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Ua mahalo nui ʻia ke koʻikoʻi o ka nānā ʻana i nā laʻana kaiapuni mai ka hoʻomaka ʻana o ka maʻi maʻi COVID-19, a ke hana ʻia nei kekahi mau hana kiaʻi ʻana me ka hoʻohana ʻana i ke kūlana gula, ʻoiai he kumukūʻai nā ʻano hana qPCR. ka hopena no ka nānā ʻana i nā laʻana wai kaiapuni ma nā ʻāina haʻahaʻa a me waena. e hoʻopau i nā electrodes PCB me ka ʻole o ka hoʻololi ʻana i ka ʻili.sex. Ua hōʻike pono ʻia ka pane ʻike electrochemical no ʻelua mau ʻāpana DNA o nā lōʻihi like ʻole (\({117}\,\hbox {bp}\) a me \({503}\,\hbox {bp}\)), a me ka ʻO ka hopena o nā paʻakai ma PCR master mixes ma ka methylene blue (MB) -DNA interactions.ʻO kā mākou hopena e hōʻike ana i ka lōʻihi o nā'āpana DNA e hoʻoholo nui i kaʻike electrochemical a hōʻike i kēia hana i ka hiki keʻike i nā amplicons lōʻihi me ka hoʻomaʻemaʻe gel o nā huahana PCR. mea nui no ke ana in situ o na laana wai.ʻO kahi hopena automate piha no ka viral load bodes maikaʻi.
Ua ʻike ʻia ka hoʻoili ʻia ʻana o ka maʻi maʻi wai ma ke ʻano he pilikia olakino lehulehu mai ka makahiki 1940, me ka hōʻike mua o ka lawe ʻia ʻana o ka maʻi polio a me ka maʻi hepa E1. ʻO nā ʻano hana ʻike e hilinaʻi i nā ʻenehana maʻamau qPCR-maʻamau, he ʻano koʻikoʻi a kikoʻī, akā koi i nā limahana akamai e hoʻāʻo i loko o ke keʻena hana me ka hoʻohana ʻana i nā mea waiwai nui. ʻO ka hoʻāʻo ʻana ka laʻana ma mua o ka nānā ʻana i ka wai kaiapuni. no laila e pale ana iā lākou mai nā hopena socioeconomic koʻikoʻi o ka maʻi maʻi. ka ʻili a me ka hybrid i ka wā e loaʻa ai kahi kaʻina like i loko o ka hāpana. Hiki ke hoʻololi ʻia kēia i hōʻailona e nā ʻenehana electrochemical like ʻole me ka hoʻohana ʻana i nā mediators redox e like me potassium iron/ferrocyanide. ua hōʻike ʻia e intercalate i loko o ka DNA stranded ʻelua (dsDNA) me ka hoʻohui ʻana i kāna mea paʻa ʻole i ka DNA5,6. sensor configurations5,6,7,8,9.ʻOiai ka intercalation o MB i DNA mea ole, a me ka specificity o keia electrochemical sensor hilinaʻi nui ma luna o ka maemae o ka primers hoʻohana no ka PCR a isothermal amplification, ua kūpono no ka hoʻokō maoli. -time electrochemical-based qPCR a i ʻole fluorescence isothermal amplification ma ke ʻano he ʻokoʻa i ke ana ʻana o ka DNA 9 . Ma kekahi o ia hoʻokō, Won et al. Ua hoʻololi ʻia ka ʻili o nā electrodes gula me 6-mercapto-1-hexanol (MCH) no ka manawa maoli. ana o ka PCR amplicons me MB me ka hoohana ana i ka differential pulse voltammetry (DPV) 9. Ma na mea e ae, Ramirez et al. Ka ike ana o SARS-CoV-2 i ka wai hoino e RT-LAMP pane me ka MB me ka pale-pa'i electrodes.Platinum electrodes ua hoi. hoʻohana 'ia e like me in situ electrodes i loko o ka microfluidic PCR anuu i hoʻolālā 'ia e electrochemically ike amplicons i ka manawa reactions 8. A pau o keia mau haʻawina koi ili hoʻololi 'ana o ka electrodes, implying hoʻonui hana a me ka hana koina ma muli o kūikawā mālama pono no ka paʻa o keia functionalized electrodes.
Schematic o ke kaʻina hana no ka ʻike electrochemical o nā amplicons i loaʻa mai nā ʻāpana viral i hoʻopaʻa ʻia i loko o nā lake wai.
Ua hōʻike hou mākou i ka ʻike electrochemical o nā amplicons SARS-CoV-2 me nā electrodes papa kaiapuni paʻi haʻahaʻa (PCB) e pili ana i ka DPV a me ka voltammetry cyclic (CV) i hoʻoulu ʻia e ka adsorption o nā paʻakikī MB-DNA ma ka ʻili o nā electrodes i hoʻololi ʻole ʻia) i nā loli i ka piko. i kēia manawa11.Hōʻike mākou ua hoʻokumu ʻia nā ʻāpana DNA lōʻihi (N1-N2, \({943}\, \hbox) me ka hoʻohana ʻana i ka N1 i manaʻo ʻia e CDC i mua a me N2 hoʻohuli i ka hoʻohālikelike ʻia me nā ʻāpana pōkole {bp}\)) hōʻike i ka linearity ʻoi aku ka maikaʻi o ka pane sensor. (N1, \(72\,\hbox {bp}\)) i hoʻokumu ʻia me ka hoʻohana ʻana i ka N1 i mua a me N1 reverse primer sets. Hōʻike ʻia kēia mau haʻawina me ka hoʻohana ʻana i nā dilutions DNA i hoʻomākaukau ʻia i ka wai nuclease-free. -2 amplicons i simulated wastewater samples (loaʻa ma ka spiking total RNA samples with SARS-CoV-2 RNA). No ka mea hiki ke RNA i ka shearing i ka wā kaʻawale a me ka hana ʻana i lalo, 12,13 paʻakikī ke hoʻonui i nā ʻāpana lōʻihi me kēia hāpana heterogeneous. No laila, ua kaupalena ʻia ka hōʻike ʻana o ka electrochemical sensing o SARS-CoV-2 amplicon i ka wai ʻino i ka ʻāpana pōkole \(72\,\hbox {bp}\) N1.
Ma kēia hana, ua noiʻi mākou i ka hiki o ka ENIG PCB-based electrochemical sensing of phage Phi6 concentrated and isolated from lake water samples (Fig. 1). Phi6 phages are comparable in size (80-100 nm) to SARS-CoV-2 and loaʻa pū kekahi membrane lipid a me kahi protein spike. No kēia mau kumu, ʻo ka bacteriophage Phi6 kahi mea kūʻai kaulana no SARS-CoV-2 a me nā maʻi RNA pathogenic ʻē aʻe i hoʻopuni ʻia14,15. Ua hoʻohana ʻia ka RNA i hoʻokaʻawale ʻia mai nā ʻāpana phage ma ke ʻano he kumu hoʻohālike no ka cDNA synthesis i ukali ʻia e PCR no ka loaʻa ʻana o ʻelua mau ʻāpana DNA o 117 a me 503 mau ʻāpana kumu i ka lōʻihi. Ma muli o ka paʻakikī o ka hoʻonui ʻana i ka \(943\,\hbox {bp}\) N1-N2 mau ʻāpana i kā mākou hana mua, ke kuhikuhi nei mākou i nā ʻāpana lōʻihi waena (\(117). \,\hbox {bp}\) a me \(503 \,\hbox {bp}\)), ma muli o nā kumu mua loaʻa. \hbox {pg}/{\upmu \hbox {l}}}\) i \({20}\, {\hbox {ng}/{\upmu \hbox {l}}}\)) No nā ʻāpana ʻelua ma ʻO ka loaʻa ʻana o MB, ua ʻike ʻia ka hopena o ka paʻakai ma ka pane sensor a ua hōʻoia ʻia e nā ana spectrophotometric. ʻO nā haʻawina nui o kēia hana penei:
ʻO ka lōʻihi o ka ʻāpana DNA a me ka loaʻa ʻana o ka paʻakai i loko o ka hāpana pili ikaika i ka naʻau.Hōʻike kā mākou mau hopena e pili ana ka hana electrochemical i nā ʻano hana like ʻole o ka launa pū ʻana o MB, DNA, a me ka sensor i ka pane voltammetric, ma muli o ka ʻike DNA a me ka lōʻihi, me ka lōʻihi o nā Fragments e hōʻike ana i ka naʻau kiʻekiʻe, ʻoiai he hopena maikaʻi ʻole ka paʻakai i nā pilina electrostatic ma waena o MB a me DNA.
Hoʻoholo ka ʻike DNA i ke ʻano o ka pilina MB-DNA i nā electrodes i hoʻololi ʻole ʻia. {l}}}\), ua ʻike mākou ua hoʻoholo nui ʻia ka pane electrochemical i kēia manawa e ka adsorption o MB-DNA ma ka electrode, ʻoiai ma nā haʻahaʻa haʻahaʻa Ma nā kiʻekiʻe DNA kiʻekiʻe, ua hoʻoholo ʻia ka pane electrochemical e ka steric inhibition o redox. ka hana ma muli o ka hoʻokomo ʻana o MB ma waena o nā pālua kumu DNA.
ENIG PCB-Based Electrochemical Sensing of Viral Nucleic Acids in Lake Water Samples Ua hōʻoia ʻia nā ʻike e ka electrochemical detection o Phi6-added \(503\,\hbox {bp}\) nā ʻāpana DNA i loaʻa mai nā ʻano wai mai Powai Lake, IIT Mumbai Campus Ka hopena hopena.
ʻO ke kumu kūʻai haʻahaʻa o ka hoʻokō a me ka hiki ke hoʻohui i nā ʻōnaehana mākaʻikaʻi piha piha, oligonucleotides a i ʻole aptamers ma nā electrodes me ka lōʻihi o ke ola.
ʻO Phage Phi6 kahi maʻi dsRNA i hoʻopuni ʻia o ka ʻohana Cytoviridae e hoʻopilikia i ka Pseudomonas syringae. Aia ka genome o Phi6 phage ma ke ʻano o 3 mau ʻāpana: S (\(2.95\,\hbox {Kb}\)), M (\(4.07) \,\hbox {Kb}\)) a me L (\ (6.37\ ,\hbox{Kb}\)) 16,17. No ka mea, hoʻopili ka Phi6 phage i kahi maʻi BSL-1 Pseudomonas strain non-pathogenic, palekana ke hoʻohana. a hiki ke ulu maʻalahi i loko o ka hale hana. Ua kūʻai ʻia ʻo Phage Phi6 a me kāna mea hoʻokipa ʻo Pseudomonas syringae mai ka Felix d'Herelle Reference Center for Bacterial Viruses, Laval University, Kanada (nā helu helu helu kikowaena HER-102 a me HER-1102, kēlā me kēia) .Phi6 phage a me kona pū'ali i ho'ōla hou 'ia e like me ke kuhikuhi 'ana e ka reference center. Phage Phi6 ua ho'oma'ema'e 'ia e ka plate lysis a me ka elution no ka loa'a 'ana o nā titers hope me \(\ma kahi o 10^{12}\,{\hbox {PFU}/\hbox { M 100}\, {{\upmu \hbox {l}}}\) a ua hooiliia ka lysate ma luna o ke kolamu milo e hiki ai i ka RNA ke hoopaa i ke kolamu resin. 50}\,{{\upmu \hbox {l}}}\) i haawiia mai e ke kit. E koho i ke ku ana o ka RNA ma o ka absorbance ma \(260\,\hbox {nm}\).Ua malamaia ka RNA iloko o na aliquots ma \ ({-80}\,{^{\circ }\hbox {C}}\) a hiki i ka hoohana hou ana.\({2}\,{\upmu \hbox {g}}\) Ka iScript cDNA Synthesis Kit (Bio -Rad Laboratories) ua hoʻohana ʻia ma ke ʻano he kumu hoʻohālike no ka cDNA synthesis ma muli o nā ʻōlelo a ka mea hana. )\({5}\,{\hbox {min} }\) , ka unuhi huli ana o \({20}\,{\hbox {min}}\) ma \({46}\,{^{\circ }\hbox {C}}\), a huli hope Aia ka mea kakau ma \({95}\,{^{\circ }\hbox {C}}\) no \({1}\,{\hbox {min. }}\).I ka holo ana ma ka 1% agarose gel, ua hoike mai ka cDNA i ekolu mau bana e pili ana i ekolu mau apana RNA i manaoia (i hoike ole ia ka ikepili). e hoʻohana ana i ka cDNA ma ke ʻano he laʻana no ka PCR i loko o kahi miniPCR® mini8 thermal cycler:
ʻO nā kumu mua no \(117\,\hbox {bp}\) a me \(503\,\hbox {bp}\) pili i nā nucleotides 1476-1575 o ka māhele M a me 458-943 nucleotides o ka māhele L, ka waika. .Ua ho'ohana 'ia nā huahana PCR i ho'onui 'ia ma ka 1% agarose gels, a ua ho'oma'ema'e 'ia ka DNA target ma ka ho'ohana 'ana i ka GeneJET Gel Extraction Kit (Thermo Fisher Scientific).
Ua hoʻohana ʻia kahi loko ma ka pā kula ʻo IIT Mumbai (Powai Lake, Powai, Mumbai) e hoʻohui i nā ʻāpana phage. nā ʻāpana i hoʻokuʻu ʻia, a laila hoʻohui ʻia ka Phi6 phage. Hoʻohui i ka \({1}\,{\hbox {ml}}\) o \(10^{6}\,{\hbox {PFU}/\hbox {ml}} \) i \({100}\ ,{\hbox {ml}}\) wai loko i kānana, ma \({4}\,{^{\circle}\hbox {C}}\). Ua hoʻāʻo mākou i ʻelua ʻano hana like ʻole e hoʻokaʻawale i nā ʻāpana virus Phi6: (1) ke ʻano alumini hydroxide adsorption-precipitation, 19 i hōʻoia ʻia no ka hoʻopaʻa ʻana o nā maʻi virus RNA i hoʻopuni ʻia mai nā laʻana kaiapuni, a (2) ) Ua hoʻololi ʻia ke ʻano o ka hoʻopaʻa ʻana o ka maʻi maʻi polyethylene glycol (PEG) mai Flood et al.20 .Ma muli o ka ʻoi aku o ka maikaʻi o ka hoʻihoʻi ʻana o ke ʻano PEG ma mua o ke ʻano o ka alumini hydroxide, ua hoʻohana ʻia ke ʻano hana PEG e hoʻopili i nā ʻāpana Phi6 mai nā lake wai.
ʻO ke ʻano hana PEG i hoʻohana ʻia penei: PEG 8000 a me \(\hbox {NaCl}\) i hoʻohui ʻia i nā lake wai loko Phi6-spiked no ka loaʻa ʻana o 8 % PEG 8000 a me \(0.2\,\hbox {M} \) \( \ hbox {NaCl}\).Hoʻomoʻa ʻia nā laʻana ma luna o kahi mea lulu\({4}\,{^{\circ }\hbox {C}}\)\({4}\,{\hbox {h}}\ ), a laila centrifuged ma \(4700 \,\hbox {g}\) ʻo \({45}\,{\hbox {min}}\). {\hbox {ml}}\) i loko o ka supernatant hoʻokahi. Ua hana ʻia nā hoʻokolohua spiking a me nā maʻi hoʻomaʻamaʻa hoʻokolohua ʻekolu. i loko o ke kit-supplied elution buffer\({40}\,{\upmu \hbox {l}}\).No ka mea, e okoa ana ka manao o ka RNA mai kekahi la'ana i ka la'ana ma ka papakolu, ka \({2}\,{\upmu \ Hoʻohana ʻia ka hbox {l}}\) o ka RNA no nā mea ʻekolu me ka nānā ʻole i kona ʻano cDNA synthesis o nā laʻana. ua hoʻohana ʻia ma ke ʻano he kumu hoʻohālike no \({20}\,{\upmu \hbox {l}}\) PCR no 35 pōʻaiapuni e hoʻonui i ka \ (117\,\hbox {bp}\) a me \(503\,\hbox { bp}\) ʻāpana. Ua hōʻike ʻia kēia mau laʻana ma ke ʻano he "1: 1", ʻo ia hoʻi me ka hoʻoheheʻe ʻole ʻia. ma ke ʻano he laʻana no ka mana maikaʻi (PC). Ua hana ʻia ka Quantitative PCR (qPCR) i kahi mea kani Stratagene Mx3000P RT-PCR me ka hoʻohana ʻana iā Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies). i wehewehe ʻia. Ua hoʻopaʻa ʻia ka paepae pōʻai (Ct) no nā laʻana a pau. \({20}\,{\upmu \hbox {l}}\) PCR no 35 mau pōʻaiapuni. Hōʻike ʻia kēia mau laʻana ma ke ʻano he “1:100″.
Hoʻokumu ʻia nā electrodes PCB me ka hoʻohana ʻana i kahi kaʻina kūʻai haʻahaʻa Electroless Nickel Immersion Gold (ENIG) me ka ʻole o ka pono o ka plating gula hou. ʻAʻole ʻōlelo ʻia ka solution piranha a i ʻole sulfuric acid cyclic voltammetry no ka mea hiki iā lākou ke hana i ka ʻili ʻana o ka ʻāpana gula lahilahi (ka mānoanoa \(\approx\) \(100\,\hbox {nm }\)) a e hōʻike i nā ʻāpana keleawe i lalo. i ka corrosion 21, 22, 23, 24, 25. No laila, e hoʻomaʻemaʻe i nā electrodes me kahi lole lint-free i hoʻomaʻemaʻe ʻia me ka IPA. ʻO ka hāpana e hoʻāʻo ʻia ua hoʻomoʻa ʻia me ka \({50}\,{\upmu \hbox {M} }\) MB i \({4}\,{^{\circ }\hbox {C}}\)\({ 1}\,{\hbox {h}}\) no ka hookomo ma'alahi. Ma kā mākou hana mua , ua ʻike mākou ua hoʻomaikaʻi ʻia ka sensitivity a me ka linearity o ka sensor ma o ka hoʻonui ʻana i ka manaʻo MB 11. Ma muli o nā optimizations i hōʻike ʻia ma kā mākou hana mua, ua hoʻohana mākou i \({50}\,{\upmu \hbox {M}}\) MB Hiki ke hoʻokō ʻia ka ʻike electrochemical o ka DNA stranded pālua (ds-DNA) me ka hoʻohana ʻana i ka anionic a i ʻole ka cationic intercalators. ʻO ka lima ʻē aʻe, nā intercalators cationic e like me MB makemake i nā manawa incubation pōkole, ma kahi o \({1}\,{\ hbox {h}}\) no ka ʻike electrochemical o ds-DNA6. ʻO kēlā me kēia ana e pili ana i ka hāʻawi ʻana i ka hāpana e hoʻāʻo ʻia ma ka electrode\({5}\,{{\upmu \hbox {l}}}\), a laila e hoʻomaʻemaʻe me ka lole IPA-moistened, ma mua o ka hoʻomaka ʻana me kekahi laʻana.Hoʻokahi ana. Ua hoʻāʻo ʻia kēlā me kēia laʻana ma 5 mau electrodes ʻokoʻa ke ʻole i ʻōlelo ʻia. Ua hana ʻia nā ana DPV a me CV me ka hoʻohana ʻana i ka PalmSens Sensit Smart potentiostat, a ua hoʻohana ʻia ka polokalamu PSTrace no ka hoʻonohonoho potentiostat a me ka loaʻa ʻana o ka ʻikepili, me nā helu helu kiʻekiʻe. no nā ana DPV a me CV:
DPV: Manawa Kaulike = \(8\,\hbox {s}\), Voltage Step = \(3\,\hbox {mV}\), Pulse Voltage = \(25\,\hbox {mV}\) , ka lōʻihi o ka pulupulu = \(50\,\hbox {ms}\), ka helu scan = \({20}\,\hbox {mV/s}\)
CV: Manawa Kaulike = \(8\,\hbox {s}\), Voltage Steps = \(3\,\hbox {mV}\), Sweep Rate = \({300}\,\hbox {mV/s }\)
Loaʻa nā ʻauwai kiʻekiʻe mai nā voltammograms o DNA paʻakikī me \({50}\,{\upmu \hbox {M}}\) MB: (a) \(503\,\hbox {bp}\) DPV , (b) \ (503\,\hbox {bp}\) CV, (c) \(117\,\hbox {bp}\) DPV, (d) \(117\,\hbox {bp}\) CV.
Ua loaʻa nā DPV a me CV voltammograms ma ENIG PCB electrodes \({50}\,{\upmu \hbox {M}}\) MB paʻakikī me DNA (ma nā hoʻopaʻa ʻana o 10–\({20}\,{\ hbox {ng). }/{\upmu \hbox {l}}}\) ie 0.13–\({0.26}\,{\upmu \hbox {M}}\) no \(117\,\hbox {bp}\ ) a me 0.03 –\({0.06}\,{\upmu \hbox {M}}\) no \(503\,\hbox {bp}\)). Hōʻike ʻia nā voltammograms lunamakaainana ma ke Kiʻi S1 ma ka ʻike hou. o nā ana DPV a me CV (peak current) me ka hoʻohana ʻana i nā huahana PCR i hoʻomaʻemaʻe ʻia i ka gel. Ke hoʻohālikelike ʻia me nā ana CV, hōʻike nā ana DPV i ke ʻano kiʻekiʻe o ka naʻau (i kēia manawa ma ke ʻano he hana o ka manaʻo DNA) no ka mea ʻo ke kāʻei capacitive hope i nā ana CV hūnā i nā au Faradaic 26 . ʻO ka ʻikepili no kēlā me kēia pahu i loko o ka boxplot nā ana mai nā electrodes 5. Hoʻohana nā ana a pau i ka hoʻonohonoho like o nā electrodes e pale i nā hewa ana ma muli o ke ʻano electrode-to-electrode. , lōʻihi (\(503\,\hbox {bp}\)) \,\hbox {bp}\ i hoʻohālikelikeʻia me \(117) ) fragment. Ua kūlike kēia me ka manaʻo i manaʻoʻia o ka electrode adsorption i hōʻikeʻia ma kā mākou hana mua. ʻO ka adsorption o ka MB-DNA complex e hoʻomaʻamaʻa i ka hoʻoili kālā ma ka electrode, kahi e hoʻonui ai i ka piʻi o ka manawa. -cytosine (GC) maʻiʻo o nā amplicons ʻelua (\(117\,\hbox {bp}\) a me \(503\,\hbox {bp}\)) ma kahi o 50%, e hōʻike ana i ka nānā ʻana. i ka lōʻihi amplicon. Akā naʻe, no ka ʻoi aku ka nui o ka DNA (\(>{2}\,{\hbox {ng}/{\upmu \hbox {l}}}\), no \(503\,\hbox {bp} \) a me \( >{10}\,{\hbox {ng}/{\upmu \hbox {l}}}\) no \(117\,\hbox {bp}\)), ike makou i elua hoonui ana. Ua ho'ēmiʻia nā kaha kiʻekiʻe o nā subs ma nā ana DPV a me CV. ʻO kēia no ka MB saturates a intercalates ma waena o nā papa kumu o DNA, e hopena i ka steric inhibition o ka hana redox o ka hui hoʻemi i 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.
ʻO nā paʻakai i loaʻa i loko o ka PCR master mixes ke keʻakeʻa i ka pilina electrostatic ma waena o MB a me DNA, no laila ma ka hoʻohui ʻana i \(2\,\hbox {mM}\) \(\hbox {MgCl }_2\) me \({50} \,{\ upmu \hbox {M}}\) MB gel-hoʻomaʻemaʻe huahana e aʻo i ka hopena o ka paʻakai ma MB-DNA launa pū. E like me ia i hōʻike ʻia ma ke Kiʻi 3, ua ʻike mākou no ka kiʻekiʻe o ka DNA (\(>{2}\,{\ hbox {ng}/{\upmu \hbox {l}}}\) (503\,\hbox {bp }\) a me \(>{10}\,{\hbox {ng}/{\upmu \hbox { l}}}\) no \(117\,\hbox {bp} \)), ma DPV a me CV ʻAʻole i pili nui ka hoʻohui ʻana o ka paʻakai i nā ana (e nānā i ke Kiʻi S2 ma ka ʻIke Hoʻohui no nā voltammograms luna). haʻahaʻa DNA hoʻohaʻahaʻa, ka hoʻohui ʻana o ka paʻakai e hoʻemi nui i ka naʻau, ka hopena ʻaʻole i hoʻololi nui ʻia i kēia manawa me ka ʻike DNA. Ua hōʻike mua ʻia nā hopena maikaʻi ʻole o ka paʻakai ma MB-DNA a me intercalation e nā mea noiʻi ʻē aʻe33,34.\(\hbox { Mg}^{2+}\) hoʻopaʻa ʻia nā cations i ka iwi kuamoʻo phosphate maikaʻi ʻole o DNA, no laila ke keʻakeʻa nei i ka pilina electrostatic ma waena o MB a me DNA. ʻaʻole e pili nui i ka pane ʻana o ka mea ʻike. ʻO ka mea nui, ʻoi aku ka maikaʻi o kēia biosensor no ka ʻike ʻana i nā koina DNA kiʻekiʻe (kakaʻikahi \({\hbox {ng}/{\upmu \hbox {l}}}\) a ʻoi aku paha), no ka hoʻopololei piha ʻana i nā laʻana wai kaiapuni, kahi hiki ʻole ke hoʻomaʻemaʻe gel o nā huahana PCR.
ʻĀpana ma lalo o ka ʻākea absorption no ka laulā hawewe 600–700 \(\hbox {nm}\) no nā ʻano ʻokoʻa like ʻole o ka DNA i paʻakikī me \({50}\,{\upmu \hbox {M}}\) MB: ( a ) \(503\,\hbox {bp}\) me ka paakai ole (\(2\,\hbox {mM}\) \(\hbox {MgCl}_2\)), (b) \( 117\, \hbox {bp}\) me ka paʻakai ʻole (\(2\,\hbox {mM}\) \(\hbox {MgCl}_2\)).\({0}\,{\hbox {pg}/ {\upmu \hbox {l}}}\) Nā hāpana DNA e pili ana i \({50}\,{\upmu \hbox {M}}\) MB samples No DNA.
No ka hōʻoia hou ʻana i nā hualoaʻa ma luna, ua hana mākou i nā ana optical me ka hoʻohana ʻana i kahi spectrophotometer UV/Vis (Thermo Scientific Multiskan GO), ua hoʻohana ʻia nā laʻana \({50}\,{{\upmu \hbox {l}}}\) no kēlā me kēia. Ana. E emi ana ka inoa absorption me ka pi'i 'ana o ka DNA, e like me ka 'ike 'ia mai ke au o ka wahi ma lalo o ka absorption curve no ka loa'a hawewe \(600\,\hbox {nm}\) a i \(700\,\hbox { nm}\) , e like me ka hoike ana ma ka Fig. 4 (absorption spectrum i hoikeia ma ka Fig. S3 ma ka 'Ikepili Ho'ohui). {l}}}\), ʻaʻohe ʻokoʻa koʻikoʻi o ka lawe ʻana ma waena o nā hōʻailona DNA a me MB wale nō (no \(503\,\hbox {bp}\) a me \(117\,\hbox {bp}\ ) nā ʻāpana lōʻihi), e hōʻike ana i ka loaʻa ʻole o ka steric inhibition o redox-active MB. Ma nā kiʻekiʻe DNA kiʻekiʻe, ua ʻike mākou i ka emi iki ʻana o ka hōʻailona absorbance a ʻike i ka emi iki o ka absorbance i mua o ka paʻakai. ka launa pū ʻana a me ka steric inhibition me ka hoʻopaʻa ʻana i ke kumu i loko o nā hybrids DNA. Ua kūlike kā mākou hopena me nā hōʻike i loko o ka puke e pili ana i nā haʻawina spectroscopic o MB-DNA intercalation e pili ana i ka hypochromaticity me ka emi ʻana o ka ikehu ma \(\pi\)–\(\pi ^*\ ) nā hoʻololi uila ma muli o ka intercalation Layer 36, 37, 38.
Agarose gel electrophoresis o ka phage Phi6: nā huahana PCR o ka lōʻihi \(117\,\hbox {bp}\) a me \(503\,\hbox {bp}\) mai nā hōʻailona wai loko.M-DNA marker;NTC-no-template control, primers loaʻa nā amplicons pili;PC mana maikaʻi;1, 2, 3-undiluted (1:1) spiked lake water samples in triplicate. A band is ike ma \(\ma 50\,\hbox {bp}\) ma muli o ka hoohana ole ia oligonucleotides i ka \(503\,\ hbox {bp}\) alahele.
Ua loiloi mākou i ka pono o ka mea ʻike me ka hoʻohana ʻana i nā laʻana wai ʻo Powai Lake i hoʻopaʻa ʻia me ka Phi6 phage. ʻO nā ʻāpana RNA i hoʻokaʻawale ʻia mai nā laʻana wai phage-spiked mai 15.8–\({19.4}\,{\upmu \hbox {g}/\hbox { M Ua hoʻololi hou ʻia ka %.RNA i loko o ka cDNA a hoʻohana ʻia ma ke ʻano he laʻana no PCR a me qPCR. Ua hōʻoia ʻia ka nui o ka huahana e ka electrophoresis gel agarose (Figure 5) ma mua o ka hoʻāʻo ʻana me ka sensor. ʻO nā waiwai Ct i hoʻopaʻa ʻia i ka wā qPCR (Table 1) ua hōʻike ʻia e pili pū me ka neʻe ʻana o ka RNA i hoʻokaʻawale ʻia mai nā ʻano wai spiked pili. ʻoi aku ma mua o ka paepae a i ʻole ka hōʻailona hope. ʻO nā koina Ct kiʻekiʻe e hōʻike ana i nā manaʻo haʻahaʻa haʻahaʻa a me ka hope. ʻO ka mana maikaʻi a me ka hōʻike hōʻike hōʻike hou e hōʻike ana kēlā me kēia hōʻike hōʻike ma kahi o 1% template i hoʻohālikelike ʻia i ka mana maikaʻi. Ua kūkākūkā mua mākou i nā amplicons lōʻihi e alakaʻi i ka sensitivity ʻoi aku ka maikaʻi. o ka hoʻohaʻahaʻa haʻahaʻa viral a me ka RNA degradation. Akā naʻe, me kā mākou virus enrichment a me PCR amplification protocol, ua hiki iā mākou ke hoʻonui i ka ʻāpana \(503\,\hbox {bp}\) no ka ʻike electrochemical.
Hōʻike ke Kiʻi 6 i nā hopena ʻike electrochemical o ka ʻāpana amplicon \(503\,\hbox {bp}\), me ka hoʻohana ʻana i ka cDNA undiluted ma ke ʻano he template (1:1) a me 100-fold diluted cDNA ma ke ʻano he template (1:100) i hana ʻia PCR , Hoʻohālikelike ʻia me NTC a me PC (e ʻike i ke Kiʻi S4 i ka ʻike ʻokoʻa no nā voltammograms ʻelele). ʻO kēlā me kēia pahu i ka boxplot ma Figure 6 aia nā ana mai ʻekolu mau mea ma 5 electrodes. -to-electrode variation.Hoʻohālikelike ʻia me nā ana CV, hōʻike nā ana DPV i ka hoʻonā ʻoi aku ka maikaʻi o ka hoʻokaʻawale ʻana i nā hōʻike hoʻāʻo a me nā PC mai nā NTC no ka mea, e like me ka mea i ʻōlelo mua ʻia, ua hūnā ʻia nā au Faradaic ma muli o nā kahawai capacitive hope i ka hope. ʻO ka mana maikaʻi ʻole (NTC) ka hopena i ka CV a me ka DPV peak kiʻekiʻe e pili ana i ka mana maikaʻi, ʻoiai nā hōʻike hoʻāʻo maikaʻi a me ka hoʻāʻo ʻole i hōʻike ʻia i nā kiʻekiʻe kiʻekiʻe o DPV peak currents. ) ho'āʻo laʻana a me ka PC hiki ke maopopo hoʻoholo 'ana mai ka sensor puka no ka NTC hāpana, oiai ka olelo hooholo no ka 1:100 diluted hāpana mea emi pronounced. (ʻaʻole i hōʻike ʻia nā alahele ma ke Kiʻi 5), a ua like nā DPV a me CV pili i nā au kiʻekiʻe me nā mea i manaʻo ʻia no NTC. Hōʻike ʻia nā hopena no ka ʻāpana \(117\,\hbox {bp}\) ma ka ʻIke Hoʻohui. ka mana i hoʻokomo i ka pane electrochemical mai ka PCB sensor ma muli o ka adsorption o ka noa MB ma ka electrode a me ka launa pū o ka MB me ka oligonucleotide primer one-stranded. peak au o ka ho'āʻo hāpana i hoʻohālikelike ʻia me ka peak au i loaʻa e ka mana maikaʻi ʻole e hoʻokō i kahi ana ʻokoʻa (pilina)39,40 e hoʻokaʻawale i ka hāpana hoʻāʻo ma ke ʻano he maikaʻi a maikaʻi ʻole paha.
(a) DPV, a me (b) CV peak current no ka ike electrochemical o \(503\,\hbox {bp}\) mau hakina i loko o ka loko wai laana. nā mana maikaʻi (PC).
Hōʻike kā mākou ʻike i nā ʻano hana like ʻole e pili ana i ka hana ʻana o nā mea ʻike electrochemical no nā amplicons o nā lōʻihi like ʻole no nā DNA like ʻole, me nā ʻike i hōʻoia ʻia e nā ana optical me ka hoʻohana ʻana i kahi spectrophotometer UV/Vis. \(500\,\hbox {bp}\) hiki ke 'ike 'ia me ka 'oi a'e ka 'ike 'ana a 'o ka loa'a 'ana o ka pa'akai i loko o ka hāpana 'a'ole 'o Sensitivity DNA ku'ina e pili ana i ka 'ike ki'eki'e (kaka'ikahi \({\hbox {ng}/{\upmu \hbox {l}}}\) a ma luna aʻe). Eia kekahi, ua noiʻi mākou i ka hopena o nā ʻano laʻana like ʻole, me nā amplicons i hoʻomaʻemaʻe ʻia me ka paʻakai a me ka ʻole o ka paʻakai, a me ka hoʻohui ʻana i nā lake wai i loko o nā ana DPV a me CV.Ua ʻike mākou ua hāʻawi ʻo DPV i ka hoʻonā ʻoi aku ka maikaʻi, no ka mea, pili pū ke ʻano capacitive hope i ke ana ʻana o ka CV, me ka emi ʻana o ka naʻau.
ʻO ka hoʻonuiʻiaʻana o nā'āpana lōʻihi e pili ana i ka pono o ka viral genomic RNA. Ua hōʻikeʻia nā haʻawina he nuiʻole ka maikaʻi o ka hoʻonuiʻana i nā'āpana lōʻihi ma muli o ka hoʻohaʻahaʻaʻana o RNA i loko o ke kaiapuni a me ka hiki ke hoʻopili i ka wā kaʻawale11,41,42,43,44 .Ua ʻike mākou ua ʻoi aku ka maikaʻi o ke ʻano o ka hoʻopaʻa ʻana i nā maʻi maʻi PEG i ka hoʻopaʻa ʻana i ka phage Phi-6 spiked i loko o nā lake wai ma mua o ka alumini hydroxide-based virus concentration method. e hoʻonui i nā kumu hoʻohālikelike lōʻihi pōkole a hōʻemi i ka hiki ke hoʻohālikelike ʻia.
ʻAʻole liʻiliʻi nā laʻana olaola, no laila pono e hoʻolālā i kahi biosensor e koi ana i nā laʻana liʻiliʻi no ka hoʻāʻo. ) nā laʻana no ka hoʻāʻo ʻana e uhi i ka wahi kūpono o nā electrodes. Eia kekahi, hiki ke hoʻohana hou ʻia ka electrode like ma hope o ka hoʻomaʻemaʻe ʻana ma mua o ka hāʻawi ʻana i ka laʻana aʻe. a me ka mea hoʻohana maʻamau. No ka mea, ke kumukūʻai o kēlā me kēia electrode e pili ana i $ 0.55 (a i ʻole INR 40) no ka hana ʻana, hiki i kēia biosensor ke lilo i kumu kūʻai maikaʻi loa i nā ʻenehana ʻike ʻike. ʻO nā ʻāpana DNA i nā ʻano like ʻole.
Ma muli o ka hilinaʻi ʻana o nā protocols electrochemical detection MB i ka kikoʻī o ka PCR, ʻo kahi palena nui o kēia ʻano ka hiki ke hoʻonui ʻole i nā ʻano like ʻole e like me ka wai ʻino a me ka wai loko a i ʻole ka hoʻohana ʻana i nā kumu hoʻomaʻemaʻe haʻahaʻa. ʻO nā ʻano hana ʻike electrochemical no ka ʻike ʻana i ka DNA o nā huahana PCR i hoʻomaʻemaʻe ʻole ʻia me ka hoʻohana ʻana i nā electrodes ENIG PCB i hoʻololi ʻole ʻia, pono e hoʻomaopopo maikaʻi i nā hewa i hoʻokomo ʻia e nā dNTP a me nā primers i hoʻohana ʻole ʻia, a no ka hoʻonui ʻana i nā kūlana hopena a me nā protocol assay. Pono e ana ʻia ka koi oxygen (BOD) o ka hāpana wai i mea e hoʻomaikaʻi ai i ka pololei o ke ana.
I ka hopena, ke hāpai nei mākou i kahi mea ʻike haʻahaʻa electrochemical ENIG PCB no ka ʻike ʻana i nā maʻi i loko o ke kaiapuni (wai wai). nā electrodes me ka lōʻihi o ke ola a ʻaʻohe pono o ka mālama ʻana a no laila kūpono no ka hoʻomohala ʻana i nā hopena ana me ka hoʻoili ʻana i ka laʻana automated i hoʻohana ʻia ma LMICs. ʻO ka mea maʻamau i nā laʻana kaiapuni e hōʻemi i ka kikoʻī o kēia ʻano ʻike ma muli o ka hoʻopaʻa ʻole ʻana o MB i ka oligonucleotides hoʻokahi a me ʻelua. a me ka DPV peak au i loaʻa mai nā laʻana i ho'āʻoʻia e pono e unuhiʻia e pili ana i nā pane i loaʻa mai ka mana maikaʻiʻole (NTC) no kēlā me kēia ho'āʻo. -ʻO ke kumu kūʻai e hiki ke hōʻiliʻili a hoʻopaʻa i nā laʻana a hoʻihoʻi i nā hopena i ke keʻena hana.
Cashdollar, J. & Wymer, L. Nā ala no ka hoʻomaka mua ʻana o nā maʻi mai nā ʻano wai: kahi loiloi a me ka meta-analysis o nā haʻawina hou.J.Application.microorganism.115, pp. 1-11 (2013).
Gall, AM, Mariñas, BJ, Lu, Y. & Shisler, JL Nā maʻi maʻi wai: Nā pale i ka wai inu palekana.PLoS Pathogens.11, E1004867 (2015).
Shrestha, S. et al.Wastewater epidemiology for cost-effective large-scale surveillance of COVID-19 in low-and middle-income country: challenges and opportunities.Water 13, 2897 (2021).
Palecek, E. & Bartosik, M. Nucleic acid electrochemistry.Chemical.Rev.112, 3427–3481 (2012).
ʻO Tani, A., Thomson, AJ & Butt, JN Methylene blue ma ke ʻano he electrochemical discriminator o nā oligonucleotides i hoʻopaʻa ʻia i hoʻokahi a me ʻelua i hoʻopaʻa ʻia ma nā substrates gula. Analyst 126, 1756-1759 (2001).
Wong, EL, Erohkin, P. & Gooding, JJ Comparison of Cation and Anion Intercalators for Electrochemical Transduction of DNA Hybridization by Long-Range Electron Transfer.Electrochemistry.comminicate.6, 648–654 (2004).
Wong, EL & Gooding, JJ Hoʻoili kālā ma o DNA: kahi koho electrochemical DNA biosensor.anus.Chemical.78, 2138–2144 (2006).
Fang, TH et al.Real-time PCR microfluidic device with concurrent electrochemical detection.biological sensor.Bioelectronics.24, 2131–2136 (2009).
Win, BY et al.Signal mechanical study and performance verification of a 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 no ka ʻike ʻana i ka sars-cov-2 i loko o nā laʻana wai ʻōpala.J.Environment.Chemical.Britain.10, 107488 (2022).
Kumar, M. et al.Electrochemical sensing of SARS-CoV-2 amplicons with PCB electrodes.The sensor is activated.B Chemistry.343, 130169 (2021).
Kitamura, K., Sadamasu, K., Muramatsu, M. & Yoshida, H. ʻIke kūpono o SARS-CoV-2 RNA i loko o ka hapa paʻa o wastewater.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. Ke ola ʻana o ka bacteriophage Phi6 i hoʻopuni ʻia (he mea pani no SARS-CoV-2) i loko o nā kulu kohu i hoʻoheheʻe ʻia i waiho ʻia ma nā ʻili aniani.science.Rep.10, 1–10 (2020).
Dey, R., Dlusskaya, E. & Ashbolt, NJ ʻO ka hoʻomau ʻana o ke kaiapuni o kahi mea pani SARS-CoV-2 (Phi6) i ka amoeba.J.Ola Ola 20, 83 (2021).
Mindich, L. Hoʻopili pololei ʻia o nā ʻāpana genomic ʻekolu o ka RNA bacteriophage ʻelua-stranded\(\varphi\)6.microorganism.Moore.biology.Rev.63, 149–160 (1999).
Pirttimaa, MJ & Bamford, ʻO ka hana lua o ka DH RNA phage\(\varphi\)6 ʻāpana ʻāpana.RNA 6, 880–889 (2000).
Bonilla, N. et al.Phages on the Tap – He protocol wikiwiki a maikaʻi no ka hoʻomākaukau ʻana i nā waihona hale hana o bacteriophages.PeerJ 4, e2261 (2016).
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