox {PFU}/\hbox { ml}}\) (iiyunithi zokwenza i-plaque / iimililitha) .I-RNA yahlukaniswa kumasuntswana e-phage ahlanjululweyo kusetyenziswa i-GenElute™ Universal Total RNA Purification Kit (Sigma-Aldrich) ngokwemiyalelo yomenzi.Ngokufutshane, i-phage ecocekileyo ye-Phi6 yokunqunyanyiswa\({{ 100}\,{{\upmu \hbox {l}}}\) yafakwa i-lysed kwaye i-lysate yalayishwa kwikholamu ejikelezayo ukuvumela i-RNA ukuba ibophelele kwikholamu yeresin .I-RNA iye yacaciswa kwisisombululo se-lution \({{ 50}\,{{\upmu \hbox {l}}}\) enikezwe yikhithi. Qikelela ukuxinwa kwe-RNA ngokufunxa kwi \(260\,\hbox {nm}\).RNA yagcinwa kwi-aliquots kwi\ ({-80}\,{^{\circ }\hbox {C}}\) kude kusetyenziswe okunye.\({2}\,{\upmu \hbox {g}}\) I-iScript cDNA Synthesis Kit (Bio -Rad Laboratories) yayisetyenziswa njenge template ye cDNA synthesis ilandela imiyalelo yomenzi.Ngokufutshane, i cDNA synthesis reaction ina 3 amanyathelo: priming at \({25}\,{^{\circ}\hbox {C}}\ )\({5}\,{\hbox {min} }\) , uguqulelo loshicilelo lwe \({20}\,{\hbox {min}}\) e\({46}\,{^{\circ }\hbhokisi {C}}\), kwaye umva Umshicileli ungaphakathi \({95}\,{^{\circ }\hbox {C}}\) ye \({1}\,{\hbox {min }}\).Xa iqhutywe kwi-gel ye-agarose ye-1%, i-cDNA ibonise iibhendi ezintathu ezihambelana neziqwenga ezintathu ezilindelekileyo ze-RNA (idatha engaboniswa) . Ezi ziqalo zilandelayo zisetyenziselwa ukukhulisa iinqununu ezimbini ze-DNA ze-117 kunye ne-503 bp ubude, usebenzisa i-cDNA njengethempleyithi yePCR kwiminiPCR® mini8 thermal cycler:
Iiprimers ze \(117\,\hbox {bp}\) kunye \(503\,\hbox {bp}\) zihambelana ne-1476-1575 nucleotides yecandelo le-M kunye ne-458-943 nucleotides yecandelo le-L, ngokulandelelana kwe-asidi. .Zonke iimveliso ze-PCR ezandisiweyo zenziwe nge-electrophoresed kwi-1% yeegels ze-agarose, kwaye i-DNA ekujoliswe kuyo eyandisiweyo yahlanjululwa kusetyenziswa i-GeneJET Gel Extraction Kit (i-Thermo Fisher Scientific).
Ichibi kwikhampasi ye-IIT Mumbai (i-Powai Lake, i-Powai, i-Mumbai) yayisetyenziselwa ukongeza i-phage particles. amasuntswana axhonyiweyo, kwaye ke Phi6 phage yongezwa.Yongeza \({1}\,{\hbox {ml}}\) ye \(10^{6}\,{\hbox {PFU}/\hbox {ml}}} \) ukuya \( {100}\ ,{\ hbox {ml}}\) ahluziweyo amanzi echibini, kwi \({4}\,{^{\ isangqa}\hbox {C}}\).I-aliquot encinci yaba Sivavanye iindlela ezimbini ezahlukeneyo zokujonga amasuntswana entsholongwane e-Phi6: (1) indlela ye-aluminiyam hydroxide adsorption-precipitation, 19 eye yaqinisekiswa kuxinzelelo lweentsholongwane ezininzi ze-RNA ezivela kwiisampulu zokusingqongileyo, kunye (2) ) I-polyethylene glycol (PEG)-based based virus ye-concentration method yatshintshwa ukusuka kuMkhukula et al.20 .Njengoko ukubuyisela ukusebenza kwe-PEG-based method kwafunyaniswa ukuba ingcono kunendlela ye-aluminium hydroxide, indlela esekelwe kwi-PEG yayisetyenziselwa ukugxila kwiinqununu ze-Phi6 ezivela kwiisampuli zamanzi echibi.
Indlela ye-PEG esetyenzisiweyo yayi ngolu hlobo lulandelayo: I-PEG 8000 kunye \(\ hbox {NaCl}\) zongezwa kwiisampuli zamanzi echibi e-Phi6-spiked ukufumana i-8 % PEG 8000 kunye \(0.2\,\hbox {M} \) \( \ hbox {NaCl}\).Iisampulu zaqanjwa kwi-shaker\({4}\,{^{\circ }\hbox {C}}\)\({4}\,{\hbox {h}}\ ), emva koko i-centrifuged ku \(4700 \,\hbox {g}\) yi \({45}\,{\hbox {min}}\).Lahla i-supernatant kwaye uphinde umise i-pellet kwi \({1}\, {\ hbox {ml}}\) kwi-supernatant efanayo.Zonke iimvavanyo zoxinaniso ze-spiking kunye nentsholongwane zenziwa kwi-triplicate.Emva koxinaniso, i-aliquot encinci yayigcinelwe ukulinganisa ukuphumelela kokubuyisela nge-plaque assay.RNA yahlukaniswa njengoko ichazwe ngaphambili kwaye yahlanjululwa kwikhithi enikeziweyo elution buffer\({40}\,{\upmu \hbox {l}}\) .Njengoko uxinaniso lweRNA luya kwahluka kwisampulu ukuya kwisampulu kathathu, i \({2}\,{\upmu \\) I-hbox {l}}\) ye-RNA isetyenziswa kuzo zontathu kungakhathaliseki ukuba i-cDNA yoxinaniso ludityaniswe neesampuli. Udibaniso lwe-cDNA lwenziwa njengoko bekuchaziwe ngaphambili.\({1}\,{\upmu \hbox {l}}\) cDNA yasetyenziswa njenge template ye \({20}\,{\upmu \hbox {l}}\) PCR ye 35 imijikelo ukukhulisa \ (117\,\hbox {bp}\) kunye \(503\,\hbox { bp}\) amaqhekeza.Ezi sampuli zimelwe njenge "1: 1", okt ngaphandle kwe-dilution.Ulawulo lwe-no-template (NTC) lwamiselwa njengolawulo olubi, ngelixa i-cDNA eyenziwe kusetyenziswa i-RNA eyodwa kwi-phage ehlanjululweyo yamiselwa. njengethempleyithi yolawulo oluhle (PC) .I-PCR yobungakanani (qPCR) yenziwe kwisixhobo se-Stratagene Mx3000P RT-PCR kusetyenziswa i-Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies) .Iimpendulo zamiselwa ngokuphindwe kathathu njengoko ngaphambili Inkcazo.Umda womjikelo (Ct) warekhodwa kuzo zonke iisampuli. Ukongeza, iisampulu ezihlanjululweyo zaye \({1}\,{\upmu \hbox {l}}\) kusetyenziswa i-cDNA exutywe 1:100 kumanzi echibi ahluziweyo njengoko \({20}\,{\ upmu \hbox {l}}\) PCR ye 35 cycles. Ezi sampuli zimelwe njenge "1:100".
I-electrode ye-PCB yenziwe kusetyenziswa inkqubo ye-electroless Nickel Immersion Gold (ENIG) efumaneka ngokurhweba ngaphandle kwesidingo segolide eyongezelelweyo.Iinkcukacha ze-electrode ze-ENIG PCB zichazwe kumsebenzi wethu wangaphambili11.Kwi-electrodes ye-PCB ye-ENIG, iindlela zokucoca i-electrode eziqhelekileyo ezifana isisombululo sepiranha okanye i-sulfuric acid cyclic voltammetry ayikhuthazwa njengoko inokubangela ukuxobula umaleko obhityileyo wegolide (ubutyebe \(\ malunga\) \(100\,\hbox {nm}\)) kwaye iveze iileya zobhedu ezisezantsi eziqheleneyo. ukuya kwi-corrosion 21, 22, 23, 24, 25. Ngoko ke, coca i-electrodes ngelaphu eli-lint-free elixutywe ne-IPA.Isampuli eza kuvavanywa ifakwe kwi-({50}\,{\upmu \hbox {M}) }\) MB kwi \({4}\,{^{\circ }\hbox {C}}\)\({1}\,{\hbox {h}}\) ukuze uyifake lula. Kumsebenzi wethu wangaphambili , siqaphele ukuba uvakalelo kunye nomgca we-sensor yaphuculwa ngokunyusa i-MB yoxinaniso 11 .Ngokusekelwe kwi-optimizations echazwe kumsebenzi wethu wangaphambili, sisebenzise \({50}\,{\upmu \hbox {M}}\) MB ukugxininiswa ukubethelela i-DNA kule sifundo.Ukufunyanwa kwe-Electrochemical ye-DNA ephindwe kabini (ds-DNA) inokufezekiswa ngokusebenzisa i-anionic okanye i-cationic intercalators.Nangona i-anionic intercalators ibona i-DNA ngokukhetha okungcono, ifuna ukuxutywa ebusuku, okubangela ukuba kubonwe ixesha elide. kwelinye icala, i-cationic intercalators njenge-MB ifuna amaxesha amafutshane okufukamela, malunga \({1}\,{\hbox {h}}\) ubhaqo lwe-electrochemical ye-ds-DNA6.Umlinganiselo ngamnye ubandakanya ukukhupha isampuli ukuba ivavanywe i-electrode \ ( {5} \ , {{\ upmu \ hbox {l}}} \), emva koko ucoca nge-IPA-moisted rag, ngaphambi kokuba uqhubeke nenye isampuli.Umlinganiselo omnye.Isampuli nganye yavavanywa kwii-electrode ezi-5 ezahlukeneyo ngaphandle kokuba kuchazwe ngenye indlela.Imilinganiselo ye-DPV kunye ne-CV yenziwa kusetyenziswa i-PalmSens Sensit Smart potentiostat, kunye ne-software ye-PSTrace isetyenziselwa ukucwangciswa kwe-potentiostat kunye nokufumana idatha, kubandakanywa nezibalo eziphezulu zangoku.Izicwangciso ezilandelayo zisetyenziswa. ye-DPV kunye ne-CV imilinganiselo:
DPV: Equilibrium Time = \(8\,\hbox {s}\), Voltage Step = \(3\,\hbox {mV}\), Pulse Voltage = \(25\,\hbox {mV}\) , Ubude bokubetha kwentliziyo = \(50\,\hbox {ms}\), izinga lokuskena = \({20}\,\hbox {mV/s}\)
I-CV: Ixesha loLingano = \(8\,\hbox {s}\), Inyathelo leVoltage = \(3\,\hbox {mV}\), iRate yokuTshayela = \({300}\,\hbox {mV/ s }\)
Incopho yemisinga efunyenwe kwiivoltammograms zeDNA edityaniswe nge \({50}\,{\upmu \hbox {M}}\) MB: (a) \(503\,\hbox {bp}\) DPV , (b) \ (503\,\hbox {bp}\) CV, (c) \(117\,\hbox {bp}\) DPV, (d) \(117\,\hbox {bp}\) CV.
I-DPV kunye ne-CV voltammograms zafunyanwa kwi-ENIG PCB electrode \({50}\,{\upmu \hbox {M}}\) MB ehlanganiswe ne-DNA (kugxininiso lwe-10–\({20}\,{\ hbox {ng) }/{\ upmu \hbox {l}}}\) okt 0.13–\({0.26}\,{\upmu \hbox {M}}\) ye \(117\,\hbox {bp}\ ) kunye ne-0.03 -\({0.06}\,{\upmu \hbox {M}}\) ye\(503\,\hbox {bp}\)).Iivoltammograms ezimele ziboniswa kuMfanekiso S1 kuLwazi oloNgezelelweyo.Umfanekiso 2 ubonisa iziphumo ye-DPV kunye ne-CV yokulinganisa (i-peak current) usebenzisa iimveliso ze-PCR ezihlanjululweyo ze-gel.Xa kuthelekiswa nemilinganiselo ye-CV, imilinganiselo ye-DPV ibonisa uvakalelo oluphezulu (olukhoyo njengomsebenzi we-DNA concentration) kuba i-background capacitive currents kwi-CV measurements ifihla i-Faradaic currents 26 .Idatha kwibhokisi nganye kwibhokisi yebhokisi iqulethe imilinganiselo esuka kwi-electrode ye-5. Zonke imilinganiselo zisebenzisa isethi efanayo yee-electrode ukuphepha iimpazamo zokulinganisa ngenxa ye-electrode-to-electrode variation.Sibone ukunyuka kwe-DPV kunye ne-CV elinganisa imisinga ye-peak currents kwi-concentrations ephantsi ye-DNA. , ixesha elide (\(503\,\hbox {bp}\)) \,\hbox {bp}\ xa kuthelekiswa ne \(117) ) iqhekeza.Oku kuhambelana nentsingiselo elindelekileyo ye-electrode adsorption echazwe kumsebenzi wethu wangaphambili. I-adsorption ye-MB-DNA complex iququzelela ukuhanjiswa kwentlawulo kwi-electrode, enegalelo ekunyuseni kwe-peak current.Olunye uphando lubonise umphumo we-oligonucleotide ubukhulu kunye nokulandelelana kwi-MB-DNA intercalation27,28,29,30.I-guanine Umxholo we-cytosine (GC) we-amplicon ezimbini (\(117\,\hbox {bp}\) kunye \(503\,\hbox {bp}\)) yayimalunga ne-50%, ebonisa ukuba ukuqwalaselwa. kubude beamplicon.Nangona kunjalo, kugxininiso lweDNA oluphezulu (\(>{2}\,{\hbox {ng}/{\upmu \hbox {l}}}\), ye \(503\,\hbox {bp} \) kunye \( > {10}\,{\ hbox {ng}/{\ upmu \hbox {l}}}\) ye \(117\,\hbox {bp}\)), siqwalasela i-amplifications ezimbini I-peak currents ye-subs iyancitshiswa kwi-DPV kunye ne-CV yokulinganisa imilinganiselo. Oku kungenxa yokuba i-MB igcwele kwaye idibanisa phakathi kweebini zesiseko se-DNA, okubangelwa ukuvinjelwa kwe-steric yomsebenzi we-redox weqela elincitshisiweyo kwi-MB31,32.
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.
Iityuwa ezikhoyo kwi-PCR master mixes ziphazamisa i-electrostatic interactions phakathi kwe-MB kunye ne-DNA, ngoko ngokongeza \(2\,\hbox {mM}\) \(\hbox {MgCl }_2\) nge \({50} \,{\ upmu \hbox {M}}\) MB imveliso ye-gel-ehlanjululwe ukufundisisa umphumo wetyuwa kwintsebenziswano ye-MB-DNA. Njengoko kubonisiwe kuMfanekiso 3, siqaphele ukuba ugxininiso lwe-DNA ephezulu (\(>{2}\,{\ hbox {ng}/{\ upmu \hbox {l}}}\) (503\,\hbox {bp}\) kunye \(>{10}\,{\hbox {ng}/{\upmu \hbox { l}}}\) ye-\(117\,\hbox {bp} \)), kwi-DPV kunye ne-CV Ukongezwa kwetyuwa akuzange kuchaphazele kakhulu imilinganiselo (jonga uMzobo S2 kwiiNkcukacha ezoNgezelelweyo zevoltammograms ezimele). ukugxininiswa kwe-DNA ephantsi, ukongezwa kwetyuwa kunciphisa kakhulu uvakalelo, okubangela ukuba akukho tshintsho oluphawulekayo lwangoku kunye noxinaniso lwe-DNA.Imiphumo emibi efanayo yetyuwa kwi-MB-DNA ukusebenzisana kunye nokudibanisa kuye kwabikwa ngaphambili ngabanye abaphandi33,34.\(\ hbox { Mg} ^ {2+}\) i-cations ibophelela kumqolo we-phosphate ongalunganga we-DNA, ngaloo ndlela ithintela ukusebenzisana kwe-electrostatic phakathi kwe-MB kunye ne-DNA. ayichaphazeli kakhulu impendulo yenzwa.Inqaku eliphambili kukuba le biosensor ifaneleka ngakumbi ukufumanisa i-DNA concentrations ephezulu (inqabile \({\ hbox {ng}/{\ upmu \hbox {l}}}\) okanye ngaphezulu), ngokupheleleyo-Ukwenziwa kokuzenzekelayo kweesampulu zamanzi okusingqongileyo, apho ukucocwa kwejeli kweemveliso zePCR kusenokungenzeki.
Ummandla ophantsi kwegophe lokufunxa kuluhlu lwamaza obude 600–700 \(\ hbox {nm}\) yoxinaniso olwahlukeneyo lweDNA oludityaniswe nge \({50}\,{\upmu \hbox {M}}\) MB: ( a ) \(503\,\hbox {bp}\) kunye nangaphandle kwetyuwa (\(2\,\hbox {m}\) \(\hbox {MgCl}_2\)), (b) \( 117\, \hbhokisi {bp}\) kunye nangaphandle kwetyuwa (\(2\,\hbox {mM}\) \(\hbox {MgCl}_2\)).\({0}\,{\hbox {pg}/) {\ upmu \hbox {l}}}\) Ugxininiso lweDNA oluhambelana ne \({50}\,{\ upmu \hbox {M}}\) MB iisampulu Akukho DNA.
Ukuqinisekisa ngakumbi ezi ziphumo zingasentla, senze imilinganiselo yamehlo sisebenzisa iUV/Vis spectrophotometer (Thermo Scientific Multiskan GO), iisampuli \({50}\,{{\upmu \hbox {l}}}\) zisetyenziselwe nganye Umlinganiselo.Isiginitsha yokufunxa iyancipha ngokunyuka koxinzelelo lwe-DNA, njengoko kunokubonwa kwintsingiselo yendawo phantsi kwegophe lokufunxa kuluhlu lwamaza obude \(600\,\hbox {nm}\) ukuya \(700\,\hbox { nm}\) , njengoko kubonisiwe kumfanekiso we-4 (i-spectrum yokufunxa iboniswe kwi-Fig. S3 kwiNkcukacha eyoNgezelelweyo) .Kwiisampulu ezinoxinzelelo lwe-DNA ngaphantsi kwe \({1}\,{\hbox {ng}/{\upmu \hbox {l}}}\), bekungekho mahluko ubalulekileyo ekuthathweni phakathi kwe-DNA-equlethe kunye neesampuli ze-MB kuphela (ye \(503\,\hbox {bp}\) kunye \(117\,\hbox {bp}\) ) amaqhekeza ubude), ebonisa ukungabikho kwe-steric inhibition ye-MB. ukusebenzisana kunye ne-steric inhibition kunye ne-base stacking kwi-DNA hybrids.Iziphumo zethu zihambelana neengxelo kuncwadi kwizifundo ze-spectroscopic ze-MB-DNA intercalation ezidibanisa i-hypochromaticity kunye nokunciphisa amanqanaba amandla kwi \(\pi\)–\(\pi ^*\ ) iinguqu ze-elektroniki ngenxa yokudityaniswa kweeLayers 36, 37, 38.
I-Agarose gel electrophoresis ye-phage Phi6: iimveliso ze-PCR zobude \(117\,\hbox {bp}\) kunye \(503\,\hbox {bp}\) ukusuka kwiisampuli zamanzi echibi.M-DNA marker;I-NTC-no-template control, iiprimers eziqulethe i-amplicon ehambelanayo;Ulawulo olulungileyo lwePC;1, 2, 3-undiluted (1:1) spiked iisampulu zamanzi echibini in triplicate.A band ibonakala kwi \(\malunga 50\,\hbox {bp}\) ngenxa oligonucleotides engasetyenziswanga kwi \(503\,\ hbox {bp}\) indlela.
Sivavanye ukusetyenziswa kwenzwa kusetyenziswa iisampulu zamanzi ze-Powai Lake ezifakwe kwi-Phi6 phage. Ugxininiso lwe-RNA oluhlukanisiweyo kwiisampuli zamanzi ze-phage-spiked ukusuka kwi-15.8-\({19.4}\,{\upmu \hbox {g}/\hbox { ml}}\), ngelixa abo bahlukanisiweyo behlanjululwe ukunqunyanyiswa kwe-phage I-RNA yaqikelelwa ukuba \({1945}\,{\upmu \hbox {g}/\hbox {ml}}\) kunye nokusebenza kakuhle kokubuyisela malunga ne-1 I-%.RNA iguqulelwe iguqulelwe kwi-cDNA kwaye isetyenziswe njenge template ye-PCR kunye ne-qPCR.Ubungakanani bemveliso buqinisekiswe yi-agarose gel electrophoresis (Umfanekiso 5) ngaphambi kokuvavanya nge-sensor.Ezi sampuli azihlanjululwanga ijeli kwaye ke ziqulethe zonke iinxalenye ze-PCR njenge kunye neeamplicon zomdla.Ixabiso le-Ct elirekhodiweyo ngexesha le-qPCR (Itheyibhile 1) iboniswe ukunxulumana noxinaniso lwe-RNA ekwanti kwiisampulu zamanzi ane-spiked ezihambelanayo.Ixabiso le-Ct libonisa inani lemijikelo efunekayo kumqondiso we-fluorescent igqithise umda okanye isignali yangasemva. Amaxabiso aphezulu e-Ct abonisa ukugxininiswa kwetemplate esezantsi kwaye ngokuphambeneyo. Amaxabiso e-Ct eesampuli ze-NTC ayephezulu njengoko bekulindelekile. Umahluko kwi- \(\ malunga ne-3\) amaxabiso e-Ct phakathi ulawulo olulungileyo kunye nesampulu yovavanyo lubonisa ngakumbi ukuba isampuli nganye yovavanyo ine-template emalunga ne-1% xa kuthelekiswa nolawulo olulungileyo.Besikhe saxoxa ngaphambili ukuba iiamplicon ezinde zikhokelela kubuntununtunu obungcono. ye-viral concentration ephantsi kunye ne-RNA degradation.Nangona kunjalo, kunye ne-virus yethu yokutyebisa kunye neprotocol yokukhulisa i-PCR, sakwazi ukukhulisa ngempumelelo \ (503 \, \ hbox {bp}\) iqhekeza le-electrochemical sensing.
Umzobo 6 ubonisa iziphumo zoluvo lwe-electrochemical sensor ye- \(503\,\hbox {bp}\) iqhekeza le-amplicon, zombini zisebenzisa i-cDNA engaxutywanga njenge template (1:1) kunye ne-100-fold diluted cDNA njenge template (1:100) eyenziwa yiPCR. , xa kuthelekiswa ne-NTC kunye ne-PC (jonga i-Figure S4 kwiiNkcukacha ezongezelelweyo kwi-voltammograms emele) .Ibhokisi nganye kwibhokisi yebhokisi kuMzobo 6 iqulethe imilinganiselo evela kwiisampuli ezintathu kwi-electrode ye-5. I-electrode efanayo yayisetyenziselwa ukulinganisa zonke iisampuli ukuphepha iimpazamo ngenxa ye-electrode -ukuya kwi-electrode variation.Xa kuthelekiswa nemilinganiselo ye-CV, imilinganiselo ye-DPV ibonisa isisombululo esingcono sokuhlukanisa iisampuli zovavanyo kunye ne-PC ezivela kwi-NTCs kuba, njengoko kukhankanyiwe ngaphambili, i-Faradaic currents ifihliwe ngenxa yemvelaphi ye-capacitive currents ekugqibeleni. ulawulo olubi (NTC) lubangele i-CV ephezulu kunye ne-DPV incopho yemisinga ngokunxulumene nolawulo olulungileyo, kanti iisampulu zovavanyo ezilungileyo nezingadityaniswanga zibonise ukuphakama okufanayo kweencopho zemisinga ye-DPV. ) isampuli yovavanyo kunye nePC inokusombululwa ngokucacileyo kwi-sensor output ye-NTC isampuli, ngelixa isisombululo se-1: i-100 isampuli ehlanjululweyo ayibonakali kakhulu. Kwi-dilution ye-100 ye-cDNA, asizange sigcine nayiphi na ibhendi ngexesha le-gel electrophoresis. (iindlela ezingaboniswanga kuMfanekiso 5), kunye ne-DPV ehambelanayo kunye ne-CV peak currents zifana nezo zilindelekileyo kwi-NTC.Iziphumo ze-(117\,\hbox {bp}\) zeqhekeza ziboniswe kuLwazi oloNgezelelweyo. Ukulawula kubangele impendulo ye-electrochemical evela kwi-PCB sensor ngenxa ye-adsorption ye-MB yamahhala kwi-electrode kunye nokusebenzisana kwe-MB kunye ne-oligonucleotide ye-primer ene-single-stranded. Incopho yangoku yesampulu yovavanyo xa kuthelekiswa nencopho yangoku efunyenwe ngolawulo olungalunganga ukufezekisa umlinganiso (isihlobo) somlinganiso39,40 ukuhlula isampula yovavanyo njengento entle okanye embi.
(a) I-DPV, kunye (b) nencopho ye-CV yangoku yokukhangela i-electrochemical fragments \(503\,\hbox {bp}\) kwisampulu zamanzi echibi. ulawulo olulungileyo (PC).
Iziphumo zethu zibonisa iindlela ezahlukeneyo ezichaphazela ukusebenza kwee-electrochemical sensors ze-amplicon zobude obahlukeneyo kwii-DNAs ezahlukeneyo, kunye nogxininiso oluqinisekiswa ngemilinganiselo ye-optical usebenzisa i-UV / Vis spectrophotometer. \(500\,\hbox {bp}\) inokufunyanwa ngovakalelo oluphezulu kunye nokuba ubukho betyuwa kwisampulu ayenzi uSensitivity DNA yoxinaniso echaphazela ubuntununtunu obuphezulu (kunqabile \({\ hbox {ng}/{\ upmu \ hbhokisi {l}}} \) nangaphezulu). Ukongeza, siye saphanda umphumo weentlobo ezahlukeneyo zeesampulu, kubandakanywa i-amplicon ecocekileyo ye-gel kunye nangaphandle kwetyuwa eyongeziweyo, kunye nokongezwa kweesampuli zamanzi echibi kwi-DPV kunye nemilinganiselo ye-CV.Siye saqaphela ukuba i-DPV ibonelele ngesisombululo esingcono, kuba i-background capacitive current ikwachaphazela umlinganiselo weCV, iyenza ingabinovakalelo.
Ukwandiswa kwamaqhekeza amade kuxhomekeke kwingqibelelo ye-viral genomic RNA.Izifundo ezininzi zibonise ukuba ukunyuswa kwamaqhekeza amade akusoloko kusebenza kakuhle ngenxa yokuthotywa kwe-RNA kwimo engqongileyo kunye nokukwazi ukudibanisa ngexesha lokuzihlukanisa11,41,42,43,44 .Siye saqaphela ukuba i-PEG-based based virus ye-concentration method yayisebenza ngakumbi ekugxininiseni i-phage Phi-6 spiked kwiisampuli zamanzi echibi kune-aluminium hydroxide-based based based virus concentration. ukukhulisa iitemplates ezinobude obufutshane kunye nokunciphisa ukuba nokwenzeka kokucaciswa okunqamlezayo.
Iisampulu zebhayoloji zinqongophele, ngoko kukho imfuneko yokuyila i-biosensor efuna iisampuli ezincinci zovavanyo.I-electrodes ye-ENIG PCB esetyenziswe kolu phononongo kufuneka kuphela \({5}\,{{\upmu \hbox {l}}}\ Iisampulu zokuvavanya ukugubungela indawo esebenzayo yee-electrodes.Ukwengeza, i-electrode efanayo ingaphinda isetyenziswe emva kokucoca ngaphambi kokuhambisa isampuli elandelayo.Iisampulu ezinyusiweyo azifuni ukongezwa kwanoma yiyiphi imichiza ngaphandle kwe-methylene blue, engabizi kakhulu. kunye neekhemikhali ezisetyenziswa ngokuqhelekileyo.Njengoko i-electrode nganye ixabisa i-$ 0.55 (okanye i-INR 40) ukuvelisa, le biosensor ingaba yindleko esebenzayo kwiiteknoloji zokubona ezikhoyo.Itheyibhile 2 ibonisa ukuthelekiswa kwalo msebenzi kunye nezinye iinzwa ezichazwe kwiincwadi ixesha elide. Amaqhekeza e-DNA kwiisampulu ezingafaniyo.
Ngenxa yokuba i-MB-based electrochemical ukubona i-electrochemical protocols ixhomekeke kwi-PCR ethile, umda omkhulu wale ndlela yinto enokubakho yokwandisa okungangqalanga kwiisampulu ezingafaniyo ezifana namanzi amdaka kunye namanzi echibi okanye ukusebenzisa iiprimers ezisezantsi. Iindlela zokubona i-electrochemical yokubona i-DNA yeemveliso ze-PCR ezingahlambulukanga zisebenzisa i-electrodes ye-ENIG PCB engaguqukiyo, kuyimfuneko ukuqonda ngcono iimpazamo eziveliswe yi-dNTP engasetyenziswanga kunye ne-primers, kunye nokuphucula iimeko zokusabela kunye neeprothokholi zokuvavanya.Iiparamitha ezongezelelweyo ze-physicochemical ezifana ne-pH, ubushushu kunye ne-biological imfuno ye-oksijini (BOD) yesampuli yamanzi nayo ingadinga ukulinganiswa ukuze kuphuculwe ukuchaneka komlinganiselo.
Ukuqukumbela, siphakamisa i-electrochemical sensor ye-ENIG PCB enexabiso eliphantsi lokubona intsholongwane kwindawo (amanzi echibi) iisampuli.Ngokungafaniyo ne-electrode ye-oligonucleotide engenakunyakaziswa okanye i-substrates yesiko lokuva i-DNA efuna ukugcinwa kwe-cryogenic ukugcina ubuntununtunu,53,54 ubuchule bethu busebenzisa i-PCB engaguqukiyo. i-electrodes enobomi obude beshelufu kwaye akukho mfuno zikhethekileyo zokugcina kwaye ngoko ke ifanelekile kuphuhliso lwezisombululo zokulinganisa kunye ne-automated sample processing efakwe kwi-LMICs.I-biosensor isebenzisa i-DNA-intercalating redox dyes (MB) engabizi kakhulu ukubhaqwa ngokukhawuleza kwe-amplicon ekujoliswe kuyo.I-amplification engabonakaliyo eziqhelekileyo kwiisampulu zokusingqongileyo kunciphisa ukuchaneka kwale ndlela yokuva ngenxa yokunganyaniseki kwe-MBs kwi-oligonucleotides enye kunye ne-double-stranded oligonucleotides.Ngoko ke, ukucaciswa kolu vavanyo kuxhomekeke ekuphuculweni kwe-primers kunye neemeko zokusabela kwe-PCR.Ukongezelela, i-CV kunye ne-DPV peak currents ezifunyenwe kwiisampuli ezivavanyiweyo kufuneka zitolikwe ngokumalunga neempendulo ezifunyenwe kulawulo olubi (NTC) kuvavanyo ngalunye.I-electrochemical sensor designs kunye neendlela ezichazwe kulo msebenzi zinokudibaniswa kunye ne-autosamplers ukuphuhlisa i-automated ngokupheleleyo kwaye iphantsi. -isisombululo seendleko esinokuqokelela kwaye sihlalutye iisampuli kunye nokuhambisa ngaphandle kwamacingo iziphumo emva kwebhubhoratri.
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Ixesha lokuposa: May-27-2022