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A na-enwe ekele dị ukwuu maka nleba anya nlele gburugburu ebe obibi kemgbe mmalite nke ọrịa COVID-19, a na-emekwa ụfọdụ mbọ nlekota site na iji ọkọlọtọ ọla edo, n'agbanyeghị na usoro qPCR dị ọnụ ahịa. N'ime ọrụ a, anyị na-egosipụta nchọpụta electrochemical nke amplicon enwetara site na Phi6 phage dịpụrụ adịpụ na mmiri mmiri ọdọ mmiri spiked (onye na-ewu ewu maka SARS-CoV-2), na-eji ENIG. iji mechaa PCB electrodes na-enweghị ngbanwe elu.sex. A na-amata nzaghachi nke electrochemical nke ọma maka iberibe DNA abụọ dị ogologo dị iche iche (\({117}\,\hbox {bp}\) na \({503}\,\hbox {bp}\)), na mmetụta nke salts na PCR master mixes on methylene blue (MB) -DNA interactions.Our results na-egosi na ogologo nke DNA iberibe budata ekpebi electrochemical uche na-egosi na ọrụ a na ike ịchọpụta ogologo amplicons enweghị gel ọcha nke PCR ngwaahịa bụ. dị mkpa maka n'ọnọdụ nlele nke mmiri.Ihe ngwọta akpaghị aka zuru oke maka ibu nje virus na-eme nke ọma.
Amara mbufe nje na-ebute mmiri dị ka ihe ize ndụ ahụike ọha na eze kemgbe 1940s, yana ihe akaebe mbụ nke mbufe ọrịa polio na ịba ọcha n'anya E1. Healthtù Ahụ Ike (wa (WHO) ewepụtala ọtụtụ nje nje na-efe efe nke mmiri nke agafeghị oke na ahụike dị elu2.Omenala nje. ụzọ nchọpụta na-adabere na ọla edo-ọkọlọtọ qPCR usoro dabeere na gold, bụ nke ukwuu mmetụta na kpọmkwem, ma na-achọ ndị ọkachamara na-arụ ọrụ iji nwalee na laabu na-eji oké ọnụ ngwá. Otú ọ dị, na ala na n'etiti mba (LMICs) na obere ego, mmadụ. Nnwale nlele nwere ike ibute ụzọ n'ihu nlekota nlele mmiri gburugburu ebe obibi. Ya mere, ụzọ ndị ọzọ dị ọnụ ala dị mkpa maka ịdịgide, nleba anya nke mmiri na mmiri na-ekpofu mmiri na mba ndị dị ala na nke etiti dị ka ịdọ aka ná ntị mbụ nke ọrịa na-apụta. si otú a na-echebe ha pụọ na mmetụta akụ na ụba siri ike nke ọrịa nje na-efe efe. Electrochemical biosensors nke dị ọnụ ala maka acid nucleic nwere ike inye ihe ngwọta na-ekwe nkwa maka mkpa a na-enweghị mkpa. elu na hybridize mgbe a kenha usoro dị na sample. Nke a nwere ike wee ghọọ a mgbaàmà site dị iche iche electrochemical usoro iji redox mediators dị ka potassium iron / ferrocyanide. Methylene blue (MB) bụ otu ndị dị otú ahụ redox-arụ ọrụ molekul, nke nwere. A kọwapụtara na ọ ga-agbakọta n'ime DNA nke nwere eriri abụọ (dsDNA) na mgbakwunye na njikọ ya na-enweghị nkọwa ya na DNA5,6 nke na-emekọrịta ihe. Ụdị njikọ nke MB iji mepụta ogige MB-DNA na-eme ka ha bụrụ nhọrọ a ma ama dị ka ndị mgbasa ozi redox na ọtụtụ DNA electrochemical. sensọ nhazi 5,6,7,8,9.Ọ bụ ezie na intercalation nke MB na DNA bụ nonspecific, na kpọmkwem nke a electrochemical sensọ na-adabere n'ụzọ dị ukwuu na ịdị ọcha nke primers eji PCR ma ọ bụ isothermal amplification, ọ bụ nke ọma ruru eru maka mmejuputa ata n'ezie. -oge electrochemical dabeere qPCR ma ọ bụ fluorescence isothermal amplification dị ka ihe ọzọ na DNA ịta nha 9 .N'ime otu ndị dị otú ahụ mmejuputa iwu, Won et al.The elu nke gold electrodes e gbanwetụrụ na 6-mercapto-1-hexanol (MCH) maka ezigbo oge. nha nke PCR amplicons na MB iji di iche iche pulse voltammetry (DPV) 9.N'ọnọdụ ndị ọzọ, Ramirez et al.Nchọpụta nke SARS-CoV-2 na mmiri mkpofu site RT-LAMP mmeghachi omume iji MB na ihuenyo-e biri ebi electrodes. Platinum electrodes nwekwara e. eji dị ka ọnọdụ electrodes na microfluidic PCR n'elu ikpo okwu nke e mere ka electrochemically chọpụta amplicons n'oge mmeghachi omume 8 . Ihe ọmụmụ ndị a niile chọrọ ngbanwe elu nke electrodes, na-egosi mmụba mmepụta na ụgwọ ọrụ n'ihi ihe nchekwa pụrụ iche chọrọ maka nkwụsi ike nke electrodes ndị a na-arụ ọrụ.
Schematic nke usoro ọrụ maka nchọpụta electrochemical nke amplicons enwetara site na ụmụ irighiri viral etinyere na nlele mmiri ọdọ.
N'oge na-adịbeghị anya, anyị gosipụtara mmetụta electrochemical nke SARS-CoV-2 amplicons nwere elektrọd sekit a na-ebi akwụkwọ dị ọnụ ala (PCB) dabere na DPV na cyclic voltammetry (CV) na-akpata site na mgbasa nke ogige MB-DNA n'elu electrodes na-agbanweghị agbanwe) na-agbanwe na elu. ugbu a11.Anyị na-akọ na iberibe DNA ogologo (N1-N2, \({943}\, \ hbox) kpụrụ site na iji CDC-atụ aro N1 n'ihu na N2 reverse primers ma e jiri ya tụnyere obere mkpirisi {bp}\)) gosipụtara akara dị mma na nzaghachi sensọ. ( N1, \ (72 \, \ hbox {bp} \)) kpụrụ site na iji N1 n'ihu na N1 reverse primer sets. A na-akọ ọmụmụ ihe ndị a site na iji DNA dilutions kwadebere na mmiri na-enweghị nuclease. A na-ejikwa ikpo okwu chọpụta SARS-CoV. -2 amplicons na simulated mmiri mkpofu sample (enwetara site spiking ngụkọta RNA samples na SARS-CoV-2 RNA) Ebe ọ bụ na RNA nwere ike ịkpacha ajị n'oge iche na ala nhazi,12,13 ọ na-esiri ike mụbaa ogologo iberibe na a heterogeneous sample. Ya mere, ngosipụta nke electrochemical sensing nke SARS-CoV-2 amplicon na mmiri mkpofu na-ejedebeghị na mkpumkpu \ (72 \, \ hbox {bp} \) N1 iberibe.
N'ime ọrụ a, anyị nyochara ike nke ENIG PCB dabeere na electrochemical sensing nke phage Phi6 concentrated na iche na ọdọ mmiri samples (Fig. 1) .Phi6 phages bụ tụnyere na size (80-100 nm) ka SARS-CoV-2 na. nwekwara akpụkpọ ahụ lipid na protein spike. N'ihi nke a, bacteriophage Phi6 bụ ihe na-ewu ewu maka SARS-CoV-2 na nje virus RNA ndị ọzọ kpuchiri ekpuchi 14,15.RNA dịpụrụ adịpụ site na phage particles e ji mee ihe dị ka template maka cDNA njikọ sochiri ya. PCR iji nweta iberibe DNA abụọ nke 117 na 503 isi ụzọ abụọ n'ogologo. Nyere ihe ịma aka nke amplifying \ (943 \, \ hbox {bp} \) N1-N2 iberibe na ọrụ anyị gara aga, anyị na-elekwasị anya n'iberibe ogologo oge (\(117) \, \ hbox {bp} \) na \ (503 \, \ hbox {bp} \)), dabere na primers dị. A na-enyocha nzaghachi electrochemical n'usoro n'usoro n'ọtụtụ ebe ịta ahụhụ (\({10}\,{ \hbox {pg}/{\upmu \hbox {l}}}\) ka \({20}\, {\hbox {ng}/{\upmu \hbox {l}}}\)) maka iberibe abụọ n'ime ọnụnọ nke MB, mmetụta nke nnu na nzaghachi sensọ bụ nke e ji mara ma kwadoro ya site na nha spectrophotometric. Isi onyinye nke ọrụ a bụ ndị a:
Ogologo iberibe DNA na ọnụnọ nnu dị na nlele ahụ na-emetụta mmetụta uche.Nsonaazụ anyị na-egosi na ọrụ electrochemical dabere na usoro dị iche iche nke mmekọrịta nke MB, DNA, na ihe mmetụta na nzaghachi voltammetric, dabere na ntinye DNA na ogologo oge, na ogologo mpempe akwụkwọ na-egosi mmetụta dị elu, ọ bụ ezie na nnu nwere mmetụta ọjọọ na mmekọrịta electrostatic n'etiti. MB na DNA.
Ntụle DNA na-ekpebi usoro nke mmekọrịta MB-DNA na electrodes na-adịghị agbanwe agbanwe Anyị na-egosi na usoro dị iche iche nke MB-DNA na-adabere na ntinye DNA. N'ebe DNA dị n'okpuru obere ego nke \ ({\hbox {ng}/{\upmu \ hbox) {l}}}\), anyị chọpụtara na nzaghachi electrochemical nke ugbu a bụ nke kachasị kpebisiri ike site na mgbasa ozi nke MB-DNA na electrode, ebe ọ bụ na ntinye uche dị ala N'ebe DNA dị elu, a na-ekpebi nzaghachi electrochemical ugbu a site na nkwụsị nke redox. ọrụ n'ihi ntinye MB n'etiti ụzọ abụọ isi DNA.
ENIG PCB-Based Electrochemical Sensing of Viral Nucleic Acids in Lake Water Samples akwadoro ihe nleba anya site na nchọpụta electrochemical nke Phi6-agbakwunyere \(503 \, \ hbox {bp}\) iberibe DNA enwetara site na nlele mmiri sitere na Powai Lake, IIT Mumbai Campus. Fage nsonaazụ.
Ọnụ ego dị ala nke mmejuputa na ikike maka ntinye n'ime sistemu nlekota akpaaka zuru oke, oligonucleotides ma ọ bụ aptamers na electrodes nwere ogologo ndụ.
Phage Phi6 bụ nje dsRNA kpuchiri ekpuchi nke ezinụlọ Cytoviridae nke na-ebute Pseudomonas syringae. Mkpụrụ ndụ ihe nketa nke Phi6 phage dị n'ụdị iberibe 3: S (\(2.95 \, \ hbox {Kb}\)), M (\(4.07) \, \ hbox {Kb} \)) na L (\ (6.37 \ , \ hbox {Kb} \)) 16,17. Ebe ọ bụ na Phi6 phage na-emetụta ụdị BSL-1 Pseudomonas na-abụghị pathogenic, ọ dị mma iji. na enwere ike itolite ngwa ngwa na ụlọ nyocha. Phage Phi6 na onye ọbịa ya Pseudomonas syringae zụtara n'aka Felix d'Herelle Reference Center for Bacterial Viruses, Laval University, Canada (ọnụọgụ ebe ndekọ aha bụ HER-102 na HER-1102, n'otu n'otu) .Phi6 phage na onye ọbịa ya tụgharịrị dị ka ụlọ ọrụ ntụaka nyere ntụziaka ya. Phi6 e mere ka ọ dị ọcha site na efere lysis na elution iji nweta titers ikpeazụ na \ (\ about 10 ^ {12} \, {\ hbox {PFU} / hbox { ml}}\) (ihe e ji emepụta nkeji/ milliliters) RNA dịpụrụ adịpụ site na irighiri ihe phage dị ọcha site na iji GenElute™ Universal Total RNA Purification Kit (Sigma-Aldrich) dị ka ntuziaka onye nrụpụta si dị. 100}\,{{\upmu \hbox {l}}}\) bụ lysed wee wụnye lysate ahụ n'elu kọlụm a na-agbagharị agbagharị iji mee ka RNA jikọta na kọlụm resin. A na-ewepụ RNA ahụ na ngwọta elution \({ 50}\,{{\upmu \hbox {l}}}\) nke kit na-enye. Tụlee mkpokọta RNA site na nnabata na \(260\,\hbox {nm}\) . A na-echekwa RNA na aliquots na \ ({-80}\,{^{\circ }\hbox {C}}\) ruo mgbe a ga-eji ya eme ihe.\({2}\,{\upmu \hbox {g}}\) The iScript cDNA Synthesis Kit (Bio) -Rad Laboratories) e ji mee ihe dị ka template maka cDNA njikọ na-eso ntụziaka nke onye mmepụta. N'ụzọ dị nkenke, a cDNA synthesis mmeghachi omume mejupụtara 3 nzọụkwụ: priming na \ ({25}\,{^{\circ }\hbox {C}}\ )\({5}\,{\hbox {min} }\) , ntụgharị ntụgharị nke \({20}\,{\hbox {min}}\) na \({46}\,{^{\circ }\hbox {C}}\), ma tụgharịa ihe ndekọ ahụ nọ na \({95}\,{^{\circ }\hbox {C}}\) maka \({1}\,{\hbox {min) }}\) . Mgbe ọ na-agba ọsọ na 1% agarose gel, cDNA gosipụtara eriri atọ kwekọrọ na mpempe RNA atọ a na-atụ anya ya (data egosighi) . A na-eji primers ndị na-esonụ mee ka mpempe DNA abụọ dị elu nke 117 na 503 bp n'ogologo, iji cDNA dị ka ndebiri maka PCR na miniPCR® mini8 cycler thermal:
The primers maka \ (117 \, \ hbox {bp} \) na \ (503 \, \ hbox {bp}\) kwekọrọ na 1476-1575 nucleotides nke M akụkụ na 458-943 nucleotides nke L nke n'otu n'otu acid. .A na-agbanye ngwaahịa PCR niile na-emepụta electrophoresed na 1% agarose gels, na-eme ka DNA dị elu dị ọcha site na iji GeneJET Gel Extraction Kit (Thermo Fisher Scientific).
A na-eji ọdọ mmiri dị na ogige ụlọ akwụkwọ IIT Mumbai (Powai Lake, Powai, Mumbai) gbakwunye phage. ihe ndị a kwụsịtụrụ, wee tinye Phi6 phage. Tinye \({1}\,{\hbox {ml}}\) nke \(10^{6}\,{\hbox {PFU}/\hbox {ml}} \) ka \( {100} \ , {\hbox {ml}} \) filtered ọdọ mmiri, na \({4}\,{^{\circle}\hbox {C}}\).Obere aliquot bụ echekwabara maka viral ibu nha site plaque assay.Anyị nwalere ụzọ abụọ dị iche iche na-etinye uche spiked Phi6 nje irighiri: (1) aluminum hydroxide adsorption-zoo mmiri usoro,19 nke e validated maka ịta nke ọtụtụ enveloped RNA nje si gburugburu ebe obibi samples, na (2)) Usoro ntinye uche nke nje virus nke dabeere na polyethylene glycol (PEG) emebere site na Iju Mmiri et al.20 .Ebe ọ bụ na a chọpụtara na nrụpụta ọrụ nke usoro PEG dị mma karịa nke aluminom hydroxide, a na-eji usoro PEG na-etinye uche na Phi6 site na mmiri mmiri ọdọ mmiri.
Ụzọ PEG e ji mee ihe bụ nke a: PEG 8000 na \(\hbox {NaCl}\) atụkwasịkwara na mmiri ọdọ mmiri Phi6-spiked iji nweta 8 % PEG 8000 na \(0.2\,\hbox {M} \) \ hbox {NaCl}\) .Agbanyere ihe nlele na shaker\({4}\,{^{\circ }\hbox {C}}\)\({4}\,{\hbox {h}}\) ), wee centrifuged na \(4700 \,\hbox {g}\) bụ \({45}\,{\hbox {min}}\) .Tụfuo supernatant wee kwụsịtụ pellet na \({1}\, {\hbox {ml}}\) n'otu ikike ahụ. A na-eme nyocha niile spiking na nje virus na ugboro atọ. Mgbe itinye uche na ya, a na-edobe obere aliquot maka ịlele arụmọrụ mgbake site na plaque assay. RNA dịpụrụ adịpụ dị ka akọwara na mbụ wee pụta ìhè. in kit-supplied elution buffer\({40}\,{\upmu \hbox {l}}\) .Ebe ọ bụ na ntinye RNA ga-adịgasị iche site na sample gaa n'ụdị n'ime atọ, \({2}\,{\upmu \) A na-eji hbox {l}}\) nke RNA mee ihe atọ ahụ n'agbanyeghị na mkpokọta cDNA na-arụ ọrụ nke sample. ejiri mee ihe dị ka template maka \({20}\,{\upmu \hbox {l}}\) PCR maka cycles 35 iji bulie \ (117\,\hbox {bp}\) na \(503\,\hbox { bp}\) iberibe. A na-anọchi anya ihe nlele ndị a dị ka "1: 1", ya bụ na-enweghị dilution. A na-edozi njikwa enweghị ụkpụrụ (NTC) dị ka njikwa na-adịghị mma, ebe cDNA mebere site na iji RNA dịpụrụ adịpụ na phage dị ọcha. dị ka ndebiri maka njikwa dị mma (PC) .Quantitative PCR (qPCR) mere na ngwa Stratagene Mx3000P RT-PCR na-eji Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies) .Edobere mmeghachi omume na atọ dị ka ọ dị na mbụ. A kọwara ya. E dekọrọ ọnụ ụzọ okirikiri (Ct) maka ihe atụ niile. Tụkwasị na nke ahụ, ihe atụ ndị a gwakọtara bụ \ ({1}\,{\upmu \ hbox {l}} \) na-eji cDNA diluted 1: 100 na mmiri ọdọ mmiri a kpochara dịka. \({20}\,{\upmu \hbox {l}}\) PCR maka okirikiri 35. A na-anọchi anya ihe nlele ndị a dị ka "1:100″.
The PCB electrodes na-emepụta site na iji azụmahịa dị ọnụ ala Electroless Nickel immersion Gold (ENIG) usoro na-enweghị mkpa ọzọ gold plating.ENIG PCB electrode nkọwa zuru ezu na anyị gara aga work11.For ENIG PCB electrodes, omenala electrode ihicha ụzọ dị ka. A naghị atụ aro ngwọta piranha ma ọ bụ sulfuric acid cyclic voltammetry n'ihi na ha nwere ike ime ka ọkpụkpụ ọla edo dị gịrịgịrị (ọkpụrụkpụ \ (\ approx \) \ (100 \, \ hbox {nm} \)) ma kpughee akwa ọla kọpa dị n'okpuru nke na-adịkarị mfe. ka corrosion 21, 22, 23, 24, 25. N'ihi ya, hichaa electrodes na lint-free ákwà moisted na IPA. The sample a ga-anwale bụ incubated na \({50}\,{\upmu \hbox {M}). }\) MB na \({4}\,{^{\circ }\hbox {C}}\)\({1}\,{\hbox {h}}\) maka ntinye dị mfe. N'ọrụ anyị gara aga. , anyị hụrụ na a na-eme ka uche na linearity nke ihe mmetụta dịkwuo mma site n'ịba ụba nke MB 11 . Dabere na njikarịcha a kọrọ na ọrụ mbụ anyị, anyị na-eji \({50}\,{\upmu \hbox {M}}\) MB. Nchoputa iji tinye DNA n'ime ihe omumu a. Enwere ike nweta nchọta electrochemical nke DNA nke nwere okpukpu abuo (ds-DNA) site na iji anionic ma obu cationic intercalators. N'aka nke ọzọ, cationic intercalators dị ka MB chọrọ mkpirisi oge incubation, ihe dịka \({1}\,{\hbox {h}}\) maka nchọpụta electrochemical nke ds-DNA6. Ntụle ọ bụla gụnyere inyefe sample ga-anwale na electrode\({5}\,{{\upmu \hbox {l}}}\), wee jiri nkịrịka mmiri mmiri IPA na-ehicha ya, tupu i were ihe nlele ọzọ gaa n'ihu.Otu nha. A nwalere nlele ọ bụla na electrodes 5 dị iche iche ọ gwụla ma ekwuru ya. A na-eme nha DPV na CV site na iji PalmSens Sensit Smart potentiostat, na PSTrace software na-eji maka nhazi potentiostat na ịnweta data, gụnyere ọnụ ọgụgụ kasị elu ugbu a. A na-eji ntọala ndị a. maka nha DPV na CV:
DPV: Oge nhata = \ (8 \, \ hbox {s} \), Nzọụkwụ voltaji = \ (3 \, \ hbox {mV} \), Pulse Voltage = \ (25 \, \ hbox {mV} \) , pulse duration = \ (50 \, \ hbox {ms} \), nyocha ọnụọgụ = \ ({20} \, \ hbox {mV/s} \)
CV: Oge nha nhata = \ (8 \, \ hbox {s} \), Nzọụkwụ voltaji = \ (3 \, \ hbox {mV} \), Sweep Rate = \ ({300} \, \ hbox {mV/ s) }\)
Ọnụ mmiri kachasị elu enwetara site na voltammogram nke DNA gbajuru ya na \({50}\,{\upmu \hbox {M}}\) MB: (a) \(503\,\hbox {bp}\) DPV , (b) \ (503 \, \ hbox {bp} \) CV, (c) \ (117 \, \ hbox {bp} \) DPV, (d) \ (117 \, \ hbox {bp} \) CV.
E nwetara DPV na CV voltammogram na ENIG PCB electrodes \({50}\,{\upmu \hbox {M}}\) MB ejiri DNA gbajuo ya (n'ogo nke 10–\({20}\,{\ hbox {ng) }/{\upmu \hbox {l}}} –\({0.06}\,{\upmu \hbox {M}}\) maka \(503\,\hbox {bp}\))).Egosipụta voltammogram ndị nnọchite anya na foto S1 na Ozi Mgbakwụnye. Foto nke 2 na-egosi nsonaazụ ya. nke DPV na CV (nke kachasị elu) site na iji ngwaahịa PCR gel-ọcha. E jiri ya tụnyere CV nha, nha DPV na-egosi mmetụta dị elu (nke dị ugbu a dị ka ọrụ nke ntinye DNA) n'ihi na n'azụ capacitive iyi na CV nha na-ezochi mmiri Faradaic 26 .The data n'ihi na igbe ọ bụla na igbe igbe nwere nha sitere na electrodes 5. Ntụle niile na-eji otu setịpụ electrodes iji zere mmejọ nha n'ihi mgbanwe mgbanwe electrode-to-electrode. Anyị hụrụ mmụba na-arịwanye elu na DPV na CV tụrụ ọnụ ọgụgụ kasị elu maka obere mkpokọta DNA. , ogologo (\(503 \, \ hbox {bp} \)) \, \ hbox {bp} \ tụnyere \ (117) ) iberibe. Nke a kwekọrọ na-atụ anya omume nke electrode adsorption kọrọ na anyị gara aga ọrụ. The adsorption nke mgbagwoju MB-DNA na-eme ka nnyefe ụgwọ na electrode, nke na-eme ka ọnụ ọgụgụ dị elu nke ugbu a dị elu. Nnyocha ndị ọzọ egosiwo mmetụta nke oligonucleotide size na usoro na MB-DNA intercalation27,28,29,30.The guanine. -cytosine (GC) ọdịnaya nke amplicon abụọ (\ (117 \, \ hbox {bp} \) na \ (503 \, \ hbox {bp} \)) bụ ihe dịka 50%, na-egosi na nleba anya ihe dị iche bụ n'ihi n'ogologo amplicon. Otú ọ dị, maka nchịkọta DNA dị elu (\(>{2}\,{\hbox {ng}/{\upmu \hbox {l}}}\), maka \(503\,\hbox {bp} \) na \( >{10}\, {\hbox {ng}/{\upmu \hbox {l}}}\) maka \(117\,\hbox {bp}\)), anyị na-edebe ihe abụọ amplifications The A na-ebelata ọnụ ọgụgụ kasị elu nke subs na ma nha DPV na CV. Nke a bụ n'ihi na MB saturates na intercalates n'etiti isi ụzọ abụọ nke DNA, na-ebute steeric mgbochi nke redox ọrụ nke reducible otu na 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.
Nnu dị na PCR master mixes na-egbochi mmekọrịta electrostatic n'etiti MB na DNA, yabụ site n'ịgbakwunye \(2 \, \ hbox {mM} \) \ (\ hbox {MgCl }_2 \) na \ ({50} \,{\) upmu \hbox {M}}\) MB gel-ọcha ngwaahịa iji mụọ mmetụta nnu na mmekọrịta nke MB-DNA. Dị ka e gosiri na foto 3, anyị hụrụ na n'ihi na elu DNA mkpokọta (\(>{2}\,{\) hbox {ng}/{\upmu \hbox {l}}}(503\,\hbox {bp}\) na \(>{10}\,{\hbox {ng}/{\upmu \hbox { l}}}\) maka \(117\,\hbox {bp} \)), na DPV na CV Mgbakwunye nnu emetụtaghị nha nha (lee foto S2 na Ozi mgbakwunye maka voltammograms nnọchite anya) Otú ọ dị, na ọnụ ọgụgụ DNA dị ala, mgbakwunye nke nnu na-ebelata mmetụta uche nke ukwuu, na-eme ka ọ dịghị mgbanwe dị ịrịba ama ugbu a na ntinye DNA. Mmetụta ọjọọ nke nnu na mmekọrịta MB-DNA na mkparịta ụka ka ndị ọzọ na-eme nchọpụta 33,34 na-akọ na mbụ.\(\ hbox { Mg}^{2+}\) cations na-ejikọta na ọkpụkpụ azụ phosphate na-adịghị mma nke DNA, si otú ahụ na-egbochi mmekọrịta electrostatic n'etiti MB na DNA. N'ihe dị elu nke DNA, mgbochi siri ike nke redox-active MBs na-eme ka mmiri dị elu dị ala, ya mere mmekọrịta electrostatic. adịghị emetụta nzaghachi sensọ nke ukwuu. Isi ihe bụ na biosensor a ka mma iji chọpụta ọnụọgụ DNA dị elu (ọ na-adịkarịghị \({\hbox {ng}/{\upmu \hbox {l}}}\) ma ọ bụ karịa), maka nhazi zuru oke nke akpaaka nke ihe nlele mmiri gburugburu ebe obibi, ebe gel ọcha nke ngwaahịa PCR nwere ike ọ gaghị ekwe omume.
Mpaghara dị n'okpuru usoro mmịpụta maka ogologo ikuku 600–700 \(\hbox {nm}\) maka nchịkọta DNA dị iche iche ejiri \({50}\,{\upmu \hbox {M}}\) gbakwụnye na MB: ( a ) \(503 \, \ hbox {bp} \) na na-enweghị nnu (\(2 \, \ hbox {mM} \) \ (\ hbox {MgCl}_2 \)), (b) \ ( 117 \, \hbox {bp}\) nwere na enweghị nnu (\(2\,\hbox {mM}\) \(\hbox {MgCl}_2 \)).\({0}\,{\hbox {pg}/) {\upmu \hbox {l}}}\) Ntụle DNA dabara na \({50}\,{\upmu \hbox {M}}\) MB samples Ọ dịghị DNA.
Iji nyochaa nsonaazụ ndị a dị n'elu, anyị jiri nyocha UV/Vis spectrophotometer (Thermo Scientific Multiskan GO), ihe atụ \({50}\,{{\upmu \hbox {l}}}\) jiri mee ihe maka nke ọ bụla. Measurement.Akara ntinye aka na-ebelata site na ntinye uche DNA na-abawanye, dị ka enwere ike ịhụ site na usoro nke mpaghara n'okpuru usoro mmịpụta maka ogologo wavelength \ (600 \, \ hbox {nm} \) ruo \ (700 \, \ hbox {) nm}\), dị ka e gosiri na Fig. 4 (absorption spectrum egosiri na Fig. S3 na Mgbakwụnye Ozi).N'ihi na samples nwere DNA mkpokọta ihe na-erughị \({1}\,{\hbox {ng}/{\upmu \hbox) {l}}}\), enweghị nnukwu ọdịiche dị na nnabata n'etiti ihe nlele DNA nwere na naanị MB (maka \(503\,hbox {bp}\) na \(117\,hbox {bp}\) ) ogologo iberibe), na-egosi na enweghi ihe mgbochi stric nke redox-active MB. N'ihe dị elu DNA dị elu, anyị na-ahụ mbelata nke nta nke nta na mgbaàmà nnabata ma chọpụta mbelata nke nta nke nta nke nta nke nta nke nta nke nta n'ihu nnu. A na-ekwu na nsonaazụ ndị a sitere na molekul. mmekọrịta na stric inhibition na isi stacking na DNA hybrids.Our results kwekọrọ na akụkọ na akwụkwọ na spectroscopic ọmụmụ nke MB-DNA intercalation na-ejikọta hypochromaticity na mbelata ume na ọkwa na \ (\ pi \) - \ (\ pi ^ * \ ) Ntugharị eletrọnịkị n'ihi njikọta nke oyi akwa 36, 37, 38.
Agarose gel electrophoresis nke phage Phi6: PCR ngwaahịa nke ogologo \ (117 \, \ hbox {bp} \) na \ (503 \, \ hbox {bp} \) si ọdọ mmiri samples.M-DNA akara;NTC-enweghị-nhazi ụkpụrụ, primers nwere amplicon kwekọrọ ekwekọ;PC ziri ezi njikwa;1, 2, 3-ndiluted (1: 1) spiked ọdọ mmiri samples na triplicate. A na-ahụ band na \ (\ about 50 \, \ hbox {bp} \) n'ihi na-ejighi oligonucleotides na \ (503\,\) hbox {bp}\) uzo.
Anyị tụlere uru nke ihe mmetụta ahụ site na iji ihe nlele mmiri Powai nke ejiri Phi6 phage. Ntụle RNA dịpụrụ adịpụ na ihe nlele mmiri phage-spiked sitere na 15.8- \ ({19.4} \, {\upmu \ hbox {g}/\ hbox { ml}}\), ebe ndị dịpụrụ adịpụ na nkwusioru phage dị ọcha A na-eche na RNA ga-abụ \({1945}\,{\upmu \hbox {g}/hbox {ml}}\) na mgbake dị ihe dị ka 1 A na-atụgharị RNA n'ime cDNA ma jiri ya mee ihe dị ka template maka PCR na qPCR. A kwadoro nha ngwaahịa site na agarose gel electrophoresis (Nyocha 5) tupu a na-anwale ihe mmetụta ahụ. Ihe nlele ndị a abụghị gel dị ọcha ma ya mere nwere ihe niile nke PCR dị ka. yana amplicons nke mmasị. The Ct ụkpụrụ e dekọrọ n'oge qPCR (Table 1) e gosiri correlate na ịta nke RNA dịpụrụ adịpụ na kwekọrọ spiked mmiri samples.The Ct uru na-ekpughe ọnụ ọgụgụ nke cycles chọrọ maka fluorescent mgbaàmà ka. gafere ọnụ ụzọ ma ọ bụ n'azụ signal. Higher Ct ụkpụrụ na-egosi ala template ịta na vice versa.The Ct ụkpụrụ nke NTC samples dị elu dị ka a tụrụ anya ya.The iche na \ (\ approximately 3 \) Ct ụkpụrụ n'etiti njikwa dị mma na ihe nlele ule n'ihu na-egosi na nlele ule ọ bụla nwere ihe dịka 1% template ma e jiri ya tụnyere njikwa dị mma. Anyị atụleworị na ogologo amplicon na-eduga n'inwe mmetụta dị mma. Mmụba nke obere iberibe dịpụrụ adịpụ site na ihe atụ gburugburu ebe obibi dị iche iche bụ ihe ịma aka nyere adịghị ike. Otú ọ dị, site na ịba ụba nje anyị na protocol amplification PCR, anyị na-enwe ike ịkwalite mpekere \ (503 \, \ hbox {bp} \) nke ọma maka nghọta electrochemical.
Ọgụgụ 6 na-egosi nsonaazụ ihe mmetụta electrochemical nke amplicon iberibe \ (503 \, \ hbox {bp} \), ha abụọ na-eji cDNA na-enweghị ntụpọ dị ka template (1: 1) na 100-fold diluted cDNA dị ka template (1: 100) rụrụ PCR. , tụnyere NTC na PC (lee Figure S4 na Ozi Mgbakwụnye maka voltammograms nnọchiteanya) . Igbe ọ bụla dị na igbe igbe dị na Figure 6 nwere nha site na nlele atọ na electrodes 5. A na-eji otu electrodes tụọ ihe niile iji zere njehie n'ihi electrode. -to-electrode variation. E jiri ya tụnyere CV nha, DPV nha na-egosi mkpebi ka mma ịmata ọdịiche nke ule na PC samples si NTCs n'ihi na, dị ka e kwuru na mbụ, Faradaic mmiri iyi na-ezo n'ihi ndabere capacitive iyi na nke ikpeazụ. Maka ogologo amplicons, anyị hụrụ na. njikwa na-adịghị mma (NTC) mere ka CV dị elu na ọnụ ọgụgụ kasị elu nke DPV n'ihe metụtara njikwa dị mma, ebe nlele ule dị mma na nke a na-ejighị n'aka gosipụtara ọnụ ọgụgụ dị elu nke elu nke elu DPV. The tụrụ pụtara na etiti ụkpụrụ maka onye ọ bụla undiluted (1: 1). ) ihe nlele ule na PC nwere ike dozie nke ọma site na mmepụta ihe mmetụta maka ihe nlele NTC, ebe mkpebi maka 1: 100 diluted sample na-adịchaghị akpọpụta. (okporo ụzọ adịghị egosi na foto 5), na DPV na CV ọnụ ọnụ ọnụ mmiri kwekọrọ na ya yiri nke a na-atụ anya maka NTC. E gosipụtara nsonaazụ maka mpekere \ (117 \, \ hbox {bp} \) na Ozi Mgbakwụnye. njikwa kpalitere nzaghachi electrochemical site na PCB sensọ n'ihi mgbasa ozi nke MB n'efu na electrode na mmekọrịta nke MB na primer oligonucleotide nke nwere otu eriri. Ya mere, oge ọ bụla a nwalere ihe nlele, a ghaghị ịgba ọsọ njikwa na-adịghị mma na ọnụ ọgụgụ kachasị elu nke ihe nlele ule ma e jiri ya tụnyere ọnụ ọgụgụ kachasị elu nke enwetara site na njikwa na-adịghị mma iji nweta nha dị iche (ihe metụtara)39,40 iji kewaa nlele ule dị ka ihe dị mma ma ọ bụ adịghị mma.
(a) DPV, na (b) CV kasị elu ugbu a maka nchọpụta electrochemical nke \(503\,\hbox {bp}\) iberibe n'ime mmiri ọdọ mmiri. A tụrụ nlele ule na ugboro atọ ma jiri ya tụnyere enweghị njikwa template (NTC) na njikwa ziri ezi (PC).
Nchọpụta anyị na-egosi usoro dị iche iche na-emetụta arụmọrụ nke sensọ electrochemical maka amplicons nke ogologo dị iche iche maka DNA dị iche iche, na ntinye anya kwadoro site na nha anya site na iji UV/Vis spectrophotometer. Ihe anyị chọpụtara na-emesi nghọta na ogologo DNA na-agbaji ruo \ (\ approx \) Enwere ike ịchọpụta \ (500 \, \ hbox {bp} \) site na nghọta dị elu yana na ọnụnọ nnu na nlele ahụ anaghị emetụta uche DNA nke na-emetụta mmetụta dị elu (ọ na-adịkarịghị \ ({\hbox {ng}/{\upmu) \ hbox {l}}} \) na n'elu).Na mgbakwunye, anyị nyochara mmetụta nke ụdị ụdị dị iche iche, gụnyere gel-ọcha amplicon na na-enweghị agbakwunyere nnu, na mgbakwunye nke mmiri ọdọ mmiri na DPV na CV nha.Anyị chọpụtara na DPV nyere mkpebi ka mma, ebe ọ bụ na azụ azụ azụ na-emetụta nha CV, na-eme ka ọ ghara inwe mmetụta.
Mmụba nke iberibe ogologo na-adabere n'iguzosi ike n'ezi ihe nke RNA genomic viral. Ọtụtụ nnyocha egosiwo na mmụba nke obere iberibe anaghị adị mma mgbe niile n'ihi mmebi nke RNA na gburugburu ebe obibi na ikike nke ịgbasa n'oge iche11,41,42,43,44 .Anyị hụrụ na PEG dabeere nje ịta usoro bụ ihe dị irè karị na itinye uche phage Phi-6 spiked na ọdọ mmiri samples karịa aluminum hydroxide dabeere nje ịta usoro.The ike ịchọpụta ogologo DNA iberibe gosiri iji merie chọrọ maka multiplex PCR. ime ka ọtụtụ ndebiri ogologo dị mkpụmkpụ dịkwuo elu ma belata ohere nke nkọwa nke obe.
Nlere anya ihe ndị dị ndụ dị ụkọ, yabụ ọ dị mkpa imepụta biosensor nke chọrọ obere nlele maka nnwale. Eletrọd ENIG PCB eji na ọmụmụ ihe a chọrọ naanị \({5}\,{{\upmu \hbox {l}}}\) ) samples maka ule iji kpuchie ebe dị irè nke electrodes. Ọzọkwa, a pụrụ iji otu electrode ahụ mee ihe mgbe ọ kpochara tupu ya ewepụta ihe na-esote. na kemịkalụ a na-ejikarị eme ihe. Ebe ọ bụ na electrode ọ bụla na-efu ihe dị ka $ 0.55 (ma ọ bụ INR 40) iji rụpụta, biosensor a nwere ike ịbụ ihe ọzọ na-akwụ ụgwọ maka teknụzụ nchọpụta dị ugbu a. Tebụl 2 na-egosi ntụnyere nke ọrụ a na ihe mmetụta ndị ọzọ a kọrọ na akwụkwọ akụkọ ogologo oge. Iberibe DNA n'ụdị ụdị dị iche iche.
Nyere na usoro nchọpụta electrochemical dabeere na MB na-adabere na kpọmkwem PCR, njedebe bụ isi nke usoro a bụ ikike maka mmụba na-abụghị kpọmkwem na ihe atụ dị iche iche dị ka mmiri mmiri na mmiri ọdọ mmiri ma ọ bụ iji primers dị ala. Ụzọ nchọpụta electrochemical maka nchọpụta DNA nke ngwaahịa PCR na-adịghị ọcha site na iji electrodes ENIG PCB na-adịghị agbanwe agbanwe, ọ dị mkpa ịghọta nke ọma njehie nke dNTP na primers webatara, na ịkwalite ọnọdụ mmeghachi omume na protocols nyocha. Ọzọ physicochemical parameters dị ka pH, okpomọkụ, na ndu. Ọchịchọ oxygen (BOD) nke ihe nlele mmiri nwekwara ike ịdị mkpa ka a tụọ ya ka o wee melite nha nha.
N'ikpeazụ, anyị na-atụ aro ihe mmetụta electrochemical ENIG PCB dị ọnụ ala maka nchọpụta nje na gburugburu ebe obibi (ọdọ mmiri) samples. N'adịghị ka electrodes oligonucleotide na-adịghị agbanwe agbanwe ma ọ bụ ihe eji eme ihe maka DNA sensing nke chọrọ nchekwa cryogenic iji nọgide na-enwe uche,53,54 usoro anyị na-eji PCB na-adịghị agbanwe agbanwe. electrodes na a ogologo ndụ ndụ na ọ dịghị kpọmkwem nchekwa chọrọ na ya mere adabara mmepe nke nha ngwọta na akpaghị aka sample nhazi deployed na LMICs.The biosensor utilizes ọnụ ọnụ DNA-intercaating redox agba (MB) maka ngwa ngwa nchọpụta nke lekwasịrị amplicon.The nonspecific amplification. nke a na-ahụkarị na ihe atụ gburugburu ebe obibi na-ebelata nkọwa nke usoro nhụta a n'ihi njikọ na-abụghị nke MB na otu oligonucleotides na-ejikọta ya na nke abụọ. Ya mere, nkọwa nke ule a na-adabere na njikarịcha nke primers na ọnọdụ mmeghachi omume PCR. Tụkwasị na nke ahụ, CV. na DPV ọnụ ọgụgụ kasị elu nke enwetara site na ihe nlele ndị a nwalere kwesịrị ịkọwa ya na nzaghachi ndị a na-enweta site na njikwa na-adịghị mma (NTC) maka ule ọ bụla. Ụdị ihe mmetụta electrochemical na usoro ndị a na-egosi na ọrụ a nwere ike ijikọta ya na autosamplers iji mepụta zuru oke na-akpaghị aka na ala. -Eri ngwọta nke nwere ike ịnakọta na nyochaa samples na ikuku na-ebufe arụpụta azụ na laabu.
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Oge nzipu: Mee-27-2022