Ua tsaug rau koj tuaj xyuas Nature.com.Qhov browser version uas koj siv tau txwv kev txhawb nqa rau CSS.Rau qhov kev paub zoo tshaj plaws, peb xav kom koj siv qhov browser tshiab (lossis tua hom kev sib raug zoo hauv Internet Explorer).Lub sijhawm no, kom paub meej txuas ntxiv kev txhawb nqa, peb yuav tso saib lub vev xaib tsis muaj qauv thiab JavaScript.
Qhov tseem ceeb ntawm kev saib xyuas ib puag ncig cov qauv tau txais txiaj ntsig zoo txij li thaum pib ntawm COVID-19 kis thoob qhov txhia chaw, thiab qee qhov kev saib xyuas tau ua raws li tus qauv kub, txawm hais tias qPCR-based cov txheej txheem yog kim.Electrochemical DNA biosensors tuaj yeem muab cov txiaj ntsig zoo. Kev daws teeb meem rau kev soj ntsuam ib puag ncig cov qauv dej hauv cov teb chaws tau nyiaj tsawg thiab nruab nrab. Hauv kev ua haujlwm no, peb ua kom pom cov electrochemical tshawb pom ntawm amplicons tau los ntawm Phi6 phage cais tawm ntawm cov qauv dej hauv pas dej (ib qho nrov surrogate rau SARS-CoV-2), siv ENIG ua kom tiav PCB electrodes yam tsis muaj kev hloov kho saum npoo.Kev sib deev.Lub tshuab hluav taws xob hluav taws xob cov lus teb tau raug qhia meej meej rau ob qhov DNA ntawm qhov ntev sib txawv (\({117}\,\hbox {bp}\) thiab \({503}\,\hbox {bp}\)), thiab Cov txiaj ntsig ntawm cov ntsev hauv PCR tus tswv sib xyaw ntawm methylene xiav (MB)-DNA kev sib cuam tshuam.Peb cov txiaj ntsig tau pom tias qhov ntev ntawm DNA tawg tau txiav txim siab txog cov tshuaj electrochemical rhiab heev thiab ua rau pom hauv txoj haujlwm no tias lub peev xwm los ntes cov amplicons ntev yam tsis muaj gel purification ntawm PCR cov khoom yog Nws yog ib qho tseem ceeb rau kev ntsuas qhov ntsuas dej.Ib tug tag nrho automated tov rau viral load bodes zoo.
Kev kis tus kab mob hauv dej tau paub tias muaj kev phom sij rau pej xeem txij li xyoo 1940, nrog thawj cov pov thawj ntawm kev kis tus kab mob polio thiab kab mob siab E1.Lub Koom Haum Saib Xyuas Kev Noj Qab Haus Huv Ntiaj Teb (WHO) tau txheeb xyuas ntau yam kab mob hauv dej ntawm cov kab mob nruab nrab mus rau kev noj qab haus huv tseem ceeb2.Tseem ceeb tus kab mob txoj kev tshawb nrhiav tau vam khom rau cov txheej txheem kub-txheej txheem qPCR-raws li cov tswv yim, uas yog cov rhiab heev thiab tshwj xeeb, tab sis xav tau cov neeg txawj ntse los kuaj hauv chav kuaj siv cov cuab yeej kim kim.Txawm li cas los xij, hauv cov tebchaws tau nyiaj tsawg thiab nruab nrab (LMICs) nrog cov peev txheej tsawg, tib neeg Kev kuaj cov qauv zoo li yuav ua tau zoo tshaj qhov kev soj ntsuam cov qauv dej ib puag ncig.Yog li ntawd, lwm txoj hauv kev siv tus nqi qis yog xav tau kom muaj kev ruaj ntseg, kev soj ntsuam xyuas dej thiab cov dej khib nyiab hauv lub tebchaws tau nyiaj tsawg thiab nruab nrab raws li kev ceeb toom ntxov ntawm cov kab mob tshwm sim, yog li tiv thaiv lawv los ntawm kev cuam tshuam loj ntawm kev lag luam ntawm tus kab mob kis thoob qhov txhia chaw.Tsawg-nqi electrochemical biosensors rau nucleic acids tuaj yeem muab cov kev cog lus muaj peev xwm daws tau rau qhov kev xav tau no.Ntau yam ntawm cov DNA biosensors no ua haujlwm los ntawm qhov tseeb tias cov DNA txuas ntxiv yog immobilized ntawm electrode. nto thiab hybridize thaum muaj qhov sib txuam ua ke nyob rau hauv cov qauv.Qhov no tuaj yeem hloov mus rau hauv lub teeb liab los ntawm ntau yam kev siv hluav taws xob siv hluav taws xob siv redox mediators xws li potassium hlau / ferrocyanide.Methylene xiav (MB) yog ib qho xws li redox-active molecule, uas muaj tau tshaj tawm rau intercalate mus rau hauv ob-stranded DNA (dsDNA) ntxiv rau nws ntau nonspecific binding rau ib leeg-stranded DNA5,6.Qhov intercalating xwm ntawm MBs los ua MB-DNA complexes ua rau lawv ib tug nrov xaiv raws li redox mediators nyob rau hauv ob peb electrochemical DNA. sensor configurations5,6,7,8,9.Txawm hais tias qhov intercalation ntawm MB rau DNA yog nonspecific, thiab qhov tshwj xeeb ntawm no electrochemical sensor nyob ntawm qhov purity ntawm primers siv rau PCR los yog isothermal amplification, nws yog zoo suited rau kev siv tiag. -lub sij hawm electrochemical-raws li qPCR los yog fluorescence isothermal amplification raws li ib tug lwm txoj rau DNA concentration ntsuas 9 .In ib tug xws li kev siv, Won li al.Qhov saum npoo ntawm kub electrodes tau hloov nrog 6-mercapto-1-hexanol (MCH) rau lub sij hawm. Kev ntsuas ntawm PCR amplicons nrog MB siv qhov sib txawv pulse voltammetry (DPV) 9.Nyob rau lwm qhov, Ramirez li al.Kev kuaj pom ntawm SARS-CoV-2 hauv cov dej khib nyiab los ntawm RT-LAMP cov tshuaj tiv thaiv siv MB nrog cov tshuaj ntsuam luam tawm electrodes.Platinum electrodes kuj tau. siv raws li nyob rau hauv situ electrodes nyob rau hauv ib tug microfluidic PCR platform tsim los electrochemically ntes amplicons thaum lub sij hawm cov tshuaj tiv thaiv 8 .Txhua yam ntawm cov kev tshawb fawb no yuav tsum tau hloov nto ntawm cov electrodes, implying nce ntau lawm thiab kev khiav hauj lwm nqi vim tshwj xeeb cia yuav tsum tau rau cov stability ntawm cov functionalized electrodes.
Schematic ntawm kev ua haujlwm rau electrochemical tshawb pom ntawm amplicons tau los ntawm cov kab mob sib xyaw ua ke hauv cov qauv dej hauv pas dej.
Peb tsis ntev los no tau qhia txog electrochemical sensing ntawm SARS-CoV-2 amplicons nrog cov nqi qis tshaj tawm Circuit Board (PCB) electrodes raws li DPV thiab cyclic voltammetry (CV) induced los ntawm adsorption ntawm MB-DNA complexes ntawm qhov chaw ntawm unmodified electrodes ) hloov nyob rau hauv ncov. tam sim no 11.Peb tshaj tawm hais tias ntev DNA fragments (N1-N2, \({943}\, \hbox) tsim siv CDC-pom zoo N1 rau pem hauv ntej thiab N2 rov qab primers piv nrog luv luv fragments {bp}\)) pom zoo linearity nyob rau hauv lub sensor teb (N1, \(72\,\hbox {bp}\)) tsim siv N1 rau pem hauv ntej thiab N1 thim rov qab primer sets.Cov kev tshawb fawb no tau tshaj tawm siv DNA dilutions npaj rau hauv cov dej tsis muaj nuclease.Lub platform kuj tau siv los kuaj SARS-CoV. -2 amplicons nyob rau hauv cov qauv simulated dej khib nyiab (tau los ntawm spiking tag nrho RNA cov qauv nrog SARS-CoV-2 RNA).Vim RNA muaj kev cuam tshuam rau shearing thaum lub sij hawm kev sib cais thiab downstream processing, 12,13 nws yog ib qho nyuaj rau amplify ntev fragments nrog no heterogeneous qauv. Yog li ntawd, kev ua qauv qhia ntawm electrochemical sensing ntawm SARS-CoV-2 amplicon nyob rau hauv cov dej khib nyiab yog txwv rau qhov luv dua \(72\,\hbox {bp}\) N1 fragment.
Hauv kev ua haujlwm no, peb tau tshawb xyuas qhov ua tau ntawm ENIG PCB-based electrochemical sensing ntawm phage Phi6 concentrated thiab cais tawm ntawm cov qauv dej hauv pas dej (Fig. 1).Phages yog piv rau qhov loj (80-100 nm) rau SARS-CoV-2 thiab Kuj muaj cov lipid daim nyias nyias thiab cov protein ntau.Rau cov laj thawj no, bacteriophage Phi6 yog ib tus neeg nyiam tshaj plaws rau SARS-CoV-2 thiab lwm cov kab mob RNA viruses14,15.RNA cais tawm ntawm phage particles tau siv los ua tus qauv rau cDNA synthesis ua raws. PCR kom tau txais ob qhov DNA ntawm 117 thiab 503 lub hauv paus khub hauv qhov ntev.Tau qhov kev sib tw ntawm amplifying \(943\,\hbox {bp}\) N1-N2 fragments nyob rau hauv peb cov hauj lwm dhau los, peb tsom nruab nrab ntev fragments (\(117 \,\hbox {bp}\) thiab \(503 \,\hbox {bp}\)), raws li muaj primers.Cov electrochemical sensor teb tau systematically kawm nyob rau hauv ib tug dav concentration ntau yam (\({10}\,{ \hbox {pg}/\upmu \hbox {l}}\) to \({20}\, {\hbox {ng}/\upmu \hbox {l}}}\)) Rau ob qho tib si fragments hauv lub xub ntiag ntawm MB, cov nyhuv ntawm ntsev ntawm cov lus teb sensor tau raug tus cwj pwm thiab hla kev siv tau los ntawm kev ntsuas spectrophotometric.Cov kev koom tes tseem ceeb ntawm txoj haujlwm no yog raws li hauv qab no:
DNA fragment ntev thiab muaj ntsev nyob rau hauv cov qauv muaj kev cuam tshuam rhiab heev.Peb cov txiaj ntsig tau pom tias electrochemical kev ua haujlwm nyob ntawm cov txheej txheem sib txawv ntawm kev sib cuam tshuam ntawm MB, DNA, thiab cov sensor nyob rau hauv cov lus teb voltammetric, nyob ntawm DNA concentration thiab ntev, nrog ntev Fragments qhia ntau dua rhiab heev, txawm tias ntsev muaj qhov tsis zoo ntawm electrostatic kev sib cuam tshuam ntawm MB thiab DNA.
DNA concentration txiav txim lub mechanism ntawm MB-DNA kev sib cuam tshuam nyob rau hauv unmodified electrodes Peb qhia tau hais tias txawv mechanisms ntawm MB-DNA kev sib cuam tshuam nyob ntawm DNA concentration.At DNA concentrations qis dua ib tug me me ntawm \({\hbox {ng}/\upmu \hbox {l}}}\), peb tau pom tias cov lus teb electrochemical tam sim no feem ntau txiav txim siab los ntawm kev nqus ntawm MB-DNA ntawm cov electrode, thaum nyob rau hauv qis dua ntawm DNA concentrations, cov lus teb electrochemical tam sim no tau txiav txim siab los ntawm steric inhibition ntawm redox. kev ua si vim MB nkag ntawm DNA puag khub.
ENIG PCB-Based Electrochemical Sensing of Viral Nucleic Acids in Lake Water Samples Cov kev soj ntsuam tau raug lees paub los ntawm kev tshawb nrhiav electrochemical ntawm Phi6-ntxiv \(503\,\hbox {bp}\) DNA fragments tau los ntawm cov qauv dej los ntawm Powai Lake, IIT Mumbai Campus Result phage.
Tus nqi qis ntawm kev siv thiab muaj peev xwm rau kev sib koom ua ke rau hauv kev tswj xyuas kev ua haujlwm siab, oligonucleotides lossis aptamers ntawm electrodes nrog lub neej ntev.
Phage Phi6 yog ib lub hnab ntim dsRNA tus kab mob ntawm tsev neeg Cytoviridae uas kis tau Pseudomonas syringae.Lub genome ntawm Phi6 phage muaj nyob rau hauv daim ntawv ntawm 3 fragments: S (\(2.95\,\hbox {Kb}\)), M (\(4.07) \,\hbox {Kb}\)) thiab L (\ (6.37\ ,\hbox{Kb}\)))16,17.Vim Phi6 phage kis tus kab mob BSL-1 Pseudomonas uas tsis yog kab mob, nws muaj kev nyab xeeb siv. thiab tuaj yeem cog tau yooj yim hauv chav kuaj.Phage Phi6 thiab nws tus tswv tsev Pseudomonas syringae tau yuav los ntawm Felix d'Herelle Reference Center for Bacterial Viruses, Laval University, Canada (chaw siv cov lej teev npe yog HER-102 thiab HER-1102, ntsig txog) .Phage Phi6 thiab nws tus tswv tsev tau rov kho dua raws li qhia los ntawm qhov chaw siv.Phage Phi6 tau ua kom huv los ntawm phaj lysis thiab elution kom tau txais titers zaum kawg nrog \(\txog 10^{12}\,\hbox {PFU}/\hbox { ml}}\) (plaque forming units / milliliters).RNA raug cais tawm ntawm cov phage purified particles siv GenElute™ Universal Total RNA Purification Kit (Sigma-Aldrich) raws li cov chaw tsim tshuaj paus cov lus qhia.Rov luv, ib tug purified phage Phi6 ncua\({ 100}\,{{\upmu \hbox {l}}}\) tau lysed thiab cov lysate tau thauj khoom mus rau kab kem kom tso cai rau RNA khi rau kab ke .Lub RNA yog tom qab ntawd eluted hauv cov tshuaj elution \({ 50}\,{{\upmu \hbox {l}}}\) muab los ntawm cov khoom siv.Xaiv qhov concentration ntawm RNA los ntawm absorbance ntawm \(260\,\hbox {nm}\).RNA tau khaws cia rau hauv aliquots hauv \ ({-80}\,{^{\circ }\hbox {C}}\) mus txog thaum siv ntxiv.\({2}\,\upmu \hbox {g}}\) iScript cDNA Synthesis Kit (Bio -Rad Laboratories) tau siv los ua tus qauv rau cDNA synthesis raws li cov chaw tsim khoom cov lus qhia.Rov qab, cov tshuaj tiv thaiv cDNA muaj 3 kauj ruam: priming ntawm \({25}\,{^{\circ }\hbox {C}}\ )\({5}\,\hbox {min} }\) , reverse transcription of \({20}\,\hbox {min}\) at \({46}\,{^{\circ }\hbox {C}}\), thiab rov qab Cov recorder yog nyob rau hauv \({95}\,{^{\circ }\hbox {C}}\) rau \({1}\,\hbox {min }}\).Thaum khiav ntawm 1% agarose gel, cDNA pom peb pawg sib txuas rau qhov xav tau peb RNA fragments (cov ntaub ntawv tsis qhia).Cov primers hauv qab no tau siv los nthuav ob DNA tawg ntawm 117 thiab 503 bp ntev, siv cDNA ua qauv rau PCR hauv miniPCR® mini8 thermal cycler:
Cov primers rau \(117\,\hbox {bp}\) thiab \(503\,\hbox {bp}\) sib raug rau 1476-1575 nucleotides ntawm M ntu thiab 458-943 nucleotides ntawm L ntu, raws li acid. .Txhua cov khoom lag luam PCR amplified tau electrophoresed ntawm 1% agarose gels, thiab amplified lub hom phiaj DNA tau purified siv GeneJET Gel Extraction Kit (Thermo Fisher Scientific).
Ib lub pas dej ntawm IIT Mumbai tsev kawm ntawv (Powai Lake, Powai, Mumbai) tau siv los ntxiv phage particles.Lub pas dej dej tau lim los ntawm \({5}\,{\upmu \hbox {m}}\) membrane kom tshem tawm. suspended particles, thiab ces Phi6 phage tau ntxiv.Add \({1}\,\hbox {ml}\) of \(10^{6}\,{\hbox {PFU}/\hbox {ml}} \) to \( {100}\ ,{\hbox {ml}}\) filtered lake water, in \({4}\,{^{\circle}\hbox {C}}\).A me aliquot yog reserved rau viral load ntsuas los ntawm plaque assay.Peb tau sim ob txoj kev sib txawv los mloog zoo rau cov kab mob Phi6 cov kab mob: (1) aluminium hydroxide adsorption-precipitation method, 19 uas tau raug lees paub rau qhov concentration ntawm ntau lub hnab ntawv RNA cov kab mob los ntawm ib puag ncig cov qauv, thiab (2)) Cov kab mob polyethylene glycol (PEG)-raws li tus kab mob concentration yog hloov los ntawm Dej nyab thiab al.20 .Txij li kev rov ua haujlwm ntawm PEG-raws li txoj kev tau pom tias zoo dua li cov txheej txheem txhuas hydroxide, PEG-raws li txoj kev tau siv los ua kom pom tseeb Phi6 hais los ntawm cov qauv dej hauv pas dej.
PEG txoj kev siv tau raws li hauv qab no: PEG 8000 thiab \(\hbox {NaCl}\) tau ntxiv rau Phi6-spiked pas dej piv txwv kom tau 8% PEG 8000 thiab \(0.2\,\hbox {M} \) \( \ hbox {NaCl}\).Samples incubated on a shaker\({4}\,{^{\circ }\hbox {C}\)\({4}\,\hbox {h}}\ ), ces centrifuged ntawm \(4700 \,\hbox {g}\) yog \({45}\,\hbox {min}\). {\hbox {ml}}\) nyob rau tib lub supernatant.Tag nrho cov spiking thiab tus kab mob concentration thwmsim tau ua nyob rau hauv triplicate.Tom qab concentration, ib tug me me aliquot tau tseg rau kev ntsuas ntawm rov ua hauj lwm efficiency los ntawm plaque assay.RNA raug cais raws li yav tas los piav thiab eluted nyob rau hauv cov khoom siv-supplyed elution buffer\({40}\,{\upmu \hbox {l}}\).Vim RNA concentration yuav txawv ntawm cov qauv rau cov qauv hauv triplicate, lub \({2}\,\upmu \ hbox {l}}\) ntawm RNA yog siv rau tag nrho peb yam tsis hais nws qhov concentration cDNA synthesis ntawm samples.cDNA synthesis tau ua raws li tau piav qhia yav dhau los.\({1}\,{\upmu \hbox {l}}\) cDNA tau siv los ua template rau \({20}\,{\upmu \hbox {l}}\) PCR rau 35 cycles rau amplify \(117\,\hbox {bp}\) thiab \(503\,\hbox { bp}\) fragments.Cov qauv no yog sawv cev raws li "1: 1", piv txwv li tsis muaj dilution.A tsis muaj tus qauv tswj (NTC) tau teeb tsa raws li kev tswj tsis zoo, thaum lub cDNA synthesized siv RNA cais los ntawm purified phage tau teeb tsa Raws li tus qauv rau kev tswj xyuas qhov zoo (PC).Quantitative PCR (qPCR) tau ua nyob rau hauv Stratagene Mx3000P RT-PCR ntsuas siv Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies). Cov tshuaj tiv thaiv tau teeb tsa hauv triplicate raws li yav dhau los piav qhia.Lub voj voog lub voj voog (Ct) raug kaw rau tag nrho cov qauv.Ntxiv mus, cov qauv diluted tau \({1}\,\upmu \hbox {l}}\) siv cDNA diluted 1:100 hauv cov dej lim dej raws li \({20}\,\upmu \hbox {l}}\) PCR rau 35 cycles.Cov qauv no yog sawv cev li “1:100″.
Lub PCB electrodes yog fabricated siv cov khoom lag luam uas muaj nqi qis Electroless Nickel Immersion Kub (ENIG) txheej txheem yam tsis tas yuav tsum tau ntxiv kub plating.ENIG PCB electrode specifications yog cov ncauj lus kom ntxaws nyob rau hauv peb yav dhau los ua hauj lwm11.For ENIG PCB electrodes, tsoos electrode tu txoj kev xws li piranha tov los yog sulfuric acid cyclic voltammetry tsis pom zoo vim lawv yuav ua rau tev ntawm cov txheej kub nyias (thickness \(\approx\) \(100\,\hbox {nm }\)) thiab nthuav tawm cov txheej tooj liab hauv qab uas yog nquag. rau corrosion 21, 22, 23, 24, 25.Yog li ntawd, ntxuav cov electrodes nrog ib daim ntaub tsis muaj lint-dawb moistened nrog IPA.Cov qauv yuav tsum tau kuaj yog incubated nrog \({50}\,{\upmu \hbox {M} }\) MB in \({4}\,{^{\circ }\hbox {C}}\)\({ 1}\,\hbox {h}}\) kom yooj yim ntxig.Nyob rau hauv peb cov hauj lwm yav dhau los , peb pom tias qhov rhiab heev thiab linearity ntawm lub sensor tau txhim kho los ntawm kev nce MB concentration 11 .Raws li kev ua kom zoo tshaj plaws hauv peb txoj haujlwm dhau los, peb siv \({50}\,{\upmu \hbox {M}}\) MB Cov concentrations rau embed DNA nyob rau hauv txoj kev tshawb no.Electrochemical nrhiav tau ntawm ob-stranded DNA (ds-DNA) yuav ua tau tiav siv anionic los yog cationic intercalators.Txawm hais tias anionic intercalators ntes DNA nrog zoo selectivity, lawv yuav tsum tau ib hmo incubation, ua rau lub sij hawm ntev nrhiav tau. qhov tod tes, cationic intercalators xws li MB yuav tsum tau luv incubation lub sij hawm, kwv yees li \({1}\, \hbox {h}}\) rau electrochemical nrhiav tau ntawm ds-DNA6.Txhua qhov kev ntsuas yuav tsum dispensing tus qauv mus kuaj ntawm lub electrode\({5}\,{{\upmu \hbox {l}}}\), tom qab ntawd ntxuav nrog IPA-moistened rag, ua ntej pib nrog lwm tus qauv.ib qho kev ntsuas.Txhua tus qauv raug sim ntawm 5 qhov sib txawv electrodes tshwj tsis yog hais tias lwm yam.DPV thiab CV ntsuas tau ua los ntawm PalmSens Sensit Smart potentiostat, thiab PSTrace software tau siv rau potentiostat configuration thiab cov ntaub ntawv nrhiav tau, suav nrog cov kev suav tam sim no.Cov chaw hauv qab no yog siv. rau DPV thiab CV ntsuas:
DPV: Equilibrium Time = \(8\,\hbox {s}\), Voltage Step = \(3\,\hbox {mV}\), Pulse Voltage = \(25\,\hbox {mV}\), pulse duration = \(50\,\hbox {ms}\), scan rate = \({20}\,\hbox {mV/s}\)
CV: Equilibrium Time = \(8\,\hbox {s}\), Voltage Step = \(3\,\hbox {mV}\), Sweep Rate = \({300}\,\hbox {mV/s }\)
Peak tam sim no tau los ntawm voltammograms ntawm DNA complexed nrog \({50}\,\upmu \hbox {M}}\) MB: (a) \(503\,\hbox {bp}\) DPV , (b) \ (503\,\hbox {bp}\) CV, (c) \(117\,\hbox {bp}\) DPV, (d) \(117\,\hbox {bp}\) CV.
DPV thiab CV voltammograms tau txais ntawm ENIG PCB electrodes \({50}\,\upmu \hbox {M}}\) MB complexed nrog DNA (ntawm concentrations ntawm 10–\({20}\, hbox {ng }/\upmu \hbox {l}}\) ie 0.13–\({0.26}\,{\upmu \hbox {M}}\) for \(117\,\hbox {bp}\ ) and 0.03 -\({0.06}\,{\upmu \hbox {M}}\) rau \(503\,\hbox {bp}\)). Cov neeg sawv cev voltammograms tau qhia hauv daim duab S1 hauv Cov Ntaub Ntawv Ntxiv.Daim duab 2 qhia cov txiaj ntsig ntawm DPV thiab CV ntsuas (nruab nrab tam sim no) siv cov khoom siv gel-purified PCR.Piv rau CV kev ntsuas, DPV kev ntsuas qhia tau ntau dua rhiab heev (tam sim no raws li kev ua haujlwm ntawm DNA concentration) vim hais tias lub keeb kwm yav dhau capacitive tam sim no hauv CV ntsuas zais Faradaic tam sim no 26 .Cov ntaub ntawv rau txhua lub thawv hauv lub thawv ntawv muaj cov kev ntsuas los ntawm 5 electrodes.Txhua qhov kev ntsuas siv tib lub electrodes kom tsis txhob ntsuas qhov yuam kev vim electrode-rau-electrode variation.Peb tau pom qhov sib txawv ntawm DPV thiab CV ntsuas peak tam sim no rau qis dua cov ntsiab lus ntawm DNA. , ntev (\(503\,\hbox {bp}\)) \,\hbox {bp}\ piv rau \(117) ) fragment.Qhov no yog raws li qhov kev cia siab ntawm electrode adsorption qhia nyob rau hauv peb yav dhau los ua hauj lwm. adsorption ntawm MB-DNA complex pab txhawb cov nqi hloov pauv ntawm cov electrode, uas ua rau muaj kev nce siab ntawm lub ncov tam sim no.Lwm cov kev tshawb fawb tau pom tias cov nyhuv ntawm oligonucleotide loj thiab ib ntus ntawm MB-DNA intercalation27,28,29,30.The guanine -cytosine (GC) cov ntsiab lus ntawm ob lub amplicons (\(117\,\hbox {bp}\) thiab \(503\,\hbox {bp}\))) yog kwv yees li 50%, qhia tias kev soj ntsuam qhov txawv yog vim rau amplicon length.Txawm li cas los xij, rau ntau dua DNA ntau dua (\(>{2}\,\hbox {ng}/\upmu \hbox {l}}}\), rau \(503\,\hbox {bp} \) thiab \(>{10}\,{\hbox {ng}/\upmu \hbox {l}}}\) rau \(117\,\hbox {bp}\)), peb soj ntsuam ob amplifications Qhov siab tshaj plaws tam sim no ntawm cov subs raug txo nyob rau hauv ob qho tib si DPV thiab CV ntsuas.Qhov no yog vim MB saturates thiab intercalates ntawm lub hauv paus khub ntawm DNA, uas ua rau steric inhibition ntawm redox kev ua ntawm pawg reducible hauv 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.
Cov ntsev tam sim no hauv PCR master sib tov cuam tshuam nrog electrostatic kev sib cuam tshuam ntawm MB thiab DNA, yog li los ntawm kev ntxiv \(2\,\hbox {mM}\) \(\hbox {MgCl }_2\) nrog \({50} \,{\ upmu \hbox {M}}\) MB gel-purified cov khoom los kawm txog cov txiaj ntsig ntawm ntsev ntawm MB-DNA kev sib cuam tshuam.Raws li pom hauv daim duab 3, peb pom tias kom muaj ntau dua DNA ntau dua (\(>{2}\,{\ hbox {ng}/\upmu \hbox {l}}\) (503\,\hbox {bp }\) and \(>{10}\,\hbox {ng}/\upmu \hbox { l}}}\) rau \(117\,\hbox {bp} \)), hauv DPV thiab CV Cov ntsev ntxiv tsis cuam tshuam rau kev ntsuas (saib daim duab S2 hauv Cov Lus Qhia Ntxiv rau cov neeg sawv cev voltammograms).Txawm li cas los xij, ntawm qis dua DNA concentrations, qhov sib ntxiv ntawm ntsev kuj txo qis rhiab heev, ua rau tsis muaj kev hloov pauv tseem ceeb hauv qhov tam sim no nrog DNA concentration.Zoo zoo li qhov tsis zoo ntawm ntsev ntawm MB-DNA kev sib cuam tshuam thiab intercalation tau tshaj tawm yav dhau los los ntawm lwm cov kws tshawb fawb33,34.\(\hbox { Mg}^{2+}\) cations khi rau qhov tsis zoo phosphate caj qaum ntawm DNA, yog li cuam tshuam cov electrostatic kev sib cuam tshuam ntawm MB thiab DNA.Thaum siab dua DNA concentrations, steric inhibition ntawm redox-active MBs ua rau qis qis dua tam sim no, yog li electrostatic kev sib cuam tshuam. Tsis txhob cuam tshuam rau cov lus teb sensor.Lub ntsiab lus tseem ceeb yog qhov biosensor no zoo dua los kuaj xyuas DNA ntau dua (tsis tshua muaj \({\hbox {ng}/{\upmu \hbox {l}}}\) lossis siab dua), rau tag nrho- Automated ua cov qauv dej ib puag ncig, qhov twg gel purification ntawm PCR cov khoom yuav tsis ua tau.
Cheeb tsam nyob rau hauv lub absorption nkhaus rau lub wavelength range 600–700 \(\hbox {nm}\) rau ntau yam concentrations ntawm DNA complexed nrog \({50}\,\upmu \hbox {M}}\) MB: (a ) \(503\,\hbox {bp}\) nrog thiab tsis muaj ntsev (\(2\,\hbox {mM}\) \(\hbox {MgCl}_2\)), (b) \( 117\, \hbox {bp}\) nrog thiab tsis muaj ntsev (\(2\,\hbox {mM}\) \(\hbox {MgCl}_2\)).\({0}\,\hbox {pg}/ {\upmu \hbox {l}}}\) DNA concentrations corresponding to \({50}\,\upmu \hbox {M}}\) MB samples No DNA.
Txhawm rau txheeb xyuas cov txiaj ntsig saum toj no, peb tau ua qhov ntsuas qhov muag siv lub UV / Vis spectrophotometer (Thermo Scientific Multiskan GO), cov qauv \({50}\,{{\upmu \hbox {l}}}\) tau siv rau txhua qhov. Kev ntsuas.Qhov kev nqus tau kos npe txo qis nrog kev nce DNA concentration, raws li pom tau los ntawm qhov sib txawv ntawm thaj chaw hauv qab qhov nkhaus ntawm qhov nqus rau lub wavelength range \(600\,\hbox {nm}\) rau \(700\,\hbox { nm}\), raws li qhia hauv daim duab 4 (kev nqus spectrum pom hauv daim duab S3 hauv Cov Lus Qhia Ntxiv).Rau cov qauv nrog DNA concentrations tsawg dua \({1}\,{\hbox {ng}/\upmu \hbox {l}}}\), tsis muaj qhov sib txawv tseem ceeb hauv kev txhawb nqa ntawm DNA-muaj thiab MB-tsuas cov qauv (rau \(503\,\hbox {bp}\) thiab \(117\,\hbox {bp}\ ) ntev fragments), qhia tias tsis muaj steric inhibition ntawm redox-active MB.Thaum siab dua DNA concentrations, peb tau pom ib tug maj mam txo nyob rau hauv lub absorbance teeb liab thiab sau tseg ib tug tsawg txo nyob rau hauv absorbance nyob rau hauv lub xub ntiag ntawm ntsev.Cov txiaj ntsim tau los ntawm molecular. Kev sib cuam tshuam thiab steric inhibition nrog lub hauv paus stacking hauv DNA hybrids.Peb cov txiaj ntsig tau zoo ib yam nrog cov ntaub ntawv hauv cov ntaub ntawv ntawm kev tshawb fawb spectroscopic ntawm MB-DNA intercalation uas koom nrog hypochromaticity nrog txo qis zog hauv \(\pi\)-\(\pi ^*\ ) Kev hloov hluav taws xob vim yog kev sib cuam tshuam txheej txheej 36, 37, 38.
Agarose gel electrophoresis ntawm phage Phi6: PCR cov khoom ntawm qhov ntev \(117\,\hbox {bp}\) thiab \(503\,\hbox {bp}\) los ntawm pas dej ua piv txwv.M-DNA marker;NTC-tsis muaj-tus qauv tswj, primers uas muaj cov amplicons sib xws;PC tswj zoo;1, 2, 3-undiluted (1: 1) spiked pas dej piv txwv hauv triplicate.A band pom ntawm \(\li 50\,\hbox {bp}\) vim tsis siv oligonucleotides hauv \(503\,\ hbox {bp}\) txoj kab.
Peb tau soj ntsuam cov nqi hluav taws xob ntawm lub ntsuas hluav taws xob siv cov qauv dej Powai Lake spiked nrog Phi6 phage.The RNA concentrations cais los ntawm phage-spiked dej kuaj ranged ntawm 15.8–\({19.4}\, {\upmu \hbox {g}/\hbox { ml}}\), thaum cov uas raug rho tawm los ntawm purified phage suspensions Lub RNA tau kwv yees tias yog \({1945}\, \upmu \hbox {g}/\hbox {ml}}\) nrog kev rov ua haujlwm ntawm kwv yees li 1 %.RNA tau thim rov qab rau hauv cDNA thiab siv los ua qauv rau PCR thiab qPCR.Cov khoom loj tau lees paub los ntawm agarose gel electrophoresis (Daim duab 5) ua ntej kuaj nrog lub sensor.Cov qauv no tsis yog gel purified thiab yog li ntawd muaj tag nrho cov khoom ntawm PCR raws li zoo li cov amplicons ntawm kev txaus siab.Cov Ct qhov tseem ceeb tau sau tseg thaum lub sij hawm qPCR (Table 1) tau pom tias muaj kev cuam tshuam nrog cov concentration ntawm RNA cais los ntawm cov qauv dej spiked.Tus nqi Ct qhia txog cov voj voog uas yuav tsum tau muaj rau lub teeb liab fluorescent rau Tshaj qhov pib lossis keeb kwm yav dhau los.Higher Ct qhov tseem ceeb qhia tias qis template concentrations thiab vice versa.Cov Ct qhov tseem ceeb ntawm NTC cov qauv tau siab raws li qhov xav tau.Qhov txawv ntawm \(\kwv yees 3\) Ct qhov tseem ceeb ntawm qhov zoo tswj thiab cov qauv kuaj ntxiv qhia tau hais tias txhua tus qauv kuaj muaj kwv yees li 1% template piv rau qhov zoo tswj.Peb tau yav tas los tham tias ntev amplicons ua rau kom zoo dua rhiab heev.Amplification ntawm ntev fragments cais los ntawm heterogeneous ib puag ncig cov qauv yog nyuaj rau qhov tsis zoo. ntawm cov kab mob qis qis thiab RNA degradation.Txawm li cas los xij, nrog peb cov kab mob enrichment thiab PCR amplification raws tu qauv, peb muaj peev xwm ua tau zoo amplify \(503\,\hbox {bp}\) fragment rau electrochemical sensing.
Daim duab 6 qhia cov txiaj ntsig ntawm electrochemical sensor ntawm \(503\,\hbox {bp}\) fragment amplicon, ob leeg siv undiluted cDNA li template (1:1) thiab 100-fold diluted cDNA li template (1:100) ua PCR , piv rau NTC thiab PC (saib daim duab S4 hauv Cov Lus Qhia Ntxiv rau cov neeg sawv cev voltammograms).Txhua lub thawv hauv lub thawv ntawv hauv daim duab 6 muaj kev ntsuas los ntawm peb cov qauv ntawm 5 electrodes.Cov electrodes tib yam tau siv los ntsuas tag nrho cov qauv kom tsis txhob ua yuam kev vim electrode. -to-electrode variation.Piv rau CV kev ntsuas, DPV kev ntsuas qhia tau zoo dua kev daws teeb meem kom paub qhov txawv ntawm kev sim thiab PC cov qauv los ntawm NTCs vim hais tias, raws li tau hais dhau los, Faradaic tam sim no tau muab zais vim muaj cov kab hluav taws xob tam sim no tom qab.Rau cov amplicons ntev dua, peb pom tias cov kev tswj tsis zoo (NTC) ua rau siab dua CV thiab DPV ncov tam sim no txheeb ze rau kev tswj qhov zoo, qhov zoo thiab tsis zoo cov qauv kuaj pom pom zoo li qhov siab siab ntawm DPV ncov tam sim no.Qhov ntsuas qhov nruab nrab thiab qhov nruab nrab rau txhua qhov tsis zoo (1: 1 ) kuaj cov qauv thiab PC tuaj yeem daws tau meej meej los ntawm cov khoom tso tawm sensor rau NTC qauv, thaum qhov kev daws teeb meem rau 1: 100 diluted qauv yog qhov tsis tshua muaj suab nrov.Rau 100-fold dilution ntawm cDNA, peb tsis tau soj ntsuam ib qho bands thaum gel electrophoresis (txoj kab tsis tau qhia hauv daim duab 5), thiab qhov sib thooj DPV thiab CV peak tam sim no zoo ib yam li cov uas xav tau rau NTC.Cov txiaj ntsig rau \(117\,\hbox {bp}\) fragment yog qhia hauv Cov Ntaub Ntawv Ntxiv.Qhov tsis zoo tswj induced ib tug electrochemical teb los ntawm PCB sensor vim lub adsorption ntawm dawb MB ntawm lub electrode thiab kev sib cuam tshuam ntawm MB nrog ib leeg-stranded primer oligonucleotide.Yog li ntawd, txhua zaus ib tug qauv raug kuaj, ib tug tsis zoo tswj yuav tsum tau khiav thiab cov ncov tam sim no ntawm cov qauv kuaj piv rau lub ncov tam sim no tau los ntawm kev tswj tsis zoo kom ua tiav qhov sib txawv (tus txheeb ze) ntsuas 39,40 los faib cov qauv ntsuas qhov zoo lossis qhov tsis zoo.
(a) DPV, thiab (b) CV peak tam sim no rau electrochemical tshawb pom ntawm \(503\,\hbox {bp}\) fragments nyob rau hauv lub pas dej ua qauv. Cov qauv kuaj tau ntsuas nyob rau hauv triplicate thiab piv rau tsis muaj template tswj (NTC) thiab kev tswj zoo (PC).
Peb qhov kev tshawb pom qhia txog cov txheej txheem sib txawv uas cuam tshuam rau kev ua haujlwm ntawm electrochemical sensors rau amplicons ntawm qhov ntev sib txawv rau cov DNAs sib txawv, nrog cov concentrations txheeb xyuas los ntawm kev ntsuas qhov muag siv lub UV / Vis spectrophotometer.Peb cov kev soj ntsuam underscore qhov kev pom tias ntev DNA tawg mus txog \(\ approx\) \(500\,\hbox {bp}\) tuaj yeem kuaj pom muaj qhov rhiab heev thiab qhov muaj ntsev hauv cov qauv tsis rhiab heev DNA concentration uas cuam tshuam rau rhiab heev (tsis tshua muaj \(\hbox {ng}/\upmu \hbox {l}}}\) thiab saum toj no). Tsis tas li ntawd, peb tau tshawb xyuas cov txiaj ntsig ntawm ntau hom qauv, suav nrog gel-purified amplicons nrog thiab tsis muaj ntsev ntxiv, thiab ntxiv cov qauv dej hauv pas dej hauv DPV thiab CV ntsuas.Peb tau pom tias DPV muab kev daws teeb meem zoo dua, txij li cov keeb kwm yav dhau los tam sim no tseem cuam tshuam rau CV ntsuas, ua rau nws tsis tshua muaj siab.
Kev nthuav dav ntawm cov fragments ntev yog nyob ntawm kev ncaj ncees ntawm tus kab mob genomic RNA.Muaj kev tshawb fawb tau pom tias qhov kev nthuav dav ntawm cov tawg ntev tsis yog ib txwm ua tau zoo vim yog qhov degradation ntawm RNA nyob rau hauv ib puag ncig thiab muaj peev xwm rau splicing thaum kev sib cais 11,41,42,43,44. .Peb pom tias PEG-raws li tus kab mob concentration txoj kev muaj txiaj ntsig zoo hauv kev tsom mus rau phage Phi-6 spiked hauv cov qauv dej hauv pas dej dua li cov kab mob txhuas hydroxide-based virus concentration method.Lub peev xwm los ntes cov DNA ntev ntev tau ua pov thawj los kov yeej qhov yuav tsum tau muaj rau multiplex PCR. kom nthuav dav ntau yam luv luv templates thiab txo qhov muaj peev xwm ntawm cross-specificity.
Cov qauv tshuaj lom neeg tsis tshua muaj, yog li yuav tsum tau tsim lub biosensor uas yuav tsum tau kuaj tsawg kawg nkaus rau kev sim.Cov ENIG PCB electrodes siv nyob rau hauv txoj kev tshawb no tsuas yog yuav tsum tau \({5}\,{{\upmu \hbox {l}}}\ ) cov qauv rau kev ntsuam xyuas kom npog thaj tsam zoo ntawm cov electrodes.Ntxiv rau, tib lub electrode tuaj yeem rov qab siv dua tom qab ntxuav ua ntej xa cov qauv tom ntej.Amplified cov qauv tsis tas yuav ntxiv cov tshuaj ntxiv uas tsis yog methylene xiav, uas yog ib qho pheej yig. thiab feem ntau siv tshuaj.Vim tias txhua lub electrode raug nqi txog $0.55 (lossis INR 40) los tsim, biosensor no tuaj yeem ua tus nqi zoo rau kev tshawb nrhiav cov thev naus laus zis uas twb muaj lawm.Table 2 qhia txog kev sib piv ntawm cov haujlwm no nrog lwm cov sensors qhia hauv cov ntaub ntawv ntev. DNA fragments nyob rau hauv heterogeneous kuaj.
Muab hais tias MB-raws li electrochemical detection protocols cia siab rau qhov tshwj xeeb ntawm PCR, ib qho kev txwv loj ntawm txoj kev no yog lub peev xwm rau kev ua kom tsis muaj zog tshwj xeeb hauv cov qauv sib txawv xws li dej khib nyiab thiab dej hauv pas dej lossis siv cov primers uas tsis muaj purity. electrochemical tshawb nrhiav txoj hauv kev rau DNA nrhiav kom tau cov khoom tsis huv PCR siv unmodified ENIG PCB electrodes, nws yog ib qho tsim nyog kom nkag siab zoo dua cov lus qhia los ntawm cov tsis siv dNTPs thiab primers, thiab ua kom zoo dua cov tshuaj tiv thaiv thiab kev soj ntsuam raws tu qauv.Ntxiv physicochemical tsis xws li pH, kub, thiab lom. oxygen thov (BOD) ntawm cov qauv dej kuj yuav tsum tau ntsuas txhawm rau txhawm rau txhim kho qhov tseeb ntawm qhov ntsuas.
Hauv kev xaus, peb xav kom tus nqi qis electrochemical ENIG PCB sensor rau kev kuaj kab mob hauv ib puag ncig (lub pas dej hauv pas dej) cov qauv.Tsis zoo li immobilized oligonucleotide electrodes los yog kev cai substrates rau DNA sensing uas yuav tsum tau cryogenic cia kom muaj rhiab heev, 53,54 peb cov txheej txheem siv unmodified PCB. Cov khoom siv hluav taws xob nrog lub neej ntev dua thiab tsis muaj qhov tshwj xeeb khaws cia thiab yog li tsim nyog rau kev txhim kho cov kev ntsuas kev daws teeb meem nrog cov qauv ua haujlwm ua haujlwm hauv LMICs.The biosensor siv pheej yig DNA-intercalating redox dyes (MB) rau kev tshawb nrhiav sai ntawm lub hom phiaj amplicons. Feem ntau nyob rau hauv ib puag ncig cov qauv txo qhov tshwj xeeb ntawm no sensing txoj kev vim lub nonspecific binding ntawm MBs rau ib leeg- thiab ob-stranded oligonucleotides.Yog li ntawd, qhov tshwj xeeb ntawm qhov kev ntsuam xyuas no nyob ntawm qhov optimization ntawm primers thiab PCR cov tshuaj tiv thaiv tej yam kev mob. Ntxiv rau, CV. thiab DPV ncov tam sim no tau txais los ntawm cov qauv kuaj yuav tsum tau txhais cov ntsiab lus ntawm cov lus teb tau los ntawm kev tswj tsis zoo (NTC) rau txhua qhov kev sim.Cov qauv siv hluav taws xob hluav taws xob thiab cov txheej txheem uas tau nthuav tawm hauv txoj haujlwm no tuaj yeem ua ke nrog autosamplers los tsim kom muaj kev ua haujlwm siab thiab qis. -Cov nqi daws teeb meem uas tuaj yeem sau thiab txheeb xyuas cov qauv thiab wirelessly xa cov txiaj ntsig rov qab mus rau chav kuaj.
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Post lub sij hawm: May-27-2022