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Akamaro ko gukurikirana icyitegererezo cy’ibidukikije cyashimiwe cyane kuva icyorezo cya COVID-19 cyatangira, kandi hari ingamba zo kugenzura zirimo gukorwa hifashishijwe urugero rwa zahabu, nubwo tekiniki zishingiye kuri qPCR zihenze. Biosensor ya ADN y’amashanyarazi ishobora gutanga ikiguzi cyiza cyane igisubizo cyo gukurikirana icyitegererezo cy’amazi y’ibidukikije mu bihugu bikennye kandi biciriritse.Muri iki gikorwa, turerekana amashanyarazi y’amashanyarazi ya amplicons yakuwe mu cyiciro cya Phi6 gitandukanijwe n’icyitegererezo cy’amazi y’ikiyaga (surrogate izwi cyane kuri SARS-CoV-2), dukoresheje ENIG kurangiza electrode ya PCB idahinduwe hejuru.igitsina. Igisubizo cya electrochemical sensor cyaranzwe neza kubice bibiri bya ADN bifite uburebure butandukanye (\ ({117} \, \ hbox {bp} \) na \ ({503} \, \ hbox {bp} \)), na Ingaruka zumunyu muri shobuja wa PCR ivanga kuri methylene yubururu (MB) -DNA imikoranire.Ibisubizo byacu byerekana ko uburebure bwibice bya ADN bugena cyane ibyiyumvo byamashanyarazi kandi bikerekana muriki gikorwa ko ubushobozi bwo kumenya amplicons ndende idafite isuku ya geli yibicuruzwa bya PCR ari ingenzi muburyo bwo gupima urugero rwamazi.Igisubizo cyuzuye cyimikorere ya virusi itwara neza.
Kwanduza virusi mu mazi bizwi ko byangiza ubuzima bw’abaturage kuva mu myaka ya za 1940, hamwe n’ibimenyetso bya mbere byerekana ko kwanduza amazi indwara y’igicuri na hepatite E1.Umuryango w’abibumbye wita ku buzima (OMS) washyize mu majwi virusi nyinshi zandurira mu mazi zifite akamaro kanini kandi zifite ubuzima bwiza.2 virusi ya gakondo uburyo bwo gutahura bushingiye ku buhanga bwa qPCR bushingiye kuri zahabu, bworoshye cyane kandi bwihariye, ariko busaba abakozi babishoboye kwipimisha muri laboratoire bakoresheje ibikoresho bihenze.Nyamara, mubihugu biciriritse kandi biciriritse (LMICs) bifite amikoro make, abantu kwipimisha byintangarugero birashoboka ko bizafata iyambere mugukurikirana icyitegererezo cy’amazi y’ibidukikije. Kubera iyo mpamvu, ubundi buryo buhendutse burakenewe kugira ngo hakurikiranwe ku buryo burambye, igihe nyacyo cy’amazi n’amazi y’amazi mu bihugu bikennye kandi buciriritse nk’umuburo hakiri kare w’indwara zanduye, bityo bikabarinda ingaruka zikomeye z’ubukungu n’ubukungu by’icyorezo cya virusi. Biosensor ya buke ya electrochemic biosensor ya acide nucleic irashobora gutanga igisubizo cyiza gishobora gukemura iki kibazo kidakenewe.Benshi muri aba biosensor ba ADN bakora bitewe nuko imirongo ya ADN yuzuzanya iba idafite imbaraga kuri electrode. hejuru no kuvanga mugihe urukurikirane ruhuye rugaragara murugero.Ibi birashobora noneho guhinduka mubimenyetso nubuhanga butandukanye bwamashanyarazi ukoresheje mediator mediator nka potassium iron / ferrocyanide.Methylene ubururu (MB) nimwe muri molekile ikora redox, ifite byavuzwe ko bihuza ADN ebyiri (dsDNA) hiyongereyeho uburyo budasanzwe bwo guhuza ADN imwe imwe 5.6.Imiterere ihuza MBs kugirango ibe inganda za MB-ADN bituma bahitamo gukundwa nkabunzi ba redox muri ADN nyinshi zamashanyarazi iboneza rya sensor 5,6,7,8,9.Nubwo intera ya MB na ADN idafite akamaro, kandi umwihariko wiyi sensororo yamashanyarazi biterwa ahanini nubuziranenge bwa primers zikoreshwa muri PCR cyangwa amplificateur isothermal, birakwiriye ko dushyira mubikorwa nyabyo -igihe cyamashanyarazi ashingiye kuri qPCR cyangwa fluorescence isothermal amplification nkuburyo bwo gupima ibipimo bya ADN 9 .Muri kimwe mubikorwa, Won et al. Ubuso bwa electrode ya zahabu bwahinduwe hamwe na 6-mercapto-1-hexanol (MCH) mugihe nyacyo gupima amplicons ya PCR hamwe na MB ukoresheje voltammetry itandukanye (DPV) 9.Mu bindi bihe, Ramirez nabandi.Kumenya SARS-CoV-2 mumazi yanduye na RT-LAMP reaction ukoresheje MB hamwe na electrode yacapishijwe ecran. Electrode ya platine nayo yabaye. ikoreshwa nko muri electrode ihagaze muri microfluidic PCR ya platform igenewe gushakisha amashanyarazi amplicons mugihe cyo kubyitwaramo 8 .Ubushakashatsi bwose busaba guhindura isura ya electrode, bivuze ko umusaruro wiyongereye hamwe nigiciro cyo gukora bitewe nibisabwa bidasanzwe byo kubika kugirango electrode ikore neza.
Igishushanyo mbonera cyakazi kugirango amashanyarazi abone amplicons yakuwe mubice bya virusi yibitse mumazi yikiyaga.
Muminsi ishize twerekanye amashanyarazi ya amplicons ya SARS-CoV-2 hamwe na electrode ihendutse yamashanyarazi (PCB) electrode ishingiye kuri DPV na cyclic voltammetry (CV) iterwa na adsorption yibigo bya MB-ADN hejuru ya electrode idahinduwe) ihinduka ryimpinga ubungubu11.Turamenyesha ko ibice birebire bya ADN (N1-N2, \ ({943} \, \ hbox) byakozwe hifashishijwe CDC yasabwe na N1 imbere na N2 revers primers ugereranije nibice bigufi {bp} \)) byagaragaje umurongo mwiza mubisubizo bya sensor (N1, \. -2 amplicons mubyitegererezo byamazi yanduye (byabonetse mugukubita urugero rwa RNA hamwe na SARS-CoV-2 RNA) .Kubera ko RNA ishobora gukemurwa mugihe cyo kwigunga no gutunganya epfo na ruguru, 12,13 biragoye kongera ibice birebire hamwe nurugero rutandukanye. Kubwibyo, kwerekana amashanyarazi yumuriro wa amplicon ya SARS-CoV-2 mumazi y’amazi bigarukira gusa ku gice kigufi \ (72 \, \ hbox {bp} \) N1.
Muri iki gikorwa, twasesenguye uburyo bushoboka bwa ENIG PCB bushingiye ku mashanyarazi ya fage Phi6 yibanze kandi itandukanijwe n’amazi y’ikiyaga (Ishusho 1) .Ibice bya Phi6 biragereranywa nubunini (80-100 nm) na SARS-CoV-2 na ufite na lipide membrane na proteine ya spike.Kubera izo mpamvu, bacteriophage Phi6 ni surrogate izwi cyane kuri SARS-CoV-2 hamwe nizindi virusi zanduza virusi RNA 14.15.RNA yitandukanije nuduce twa fage yakoreshejwe nk'icyitegererezo cya synthesis ya cDNA ikurikirwa PCR kugirango ibone ibice bibiri bya ADN ya 117 na 503 byombi byuburebure. Tanga ikibazo cyo kongera \ (943 \, \ hbox {bp} \) N1-N2 ibice byakazi kacu kashize, twibasiye ibice birebire (\ (117) \. \ hbox {pg} / {\ upmu \ hbox {l}}} \) kugeza \ ({20} \, {\ hbox {ng} / {\ upmu \ hbox {l}}} \)) Kubice byombi muri kuba MB, ingaruka zumunyu kubisubizo bya sensor yaranzwe kandi byemejwe no gupimwa na spekitifotometometrike.Imisanzu nyamukuru yiki gikorwa niyi ikurikira:
Uburebure bwa ADN hamwe no kuba umunyu murugero bigira ingaruka zikomeye kubyumva.Ibisubizo byacu byerekana ko ibikorwa byamashanyarazi biterwa nuburyo butandukanye bwimikoranire ya MB, ADN, hamwe na sensor mugisubizo cya voltammetric, bitewe nubunini bwa ADN hamwe nuburebure, hamwe nibice birebire byerekana sensibilité nyinshi, nubwo umunyu ugira ingaruka mbi kumikoranire ya electrostatique hagati MB na ADN.
Kwibanda kwa ADN bigena uburyo bwimikoranire ya MB-ADN muri electrode idahinduwe Twerekana ko uburyo butandukanye bwimikoranire ya MB-ADN buterwa nubushakashatsi bwa ADN.Ku kwibanda kwa ADN munsi yumubare muto wa \ ({\ hbox {ng} / {\ upmu \ hbox {}} ibikorwa kubera MB kwinjiza hagati ya ADN ibice byombi.
ENIG PCB ishingiye kuri Electrochemical Sensing ya Virus Nucleic Acide mu byitegererezo by’amazi yo mu kiyaga Ubushakashatsi bwakozwe bwemejwe no kumenya amashanyarazi ya Phi6 yongeyeho \ (503 \, \ hbox {bp} \) ADN ibice byakuwe mu ngero z’amazi yavuye mu kiyaga cya Powai, IIT Mumbai Campus Icyiciro cyibisubizo.
Igiciro gito cyo gushyira mubikorwa hamwe nubushobozi bwo kwinjiza muri sisitemu yo kugenzura yuzuye, oligonucleotide cyangwa aptamers kuri electrode ifite ubuzima burebure.
Phage Phi6 ni virusi ya dsRNA ihishe mumuryango wa Cytoviridae yanduza syringae ya Pseudomonas. Genome yicyiciro cya Phi6 ibaho muburyo bwibice 3: S (\ (2.95 \, \ hbox {Kb} \)), M (\ (4.07) \. kandi irashobora guhingwa byoroshye muri laboratoire.Phage Phi6 hamwe na syringae yakiriye Pseudomonas yaguzwe mu kigo cyitwa Felix d'Herelle Reference Centre cya virusi ya bagiteri, kaminuza ya Laval, muri Kanada (nomero yerekana urutonde ni HER-102 na HER-1102) .Icyiciro cya Phi6 hamwe nuwakiriye byagarutsweho nkuko byayobowe n’ikigo cyerekanwe.Phage Phi6 yahanaguwe na plaque lysis hamwe na elution kugirango ibone titeri yanyuma hamwe na \ (\ hafi 10 ^ {12} \, {\ hbox {PFU} / \ hbox { ml}} \). 100} \, {{\ hejuru 50} \, {{\ upmu \ hbox {l}}} \) yatanzwe nigikoresho. Gereranya ubunini bwa RNA ukoresheje kwinjiza (\ 260 \, \ hbox {nm} \). ({-80} \, {^ {\ umuzenguruko} \ hbox {C}} \) kugeza igihe bizakoreshwa. \ -Rad Laboratories) yakoreshejwe nk'icyitegererezo cya cDNA synthesis ikurikiza amabwiriza yabakozwe.Muri make, reaction ya cDNA igizwe nintambwe 3: gutangira kuri \ ({25} \, {^ {\ umuzenguruko} \ hbox {C}} \ ) \ ({5} \, {\ hbox {min}} \), guhindura inyandiko ya \ ({20} \, {\ hbox {min}} \) kuri \ } \ hbox {C}} \), hanyuma uhindure Icyuma gifata amajwi kiri muri \ ({95} \, {^ {\ umuzenguruko} \ hbox {C}} \) kuri \ ({1} \, {\ hbox {min . ukoresheje cDNA nkicyitegererezo cya PCR muri miniPCR® mini8 cycler yumuriro:
Intangiriro ya \ (117 \, \ hbox {bp} \) na \ (503 \, \ hbox {bp} \) ihuye na nucleotide 1476-1575 yo mu gice cya M na nucleotide 458-943 yo mu gice cya L, aside .Ibicuruzwa byose byongerewe ingufu bya PCR byahawe amashanyarazi kuri geles ya agarose 1%, kandi intego yagenewe ADN yasukuwe hifashishijwe ibikoresho byo gukuramo GeneJET Gel (Thermo Fisher Scientific).
Ikiyaga cyo mu kigo cya IIT Mumbai (Ikiyaga cya Powai, Powai, Mumbai) cyakoreshejwe mu kongeramo ibice bya fage. Amazi yo mu kiyaga yayungurujwe binyuze muri \ ({5} \, {\ upmu \ hbox {m}} \) kugirango akureho ibice byahagaritswe, hanyuma hiyongeraho icyiciro cya Phi6. Ongeraho \ ({1} \, {\ hbox {ml}} \) ya \ (10 ^ {6} \, {\ hbox {PFU} / \ hbox {ml}} \) kugeza \ ({100} \, {\ hbox {ml}} \) yungurujwe amazi yikiyaga, muri \ ({4} \, {^ {\ umuzingi} \ hbox {C}} \). yagenewe gupima virusi yapimwe na plaque assay. Twagerageje uburyo bubiri butandukanye kugirango twibande kuri virusi ya virusi ya Phi6: (1) uburyo bwa aluminium hydroxide adsorption-imvura igwa, 19 byemejwe ko hashyizweho virusi nyinshi za RNA zifunze ziva mubidukikije, kandi .20 .Kubera ko uburyo bwo kugarura uburyo bwa PEG bwagaragaye ko ari bwiza kuruta ubwo buryo bwa hydroxide ya aluminium, uburyo bushingiye kuri PEG bwakoreshejwe mu guhuriza hamwe ibice bya Phi6 biva mu mazi y’ikiyaga.
Uburyo bwa PEG bwakoreshejwe bwari ubu bukurikira: PEG 8000 na \ (\ hbox {NaCl} \) byongewe ku cyitegererezo cy’amazi yo mu kiyaga cya Phi6 kugira ngo babone 8% PEG 8000 na \ (0.2 \, \ hbox {M} \) \ ( \ hbox {NaCl} \). Ingero zashyizwe kuri shake \ ({4} \, {^ {\ umuzenguruko} \ hbox {C}} \) \ ({4} \, {\ hbox {h}} \ ), hanyuma centrifuged kuri \ (4700 \, \ hbox {g} \) ni \ ({45} \, {\ hbox {min}} \). Hagarika ndengakamere hanyuma uhagarike pellet muri \ ({1} \, {\ hbox {ml}} \) muburyo bumwe ndengakamere.Ubushakashatsi bwose bwo gutera spiking na virusi bwakorewe muri bitatu. muri kit-yatanzwe na elution buffer \ ({40} \, {\ upmu \ hbox {l}} \). Kubera ko kwibanda kwa RNA bizatandukana bitewe nicyitegererezo kuri bitatu, \ ({2} \, {\ upmu \ hbox {l}} \) ya RNA ikoreshwa kuri bose uko ari batatu hatitawe ku kwibanda kuri cDNA synthesis yintangarugero.CDNA synthesis yakozwe nkuko byasobanuwe mbere. \ ({1} \, {\ upmu \ hbox {l}} \) cDNA yakoreshejwe nk'icyitegererezo cya \ ({20} \, {\ upmu \ hbox {l}} \) PCR kuri cycle 35 kugirango yongere \ (117 \, \ hbox {bp} \) na \ (503 \, \ hbox { ibice bya bp} \) nk'icyitegererezo cyo kugenzura neza (PC) .Quantitative PCR (qPCR) yakorewe mu gikoresho cya Stratagene Mx3000P RT-PCR ikoresheje Brilliant III Ultra-Byihuta SYBR Icyatsi cya QPCR Master mix (Agilent Technologies) .Ibisubizo byashyizweho muri bitatu nkuko byari bisanzwe byasobanuwe.Urugero rw'umuzenguruko (Ct) rwanditswe ku ngero zose. Byongeye kandi, ingero zivanze ni \ ({1} \, {\ upmu \ hbox {l}} \) ukoresheje cDNA ivanze 1: 100 mu mazi yo mu kiyaga cyungurujwe nka \ ({20} \, {\ upmu \ hbox {l}} \) PCR kumuzingo 35. Izi ngero zigaragazwa nka "1: 100 ″.
Electrode ya PCB yahimbwe hifashishijwe ubucuruzi buboneka buhendutse buhendutse bwa Electroless Nickel Immersion Gold (ENIG) bidakenewe ko hashyirwaho izindi zahabu.Ibisobanuro bya electrode ya ENIG PCB birambuye mubikorwa byacu byashize11.Ku buryo bwa elegitoronike ya ENIG PCB, uburyo bwo koza electrode gakondo nka igisubizo cya piranha cyangwa acide sulfurike cyclic voltammetry ntibisabwa kuko bishobora gutera igishishwa cya zahabu yoroheje (uburebure \ kwangirika 21, 22, 23, 24, 25.Nuko rero, sukura electrode ukoresheje umwenda utagira linti wavanze na IPA.Urugero ruzageragezwa rwashyizwemo \ ({50} \, {\ upmu \ hbox {M} MB \) MB muri \ ({4} \, {^ {\ umuzenguruko} \ hbox {C}} \) \ . kwibanda ku gushira ADN muri ubu bushakashatsi. Gutahura amashanyarazi ya ADN ifite imirongo ibiri (ds-ADN) irashobora kugerwaho hifashishijwe intercalator ya anionic cyangwa cationic.Nubwo intercalator ya anionic itahura ADN hamwe no guhitamo neza, bisaba ko habaho ijoro ryose. kurundi ruhande, intercalator ya cationic nka MB isaba igihe gito cyo gukora, hafi \ ({1} \, {\ hbox {h}} \) kugirango tumenye amashanyarazi ya ds-ADN6. Buri gipimo gikubiyemo gutanga icyitegererezo kizageragezwa kuri electrode \ ({5} \, {{\ upmu \ hbox {l}}} \), hanyuma usukure ukoresheje imyenda ya IPA, mbere yo gukomeza urundi rugero.igipimo kimwe. Buri cyitegererezo cyageragejwe kuri electrode 5 zitandukanye keretse bivuzwe ukundi. Ibipimo bya DPV na CV byakozwe hakoreshejwe PalmSens Sensit Smart potentiostat, na software ya PSTrace yakoreshejwe muburyo bwa potentiostat no kubona amakuru, harimo kubara kuri ubu. Igenamiterere rikurikira rirakoreshwa kubipimo bya DPV na CV:
DPV: Igihe kingana = \ (8 \, \ hbox {s} \), Intambwe ya Voltage = \ (3 \, \ hbox {mV} \), Umuvuduko wa Pulse = \ (25 \, \ hbox {mV} \), igihe impiswi yamara = \ (50 \, \ hbox {ms} \), igipimo cya scan = \ ({20} \, \ hbox {mV / s} \)
CV: Igihe kingana = \ (8 \, \ hbox {s} \), Intambwe ya Voltage = \ (3 \, \ hbox {mV} \), Igipimo cyo Kwikuramo = \ ({300} \, \ hbox {mV / s } \)
Imiyoboro ihanitse yabonetse muri voltammogrammes ya ADN igizwe 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.
DPV na CV voltammograms zabonetse kuri electrode ya ENIG PCB \ ({50} \, {\ upmu \ hbox {M}} \) MB igizwe na ADN (kuri concentration ya 10 - \ ({20} \, {\ hbox {ng } / {\ upmu \ hbox {l}}} \) ni ukuvuga 0.13 - \ ({0.26} \, {\ upmu \ hbox {M}} \) kuri \ (117 \, \ hbox {bp} \) na 0.03 - \ ({0.06} \, {\ upmu \ hbox {M}} \) kuri \ (503 \, \ hbox {bp} \)). by'ibipimo bya DPV na CV (impinga ya pex) ukoresheje ibicuruzwa bya PCR bisukuwe neza. Ugereranije n'ibipimo bya CV, ibipimo bya DPV byerekana sensibilité yo hejuru (ikigezweho nk'umurimo wo kwibanda kuri ADN) kubera ko imiyoboro ya capacitifike yibipimo bya CV ihisha imigezi ya Faradaic 26 .Amakuru kuri buri gasanduku kari muri bokisi karimo ibipimo biva kuri electrode 5. Ibipimo byose bifashisha icyiciro kimwe cya electrode kugirango wirinde amakosa yo gupimwa bitewe na electrode-kuri-electrode. Twabonye uburyo bugenda bwiyongera muri DPV na CV bapimye imigezi yo hejuru kugirango yibanze kuri ADN , birebire (\ (503 \, \ hbox {bp} \)) \, \ hbox {bp} \ ugereranije nigice cya \ (117)) Ibi. adsorption yikigo cya MB-ADN yorohereza ihererekanyabubasha kuri electrode, igira uruhare mukwiyongera kwimpinga yimpanuka.Ubundi bushakashatsi bwerekanye ingaruka zubunini bwa oligonucleotide hamwe nurutonde kuri MB-ADN hagati ya 27,28,29,30. Guanine -cytosine (GC) ibiri muri amplicons ebyiri (\ (117 \, \ hbox {bp} \) na \ (503 \, \ hbox {bp} \)) byari hafi 50%, byerekana ko kwitegereza Itandukaniro rigomba uburebure bwa amplicon.Nyamara, kubushakashatsi bwa ADN burenze (\ (> {2} \, {\ hbox {ng} / {\ upmu \ hbox {l}}} \), kuri \ (503 \, \ hbox {bp} \) na \ (> {10} \, {\ hbox {ng} / {\ upmu \ hbox {l}}} \) kuri \ (117 \, \ hbox {bp} \)), twitegereje ibyongerewe bibiri The imigezi yimisozi yagabanutse mubipimo byombi bya DPV na CV.Ibi ni ukubera ko MB yuzuzanya kandi igahuza hagati y’ibice fatizo bya ADN, bikaviramo guhagarika ibikorwa bya redox byitsinda rigabanuka muri 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。
Umunyu uboneka muri shobuja wa PCR uvanga bibangamira imikoranire ya electrostatike hagati ya MB na ADN, bityo ukongeraho \ (2 \, \ hbox {mM} \) \ (\ hbox {MgCl} _2 \) hamwe na \ ({50} \, {\ upmu \ hbox {M}} \) MB ibicuruzwa bisukuye gel kugirango bige ku ngaruka zumunyu ku mikoranire ya MB-ADN.Nkuko bigaragara ku gishushanyo cya 3, twabonye ko kubushakashatsi bwa ADN bwinshi (\ (> {2} \, {\ hbox {ng} / {\ upmu \ hbox {l}}} \) (503 \, \ hbox {bp} \) na \ (> {10} \, {\ hbox {ng} / {\ upmu \ hbox { l}}} \) kuri \ (117 \, \ hbox {bp} \)), muri DPV na CV Kwiyongera k'umunyu ntabwo byagize ingaruka zikomeye kubipimo (reba Ishusho S2 mumakuru yinyongera kuri voltammogramu zihagarariye) .Nyamara, kuri munsi ya ADN yibanze, kongeramo umunyu bigabanya cyane sensibilité, bigatuma nta mpinduka nini ihari hamwe nubushakashatsi bwa ADN.Ingaruka mbi zumunyu kumikoranire ya MB-ADN no guhuza intercalation byavuzwe mbere nabandi bashakashatsi33,34. \ (\ hbox { Mg} ^ {+ 2 ntugire ingaruka cyane kubisubizo bya sensor. Ingingo y'ingenzi ni uko iyi biosensor ikwiranye no kumenya ADN nyinshi (gake \ ({\ hbox {ng} / {\ upmu \ hbox {l}}} \) cyangwa irenga), kubwuzuye- Gutunganya byikora byamazi y ibidukikije, aho gel yogusukura ibicuruzwa bya PCR ntibishoboka.
Agace kari munsi yumurongo wumurongo wuburebure bwa 600-700 \ (\ hbox {nm} \) kubintu bitandukanye bya ADN bigoye hamwe na \ ({50} \, {\ upmu \ hbox {M}} \) MB: (a ) \ (503 \, \ hbox {bp} \) hamwe n'umunyu (\ (2 \, \ hbox {mM} \) \ (\ hbox {MgCl} _2 \)), (b) \ (117 \, \ hbox {bp} \) hamwe n'umunyu (\ (2 \, \ hbox {mM} \) \ (\ hbox {MgCl} _2 \)). \ ({0} \, {\ hbox {pg} / {\ upmu \ hbox {l}}} \) Ubunini bwa ADN buhuye na \ ({50} \, {\ upmu \ hbox {M}} \) MB ntangarugero MB Nta ADN.
Kugirango turusheho kugenzura ibisubizo byavuzwe haruguru, twakoze ibipimo bya optique dukoresheje UV / Vis spectrophotometero (Thermo Scientific Multiskan GO), ibyitegererezo \ ({50} \, {{\ upmu \ hbox {l}}} \) byakoreshejwe kuri buri Igipimo.Umukono winjira ugabanuka hamwe no kwiyongera kwa ADN, nkuko bigaragara mubyerekezo byahantu munsi yumurongo wo kwinjirira kumurongo wumurambararo \ (600 \, \ hbox {nm} \) kugeza \ (700 \, \ hbox { nm} \), nkuko bigaragara ku gishushanyo 4 {}}} ) ibice by'uburebure), byerekana ko hatabayeho kubuza MB-redox ikora MB.Mu rwego rwo hejuru rwa ADN, twabonye ko igabanuka rya gahoro gahoro ibimenyetso byinjira kandi twabonye ko kugabanuka kwinshi kwinjirira imbere yumunyu.Ibisubizo byatewe na molekile imikoranire no kubuza steric hamwe no gutondekanya shingiro muri Hybrid ya ADN.Ibisubizo byacu bihuye na raporo ziri mu bitabo byerekeranye n’ubushakashatsi bwa spekitroscopique y’ubushakashatsi bwa MB-ADN buhuza hypochromaticité no kugabanuka kwingufu muri \ (\ pi \) - \ (\ pi ^ * \ ) inzibacyuho ya elegitoronike kubera intercalation Imirongo 36, 37, 38.
Agarose gel electrophorei ya fage Phi6: Ibicuruzwa bya PCR by'uburebure \ (117 \, \ hbox {bp} \) na \ (503 \, \ hbox {bp} \) biva mu byitegererezo by'amazi yo mu kiyaga. Ikimenyetso cya M-ADN;NTC-nta-shusho igenzura, primers irimo amplicons ihuye;Kugenzura PC neza;1, 2, 3-idasukuye (1: 1) icyitegererezo cyamazi yikiyaga cyikubye gatatu. Itsinda rigaragara kuri \ (\ hafi 50 \, \ hbox {bp} \) kubera oligonucleotide idakoreshwa muri \ (503 \, \ hbox {bp} \) umurongo.
Twasuzumye akamaro ka sensor dukoresheje icyitegererezo cyamazi yikiyaga cya Powai cyerekanwe nicyiciro cya Phi6. Ubushuhe bwa RNA bwitandukanije nicyitegererezo cyamazi yatandukanijwe kuva 15.8 - \ ({19.4} \, {\ upmu \ hbox {g} / \ hbox { ml}} \), mugihe abitandukanije nibihagarikwa byicyiciro cya RNA byagereranijwe ko \ ({1945} \, {\ upmu \ hbox {g} / \ hbox {ml}} \) hamwe nubushobozi bwo gukira hafi 1 %। kimwe na amplicons yinyungu. Indangagaciro za Ct zanditswe mugihe cya qPCR (Imbonerahamwe 1) zerekanwe ko zifitanye isano nubushuhe bwa RNA butandukanijwe nicyitegererezo cy’amazi meza. Agaciro Ct kagaragaza umubare wizunguruka zisabwa kugirango ibimenyetso bya fluorescent to kurenga imbibi cyangwa ibimenyetso byinyuma. Indangagaciro za Ct zerekana inyandikorugero yo hasi yibitekerezo hamwe nibindi. Indangagaciro za Ct zintangarugero za NTC zari hejuru nkuko byari byitezwe. Itandukaniro muri \ (\ hafi 3 \) Ct indangagaciro hagati igenzura ryiza hamwe nicyitegererezo cyibizamini byerekana kandi ko buri cyitegererezo cyikigereranyo gifite icyitegererezo cya 1% ugereranije nigenzura ryiza. Twabanje kuganira ko amplicons ndende iganisha ku kumva neza.Gushimangira ibice birebire bitandukanijwe n’ibidukikije by’ibidukikije biragoye bitewe n’ibibi. ya virusi nkeya hamwe no kwangirika kwa RNA.Nyamara, hamwe no gukungahaza virusi hamwe na PCR amplification protocole, twashoboye kwagura neza igice cya \ (503 \, \ hbox {bp} \) kugirango twumve amashanyarazi.
Igishushanyo cya 6 cyerekana ibyuma byerekana amashanyarazi ibisubizo bya \ (503 \, \ hbox {bp} \) agace ka amplicon, byombi bikoresha cDNA idasukuye nkicyitegererezo (1: 1) hamwe na cDNA inshuro 100 zivanze nkicyitegererezo (1: 100) zakoze PCR , ugereranije na NTC na PC (reba Ishusho ya S4 mu makuru yinyongera kuri voltammogramu zihagarariye) .Buri gasanduku kari mu gasanduku kari ku gishushanyo cya 6 karimo ibipimo biva mu ngero eshatu kuri electrode 5. Electrode imwe yakoreshejwe mu gupima ingero zose kugirango birinde amakosa bitewe na electrode -kugera kuri electrode itandukanye. Ugereranije n'ibipimo bya CV, ibipimo bya DPV byerekana icyemezo cyiza cyo gutandukanya ibizamini na PC bya PC na NTC kuko, nkuko byavuzwe haruguru, imigezi ya Faradaic ihishwa kubera imiyoboro ya capacitif ya fonctionnement ya nyuma. Kuri amplicons ndende, twabonye ko igenzura ribi (NTC) ryatumye imiyoboro ya CV na DPV ihanitse ugereranije nigenzura ryiza, mugihe icyitegererezo cyiza kandi kidahwitse cyerekanaga uburebure busa bwimisozi ya DPV. . . igenzura ryateje amashanyarazi amashanyarazi ya sensor ya PCB bitewe na adsorption ya MB yubusa kuri electrode hamwe n’imikoranire ya MB na primer oligonucleotide imwe gusa. Kubera iyo mpamvu, burigihe buri gihe icyitegererezo gipimishijwe, hagomba gukoreshwa igenzura ribi kandi Impinga yikigereranyo cyikigereranyo ugereranije nimpinga yabonetse kubwo kugenzura nabi kugirango ugere kubipimo bitandukanye (ugereranije) 39,40 kugirango ushireho icyitegererezo cyibizamini nkibyiza cyangwa bibi.
. kugenzura neza (PC).
Ibyavuye mu bushakashatsi byerekana uburyo butandukanye bugira ingaruka ku mikorere ya sensororo ya electrochemiki ya amplicons z'uburebure butandukanye kuri ADN zitandukanye, hamwe nibitekerezo byagenzuwe n'ibipimo bya optique ukoresheje UV / Vis spectrophotometer.Ubushakashatsi bwacu bushimangira ubushishozi ko ibice bya ADN birebire kugeza kuri \ (\ hafi \) (500 ( \ hbox {l}}} \) no hejuru). Twongeyeho, twakoze iperereza ku ngaruka zubwoko butandukanye bwintangarugero, harimo amplicons yasukuwe na gel hamwe nta munyu wongeyeho, no kongeramo urugero rwamazi yikiyaga mubipimo bya DPV na CV.Twabonye ko DPV yatanze igisubizo cyiza, kubera ko imiterere ya capacitif yimbere nayo igira ingaruka kubipimo bya CV, bigatuma itumva neza.
Kwiyongera kw'ibice birebire biterwa n'ubunyangamugayo bwa virusi ya RNA.Ubushakashatsi bwakozwe bwerekanye ko kwongera ibice birebire bitajya bikorwa neza bitewe no kwangirika kwa RNA mu bidukikije ndetse n'ubushobozi bwo gutera mu bwigunge11,41,42,43,44 .Twabonye ko uburyo bwo kwibanda kuri virusi ya PEG bwagize akamaro kanini mu kwibanda ku cyiciro cya Phi-6 cyagaragaye mu cyitegererezo cy’amazi yo mu kiyaga kuruta uburyo bwo gukwirakwiza virusi ya hydroxide ya aluminium. Ubushobozi bwo kumenya ibice birebire bya ADN byagaragaye ko byatsinze ibisabwa kuri multiplex PCR kwagura byinshi bigufi birebire byerekana kandi bigabanya amahirwe yo kwambukiranya.
Ingero z'ibinyabuzima ni nkeya, bityo rero hakenewe gushushanya biosensor isaba ingero ntoya yo kwipimisha. Electrode ya ENIG PCB ikoreshwa muri ubu bushakashatsi isabwa gusa \ ({5} \, {{\ upmu \ hbox {l}}} \ . kandi imiti ikoreshwa cyane.Kuko buri electrode igura amadolari 0.55 (cyangwa INR 40) kugirango ikore, iyi biosensor irashobora kuba igiciro cyinshi muburyo bwa tekinoroji yo gutahura. Imbonerahamwe ya 2 irerekana igereranya ryiki gikorwa nizindi sensor zivugwa mubitabo igihe kirekire Ibice bya ADN mubice bitandukanye.
Urebye ko protocole ya MB ishingiye kuri electrochemical detection protocole ishingiye ku mwihariko wa PCR, imbogamizi nyamukuru yubu buryo ni ubushobozi bwo kongera imbaraga zidasanzwe mu ngero z’amoko atandukanye nk’amazi y’amazi n’amazi yo mu kiyaga cyangwa gukoresha primers zifite isuku nke.Gutezimbere ibyiyumvo bya uburyo bwa electrochemical detection for ADN kumenya ibicuruzwa bya PCR bidafite ibyangombwa ukoresheje electrode ya ENIG PCB idahinduwe, birakenewe ko dusobanukirwa neza amakosa yatangijwe na dNTP idakoreshwa na primers, no kunoza imiterere yimyitwarire no gusuzuma protocole.Ibipimo bya fiziki ya chimique nka pH, ubushyuhe, na biologiya okisijene ikenewe (BOD) y'icyitegererezo cy'amazi irashobora kandi gukenera gupimwa kugirango tunonosore neza ibipimo.
Mu gusoza, turasaba icyerekezo gito cya electrochemic ENIG PCB sensor kugirango tumenye virusi mubidukikije (amazi yikiyaga ).Ntabwo bimeze nka electrode ya oligonucleotide ya immobilizasique cyangwa insimburangingo yihariye ya ADN isaba ububiko bwa kirogenike kugirango ikomeze kumva, 53,54 tekinike yacu ikoresha PCB idahinduwe. electrode ifite igihe kirekire cyo kubaho kandi nta bisabwa byihariye byo kubika bityo ikaba ikwiriye gutezimbere ibisubizo byo gupimwa hamwe no gutunganya ibyitegererezo byikora byoherejwe muri LMIC. Biosensor ikoresha ADN ihendutse ya ADN ihuza irangi rya redox (MB) kugirango hamenyekane vuba amplicons. ibisanzwe mubidukikije bigabanya umwihariko wubu buryo bwo kwiyumvisha bitewe nuburyo budasanzwe bwo guhuza MBs na oligonucleotide imwe-imwe-ebyiri. Kubera iyo mpamvu, umwihariko wiki kizamini uterwa no gutezimbere primers hamwe nuburyo PCR ikora. Byongeye kandi, CV Imiyoboro ya DPV yabonetse kuburugero rwageragejwe igomba gusobanurwa ugereranije nibisubizo byatanzwe bivuye kugenzura nabi (NTC) kuri buri kizamini.Ibishushanyo mbonera bya electrochemical sensor nuburyo bwerekanwe muriki gikorwa birashobora guhuzwa na autosampler kugirango biteze imbere byuzuye kandi biri hasi -igisubizo gishobora gukusanya no gusesengura ingero no kohereza ibisubizo bidasubirwaho muri laboratoire.
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Igihe cyo kohereza: Gicurasi-27-2022