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An yaba da mahimmancin sa ido kan samfuran muhalli tun farkon cutar ta COVID-19, kuma ana yin wasu ƙoƙarce-ƙoƙarce ta hanyar amfani da ma'aunin zinare, kodayake dabarun tushen qPCR suna da tsada. Magani don saka idanu samfuran ruwan muhalli a cikin ƙananan ƙasashe masu tasowa da masu shiga tsakani.A cikin wannan aikin, muna nuna gano electrochemical na amplicons da aka samu daga Phi6 phage ware daga samfuran ruwan tafkin spiked (wani sanannen maye ga SARS-CoV-2), ta amfani da ENIG. don kammala na'urorin lantarki na PCB ba tare da gyaran fuska ba.Jima'i. An kwatanta martanin firikwensin electrochemical sosai ga guntuwar DNA guda biyu masu tsayi daban-daban (\({117}\,\hbox {bp}\) da \({503}\,\hbox {bp}\)), da kuma Sakamakon salts a cikin PCR master mixes on methylene blue (MB) -DNA interactions.Our results ya nuna cewa tsawon DNA gutsuttsura muhimmanci kayyade electrochemical ji na ƙwarai da kuma nuna a cikin wannan aikin cewa ikon gane dogon amplicons ba tare da gel tsarkakewa na PCR kayayyakin ne. mahimmanci don auna samfuran ruwa a wurin.Cikakken bayani mai sarrafa kansa don ɗaukar hoto mai hoto da kyau.
An san watsa kwayar cutar ta ruwa a matsayin haɗari ga lafiyar jama'a tun cikin shekarun 1940, tare da shaidar farko na yaduwar cutar shan inna da cutar hanta ta E1.Hukumar Lafiya ta Duniya (WHO) ta ware wasu ƙwayoyin cuta masu kamuwa da ruwa masu matsakaici zuwa babban mahimmancin lafiya2.Traditional Virus Hanyoyin ganowa sun dogara da fasaha na tushen qPCR na zinari, waɗanda suke da matukar damuwa da ƙayyadaddun, amma suna buƙatar ƙwararrun ma'aikata don gwadawa a cikin dakin gwaje-gwaje ta amfani da kayan aiki masu tsada. Duk da haka, a cikin ƙananan kasashe masu tasowa (LMICs) tare da iyakacin albarkatu, ɗan adam gwajin samfurin zai iya zama fifiko a kan sa ido kan samfurin ruwan muhalli.Saboda haka, ana buƙatar wasu hanyoyin da ba su da tsada don dorewa, sa ido kan samfuran ruwa da na ruwa a cikin ƙasa masu ƙasa da matsakaita a matsayin gargaɗin farko na barkewar cututtuka, don haka kare su daga mummunan tasirin tattalin arziki na zamantakewar cutar ta cutar. Na'urorin lantarki masu ƙarancin tsada don ƙwayoyin nucleic acid na iya samar da kyakkyawar mafita ga wannan buƙatun da ba a cika buƙata ba. Yawancin waɗannan na'urori na DNA suna aiki ta gaskiyar cewa ƙwayoyin DNA na haɗin gwiwa ba su motsi a kan lantarki. surface da hybridize lokacin da wani matching jerin ya kasance a cikin samfurin.Wannan za a iya canza zuwa sigina ta daban-daban electrochemical dabaru ta amfani da redox matsakanci kamar potassium iron/ferrocyanide.Methylene blue (MB) daya ne irin wannan redox-active kwayoyin halitta, wanda yana da. An ba da rahoton cewa za su shiga cikin DNA mai ɗaure biyu (dsDNA) ban da ƙarin ƙayyadaddun abin da ba shi da takamaiman ɗaurinsa ga DNA5,6 mai ma'ana guda ɗaya. Halin tsaka-tsaki na MBs don samar da rukunin MB-DNA ya sa su zama mashahurin zaɓi a matsayin masu shiga tsakani a cikin DNA na lantarki da yawa. firikwensin saituna5,6,7,8,9.Ko da yake intercalation na MB zuwa DNA ne nonspecific, da kuma musamman na wannan electrochemical firikwensin ya dogara da yawa a kan tsarki na primers amfani da PCR ko isothermal amplification, shi ne da kyau dace don aiwatar da ainihin. -lokaci electrochemical-tushen qPCR ko fluorescence isothermal amplification a matsayin madadin DNA maida hankali auna 9 .A daya daga cikin aiwatar da, Won et al.The surface na zinariya electrodes aka modified da 6-mercapto-1-hexanol (MCH) don ainihin-lokaci. Aunawa na PCR amplicons tare da MB ta amfani da bambancin bugun jini voltammetry (DPV) 9. A wasu lokuta, Ramirez et al. Gano SARS-CoV-2 a cikin ruwan sharar gida ta hanyar RT-LAMP ta amfani da MB tare da na'urorin lantarki da aka buga. ana amfani da su kamar yadda ake amfani da wutar lantarki a cikin tsarin PCR na microfluidic da aka tsara don gano amplicons a lokacin halayen 8. Duk waɗannan karatun suna buƙatar gyaran gyare-gyare na lantarki, yana nuna karuwar samarwa da farashin aiki saboda bukatun ajiya na musamman don kwanciyar hankali na waɗannan lantarki masu aiki.
Tsarin tsarin aikin don gano abubuwan lantarki na amplicons da aka samo daga ƙwayoyin cuta masu kama da juna a cikin samfuran ruwan tafkin.
Kwanan nan mun nuna ji na electrochemical na SARS-CoV-2 amplicons tare da rahusa bugu da aka buga (PCB) lantarki dangane da DPV da cyclic voltammetry (CV) jawo ta hanyar adsorption na MB-DNA hadaddun a kan saman unmodified electrodes) canje-canje a kololuwa. halin yanzu 11.Mun bayar da rahoton cewa guntuwar DNA masu tsayi (N1-N2, \({943}\, \hbox) da aka kafa ta amfani da CDC-shawarar N1 gaba da N2 na baya idan aka kwatanta da guntun guntu {bp}\)) sun nuna mafi kyawun layi a cikin martanin firikwensin. ( N1, \ (72 \, \ hbox {bp} \)) da aka kafa ta amfani da N1 gaba da kuma N1 reverse primer sets. An bayar da rahoton waɗannan binciken ta amfani da dilutions DNA da aka shirya a cikin ruwa maras lafiya. An kuma yi amfani da dandamali don gano SARS-CoV. -2 amplicons a cikin samfuran ruwan sharar gida da aka kwaikwaya (wanda aka samu ta hanyar zuga jimlar samfuran RNA tare da SARS-CoV-2 RNA) Tun da RNA yana da sauƙin yankewa yayin keɓewa da sarrafa ƙasa, 12,13 yana da wahala a haɓaka guntu mai tsayi tare da wannan samfurin iri-iri. Saboda haka, nunin electrochemical ji na SARS-CoV-2 amplicon a cikin ruwan sharar gida an iyakance ga guntu \(72 \, \ hbox {bp} \) N1 guntu.
A cikin wannan aikin, mun bincika yiwuwar ENIG PCB na tushen electrochemical ji na phage Phi6 mayar da hankali da kuma ware daga lake ruwa samfurori (Fig. 1) .Phi6 phages ne kwatankwacin girman (80-100 nm) zuwa SARS-CoV-2 da kuma Hakanan suna da membrane na lipid da furotin mai karu.Saboda waɗannan dalilai, bacteriophage Phi6 sanannen wurin maye ne don SARS-CoV-2 da sauran ƙwayoyin cuta na RNA masu lulluɓe14,15.RNA waɗanda ke ware daga ƙwayoyin phage an yi amfani da su azaman samfuri don haɗin cDNA wanda ya biyo baya. PCR don samun guntuwar DNA guda biyu na 117 da 503 tushe nau'i-nau'i a tsayi. Idan aka ba da ƙalubalen haɓaka \ (943 \, \ hbox {bp} \) N1-N2 guntu a cikin aikinmu na baya, mun yi niyya ga guntu tsawon matsakaici (\(117) \,\hbox {bp} \hbox {pg}/{\upmu \hbox {l}}}\) zuwa \({20}\, {\hbox {ng}/{\upmu \hbox {l}}}\)) Domin duka gutsuttsura a ciki kasancewar MB, tasirin gishiri a kan amsawar firikwensin ya kasance an kwatanta shi kuma an daidaita shi ta hanyar ma'auni na spectrophotometric. Babban gudunmawar wannan aikin shine kamar haka:
Tsawon gutsure DNA da kasancewar gishiri a cikin samfurin yana tasiri sosai ga hankali.Sakamakonmu ya nuna cewa aikin electrochemical ya dogara da hanyoyi daban-daban na hulɗar MB, DNA, da kuma firikwensin a cikin amsawar voltammetric, dangane da tattarawar DNA da tsayi, tare da tsayin daka yana nuna mafi girman hankali, kodayake gishiri yana da mummunan tasiri akan hulɗar electrostatic tsakanin. MB dan DNA.
Tattaunawar DNA tana ƙayyade tsarin hulɗar MB-DNA a cikin na'urorin lantarki da ba a canza su ba Mun nuna cewa hanyoyi daban-daban na hulɗar MB-DNA sun dogara ne akan ƙaddamar da DNA. {l}}}\), mun lura cewa amsawar electrochemical na yanzu an ƙaddara shi ne ta hanyar adsorption na MB-DNA akan lantarki, yayin da a ƙananan ƙididdiga A babban adadin DNA, amsawar electrochemical na yanzu an ƙaddara ta hanyar hanawa na redox. aiki saboda shigar MB tsakanin nau'ikan tushe na DNA.
ENIG PCB-Based Electrochemical Sensing of Viral Nucleic Acids in Lake Water Samfuran Abubuwan da aka lura an inganta su ta hanyar ganowa na electrochemical na Phi6-added \(503 \, \ hbox {bp} \) gutsutsayen DNA da aka samu daga samfuran ruwa daga tafkin Powai, IIT Mumbai Campus Sakamakon sakamako.
Ƙananan farashin aiwatarwa da yuwuwar haɗawa cikin cikakken tsarin sa ido mai sarrafa kansa, oligonucleotides ko aptamers akan na'urorin lantarki tare da tsawon rayuwar shiryayye.
Phage Phi6 kwayar cuta ce ta dsRNA da aka lullube ta dangin Cytoviridae wacce ke cutar da sirinji na Pseudomonas. Halin halittar Phi6 phage yana wanzuwa a cikin nau'i guda 3: S (\ (2.95 \, hbox {Kb}\)), M (\(4.07) \,\hbox {Kb} kuma ana iya girma cikin sauƙi a cikin dakin gwaje-gwaje. An saya Phage Phi6 da mai masaukin baki Pseudomonas syringae daga Felix d'Herelle Reference Center for Bacterial Viruses, Jami'ar Laval, Kanada (lambobin kasida na cibiyar sune HER-102 da HER-1102, bi da bi) .Phi6 phage da mai masaukin baki an sake farfado da su kamar yadda cibiyar bincike ta umarce su. An tsarkake Phage Phi6 ta hanyar farantin karfe lysis da elution don samun titers na ƙarshe tare da \ (\ game da 10 ^ {12} \, {\ hbox {PFU} / \ hbox { ml}}\) (Plaque forming units/ milliliters)) RNA an ware shi daga ɓangarorin phage da aka tsarkake ta amfani da GenElute™ Universal Total RNA Purification Kit (Sigma-Aldrich) bisa ga umarnin masana'anta. 100} \, {{\upmu \ hbox {l}}} an lysed kuma an ɗora lysate ɗin a kan ginshiƙi na jujjuya don ba da damar RNA ta ɗaure ga ginshiƙi na guduro. Sannan RNA an ɓoye shi a cikin mafita mai haske \({ 50}\,{{\upmu \hbox {l}}} ({-80}\,{^{\circ }\hbox {C}}\) har sai an kara amfani.\({2}\,{\upmu \hbox {g}}\) IScript cDNA Synthesis Kit (Bio) -Rad Laboratories) an yi amfani dashi azaman samfuri don haɗin cDNA yana bin umarnin masana'anta. )\({5}\,{\hbox {min} } \) , juyar da rubutun \({20}\,{\hbox {min}} }\hbox {C}}\), kuma baya Mai rikodin yana cikin \({95}\,{^{\circ }\hbox {C}}\) don \({1}\,{\hbox {min) }}\) .Lokacin da aka yi amfani da gel na agarose 1%, cDNA ya nuna nau'i-nau'i guda uku da suka dace da ragowar RNA guda uku da ake tsammani (bayanan da ba a nuna ba). ta amfani da cDNA azaman samfuri don PCR a cikin miniPCR® mini8 mai hawan keke:
Abubuwan da ake buƙata don \ (117 \, \ hbox {bp} \) da \ (503 \, \ hbox {bp} \) sun dace da 1476-1575 nucleotides na ɓangaren M da 458-943 nucleotides na L sashi, bi da bi acid. .Dukkan samfuran PCR da aka haɓaka an sanya su a kan 1% agarose gels, kuma an tsarkake DNA ɗin da aka haɓaka ta hanyar amfani da Kit ɗin Extraction Gel na GeneJET (Thermo Fisher Scientific).
An yi amfani da wani tabki a harabar IIT Mumbai ( Lake Powai, Powai, Mumbai) don ƙara ɓangarorin phage. An tace ruwan tafkin ta hanyar membrane \ ({5}\,{\upmu \ hbox {m}}) don cirewa. ɓangarorin da aka dakatar, sannan an ƙara Phi6 phage. Ƙara \({1}\,{\hbox {ml}}\) na \(10^{6}\,{\hbox {PFU}/\hbox {ml}} \) zuwa \( {100}\ , {\hbox {ml}} \) tace ruwa tafki, a cikin \({4}\,{^{\circle}\hbox {C}}). Mun gwada hanyoyi daban-daban guda biyu don tattara ɓoyayyun ƙwayoyin cuta na Phi6: (1) hanyar hazo-hazo na aluminum hydroxide, 19 wanda aka inganta don tattara ƙwayoyin RNA da yawa da aka lulluɓe daga samfuran muhalli, kuma (2)) Hanyar ƙwayar ƙwayar cuta ta polyethylene glycol (PEG) an daidaita shi daga ambaliyar ruwa et al.20 .Tun tun lokacin da aka gano ingantaccen farfadowa na hanyar tushen PEG mafi kyau fiye da na hanyar aluminum hydroxide, an yi amfani da hanyar da aka yi amfani da PEG don ƙaddamar da ƙwayoyin Phi6 daga samfurori na ruwa na tafkin.
Hanyar PEG da aka yi amfani da ita ita ce kamar haka: PEG 8000 da \ (\ hbox {NaCl} \) an saka su a cikin samfuran ruwan tafkin Phi6-spiked don samun 8 % PEG 8000 da \ (0.2 \, \ hbox {M} \) \( \ hbox {NaCl}\) .Samples an sanya su a kan shaker\({4}\,{^{\circ }\hbox {C}}\)\({4}\,{\hbox {h}}\ ), sa'an nan centrifuged a \(4700 \,\hbox {g}\) shine \({45}\,{\hbox {min}}\)) .Kwatar da supernatant kuma a mayar da pellet a cikin \({1}\, {\hbox {ml}}\) a cikin wannan supernatant. Dukkan gwaje-gwajen ƙwayar cuta da ƙwayar cuta an yi su a cikin nau'i uku. Bayan maida hankali, an tanadi ƙaramin aliquot don auna ingancin farfadowa ta hanyar plaque assay. RNA ta keɓe kamar yadda aka bayyana a baya kuma an eluted in kit-supplied elution buffer\({40}\,{\upmu \hbox {l}}\) .Tunda tarin RNA zai bambanta daga samfurin zuwa samfurin a cikin sau uku, \({2}\,{\upmu \) hbox {l}}\) na RNA ana amfani da shi ga duka ukun ba tare da la'akari da tattarawar cDNA na samfurori ba. An yi amfani da shi azaman samfuri don \ ({20}\,{\upmu \ hbox {l}} \) PCR don hawan keke 35 don ƙarawa \ (117 \, \ hbox {bp} \) da \ (503 \, \ hbox { bp}\) gutsuttsura.Waɗannan samfurori ana wakilta su a matsayin “1:1″, watau ba tare da dilution ba. An saita ikon sarrafa tsari (NTC) azaman iko mara kyau, yayin da aka haɗa cDNA ta amfani da RNA da aka keɓe daga phage mai tsabta an saita shi. a matsayin samfuri don ingantaccen iko (PC) .Quantitative PCR (qPCR) an yi shi a cikin kayan aikin Stratagene Mx3000P RT-PCR ta amfani da Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies) .An saita amsa a cikin sau uku kamar yadda a baya. An bayyana madaidaicin zagayowar (Ct) don duk samfuran. Bugu da ƙari, samfuran da aka diluted sune \ ({1}\,{\upmu \ hbox {l}} \) ta amfani da cDNA diluted 1: 100 a cikin ruwan tafkin da aka tace kamar yadda \({20}\,{\upmu \hbox {l}}\) PCR na hawan keke 35. Ana wakilta waɗannan samfuran a matsayin "1:100″.
Ana ƙera na'urorin lantarki na PCB ta amfani da tsarin kasuwanci mara ƙarancin kuɗi na Electroless Nickel Immersion Gold (ENIG) ba tare da buƙatar ƙarin plating na zinari ba. Maganin piranha ko sulfuric acid cyclic voltammetry ba a ba da shawarar ba saboda suna iya haifar da peeling na bakin ciki na zinariya (kauri \ (\ kusan \) \ (100 \, \ hbox {nm } \)) da kuma fallasa ƙananan yadudduka na tagulla waɗanda ke da haɗari. don lalata 21, 22, 23, 24, 25. Don haka, tsaftace electrodes tare da zane mai laushi wanda aka jika da IPA. Samfurin da za a gwada an haɗa shi da \({50}\,{\upmu \hbox {M}) }\) MB a cikin \({4}\,{^{\circ }\hbox {C}}\)\({1}\,{\hbox {h}}\) domin sauƙaƙan shigarwa. A cikin aikinmu na baya , mun lura cewa an inganta hankali da kuma layi na firikwensin ta hanyar ƙara yawan MB na 11. Bisa ga ingantawa da aka ruwaito a cikin aikinmu na farko, mun yi amfani da \({50}\,{\upmu \hbox {M}}\) MB Za'a iya samun damar gano DNA mai ma'ana guda biyu (ds-DNA) ta amfani da intercalators anionic ko cationic. A daya hannun, cationic intercalators kamar MB na bukatar guntun lokacin shiryawa, kusan \({1}\,{\hbox {h}}\) don gano electrochemical na ds-DNA6. Kowane ma'auni ya ƙunshi rarraba samfurin da za a gwada akan electrode\({5}\,{{\upmu \hbox {l}}}\), sannan tsaftacewa da tsumma mai ruwan IPA, kafin a ci gaba da wani samfurin.An gwada kowane samfurin akan na'urori daban-daban guda 5 sai dai idan an faɗi haka. An yi ma'aunin DPV da CV ta amfani da PalmSens Sensit Smart potentiostat, kuma an yi amfani da software na PSTrace don daidaitawar potentiostat da kuma samun bayanai, gami da ƙididdigar ƙididdiga na yanzu. Ana amfani da saitunan masu zuwa. don ma'aunin DPV da CV:
DPV: Lokacin Daidaitawa = \ (8 \, \ hbox {s} \), Matsayin ƙarfin lantarki = (3 \, \ hbox {mV} \), Pulse Voltage = \ (25 \, \ hbox {mV} \) , pulse duration = \ (50 \, \ hbox {ms} \), duban adadin = \ ({20} \, \ hbox {mV/s} \)
CV: Lokacin Ma'auni = \ (8 \, \ hbox {s} \), Matsayin ƙarfin lantarki = (3 \, \ hbox {mV} \), Ƙimar Sweep = \ ({300} \, \ hbox {mV/ s) }\)
Kololuwar igiyoyin da aka samu daga voltammogram na DNA da aka haɗe da \({50}\,{\upmu \hbox {M}}\) MB: (a) \(503\,\hbox {bp}\) DPV , (b) \ (503 \, \ hbox {bp} \) CV, (c) \ (117 \, \ hbox {bp} \) DPV, (d) \ (117 \, \ hbox {bp} \) CV.
DPV da CV voltammograms an samu a kan ENIG PCB electrodes \({50}\,{\upmu \ hbox {M}}\) MB hadaddun da DNA (a taro na 10–\({20}\,{\ hbox {ng) }/{\upmu \hbox {l}}} \) wato 0.13–\({0.26}\,{\upmu \hbox {M}} -\({0.06}\,{\upmu \hbox {M}}\) na \(503\,\hbox {bp}\))) .Wakilin voltammograms ana nuna su a Hoto S1 a Ƙarin Bayani. Hoto na 2 yana nuna sakamakon. na ma'auni na DPV da CV (kololuwar halin yanzu) ta amfani da samfuran PCR masu tsarkake gel. Idan aka kwatanta da ma'aunin CV, ma'aunin DPV yana nuna mafi girman hankali (a halin yanzu a matsayin aikin maida hankali na DNA) saboda bayanan capacitive na baya a cikin ma'aunin CV yana ɓoye ma'aunin Faradaic 26. Bayanan bayanan ga kowane akwati a cikin akwatin akwatin ya ƙunshi ma'auni daga na'urorin lantarki na 5. Duk ma'auni suna amfani da saitin na'urorin lantarki guda ɗaya don kauce wa kuskuren ma'auni saboda bambancin electrode-to-electrode. Mun lura da karuwa a cikin DPV da CV auna ma'auni mafi girma don ƙananan ƙididdiga na DNA. , tsayi (\ (503 \, \ hbox {bp} \)) \, \ hbox {bp} \ idan aka kwatanta da \ (117) ) guntu. Wannan ya dace da yanayin da ake tsammani na adsorption electrode da aka ruwaito a cikin aikinmu na baya. adsorption na MB-DNA hadaddun yana sauƙaƙe canja wurin cajin akan electrode, wanda ke taimakawa wajen haɓaka mafi girma a halin yanzu.Wasu nazarin sun nuna tasirin oligonucleotide size da jerin akan MB-DNA intercalation27,28,29,30.The guanine. -cytosine (GC) abun ciki na amplicons guda biyu (\ (117 \, \ hbox {bp} \) da \ (503 \, \ hbox {bp} \)) ya kasance kusan 50%, yana nuna cewa lura Bambanci ya faru. zuwa tsayin amplicon. Duk da haka, don mafi girman adadin DNA (\(>{2}\,{\hbox {ng}/{\upmu \hbox {l}}}\), don \(503\,\hbox {bp} \) da \(>{10}\,{\hbox {ng}/{\upmu \hbox {l}}}\) na \(117\,\hbox {bp}\)), mun lura da karawa biyu An rage kololuwar magudanar ruwa a duka ma'aunin DPV da CV. Wannan saboda MB ya cika kuma yana yin tsaka-tsaki tsakanin nau'ikan DNA na tushe, wanda ya haifar da hanawa mai tsauri na ayyukan redox na rukunin da za a iya ragewa a cikin 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.
Gishiri da ke cikin haɗe-haɗe na babban PCR suna tsoma baki tare da hulɗar lantarki tsakanin MB da DNA, don haka ta ƙara \(2 \, \ hbox {mM} \) \ (\ hbox {MgCl }_2 \) tare da \ ({50} \,{\) upmu \hbox {M}}\) Samfurin da aka tsarkake na MB don nazarin tasirin gishiri akan hulɗar MB-DNA. Kamar yadda aka nuna a cikin Hoto na 3, mun lura cewa don yawan adadin DNA (\(>{2}\,{\) hbox {ng}/{\upmu \hbox {l}}}(503\,\hbox {bp}\) da \(>{10}\,{\hbox {ng}/{\upmu \hbox { l}}} \) na \ (117 \, \ hbox {bp} \)), a cikin DPV da CV Ƙarin gishiri bai shafi ma'auni ba (duba Hoto S2 a Ƙarin Bayani don wakilin voltammograms).Duk da haka, a ƙananan ƙwayoyin DNA, ƙari na gishiri yana rage yawan hankali, yana haifar da wani canji mai mahimmanci a halin yanzu tare da maida hankali na DNA. Irin wannan mummunan tasiri na gishiri akan hulɗar MB-DNA da haɗin kai an riga an ruwaito ta wasu masu bincike33,34. \ (\ hbox { Mg}^{2+}\) cations suna ɗaure ga kashin baya na phosphate mara kyau na DNA, don haka yana hana hulɗar electrostatic tsakanin MB da DNA. Kada ku yi tasiri sosai kan amsawar firikwensin. Babban mahimmin batu shine wannan biosensor ya fi dacewa don gano mafi girman adadin DNA (da wuya \({\hbox {ng}/{\upmu \hbox {l}}}) ko mafi girma), don cikakken sarrafawa ta atomatik na samfuran ruwan muhalli, inda tsabtace gel na samfuran PCR bazai yuwu ba.
Wurin da ke ƙarƙashin madaidaicin sha don kewayon tsayin raƙuman ruwa 600-700 \(\hbox {nm} ) \(503\,\hbox {bp}\) tare da gishiri (\(2\,\hbox {mM}\) \(\hbox {MgCl}_2 \)), (b) \( 117\, = hbox {bp} {\upmu \ hbox {l}}}
Don ƙara tabbatar da sakamakon da ke sama, mun yi ma'aunin gani ta amfani da UV/Vis spectrophotometer (Thermo Scientific Multiskan GO), samfuran \({50}\,{{\upmu \ hbox {l}}}) an yi amfani da su ga kowannensu. Measurement.Sa hannu na sha yana raguwa tare da ƙara yawan tattarawar DNA, kamar yadda ake iya gani daga yanayin yankin da ke ƙarƙashin lanƙwan sha don kewayon tsayi \ (600 \, \ hbox {nm} \) zuwa \ (700 \, \ hbox {) nm} \) , kamar yadda aka nuna a cikin hoto 4 (shafi bakan da aka nuna a cikin siffa S3 a cikin Ƙarin Bayani).Don samfurori tare da ƙididdigar DNA ƙasa da \ ({1}\, {\hbox {ng}/{\upmu \ hbox {l}}}\), babu wani gagarumin bambanci a cikin ɗauka tsakanin samfuran da ke ɗauke da DNA da na MB-kawai (na \(503\,\hbox {bp}\) da \(117\,\hbox {bp}\) ) tsayin gutsuttsura), yana nuna rashin ƙarancin hanawa na redox-active MB.A mafi girma yawan adadin DNA, mun lura da raguwa a hankali a cikin siginar sha kuma mun lura da raguwar raguwa a gaban gishiri. Wadannan sakamakon an danganta su ga kwayoyin halitta. mu'amala da steric hanawa tare da tushe stacking a cikin DNA hybrids.Sakamakonmu sun yi daidai da rahotanni a cikin wallafe-wallafe game da nazarin spectroscopic na MB-DNA intercalation wanda ke danganta hypochromaticity tare da rage yawan makamashi a cikin \ (\ pi \) - \ (\ pi ^ * \ ) Canje-canje na lantarki saboda haɗin kai Layers 36, 37, 38.
Agarose gel electrophoresis na phage Phi6: PCR kayayyakin tsawon \ (117 \, \ hbox {bp} \) da \ (503 \, \ hbox {bp} \) daga lake ruwa samfurori.M-DNA alama;NTC-no-template iko, na'urorin da ke dauke da amplicons masu dacewa;PC tabbatacce iko;1, 2, 3-undiluted (1:1) samfuran ruwan tafkin spiked a cikin nau'i uku. Ana iya ganin bandeji a \ (\ about 50 \, \ hbox {bp} \) saboda rashin amfani da oligonucleotides a cikin \(503\,\) hbox {bp}\) layi.
Mun kimanta amfanin na'urar firikwensin ta amfani da samfuran ruwan tafkin Powai wanda aka spiked tare da Phi6 phage. Yawan RNA da ke ware daga samfuran ruwan phage-spiked sun fito ne daga 15.8- \ ({19.4} \,{\upmu \ hbox {g}/\hbox) ml}}\), yayin da waɗanda aka keɓe daga tsararren tsararren phage An ƙiyasta RNA su zama \({1945}\,{\upmu \hbox {g}/hbox {ml}}\) tare da ingantaccen farfadowa na kusan 1 % RNA an juya baya cikin cDNA kuma an yi amfani da shi azaman samfuri don PCR da qPCR. An tabbatar da girman samfurin ta hanyar agarose gel electrophoresis (Hoto 5) kafin a gwada tare da firikwensin. Waɗannan samfuran ba su da tsabtace gel kuma saboda haka sun ƙunshi duk abubuwan PCR kamar da amplicons na sha'awa.The Ct dabi'u da aka rikodi a lokacin qPCR (Table 1) an nuna su daidaita tare da maida hankali na RNA ware daga daidai spiked ruwa samfurori.The Ct darajar ya bayyana adadin hawan keke da ake bukata domin mai kyalli siginar zuwa. wuce madaidaicin kofa ko siginar baya.Mafi girma Ct dabi'u suna nuna ƙananan ƙididdigar samfuri da akasin haka. Ƙimar Ct na samfuran NTC sun kasance kamar yadda ake tsammani. Bambanci a cikin \ (\ kimanin 3 \) Ct dabi'u tsakanin ingantaccen iko da samfurin gwajin ya kara nuna cewa kowane samfurin gwajin yana da kusan 1% samfuri idan aka kwatanta da ingantaccen iko. Mun tattauna a baya cewa amplicons masu tsayi suna haifar da mafi kyawun hankali.Amplification na guntu mai tsayi wanda aka ware daga samfuran muhalli iri-iri yana da kalubale idan aka ba da rashin amfani. Duk da haka, tare da haɓaka ƙwayoyin cuta da ƙa'idodin haɓakawa na PCR, mun sami nasarar haɓaka guntun \(503 \, \ hbox {bp} \) don ganowa na lantarki.
Hoto na 6 yana nuna sakamakon firikwensin lantarki na \(503 \, \ hbox {bp} \) amplicon guntu, dukansu suna amfani da cDNA da ba a cika su ba azaman samfuri (1:1) da 100-ninka diluted cDNA azaman samfuri (1:100) da aka yi PCR , idan aka kwatanta da NTC da PC (duba Figure S4 a Ƙarin Bayani don wakilcin voltammograms) .Kowane akwati a cikin akwatin akwatin a cikin Hoto 6 ya ƙunshi ma'auni daga samfurori guda uku a 5 electrodes. An yi amfani da na'urorin lantarki guda ɗaya don auna duk samfurori don kauce wa kurakurai saboda electrode. -to-electrode variation.Idan aka kwatanta da ma'auni na CV, ma'aunin DPV yana nuna mafi kyawun ƙuduri don rarrabe gwaji da samfurori na PC daga NTCs saboda, kamar yadda aka ambata a baya, ana ɓoye Faradaic igiyoyin ruwa saboda bayanan capacitive igiyoyin a cikin karshen. Domin tsawon amplicons, mun lura cewa. mummunan iko (NTC) ya haifar da mafi girma CV da DPV kololuwar igiyoyin ruwa dangane da ingantaccen iko, yayin da samfurori masu inganci da marasa daidaituwa sun nuna irin wannan tsayin tsayin kololuwar DPV. ) samfurin gwaji da PC za a iya warware su a fili daga firikwensin firikwensin don samfurin NTC, yayin da ƙuduri na 1: 100 diluted samfurin ba shi da ma'ana. Domin 100-ninka dilution na cDNA, ba mu lura da wani bandeji a lokacin gel electrophoresis. (Hanyoyin da ba a nuna su a cikin Hoto na 5 ba), kuma madaidaicin DPV da CV kololuwar igiyoyin ruwa sun yi kama da waɗanda ake tsammani don NTC. Ana nuna sakamakon guntun \ (117 \, \ hbox {bp} \) a cikin Ƙarin Bayani. sarrafawa ya haifar da amsawar electrochemical daga na'urar firikwensin PCB saboda tallan MB kyauta akan electrode da kuma hulɗar MB tare da oligonucleotide mai ma'auni guda ɗaya.Saboda haka, duk lokacin da aka gwada samfurin, dole ne a gudanar da kulawa mara kyau kuma mafi girman halin yanzu na samfurin gwajin idan aka kwatanta da kololuwar halin yanzu da aka samu ta hanyar sarrafawa mara kyau don cimma ma'aunin bambanci (dangi)39,40 don rarraba samfurin gwajin azaman tabbatacce ko mara kyau.
(a) DPV, da (b) CV kololuwar halin yanzu don gano electrochemical na \(503\,\hbox {bp}\) gutsuttsura a cikin samfuran ruwan tafkin. An auna samfuran gwaji a cikin sau uku kuma idan aka kwatanta da babu sarrafa samfuri (NTC) da tabbatacce controls (PC).
Abubuwan da muka gano sun kwatanta hanyoyi daban-daban da ke shafar aikin firikwensin lantarki don amplicons na tsayi daban-daban don DNA daban-daban, tare da ƙididdiga waɗanda aka tabbatar ta hanyar ma'aunin gani ta hanyar amfani da na'urar gani ta UV/Vis. Abubuwan da muka lura sun nuna fahimtar cewa DNA mai tsayi ya gutsuttsura har zuwa \ (\ kimanin \) \(500 \, \ hbox {bp} \) za a iya gano shi tare da mafi girma hankali kuma cewa kasancewar gishiri a cikin samfurin ba ya da hankali ga DNA maida hankali wanda ke rinjayar mafi girma hankali (da wuya \({\hbox {ng}/{\upmu) \ hbox {l}}} \) da kuma sama). Bugu da ƙari, mun bincika sakamakon nau'ikan samfurori daban-daban, ciki har da amplicon da aka tsarkake da gel tare da kuma ba tare da ƙara gishiri ba, da kuma ƙara samfurin ruwan tafkin a cikin DPV da ma'aunin CV.Mun lura cewa DPV ya ba da mafi kyawun ƙuduri, tun da na baya capacitive halin yanzu yana shafar ma'aunin CV, yana mai da shi ƙasa da hankali.
Ƙirƙirar guntu masu tsayi ya dogara da amincin kwayar cutar kwayar cutar kwayar cutar kwayar cutar ta RNA. Yawancin bincike sun nuna cewa ƙara yawan guntuwar guntuwa ba koyaushe yake da inganci ba saboda lalatawar RNA a cikin mahalli da yuwuwar splicing yayin ware11,41,42,43,44 .Mun lura cewa hanyar PEG-tushen ƙwayar cuta ta kasance mafi tasiri a cikin mayar da hankali ga phage Phi-6 spiked a cikin ruwan tafkin ruwa fiye da aluminum hydroxide-tushen ƙwayar cuta hanya.The ikon gano dogon DNA gutsuttsura tabbatar da shawo kan da ake bukata domin multiplex PCR. don haɓaka samfuran gajerun tsayi da yawa da rage yuwuwar ƙayyadaddun giciye.
Samfuran halittu ba su da yawa, don haka akwai buƙatar ƙira biosensor wanda ke buƙatar samfura kaɗan don gwaji. Na'urorin lantarki na ENIG PCB da aka yi amfani da su a cikin wannan binciken kawai suna buƙatar \({5}\,{{\upmu \hbox {l}}}\) ) samfurori don gwaji don rufe tasiri mai tasiri na electrodes. Bugu da ƙari, za'a iya sake amfani da wutar lantarki guda ɗaya bayan tsaftacewa kafin a ba da samfurin na gaba. kuma ana amfani da sinadarai da yawa.Tunda kowane lantarki yana kashe kimanin $0.55 (ko INR 40) don kera, wannan biosensor na iya zama madadin farashi mai tsada ga fasahar gano data kasance.Table 2 yana nuna kwatancen wannan aikin tare da sauran na'urori masu auna firikwensin da aka ruwaito a cikin wallafe-wallafe na dogon lokaci. Gutsun DNA a cikin samfurori iri-iri.
Ganin cewa ka'idodin gano electrochemical na tushen MB sun dogara da ƙayyadaddun PCR, babban ƙayyadaddun wannan hanya shine yuwuwar haɓakawa mara iyaka a cikin samfuran iri-iri kamar ruwan sharar ruwa da ruwan tafkin ko amfani da ƙa'idodi masu tsafta.Don haɓaka hankali na Hanyoyin gano electrochemical don gano DNA na samfuran PCR da ba a tsarkake ba ta amfani da na'urorin lantarki na ENIG PCB da ba a canza su ba, yana da mahimmanci don fahimtar kurakurai da aka gabatar ta hanyar dNTPs da abubuwan da ba a yi amfani da su ba, kuma don inganta yanayin amsawa da ka'idojin ƙididdiga.Ƙarin sigogi na physicochemical kamar pH, zafin jiki, da nazarin halittu. buƙatar iskar oxygen (BOD) na samfurin ruwa na iya buƙatar auna shi don inganta daidaiton ma'aunin.
A ƙarshe, muna ba da shawarar wani firikwensin ENIG PCB mai ƙarancin farashi don gano ƙwayoyin cuta a cikin samfuran muhalli (lake ruwa) samfuran.Ba kamar na'urorin lantarki na oligonucleotide da ba a daidaita su ba ko abubuwan da aka saba amfani da su don fahimtar DNA wanda ke buƙatar ajiyar ajiyar cryogenic don kula da hankali, 53,54 dabararmu tana amfani da PCB mara kyau. na'urorin lantarki tare da tsawon rai mai tsayi kuma babu takamaiman buƙatun ajiya don haka dace da haɓaka hanyoyin aunawa tare da sarrafa samfurin sarrafa kansa da aka tura a cikin LMICs.The biosensor yana amfani da dyes DNA mai tsada mara tsada (MB) don saurin gano amplicons.The nonspecific amplification. na kowa a cikin samfurori na muhalli yana rage ƙayyadaddun wannan hanyar ganewa saboda rashin ƙayyadaddun ɗaurin MBs zuwa oligonucleotides guda ɗaya da sau biyu.Saboda haka, ƙayyadaddun wannan gwajin ya dogara ne akan ingantawa na firamare da yanayin halayen PCR. Bugu da ƙari, CV ɗin. da DPV kololuwar kololuwar da aka samu daga samfuran da aka gwada ya kamata a fassara su dangane da martanin da aka samu daga iko mara kyau (NTC) don kowane gwaji. Za a iya haɗawa da ƙirar firikwensin firikwensin lantarki da hanyoyin da aka gabatar a cikin wannan aikin tare da autosamplers don haɓaka cikakken sarrafa kansa da ƙasa. Maganin tsadar kuɗi wanda zai iya tattarawa da bincika samfurori da kuma watsa sakamakon mara waya zuwa dakin gwaje-gwaje.
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Lokacin aikawa: Mayu-27-2022