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Kufunika kowunika zitsanzo zachilengedwe kwayamikiridwa kwambiri kuyambira chiyambi cha mliri wa COVID-19, ndipo zoyeserera zina zikuchitidwa pogwiritsa ntchito muyezo wa golide, ngakhale njira zozikidwa pa qPCR ndizokwera mtengo. Njira yothetsera kuwunika zitsanzo zamadzi achilengedwe m'maiko opeza ndalama zotsika komanso zapakati. Mu ntchitoyi, tikuwonetsa kuzindikirika kwa ma amplicons a electrochemical ku Phi6 phage otalikirana ndi zitsanzo zamadzi am'madzi a spiked (wodziwika bwino wa SARS-CoV-2), pogwiritsa ntchito ENIG. kumaliza maelekitirodi a PCB popanda kusinthidwa pamwamba.sex.Mayankhidwe a sensor ya electrochemical anali odziwika bwino pazidutswa ziwiri za DNA za utali wosiyana (\({117}\,\hbox {bp}\) ndi \({503}\,\hbox {bp}\)), ndi zotsatira za mchere mu PCR master mixes pa methylene blue (MB) -DNA interactions.Zotsatira zathu zimasonyeza kuti kutalika kwa zidutswa za DNA kumatsimikizira kwambiri mphamvu ya electrochemical ndikuwonetsa mu ntchitoyi kuti kutha kuzindikira amplicons aatali popanda kuyeretsedwa kwa gel kwa zinthu za PCR. zofunika pakuyezera mu situ zitsanzo za madzi.Njira yodzichitira yokha ya kuchuluka kwa ma virus imakhala bwino.
Kufala kwa kachirombo ka m'madzi kwadziwika ngati koopsa kwa thanzi la anthu kuyambira zaka za m'ma 1940, ndi umboni woyamba wa kufala kwa poliyo ndi matenda a chiwindi E1. Bungwe la World Health Organization (WHO) lasankha tizilombo toyambitsa matenda tomwe timachokera m'madzi timene timayambira pa thanzi labwino2.Traditional virus njira zodziwira zimadalira njira za golide zozikidwa pa qPCR, zomwe zimakhala zovuta kwambiri komanso zenizeni, koma zimafuna anthu aluso kuti ayese mu labotale pogwiritsa ntchito zida zodula. Kuyesa kwachitsanzo kuyenera kukhala patsogolo kuposa kuyang'anira zitsanzo za madzi zachilengedwe.Choncho, njira zina zotsika mtengo ndizofunikira kuti zitsimikizidwe zokhazikika, zenizeni zenizeni za madzi ndi madzi onyansa m'mayiko otsika ndi apakati monga machenjezo oyambirira a kubuka kwa matenda, potero kuwateteza ku mavuto aakulu azachuma a mliri wa kachilombo ka HIV.Maselo otsika mtengo a electrochemical biosensors a nucleic acids angapereke njira yodalirika yothetsera vuto lomwe silinakwaniritsidwe.Ambiri mwa ma biosensors a DNAwa amagwira ntchito chifukwa chakuti zingwe zowonjezera za DNA sizikuyenda bwino pa electrode. pamwamba ndi hybridize pamene ndondomeko yofananira ilipo mu sampuli.Izi zikhoza kusinthidwa kukhala chizindikiro ndi njira zosiyanasiyana za electrochemical pogwiritsa ntchito oyimira redox monga potaziyamu iron/ferrocyanide.Methylene blue (MB) ndi molekyulu imodzi yotereyi, yomwe ili adanenedwa kuti amalowa mu DNA yamitundu iwiri (dsDNA) kuwonjezera pa kumangiriza kwake mosadziwika bwino kwa DNA ya chingwe chimodzi5,6. Kuphatikizika kwa MBs kupanga ma MB-DNA complexes kumawapangitsa kukhala chisankho chodziwika ngati oyimira redox mu ma electrochemical DNA angapo. Sensor configurations5,6,7,8,9.Ngakhale kusakanikirana kwa MB ku DNA sikudziwika, ndipo kutsimikizika kwa sensor ya electrochemical iyi kumadalira kwambiri chiyero cha zoyambira zomwe zimagwiritsidwa ntchito pa PCR kapena isothermal amplification, ndizoyenera kugwiritsa ntchito zenizeni. -nthawi ya electrochemical-based qPCR kapena fluorescence isothermal amplification ngati njira ina ya DNA concentration 9 .Mukukhazikitsa kumodzi kotereku, Won et al.Pamwamba pa ma electrode a golide adasinthidwa ndi 6-mercapto-1-hexanol (MCH) nthawi yeniyeni. kuyeza kwa PCR amplicons ndi MB pogwiritsa ntchito differential pulse voltammetry (DPV) 9. Nthawi zina, Ramirez et al. Kuzindikira SARS-CoV-2 m'madzi onyansa ndi RT-LAMP reaction pogwiritsa ntchito MB yokhala ndi ma electrode osindikizidwa pa skrini. amagwiritsidwa ntchito monga mu situ electrodes mu microfluidic PCR nsanja yopangidwa kuti iwonetsere electrochemically amplicons panthawi ya zochita 8. Maphunziro onsewa amafuna kusintha kwapamwamba kwa ma electrode, kutanthauza kuwonjezereka kwa kupanga ndi kugwiritsira ntchito ndalama chifukwa cha zofunikira zosungirako zapadera kuti zikhale zokhazikika za ma electrode ogwiritsidwa ntchitowa.
Schematic of workflow for electrochemical kuzindikira ma amplicons otengedwa kuchokera ku tinthu tambiri ta virus mu zitsanzo zamadzi a m'nyanja.
Posachedwa tawonetsa ma electrochemical sensing a SARS-CoV-2 amplicon okhala ndi ma elekitirodi otsika mtengo osindikizidwa (PCB) kutengera DPV ndi cyclic voltammetry (CV) motsogozedwa ndi ma adsorption a MB-DNA complexes pamwamba pa maelekitirodi osasinthidwa ) panopa11.Tikunena kuti zidutswa za DNA zazitali (N1-N2, \({943}\, \hbox) zopangidwa pogwiritsa ntchito CDC-yomwe ikulimbikitsidwa N1 patsogolo ndi N2 reverse primers poyerekeza ndi zidutswa zazifupi {bp}\)) zikuwonetsa mzere wabwinoko pakuyankha kwa sensa. ( N1, \(72\,\hbox {bp}\)) yopangidwa pogwiritsa ntchito N1 forward and N1 reverse primer sets.Kafukufukuyu akunenedwa pogwiritsa ntchito DNA dilutions yokonzedwa m'madzi opanda nuclease.Pulatifomu idagwiritsidwanso ntchito kuzindikira SARS-CoV -2 amplicons mu zitsanzo za madzi oipa (zopezedwa ndi spiking zonse za RNA zitsanzo ndi SARS-CoV-2 RNA). Popeza RNA imatha kumeta ubweya panthawi yodzipatula komanso kunsi kwa mtsinje, 12,13 ndizovuta kukulitsa zidutswa zazitali ndi zitsanzo zosawerengekazi. Chifukwa chake, kuwonetsera kwa electrochemical sensing ya SARS-CoV-2 amplicon m'madzi onyansa kumangokhala ndi kachidutswa kakang'ono \(72\,\hbox {bp}\) N1.
Mu ntchito iyi, ife anafufuza kuthekera kwa ENIG PCB ofotokoza electrochemical sensing wa phage Phi6 anaikira ndi olekanitsidwa ndi zitsanzo madzi nyanja (mkuyu 1) . Pazifukwa izi, bacteriophage Phi6 ndiwodziwika bwino wa SARS-CoV-2 ndi ma virus ena a RNA okhala ndi kachilombo14,15.RNA otalikirana ndi tinthu tating'onoting'ono adagwiritsidwa ntchito ngati template ya cDNA synthesis yotsatiridwa ndi PCR kuti ipeze zidutswa za DNA ziwiri za 117 ndi 503 m'litali. \,\hbox {bp}\) ndi \(503 \,\hbox {bp}\)), kutengera zoyambira zomwe zilipo.Kuyankha kwa sensa ya electrochemical kunaphunziridwa mwadongosolo mosiyanasiyana (\({10}\,{ \hbox {pg}/{\upmu \hbox {l}}}\) ku \({20}\, {\hbox {ng}/{\upmu \hbox {l}}}}\)) Pazidutswa zonse ziwiri mu kukhalapo kwa MB, zotsatira za mchere pa yankho la sensa zakhala zikudziwika ndikutsimikiziridwa ndi miyeso ya spectrophotometric.Zopereka zazikulu za ntchitoyi ndi izi:
DNA fragment kutalika ndi kupezeka kwa mchere mu chitsanzo kwambiri zimakhudza tilinazo.Zotsatira zathu zikuwonetsa kuti ntchito zama electrochemical zimatengera njira zosiyanasiyana zolumikizirana za MB, DNA, ndi sensa mu yankho la voltammetric, kutengera ndende ya DNA ndi kutalika, ndi Zidutswa zazitali zikuwonetsa kukhudzika kwakukulu, ngakhale kuti mchere uli ndi vuto loyipa pakulumikizana kwamagetsi pakati pawo. MB ndi DNA.
Kuchuluka kwa DNA kumatsimikizira momwe MB-DNA imagwirira ntchito mu maelekitirodi osasinthidwa Timasonyeza kuti njira zosiyanasiyana za MB-DNA zimadalira kuchuluka kwa DNA. {l}}}\), tinawona kuti kuyankha kwa electrochemical panopa kumatsimikiziridwa makamaka ndi kutengeka kwa MB-DNA pa electrode, pamene m'munsi mwake Pamagulu akuluakulu a DNA, yankho la electrochemical panopa linatsimikiziridwa ndi steric inhibition ya redox. ntchito chifukwa cha kuyika kwa MB pakati pa DNA base pairs.
ENIG PCB-Based Electrochemical Sensing of Viral Nucleic Acids in Lake Water Samples Zomwe adaziwona zidatsimikiziridwa ndi kuzindikira kwa electrochemical kwa Phi6-added \(503\,\hbox {bp}\) zidutswa za DNA zotengedwa ku zitsanzo zamadzi kuchokera ku Powai Lake, IIT Mumbai Campus. Result phage.
Mtengo wotsika wa kukhazikitsa ndi kuthekera kophatikizana ndi machitidwe owunikira okha, oligonucleotides kapena ma aptamers pamagetsi okhala ndi alumali yayitali.
Phage Phi6 ndi kachirombo ka dsRNA komwe kamakhala m'banja la Cytoviridae komwe kamakhudza Pseudomonas syringae. Gulu la Phi6 phage lilipo mu mawonekedwe a zidutswa zitatu: S (\(2.95\,\hbox {Kb}\)), M (\(4.07) \,\hbox {Kb}\)) ndi L (\ (6.37\ ,\hbox{Kb}\))16,17.Popeza Phi6 phage imakhudza mtundu wa BSL-1 Pseudomonas womwe siwoyambitsa matenda, ndizotetezeka kugwiritsa ntchito. ndipo akhoza kulimidwa mosavuta mu labotale.Phage Phi6 ndi mwini wake Pseudomonas syringae anagulidwa kuchokera ku Felix d'Herelle Reference Center for Bacterial Viruss, Laval University, Canada (manambala a kalozera wa malo owonetsera ndi HER-102 ndi HER-1102, motsatana) .Phi6 phage ndi host wake adatsitsimutsidwa monga momwe adanenera.Phage Phi6 idayeretsedwa ndi plate lysis and elution kuti ipeze ma final titers ndi \(\za 10^{12}\,{\hbox {PFU}/\hbox { ml}}\) (mayunitsi opangira mapepala / milliliters).RNA inasiyanitsidwa ndi tinthu tating'ono ta phage toyeretsedwa pogwiritsa ntchito GenElute™ Universal Total RNA Purification Kit (Sigma-Aldrich) molingana ndi malangizo a wopanga.Mwachidule, kuyimitsidwa kwa phage Phi6 yoyeretsedwa\({{ 100}\,{{\upmu \hbox {l}}}\) idawululidwa ndipo lysate idakwezedwa pagawo lozungulira kuti RNA imangirire kugawo la utomoni. 50}\,{{\upmu \hbox {l}}}\) yoperekedwa ndi kit.Yerekezerani kuchuluka kwa RNA mwa kuyamwa pa \(260\,\hbox {nm}\).RNA inasungidwa mu aliquots mu \(260\,\hbox {nm}\). ({-80}\,{^{\circ }\hbox {C}}\) mpaka kugwiritsidwanso ntchito.\({2}\,{\upmu \hbox {g}}\) The iScript cDNA Synthesis Kit (Bio -Rad Laboratories) idagwiritsidwa ntchito ngati template ya cDNA synthesis motsatira malangizo a wopanga. )\({5}\,{\hbox {min} }\) , tembenuzani mawu a \({20}\,{\hbox {min}}\) pa \({46}\,{^{\circ }\hbox {C}}\), ndi kubwerera kumbuyo Chojambulira chili mu \({95}\,{^{\circ }\hbox {C}}\) kwa \({1}\,{\hbox {min }}\).Pamene imayendetsedwa pa 1% gel agarose, cDNA inawonetsa magulu atatu ofanana ndi zidutswa zitatu za RNA zomwe zimayembekezeredwa (deta yosasonyezedwa). Zoyambira zotsatirazi zinagwiritsidwa ntchito kukulitsa zidutswa ziwiri za DNA za 117 ndi 503 bp m'litali, kugwiritsa ntchito cDNA ngati template ya PCR mu miniPCR® mini8 thermal cycler:
Zoyambira za \(117\,\hbox {bp}\) ndi \(503\,\hbox {bp}\) zimagwirizana ndi 1476-1575 nucleotides ya gawo la M ndi 458-943 nucleotides ya gawo L, motsatana asidi. .Zinthu zonse za PCR zokulitsa zidapangidwa ndi electrophores pa 1% agarose gels, ndipo chandamale chokulitsa DNA chinayeretsedwa pogwiritsa ntchito GeneJET Gel Extraction Kit (Thermo Fisher Scientific).
Nyanja yomwe ili pa kampu ya IIT Mumbai (Powai Lake, Powai, Mumbai) idagwiritsidwa ntchito kuwonjezera tinthu tating'onoting'ono. particles suspended, kenako Phi6 phage anawonjezedwa. Add \({1}\,{\hbox {ml}}\) ya \(10^{6}\,{\hbox {PFU}/\hbox {ml}} \) ku \( {100}\ ,{\hbox {ml}}\) osefedwa madzi a m'nyanja, mu \({4}\,{^{\ circle}\hbox {C}}\).Aliquot yaying'ono inali Tinayesa njira ziwiri zosiyana kuti tiyike tizilombo toyambitsa matenda a Phi6: (1) njira ya aluminiyamu ya hydroxide adsorption-precipitation, 19 yomwe yatsimikiziridwa chifukwa cha kuchuluka kwa ma virus angapo a RNA ochokera ku zitsanzo zachilengedwe, ndi (2) ) The polyethylene glycol (PEG) -based virus concentration method inasinthidwa kuchokera ku Chigumula et al.20 .Popeza kuti kubwezeretsedwa kwa njira yopangira PEG kunapezeka kuti ndi yabwino kuposa njira ya aluminiyamu hydroxide, njira ya PEG inagwiritsidwa ntchito kuyika ma particles a Phi6 kuchokera ku zitsanzo za madzi a m'nyanja.
Njira ya PEG yomwe idagwiritsidwa ntchito inali motere: PEG 8000 ndi \(\hbox {NaCl}\) anawonjezedwa ku zitsanzo za madzi a m'nyanja ya Phi6-spiked kuti apeze 8 % PEG 8000 ndi \(0.2\,\hbox {M} \) \( \ hbox {NaCl}\).Zitsanzo zidakulungidwa pa shaker\({4}\,{^{\circ }\hbox {C}}\)\({4}\,{\hbox {h}}\ ), kenako centrifuged pa \(4700 \,\hbox {g}\) ndi \({45}\,{\hbox {min}}\).Tayani chapamwamba ndikuyimitsanso pellet mu \({1}\, {\ hbox {ml}}\) mu supernatant yemweyo.Zoyesera zonse za spiking ndi virus ndende zidachitika mu triplicate.After concentration, aliquot yaying'ono idasungidwira kuyeza kuchira bwino ndi plaque assay.RNA idasiyidwa monga momwe idafotokozedwera kale ndikusinthidwa. mu kit-supplied elution buffer\({40}\,{\upmu \hbox {l}}\). Popeza kuchuluka kwa RNA kudzasiyana kuchokera ku zitsanzo kupita ku zitsanzo patatu, \({2}\,{\upmu \\) hbox {l}}\) ya RNA imagwiritsidwa ntchito pa atatu onse mosasamala kanthu za kuchuluka kwake kwa cDNA synthesis of samples.Kuphatikizika kwa cDNA kunachitika monga tafotokozera kale.\({1}\,{\upmu \hbox {l}}\) cDNA idagwiritsidwa ntchito ngati template ya \({20}\,{\upmu \hbox {l}}\) PCR kwa mizungu 35 kuti ikulitse \ (117\,\hbox {bp}\) ndi \(503\,\hbox { bp}\) zidutswa.Zitsanzozi zikuimiridwa ngati "1: 1", mwachitsanzo popanda dilution.A no-template control (NTC) inakhazikitsidwa ngati kulamulira kolakwika, pamene cDNA yopangidwa pogwiritsa ntchito RNA yolekanitsidwa ndi phage yoyeretsedwa inakhazikitsidwa. monga template ya ulamuliro wabwino (PC).Quantitative PCR (qPCR) inachitidwa mu chida cha Stratagene Mx3000P RT-PCR pogwiritsa ntchito Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies). Zochita zinakhazikitsidwa katatu monga kale. anafotokoza.The cycle threshold (Ct) inalembedwa pa zitsanzo zonse.Kuonjezera apo, zitsanzo zosungunuka zinali \({1}\,{\upmu \hbox {l}}\) pogwiritsa ntchito cDNA yosungunuka 1:100 m'madzi osefa a m'nyanja monga \({20}\,{\upmu \hbox {l}}\) PCR kwa mikombero 35. Zitsanzozi zikuimiridwa ngati "1:100".
Ma electrode a PCB amapangidwa pogwiritsa ntchito njira yamalonda yotsika mtengo ya Electroless Nickel Immersion Gold (ENIG) popanda kufunikira kwa golide wowonjezera.Zolemba za electrode ENIG PCB zafotokozedwa mwatsatanetsatane mu ntchito yathu yapitayi11.Kwa ma electrode a ENIG PCB, njira zamakono zoyeretsera ma electrode monga piranha solution kapena sulfuric acid cyclic voltammetry sikulimbikitsidwa chifukwa angayambitse kusenda kwa golide wosanjikiza (kukhuthala \(\ pafupifupi\) \(100\,\hbox {nm}\)) ndikuwonetsa zigawo zamkuwa zomwe zimakhala zosavuta. ku dzimbiri 21, 22, 23, 24, 25. Chifukwa chake, yeretsani maelekitirodi ndi nsalu yopanda lint yonyowa ndi IPA. Chitsanzo choyesedwa chinali chopangidwa ndi \({50}\,{\upmu \hbox {M}) }\) MB mu \({4}\,{^{\circ }\hbox {C}}\)\({ 1}\,{\hbox {h}}\) kuti tiyike mosavuta. M'ntchito yathu yam'mbuyomu , tawona kuti kukhudzidwa ndi kusinthasintha kwa sensa kunasinthidwa powonjezera chiwerengero cha MB 11 .Kutengera kukhathamiritsa komwe kunanenedwa mu ntchito yathu yoyambirira, tinagwiritsa ntchito \({50}\,{\upmu \hbox {M}}\) MB kuyika kwa DNA mu phunziroli.Kuzindikira kwa Electrochemical kwa DNA yamitundu iwiri (ds-DNA) kungathe kupezedwa pogwiritsa ntchito anionic kapena cationic intercalators. Komano, ma cationic intercalators monga MB amafunikira nthawi zazifupi za incubation, pafupifupi \({1}\,{\hbox {h}}\) pakuzindikira kwa electrochemical ds-DNA6. electrode\({5}\,{{\upmu \hbox {l}}}\), ndiye kuyeretsa ndi chiguduli chonyowa IPA, musanayambe ndi chitsanzo china.muyeso umodzi.Chitsanzo chilichonse chinayesedwa pa ma electrode 5 osiyana pokhapokha atanenedwa zina.Miyezo ya DPV ndi CV inagwiritsidwa ntchito pogwiritsa ntchito PalmSens Sensit Smart potentiostat, ndipo pulogalamu ya PSTrace inagwiritsidwa ntchito pokonzekera potentiostat ndi kupeza deta, kuphatikizapo chiwerengero chapamwamba chamakono.Zosintha zotsatirazi zikugwiritsidwa ntchito. pamiyezo ya DPV ndi CV:
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 Step = \(3\,\hbox {mV}\), Sweep Rate = \({300}\,\hbox {mV/ s }\)
Mafunde apamwamba otengedwa ku ma voltammograms a DNA opangidwa ndi \({50}\,{\upmu \hbox {M}}\) MB: (a) \(503\,\hbox {bp}\) DPV , (b) \ (503\,\hbox {bp}\) CV, (c) \(117\,\hbox {bp}\) DPV, (d) \(117\,\hbox {bp}\) CV.
Ma DPV ndi ma voltammogram a CV adapezedwa pa ma elekitirodi a ENIG PCB \({50}\,{\upmu \hbox {M}}\) MB opangidwa ndi DNA (pazokhazikika za 10–\({20}\,{\ hbox {ng) }/{\upmu \hbox {l}}}\) ie 0.13–\({0.26}\,{\upmu \hbox {M}}\) ya \(117\,\hbox {bp}\ ) ndi 0.03 -\({0.06}\,{\upmu \hbox {M}}\) ya \(503\,\hbox {bp}\)).Ma voltammogram oyimira akuwonetsedwa mu Chithunzi S1 mu Zowonjezera Zowonjezera. Chithunzi 2 chikuwonetsa zotsatira ya miyeso ya DPV ndi CV (pamwamba pakali pano) pogwiritsa ntchito mankhwala a PCR oyeretsedwa ndi gel. Poyerekeza ndi miyeso ya CV, miyeso ya DPV imasonyeza kukhudzidwa kwapamwamba (panopa ngati ntchito ya DNA concentration) chifukwa maziko a capacitive currents mu miyeso ya CV amabisa mafunde a Faradaic 26 .Deta pabokosi lililonse la bokosilo lili ndi miyeso yochokera ku maelekitirodi 5. Miyezo yonse imagwiritsa ntchito seti yofanana ya maelekitirodi kupeŵa zolakwika zoyezera chifukwa cha kusintha kwa electrode-to-electrode. , yaitali (\(503\,\hbox {bp}\)) \,\hbox {bp}\ poyerekeza ndi \(117) ) fragment.Izi zikugwirizana ndi mchitidwe woyembekezeredwa wa electrode adsorption yomwe inanenedwa m'ntchito yathu yapitayi.The adsorption ya MB-DNA complex imathandizira kusamutsidwa kwa ndalama pa electrode, zomwe zimathandiza kuti pakhale kuwonjezeka kwapamwamba. -cytosine (GC) zomwe zili m'maamplicons awiri (\(117\,\hbox {bp}\) ndi \(503\,\hbox {bp}\)) zinali pafupifupi 50%, kusonyeza kuti kuwonetsetsa Kusiyanitsa kumachokera ku kutalika kwa amplicon. \) ndi \( >{10}\,{\hbox {ng}/{\upmu \hbox {l}}}\) ya \(117\,\hbox {bp}\)), timawona makulidwe awiri nsonga zapamwamba za subs zimachepetsedwa muzitsulo zonse za DPV ndi CV.Izi ndichifukwa chakuti MB imadzaza ndi kusakanikirana pakati pa awiriawiri a DNA, zomwe zimapangitsa kuti steric inhibition ya ntchito ya redox ya gulu lochepetsetsa mu 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.
Mchere womwe ulipo mu PCR master mixes umasokoneza ma electrostatic interactions pakati pa MB ndi DNA, kotero powonjezera \(2\,\hbox {mM}\) \(\hbox {MgCl }_2\) ndi \({50} \,{\ upmu \hbox {M}}\) MB mankhwala oyeretsedwa ndi gel kuti aphunzire momwe mchere umagwirira ntchito pa MB-DNA. Monga momwe tawonetsera pa Chithunzi 3, tidawona kuti kuchuluka kwa DNA kwapamwamba (\(>{2}\,{\ hbox {ng}/{\upmu \hbox {l}}}\) (503\,\hbox {bp}\) ndi \(>{10}\,{\hbox {ng}/{\upmu \hbox { l}}}\) kwa \(117\,\hbox {bp} \)), mu DPV ndi CV Kuwonjezera mchere sikunakhudze kwambiri miyeso (onani Chithunzi S2 mu Supplementary Information kwa oimira voltammograms). Komabe, pa kuchepa kwa DNA, kuwonjezera mchere kumachepetsa kwambiri kukhudzidwa, zomwe zimapangitsa kuti pasakhale kusintha kwakukulu pakali pano ndi DNA yokhazikika. Zotsatira zoyipa zofanana za mchere pa kuyanjana kwa MB-DNA ndi kugwirizanitsa zanenedwa kale ndi ofufuza ena33,34.\(\ hbox { Mg}^{2+}\) ma cations amamanga ku msana wa phosphate wa phosphate, motero amalepheretsa kuyanjana kwa electrostatic pakati pa MB ndi DNA. Pakachulukidwe ka DNA, kuletsa kwa steric kulepheretsa ma MBs a redox-active kumapangitsa kuti mafunde achepetse kwambiri, motero ma electrostatic interactions Mfundo yofunika kwambiri ndi yakuti biosensor iyi ndi yoyenera kuti izindikire kuchuluka kwa DNA (kawirikawiri \({\ hbox {ng}/{\ upmu \hbox {l}}}\) kapena apamwamba), kwathunthu- Kukonza zodziwikiratu za zitsanzo zamadzi zachilengedwe, pomwe kuyeretsedwa kwa gel opangira PCR sikungatheke.
Dera lomwe lili pansi pa mayamwidwe opindika a kutalika kwa mafunde 600–700 \(\ hbox {nm}\) pamagulu osiyanasiyana a DNA opangidwa ndi \({50}\,{\upmu \hbox {M}}\) MB: ( a ) \(503\,\hbox {bp}\) wokhala ndi mchere komanso wopanda mchere (\(2\,\hbox {m}\) \(\hbox {MgCl}_2\)), (b) \( 117\, \hbox {bp}\) wokhala ndi mchere komanso wopanda mchere (\(2\,\hbox {mM}\) \(\hbox {MgCl}_2\)).\({0}\,{\hbox {pg}/) {\upmu \hbox {l}}}\) Magulu a DNA ofanana ndi \({50}\,{\upmu \hbox {M}}\) MB zitsanzo Palibe DNA.
Kuti titsimikizenso zotsatira zomwe zili pamwambazi, tidayesa zowunikira pogwiritsa ntchito UV/Vis spectrophotometer (Thermo Scientific Multiskan GO), zitsanzo \({50}\,{{\upmu \hbox {l}}}\) zinagwiritsidwa ntchito pa chilichonse. Kuyeza.Siginecha yoyamwitsa imachepa pakuchulukirachulukira kwa DNA, monga momwe tingawonere kuchokera kumadera omwe ali pansi pa mayamwidwe amapindika amtundu wa wavelength \(600\,\hbox {nm}\) mpaka \(700\,\hbox { nm}\) , monga momwe zasonyezedwera mkuyu. 4 (mayamwidwe sipekitiramu akuwonetsedwa mkuyu. {l}}}\), panalibe kusiyana kwakukulu pakutenga pakati pa zitsanzo zomwe zili ndi DNA ndi MB zokha (za \(503\,\hbox {bp}\) ndi \(117\,\hbox {bp}\) ) kutalika kwa zidutswa), kusonyeza kusakhalapo kwa steric kuletsa kwa redox-active MB. Pa kuchuluka kwa DNA, tinawona kuchepa kwapang'onopang'ono kwa chizindikiro cha kuyamwa ndipo tinawona kuchepa pang'ono kwa kuyamwa pamaso pa mchere. Zotsatira zathu zimagwirizana ndi malipoti a m'mabuku okhudzana ndi kafukufuku wa MB-DNA intercalation omwe amagwirizanitsa hypochromaticity ndi kuchepa kwa mphamvu mu \(\pi\)–\(\pi ^*\ ) kusintha kwamagetsi chifukwa cha intercalation Magawo 36, 37, 38.
Agarose gel electrophoresis of phage Phi6: PCR product of length \(117\,\hbox {bp}\) and \(503\,\hbox {bp}\) from the lake water samples.M-DNA marker;Kuwongolera kwa NTC-no-template, zoyambira zomwe zili ndi amplicons ofanana;Kuwongolera kwabwino kwa PC;1. hbox {bp}\) msewu.
Tidayesa kugwiritsa ntchito sensa pogwiritsa ntchito zitsanzo zamadzi a Powai Lake omwe adawongoleredwa ndi Phi6 phage. Ma RNA otalikirana ndi madzi opangidwa ndi phage-spiked adachokera ku 15.8–\({19.4}\,{\upmu \hbox {g}/\hbox { ml}}\), pomwe iwo olekanitsidwa ndi kuyimitsidwa kwa phage koyeretsedwa The RNA akuti ndi \({1945}\,{\upmu \hbox {g}/\hbox {ml}}\) yokhala ndi mphamvu yochira pafupifupi 1. %.RNA inasinthidwa kukhala cDNA ndipo imagwiritsidwa ntchito ngati template ya PCR ndi qPCR. Kukula kwa mankhwala kunatsimikiziridwa ndi agarose gel electrophoresis (Chithunzi 5) asanayesedwe ndi sensa.Zitsanzozi siziyeretsedwa ndi gel choncho zili ndi zigawo zonse za PCR monga Miyezo ya Ct yojambulidwa pa qPCR (Table 1) idawonetsedwa kuti ikugwirizana ndi kuchuluka kwa RNA komwe kumasiyanitsidwa ndi zitsanzo zamadzi opindika. Kupitilira malire kapena chizindikiro chakumbuyo.Makhalidwe apamwamba a Ct amawonetsa kuchuluka kwa ma template otsika komanso mosinthanitsa. Ma Ct a zitsanzo za NTC anali okwera momwe amayembekezeredwa. Kusiyana kwa \(\ pafupifupi 3\) Ct pakati Kuwongolera kwabwino ndi chitsanzo choyesera chikuwonetsanso kuti chitsanzo chilichonse choyesa chimakhala ndi template ya 1% poyerekeza ndi kuwongolera koyenera. wa kuchepa kwa ma virus komanso kuwonongeka kwa RNA.Komabe, ndi kuchulukitsa kwa ma virus athu ndi PCR amplification protocol, tinatha kukulitsa bwino \(503\,\hbox {bp}\) fragment for electrochemical sensing.
Chithunzi 6 chikuwonetsa zotsatira za sensa ya electrochemical ya \(503\,\hbox {bp}\) fragment amplicon, onse pogwiritsa ntchito cDNA yosapangidwa monga template (1:1) ndi cDNA yochepetsedwa ka 100 monga template (1:100 ) anachita PCR , poyerekeza ndi NTC ndi PC (onani Chithunzi S4 mu Supplementary Information kwa oimira voltammograms) .Bokosi lirilonse mu bokosi la bokosi mu Chithunzi 6 lili ndi miyeso yochokera ku zitsanzo zitatu pa ma electrode a 5. Ma electrode omwewo adagwiritsidwa ntchito poyesa zitsanzo zonse kuti apewe zolakwika chifukwa cha electrode Poyerekeza ndi miyeso ya CV, miyeso ya DPV ikuwonetsa bwino kusiyanitsa zitsanzo za mayeso ndi ma PC kuchokera ku NTCs chifukwa, monga tanena kale, mafunde a Faradaic amabisika chifukwa cha mafunde am'mbuyo a capacitive pomaliza. Kuwongolera koyipa (NTC) kudapangitsa kuti ma CV ndi ma DPV achuluke kwambiri potengera kuwongolera kwabwino, pomwe zitsanzo zoyeserera zoyeserera zidawonetsa nsonga zofananira za DPV pachimake mafunde. ) chitsanzo choyesera ndi PC zikhoza kuthetsedwa momveka bwino kuchokera ku sensa ya NTC, pamene chigamulo cha 1: 100 sampuli yowonongeka sichidziwika bwino. (njira zomwe sizinawonetsedwe mu Chithunzi 5), ndi DPV yofananira ndi mafunde a CV anali ofanana ndi omwe amayembekezeredwa ku NTC.Zotsatira za \(117\,\hbox {bp}\) fragment zikuwonetsedwa mu Supplementary Information.The negative Kuwongolera kunayambitsa kuyankha kwa electrochemical kuchokera ku sensa ya PCB chifukwa cha kutengeka kwa MB yaulere pa electrode ndi kuyanjana kwa MB ndi oligonucleotide yoyambira yomwe ili ndi chingwe chimodzi. nsonga yamakono ya chitsanzo choyesera poyerekeza ndi chiwerengero chapamwamba chomwe chimapezedwa ndi kuwongolera kolakwika kuti mukwaniritse muyeso wosiyana (wachibale)39,40 kuti mugawire chitsanzo choyesa ngati chabwino kapena choipa.
(a) DPV, ndi (b) CV peak current for electrochemical diagnosis ya \(503\,\hbox {bp}\) zidutswa mu zitsanzo za madzi a m'nyanja. Zitsanzo zoyesera zinayesedwa katatu ndi kuyerekeza ndi no template controls (NTC) ndi zowongolera zabwino (PC).
Zomwe tapeza zikuwonetsa njira zosiyanasiyana zomwe zimakhudza magwiridwe antchito a masensa a electrochemical a ma amplicon a utali wosiyanasiyana wa ma DNA osiyanasiyana, ndikuwunika komwe kumatsimikiziridwa ndi miyeso ya kuwala pogwiritsa ntchito UV/Vis spectrophotometer. \(500\,\hbox {bp}\) imatha kuzindikirika ndi chidwi chambiri komanso kuti kupezeka kwa mchere muzachitsanzo sikumakhudza kukhudzidwa kwa DNA komwe kumakhudza kukhudzika kwakukulu (kawirikawiri \({\hbox {ng}/{\upmu) \hbox {l}}}\) ndi pamwamba).Kuonjezera apo, tinafufuza zotsatira za mitundu yosiyanasiyana ya zitsanzo, kuphatikizapo amplicon oyeretsedwa ndi gel osakaniza ndi opanda mchere, ndi kuwonjezera zitsanzo za madzi a m'nyanja mu DPV ndi CV miyeso.Tidawona kuti DPV idapereka kusintha kwabwinoko, chifukwa chakumbuyo kwa capacitive pano kumakhudzanso muyeso wa CV, ndikupangitsa kuti isavutike.
Kukulitsa kwa tizidutswa tautali kumadalira kukhulupirika kwa ma virus genomic RNA.Kafukufuku angapo awonetsa kuti kukulitsa kwa zidutswa zazitali sikuli kothandiza nthawi zonse chifukwa cha kuwonongeka kwa RNA m'chilengedwe komanso kuthekera kwapang'onopang'ono pakudzipatula11,41,42,43,44 .Tinaona kuti njira ya PEG yochokera ku kachilombo ka HIV inali yothandiza kwambiri poika phage Phi-6 spiked mu zitsanzo za madzi a m'nyanja kusiyana ndi njira ya aluminium hydroxide-based virus concentration. kukulitsa ma tempulo amtali amfupi angapo ndikuchepetsa kuthekera kwa kusiyanasiyana.
Zitsanzo za chilengedwe ndizosowa, kotero pakufunika kupanga biosensor yomwe imafunika zitsanzo zochepa kuti ziyesedwe.Ma electrode a ENIG PCB omwe amagwiritsidwa ntchito mu kafukufukuyu amangofunika \({5}\,{{\upmu \hbox {l}}}\\ ) zitsanzo zoyezetsa kuphimba malo ogwira ntchito a electrode.Kuonjezera apo, electrode yomweyi ikhoza kugwiritsidwanso ntchito pambuyo poyeretsa musanapereke chitsanzo chotsatira.Zitsanzo za Amplified sizifuna kuwonjezera mankhwala ena kupatulapo methylene blue, yomwe ndi yotsika mtengo. ndi mankhwala omwe amagwiritsidwa ntchito kawirikawiri.Popeza kuti electrode iliyonse imawononga pafupifupi $ 0.55 (kapena INR 40) kuti ipangidwe, biosensor iyi ikhoza kukhala yotsika mtengo kusiyana ndi matekinoloje omwe alipo kale. DNA zidutswa mu zitsanzo heterogeneous.
Poganizira kuti njira zozindikirira ma electrochemical za MB zimadalira kutsimikizika kwa PCR, cholepheretsa chachikulu cha njirayi ndikuthekera kwa kukulitsa mosakhazikika mu zitsanzo zosiyanasiyana monga madzi onyansa ndi madzi a m'nyanja kapena kugwiritsa ntchito zoyambira zotsika. Njira zodziwira ma electrochemical pozindikira DNA ya zinthu zosayeretsedwa za PCR pogwiritsa ntchito maelekitirodi osasinthidwa a ENIG PCB, ndikofunikira kumvetsetsa zolakwika zomwe zidayambitsidwa ndi ma dNTP osagwiritsidwa ntchito ndi oyambira, komanso kukhathamiritsa momwe zinthu zimachitikira ndi ma assay protocols.Zowonjezera za physicochemical monga pH, kutentha, ndi biological. Kufunika kwa okosijeni (BOD) kwachitsanzo cha madzi kungafunikirenso kuyezedwa kuti muyesowo ukhale wolondola.
Pomaliza, tikupangira makina otsika mtengo a electrochemical ENIG PCB sensor kuti azindikire kachilomboka m'masampula achilengedwe (madzi a m'nyanja). Mosiyana ndi ma elekitirodi a oligonucleotide osasunthika kapena magawo amtundu wa DNA omwe amafunikira kusungidwa kwa cryogenic kuti asunge kumva,53,54 njira yathu imagwiritsa ntchito PCB yosasinthika. ma elekitirodi okhala ndi alumali nthawi yayitali komanso osafunikira kusungirako zofunikira ndipo motero ndi oyenera kupanga njira zoyezera pogwiritsa ntchito makina opangira ma LMICs. The biosensor imagwiritsa ntchito utoto wotsika mtengo wa DNA-intercalating redox dyes (MB) kuti uzindikire mwachangu amplicon chandamale. zomwe zimapezeka mu zitsanzo zachilengedwe zimachepetsa kutsimikizika kwa njira yodziwikirayi chifukwa cha kumangiriza kosadziwika kwa MBs ku oligonucleotides imodzi ndi iwiri. ndi mafunde apamwamba a DPV omwe atengedwa kuchokera ku zitsanzo zoyesedwa ayenera kutanthauziridwa mogwirizana ndi mayankho omwe atengedwa kuchokera ku ulamuliro woipa (NTC) pa mayesero aliwonse.Mapangidwe a electrochemical sensor ndi njira zomwe zimaperekedwa mu ntchitoyi zikhoza kuphatikizidwa ndi ma autosamplers kuti apange makina odzipangira okha komanso otsika. -cost solution yomwe imatha kusonkhanitsa ndikusanthula zitsanzo ndikutumiza zotsatira ku labotale popanda zingwe.
Cashdollar, J. & Wymer, L. Njira zoyambira ma virus kuchokera ku zitsanzo zamadzi: kuwunika ndi kusanthula meta kwa maphunziro aposachedwa.J.Application.microorganism.115, pp. 1-11 (2013).
Gall, AM, Mariñas, BJ, Lu, Y. & Shisler, JL Mavairasi amadzimadzi: Zolepheretsa madzi akumwa abwino.PLoS Pathogens.11, E1004867 (2015).
Shrestha, S. et al.Wastewater epidemiology pakuwunika kotsika mtengo kwakukulu kwa COVID-19 m'maiko omwe ali ndi ndalama zochepa komanso zapakati: zovuta ndi mwayi.Water 13, 2897 (2021).
Palecek, E. & Bartosik, M. Nucleic acid electrochemistry.Chemical.Rev.112, 3427-3481 (2012).
Tani, A., Thomson, AJ & Butt, JN Methylene blue as an electrochemical discriminator of single-stranded double-stranded oligonucleotides immobilized on gold substrates.Analyst 126, 1756–1759 (2001).
Wong, EL, Erohkin, P. & Gooding, JJ Kuyerekeza kwa Cation ndi Anion Intercalators kwa Electrochemical Transduction of DNA Hybridization by Long-Range Electron Transfer.Electrochemistry.comminicate.6, 648-654 (2004).
Wong, EL & Gooding, JJ Charge transfer through DNA: a selective electrochemical DNA biosensor.anus.Chemical.78, 2138-2144 (2006).
Fang, TH et al.Real-time PCR microfluidic device yokhala ndi electrochemical diagnosis.biological sensor.Bioelectronics.24, 2131-2136 (2009).
Win, BY et al.Signing mechanism study and performance verification of an electrochemical real-time PCR system based on the interaction of methylene blue with DNA.Analyst 136, 1573-1579 (2011).
Ramirez-Chavarria, RG et al.Loop-mediated isothermal amplification-based electrochemical sensor kuti azindikire sars-cov-2 mu zitsanzo zamadzi onyansa.J.Environment.Chemical.Britain.10, 107488 (2022).
Kumar, M. et al. Electrochemical sensing of SARS-CoV-2 amplicon with PCB electrodes.The sensor is activated.B Chemistry.343, 130169 (2021).
Kitamura, K., Sadamasu, K., Muramatsu, M. & Yoshida, H. Kuzindikira mogwira mtima kwa SARS-CoV-2 RNA mu gawo lolimba la madzi onyansa.science.general environment.763, 144587 (2021).
Alygizakis, N. et al.Analytical Methods for SARS-CoV-2 Detection in Wastewater: Protocol and future Perspectives.TraC trending anal.Chemical.134, 116125 (2020).
Fedorenko, A., Grinberg, M., Orevi, T. & Kashtan, N. Kupulumuka kwa bakteriophage Phi6 (wothandizira wa SARS-CoV-2) m'malovu osungunuka omwe amaikidwa pagalasi.science.Rep.10, 1-10 (2020).
Dey, R., Dlusskaya, E. & Ashbolt, NJ Kulimbikira kwa chilengedwe kwa SARS-CoV-2 surrogate (Phi6) mu free-living amoeba.J.Water Health 20, 83 (2021).
Mindich, L. Kupaka molondola kwa zidutswa zitatu za genomic za RNA bacteriophage ya nsonga ziwiri\(\varphi\)6.microorganism.Moore.biology.Rev.63, 149-160 (1999).
Pirttimaa, MJ & Bamford, Kapangidwe kachiwiri ka DH RNA phage\(\varphi\)6 chigawo cholongedza.RNA 6, 880–889 (2000).
Bonilla, N. et al.Phages on the Tap - Protocol yofulumira komanso yothandiza pokonzekera ma labotale a bacteriophages.PeerJ 4, e2261 (2016).
Nthawi yotumiza: May-27-2022