Iindaba

Izicwangciso zendabuko zokuxilonga zokufumanisa izifo ezithathelwanayo zifuna ukusetyenziswa kwezixhobo ze-benchtop ezingafanelekanga ukuhlolwa kwe-point-of-care test (POCT).I-microfluidics ekhulayo yitekhnoloji encinci, ezenzekelayo, kunye nedibeneyo enokuthi ibe yindlela enokuthi ibekho kwiindlela zemveli zokuxilongwa ngokukhawuleza, ngexabiso eliphantsi, elichanekileyo kwindawo.Iindlela zokuxilonga iimolekyuli zisetyenziswa ngokubanzi kwizixhobo ze-microfluidic njengezona ndlela zisebenzayo zokufumanisa i-pathogen.Olu phononongo lushwankathela inkqubela phambili yamva nje kwi-microfluidic-based based diagnostics yezifo ezosulelayo ukusuka kumbono wezemfundo kunye noshishino.Okokuqala, sichaza ukusetyenzwa kwe-chip eqhelekileyo ye-nucleic acid, kubandakanya ukunyangwa kwangaphambili kwesampulu, ukukhulisa, kunye nokufunda umqondiso.Iimpawu, iingenelo kunye nokungonakali kweentlobo ezine zamaqonga e-microfluidic zithelekiswe ke.Emva koko, siza kuxubusha ukusetyenziswa kwee-assays zedijithali ze-quantification epheleleyo ye-nucleic acids.Zombini izixhobo zokuxilonga iimolekyuli zamandulo kunye nezamva nje zorhwebo zishwankathelwa njengobungqina bemeko yangoku yentengiso.Ekugqibeleni, siphakamisa izikhokelo zexesha elizayo zokuxilongwa kwe-microfluidic yezifo ezosulelayo.
Izifo ezosulelayo zibangelwa ziintsholongwane, eziquka iintsholongwane, iintsholongwane, nezifunxi-gazi, ezisasazwa kulo lonke ihlabathi.Ngokungafaniyo nezinye izifo, ii-pathogens zikhawuleza zisuleleke kwaye zisasazeke phakathi kwabantu kunye nezilwanyana ezisingethe nge-inoculation, umoya kunye nemithombo yamanzi [1].Uthintelo lwezifo ezosulelayo lubalulekile njengomlinganiselo wempilo yoluntu.Amacebo amathathu aphambili okulwa nezifo ezosulelayo: (1) ukulawula umthombo wosulelo;(2) ukuphazamiseka kwendlela yothumelo;(3) ukukhuselwa kwabantu abasesichengeni.Phakathi kwezicwangciso eziphambili, ukulawulwa komthombo wosulelo kuthathwa njengeyona ndlela ibalulekileyo ngenxa yokulula kwayo kunye neendleko eziphantsi.Uxilongo olukhawulezileyo, ukubekwa wedwa, kunye nonyango lwabantu abosulelekileyo lubalulekile, lufuna iindlela ezikhawulezayo, ezibuthathaka, nezichanekileyo zokuxilonga [2].Uxilongo lwangoku lwezifo ezosulelayo ludla ngokudibanisa uvavanyo lweklinikhi olusekelwe kwimiqondiso kunye neempawu kunye nezifundo zaselabhoratri ezifana nenkcubeko yeeseli kunye nokuxilongwa kwe-molecular diagnostics, efuna abasebenzi abaqeqeshiweyo, iinkqubo ezinzima zabasebenzi, kunye nezixhobo zokuvavanya ezibizayo [3, 4].Ukuthintelwa kokuqhambuka kwezifo ezithathelwanayo kufuna ukuxilongwa ngokukhawuleza, okungabizi, kunye nokuchanekileyo kwendawo, ngakumbi kwiindawo ezinobuncwane apho izifo ezithathelwanayo zixhaphake kwaye zinzima [5], kunye nonyango entlango okanye kwindawo yokulwa, apho iimeko eziphuthumayo zingalindelekanga..ukhathalelo lwezonyango lulinganiselwe [6].Kulo mongo, i-microfluidics iteknoloji edibanisa ubuchwepheshe beenkqubo ze-microelectromechanical, i-nanotechnology, okanye isayensi yezinto eziphathekayo ngokuchanekileyo ukuguqulwa kwamanzi [7,8,9,10], ukubonelela ngamathuba amatsha okufumana indawo yokunyamekela (POCT).) ii-arhente ezosulelayo ngaphandle kwezibhedlele kunye neelebhu.Xa kuthelekiswa noxilongo oluthatha ixesha eliqhelekileyo, itekhnoloji ye-microfluidic ibonelela ngesampulu kunye nogcino lweendleko zokuxilonga iimolekyuli ngexesha lokuqhambuka kwezifo.Ukusasazeka kwihlabathi jikelele kwesifo se-coronavirus ka-2019 (COVID-19) kubangelwa sisifo esibukhali sokuphefumla i-coronavirus 2 (SARS-CoV-2), ke ukubaluleka kwe-microfluidics kuthintelo kwangexesha kunye nokulawula ubhubhane kuphinda kugxininiswe [11, 12 , 13].Ngokungafaniyo noxilongo lwendabuko, i-microfluidic POCT isebenzisa izixhobo eziphathwayo ezincinci ukusuka kwi-benchtop analyzers ukuya kwi-strips encinci yokuvavanya i-sidestream ukuvavanya kufutshane nendawo yesampulu [14].Olu vavanyo lubonisa ngokulula okanye akukho lungiselelo lwesampulu, ukukhulisa umqondiso okhawulezayo, kunye nokufundwa komqondiso obuthathaka okukhokelela kubude bexesha elifutshane kunye neziphumo ezichanekileyo kwimizuzu.Ubukho kunye nobuninzi bemveliso yezixhobo zonyango ezisekelwe kwi-microfluidic ziye zandisa izicelo zabo zokuxilonga ezingabizi kakhulu kunye nezithe ngqo ngaphandle kwesibhedlele, kufuphi nesigulane, kunye nasekhaya.
Phakathi kwezicwangciso ezikhoyo zokuxilonga izifo ezithathelwanayo, i-molecular diagnostics enye yeyona nto ibuhlungu kakhulu [15, 16].Ukongeza, uxilongo lwe-molekyuli luhlala lusetyenziswa njengomgangatho wegolide wokufunyanwa rhoqo kwe-COVID-19, evumela ukuchongwa ngokuthe ngqo kwemimandla ethile yentsholongwane ye-RNA okanye i-DNA ngaphambi kokuqala kokuphendula komzimba [17, 18].Kuphononongo lwangoku, sibonisa inkqubela phambili yamva nje kwiinkqubo zokuxilonga iimolekyuli ezisekelwe kwi-microfluidics kwizifo ezithathelwanayo, ukusuka kumbono wezemfundo ukuya kwiimbono zamashishini ezizayo (umzobo 1).Siza kuqala ngamanyathelo amathathu aphambili ekubhaqweni kwe-nucleic acid: kwisampulu yesampulu ye-chip, i-nucleic acid amplification, kunye nokufunda umqondiso.Emva koko siqhathanise iintlobo ezahlukeneyo zamaqonga e-microfluidic kunye nesakhiwo kunye nomsebenzi wabo, ebonisa iimpawu ezizodwa (amandla kunye nobuthathaka).Ukufunyaniswa kwe-nucleic acid yeDijithali kuxoxwa ngakumbi kwaye kunikwe njengomzekelo weteknoloji yesizukulwana sesithathu malunga nobungakanani obupheleleyo beemolekyuli zepathogen ezosulelayo.Ukongeza, izixhobo ezininzi zePOCT eziqhelekileyo kunye nezamva nje ziya kuboniswa ukubonisa imeko yangoku ye-microfluidic POCT imakethi yokuxilongwa kweemolekyuli.Siza kuxoxa kwakhona kwaye sichaze umbono wethu wezicelo ezizayo.
Iimodyuli ze-microfluidic chips zokufumanisa i-nucleic acid zinokwahlulwa zibe ngamacandelo amathathu (isampuli, ukuqaphela, kunye nomqondiso) ngokwemisebenzi yabo [19].Phakathi kwezi modyuli, imodyuli yesampulu ikakhulu iqonda isampula ye-lysis kunye ne-nucleic acid extraction.Imodyuli yenzwa ilawula ikakhulu ukuguqulwa kunye nokukhulisa iimpawu ze-nucleic acid.Imodyuli yokubonisa ibona umqondiso oguqulwayo kwaye uqhutywe yimodyuli yokuva.Ngokusekelwe kwinkqubo yokufumana i-nucleic acids kwi-chip, siya kushwankathela iichips ezahlukeneyo ezinokuqonda umsebenzi "wokufaka kunye nokuphuma".
Inyathelo lokuqala ekubhaqweni kwe-nucleic acid kukutsalwa kwe-nucleic acid, okt ukwahlula i-nucleic acid ekujoliswe kuyo kwisampulu yokuqala.I-Nucleic acid extraction yenziwa ukuhlambulula i-nucleic acids kwezinye izinto ezingcolileyo ze-molecular, ukuqinisekisa ukunyaniseka kwesakhiwo esisisiseko se-nucleic acid, kunye nokwandisa iziphumo.I-Nucleic acid extraction ifuna i-sample lysis efunekayo kunye ne-nucleic acid yokubamba, umgangatho kunye nokusebenza kakuhle okunempembelelo enkulu kuphando kunye neziphumo zokuxilonga.Naziphi na iziphumo ebezingalindelekanga ezifihlakeleyo ngexesha lokutsalwa zinganciphisa ukubhaqwa okungaphaya.Ngokomzekelo, iindlela ze-polymerase chain reaction (PCR) kunye ne-loop isothermal amplification (LAMP) iindlela zithintelwe ngezinye izinyibilikisi ze-organic ezifana ne-ethanol kunye ne-isopropanol kwi-nucleic acid isolation reagents [20].Ukutsalwa kolwelo kunye nesigaba esiqinileyo zezona ndlela zidumileyo zokwahlula i-nucleic acids [21], nangona kunjalo, ukutsalwa kolwelo-olwelo kwitshiphu kunqongophele kakhulu, kuba iirejenti ezisetyenziswa ekutsalweni kolwelo-olwelwe zibangela ukubola kwezona chips zincinci zincinci. .Apha, sigxininisa iindlela zokutsalwa kwesigaba esisekwe kwi-microarray kwaye sithelekise izinto eziluncedo kunye nezingeloncedo.
I-Silicon yi-substrate material ehambelana ne-nucleic acids ngenxa ye-biocompatibility yayo, ukuzinza, kunye nokulula kokuguqulwa [22].Okubalulekileyo, xa ilungiswa nge-silica okanye ezinye izinto, le ngqungquthela ibonisa iipropathi zokubhengeza i-nucleic acids ehlawuliswa kakubi phantsi kwe-pH ephantsi, iimeko zetyuwa eziphezulu ngelixa zikhupha nge-pH ephezulu, izisombululo zetyuwa ephantsi.Ngokusekelwe kule nto, kunokwenzeka ukuhlambulula i-nucleic acid.
Iintlobo ezahlukeneyo zezinto ezisekelwe kwi-silica zisetyenziselwe ukukhutshwa kwe-nucleic acid kwi-microfluidics, njenge-silica beads, i-powders, i-microfiber filters, kunye ne-silica membranes [23, 24, 25, 26].Ngokuxhomekeke kwiipropati zezinto eziphathekayo, izinto ezisekelwe kwi-silicon zingasetyenziselwa kwii-microcircuits ngeendlela ezahlukeneyo.Ngokomzekelo, i-silica granules, i-powders, kunye ne-nanofilters zorhwebo zinokuthi zifakwe kwi-pores okanye i-microchannels ye-microfluidic chips kwaye incede ukukhupha i-nucleic acids kwiisampuli [27, 28, 29].Iimbrane ze-silica eziguquliweyo zingasetyenziselwa ukucoca ngokukhawuleza i-DNA kwi-pathogens ngexabiso eliphantsi.Ngokomzekelo, uWang et al.[30] Ngokudibanisa i-denaturing reactions ye-amplification kunye ne-vesicle-mediated chain chain kunye ne-silica membranes ehlanganiswe ne-chitosan oligosaccharides, inkqubo ephathekayo ephathekayo yaziswa ukuba ibone ngempumelelo i-102-108 i-coloni eyenza iiyunithi.(CFU)/ml Vibrio parahaemolyticus., kwaye ubukho bentsholongwane babubonakala lula.UPowell et al.[31] Silicon-based microarrays zaye zasetyenziselwa ukubona intsholongwane ye-hepatitis C (HCV), intsholongwane ye-immunodeficiency (HIV), intsholongwane yeZika, kunye ne-papillomavirus yabantu kunye nokusabalalisa okuzenzekelayo, apho i-1.3 μl i-tortuous microreactor yaphuhliswa ukuba ibambe iintsholongwane ze-RNA.kwaye enze i-situ amplification.Ukongeza kwezi ndlela, ii-microcolumns ze-silica-modified surface-modified surface nazo zidlala indima ephambili kwi-nucleic acid extraction, njengoko ijometri kunye neepropathi zezinto eziguquguqukayo zandisa kakhulu ukusebenza kakuhle.UChen et al.[32] icebise iqonga le-microfluidic lokuhlukaniswa kwe-RNA ephantsi yoxinaniso esekelwe kwi-amino-coated silicon microcolumns.Esi sixhobo se-microfluidic sidibanisa uluhlu lwe-0.25 cm2 micropillars kwi-silicon substrate ukuphumeza ukusebenza kakuhle kokutsalwa kwendawo ephezulu ukuya kuyilo lobungakanani bomthamo.Inzuzo yolu yilo kukuba isixhobo se-microfluidic sinokufikelela kwi-95% ye-nucleic acid extraction performance.Ezi zicwangciso ezisekelwe kwi-silicon zibonisa ixabiso lokuhlukanisa ngokukhawuleza i-nucleic acids ngexabiso eliphantsi.Ngokudityaniswa neechips ze-microfluidic, izicwangciso ze-silicon-based extraction azikwazi nje ukwandisa ukusebenza kakuhle kokufunyanwa kwe-nucleic acid, kodwa kuququzelele ukuguqulwa kwe-miniaturization kunye nokudityaniswa kwezixhobo zohlalutyo [20].
Iindlela zokwahlula imagnethi zisebenzisa amasuntswana omagnetic ukwahlula ii-nucleic acids kubukho bemagnethi yangaphandle.Iinqununu ezisetyenziswa ngokuqhelekileyo zibandakanya i-Fe3O4 okanye i-γ-Fe2O3 i-magnetic particles ehlanganiswe ne-silica, amino kunye ne-carboxyl [33,34,35,36].Isici esahlulayo samagnetic particles xa kuthelekiswa neendlela ze-silicon-based SPE kukukhululeka kokuphathwa kunye nokulawula kunye neemagnethi zangaphandle.
Ukusebenzisa i-electrostatic interaction phakathi kwe-nucleic acids kunye ne-silica, phantsi kweemeko zetyuwa ephezulu kunye ne-pH ephantsi, i-nucleic acids ibhengezwa kumphezulu we-silica-coated magnetic particles, ngelixa phantsi kweemeko zetyuwa ephantsi kunye ne-pH ephezulu, iimolekyuli zinokuhlanjwa. kwakhona..Amaso kazibuthe aqatywe ngesilica enza kube lula ukukhupha iDNA kwisampulu zevolumu enkulu (400 μL) kusetyenziswa isindululo esilawulwa ngomagnetic [37].Njengomboniso, uRodriguez-Mateos et al.[38] wasebenzisa iimagnethi ezinokuguqulelwa ukulawula ukuhanjiswa kwamaso kazibuthe kumagumbi ahlukeneyo.Ngokusekwe kwi-silica-coated magnetic particles, iikopi ezingama-470/mL ze-SARS-CoV-2 genomic RNA zinokutsalwa kwiisampulu zamanzi amdaka ukuze kufunyanwe i-LAMP reverse transcription (RT-LAMP) kwaye impendulo inokufundwa kwiyure enye.iliso lenyama (Umfanekiso 2a).
Izixhobo ezisekelwe kwizinto ezinobuchwephesha kunye ne-porous.Umzobo wengqikelelo wesixhobo se-IFAST RT-LAMP microfluidic ye-SARS-CoV-2 RNA ubhaqo (ilungiswe ukusuka [38]).b isixhobo esincinci se-Centrifugal se-dSPE ye-buccal swab nucleic acid (ethathwe kwi- [39]).c Isampulu yesampulu ekwaziyo ukuzenzela amandla kusetyenziswa ikhadi le-FTA® (lithatyathwe kwi- [50]).d Iphepha lokucoca i-Fusion 5 elitshintshwe nge-chitosan (litshintshwe ukusuka kwi- [51]).I-SARS-CoV-2 i-acute ebukhali yokuphefumla i-coronavirus 2, i-RT-LAMP reverse transcription loop mediated isothermal amplification, iFTA finders amaqabane etekhnoloji, i-NA nucleic acid
Amasuntswana kazibuthe achajiwe kakuhle alungele ukuncamathisela umqolo wephosphate we-nucleic acid.Kwinqanaba elithile letyuwa, amaqela e-phosphate ahlawuliswa kakubi we-nucleic acids anokuthi ahlawulwe ngokufanelekileyo kumphezulu we-particle magnetic composite.Ke ngoko, i-nanoparticles yamagnetic enomhlaba orhabaxa kunye noxinzelelo oluphezulu lwamaqela e-amino zaphuhliswa ukutsalwa kwe-nucleic acid.Emva kokuhlukana kwamagnetic kunye nokuthintela, i-nanoparticles yamagnetic kunye ne-DNA complexes inokusetyenziswa ngokuthe ngqo kwi-PCR, ephelisa imfuno yokucoca kunye nokuchitha ixesha kunye nokusebenza kwe-lution [35].I-Magnetic nanoparticles egqunywe ngamaqela e-carboxyl engalunganga nayo isetyenzisiwe ukwahlula i-nucleic acids adsorbed kwindawo ephezulu yoxinzelelo lwe-polyethylene glycol kunye nezisombululo zesodium chloride [36].Ngala maso aguquguqukayo amagnetic, i-DNA extraction iyahambelana nokukhulisa okulandelayo.Dignan et al.[39] ichaze iqonga elizenzekelayo neliphathwayo le-centrifugal microfluidic ye-nucleic acid pretreatment, evumela abasebenzi abangengabo ubuchwepheshe ukuba bayisebenzise kwisiza.Ukongezelela, ukuhambelana kwe-DNA eyodwa kunye ne-LAMP, indlela efanelekileyo yokuhlalutya i-nucleic acid ye-point-of-care, ibonisa ngakumbi iimfuno zezixhobo ezincinci kunye nokufaneleka kovavanyo lwe-colorimetric (Fig. 2b).
Iindlela ze-Magnetic bead zinika ithuba lokutsalwa okuzenzekelayo, ezinye zazo ezikhoyo kwii-extractor ze-nucleic acid ezizenzekelayo [i-KingFisher;ThermoFisher (Waltham, MA, USA), QIAcube® HT;I-CapitalBio (eBeijing, eChina) kunye neBiomek®;Beckman (Miami, USA).), eFlorida, eMelika)].Iingenelo zokudibanisa amaso kazibuthe kunye ne-microfluidics inokusetyenziselwa ukutsalwa okuzihambelayo kwee-nucleic acids, ezinokuthi zibenakho ukuqhubela phambili uphuhliso loxilongo lwemolekyuli;nangona kunjalo, indibaniselwano yamaso kazibuthe anemicrofluidics isaxhomekeke kakhulu kwiisistim ezintsonkothileyo zolawulo lokucofa amaso kazibuthe, nto leyo echaza ukuthandwa kweemveliso zorhwebo zibe nkulu kwaye zibiza imali eninzi, nto leyo enqanda usetyenziso olongezelelweyo lwamaso emagnethi kwiPOCT.
Izinto ezininzi ezixubileyo ezifana nezihluzo ze-nitrocellulose ezilungisiweyo, amakhadi e-Finders Technology Associates (FTA), amaphepha okucoca asekelwe kwi-polyethersulfone, kunye nezinto ezifakwe kwi-glycan nazo zisetyenziselwe ukufunyanwa kwe-nucleic acid [40, 41, 42, 43, 44].Iintsinga zemichiza ezifana nephepha elineentsinga zaqala zasetyenziselwa ukwahlula i-DNA ngokubhijela iimolekyuli zeDNA ezinemisonto emide ngeentsinga.Iipores ezincinci zikhokelela kwisithintelo esinamandla somzimba we-molecules ze-DNA, ezichaphazela ngokufanelekileyo ukutsalwa kwe-DNA.Ngenxa yobukhulu obuhlukeneyo bepore bephepha le-fibrous, ukusebenza kakuhle kokutsalwa akukwazi ukuhlangabezana neemfuno ze-DNA yokukhulisa [45, 46].Ikhadi le-FTA liphepha lokucoca urhwebo elisetyenziswa kwintsimi yeyeza le-forensic kwaye lisetyenziswa ngokubanzi kwezinye iindawo ze-molecular diagnostics.Ngokusetyenziswa kwephepha lokucoca i-cellulose elifakwe kwiikhemikhali ezahlukeneyo ukuze i-lyse iimbumba zeseli kwisampulu, i-DNA ekhutshiweyo ikhuselwe ekuthotyweni ukuya kutsho kwiminyaka emi-2.Kutshanje, iphepha leselulosi elifakwe ngaphakathi liphuhliselwe ukuchongwa kwemolekyuli yeentsholongwane ezahlukeneyo, kubandakanya i-SARS-CoV-2, i-leishmaniasis, kunye nesifo seengcongconi [47,48,49].I-HIV kwi-plasma esecaleni i-lysed ngokuthe ngqo, kwaye i-viral nucleic acid ityetyiswe kwi-FTA® flow membrane eyakhelwe kwi-concentrator, evumela ukuveliswa ngokufanelekileyo kwe-nucleic acid [50] (Umfanekiso 2c).Ingxaki ephambili ekubhaqweni kwe-nucleic acid usebenzisa amakhadi e-FTA kukuba iikhemikhali ezifana ne-guanidine kunye ne-isopropanol zivimbela ukuphendulwa kwe-amplification elandelayo.Ukuyicombulula le ngxaki, siphuhlise i-Fusion 5 ye-chitosan-modified filter paper, edibanisa izibonelelo zokudityaniswa kwee-molecule ze-DNA kunye ne-fibrous filter paper, kunye ne-electrostatic adsorption ye-DNA kwi-chitosan-modified compounds ukuze kuphunyezwe ukusebenza kakuhle kwe-nucleic acid extraction. ..iintsinga zokucoca [51] (Fig. 2d).Ngokufanayo, uZhu et al.[52] ibonise indlela ye-PCR ye-chitosan-modified esekelwe kwi-in situ capillary microfluidic system yokwahlula ngokukhawuleza kunye nokufunyanwa kwe-Zika virus RNA.I-Nucleic acids inokubhengezwa / ifakwe kwi-mixed lysate / PCR medium, ngokulandelanayo, ngokusekelwe kwipropati yokucima / yokucima i-chitosan.on and off”, esabela kwi pH.
Njengoko kukhankanyiwe ngasentla, ezi zicwangciso zidibanisa izibonelelo zezinto ezahlukeneyo zesigaba esiqinileyo kunye nokwandisa ukusebenza kakuhle kwe-nucleic acid extraction kwi-microfluidics.Kwizicelo ezisebenzayo, ukusetyenziswa kwezi zixhobo ngobuninzi obukhulu akunamali, kwaye unyango olufanelekileyo lomphezulu okanye ukulungiswa kwendawo yezinto eziqhelekileyo kunye nezi zinto kunokugcina umsebenzi wazo.Ngoko ke, kukholelwa ukuba ukuphunyezwa kwezi zicwangciso emva kophando lokulinga kunokunciphisa iindleko.
Uvavanyo lwe-Nucleic acid kwiiplatifti ze-microfluidic zihlala zisebenzisa iisampulu ezincinci (<100 µl), ngoko ke zifuna ukwandiswa kwethagethi ye-nucleic acids kunye ne-probes ethile yokuguqulwa kumqondiso olungele ukubhaqwa komlambo (i-optical, umbane, kunye nemagnethi) [53, 54]. Uvavanyo lwe-Nucleic acid kwiiplatifti ze-microfluidic zihlala zisebenzisa iisampulu ezincinci (<100 µl), ngoko ke zifuna ukwandiswa kwethagethi ye-nucleic acids kunye ne-probes ethile yokuguqulwa kumqondiso olungele ukubhaqwa komlambo (i-optical, umbane, kunye nemagnethi) [53, 54]. При тестировании нуклеиновых кислот на микрожидкостных платформах часто используются небольшие объемы образцов (< 100 мкл), поэтому требуется амплификация целевых нуклеиновых кислот с помощью специальных зондов для преобразования в сигнал, удобный для последующего обнаружения (оптического, электрического и магнитного) [53, 54]. Xa kuhlolwa i-nucleic acids kumaqonga e-microfluidic, imithamo yesampulu encinci (<100 µL) isoloko isetyenziswa, ngoko ke ukwandiswa kwethagethi ye-nucleic acids kunye ne-probes ekhethekileyo iyafuneka ukuyiguqula ibe luphawu olulungele ukubhaqwa okulandelayo (i-optical, umbane, kunye nemagnethi) [53, 54].微流控 平台 上 上 的 使用 使用 使用 小样本量 小样本量 小样本量 小样本量 特定 特定 特定 探针 扩增 目标 目标 目标, 以 转换 为 为 便于下游].微流控 平台 上 上 核酸 检测 检测 使用 小样本量使用 使用使用 使用使用使用小样本量小样本量小样本量小样本量使用,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 小样本量使用 )特定) 特定探针 探针 扩增扩增, 以 转换 为 为 下游 下游 的 的, 光学 电学电学) 的 的 的 的 [53, 54, 54, 54 ]. Обнаружение нуклеиновых кислот на микрожидкостных платформах обычно использует небольшие объемы образцов (<100 мкл), что требует амплификации целевых нуклеиновых кислот с помощью специальных зондов для преобразования в сигналы для последующего обнаружения (оптического, электрического и магнитного) [53, 54]]. Ukufunyanwa kwe-nucleic acids kumaqonga e-microfluidic ngokuqhelekileyo kusebenzisa iisampulu ezincinci (<100 μl), ezifuna ukukhulisa i-nucleic acids ekujoliswe kuyo kunye ne-probes ekhethekileyo ukuyiguqula ibe yimiqondiso yokufunyanwa okulandelayo (i-optical, umbane, kunye nemagnethi) [53, 54]] .I-Nucleic acid amplification kwi-microfluidics inokukhawulezisa ukusabela, ukwandisa imida yokubona, ukunciphisa iimfuno zesampulu, kunye nokuphucula ukuchaneka kokubona [55, 56].Kwiminyaka yakutshanje, ngokuqondwa ngokukhawuleza nangokuchanekileyo, iindlela ezahlukeneyo ze-nucleic acid amplification ziye zasetyenziswa kwi-microfluidics, kubandakanya i-PCR kunye nezinye i-isothermal amplification reactions.Eli candelo liza kushwankathela iindlela zokufumanisa i-nucleic acid esekelwe kwiinkqubo ze-microfluidic.
I-PCR yindlela yokuphindaphinda inkqubo ye-DNA yezinto eziphilayo, ithiyori echazwe ngokubanzi kwenye indawo kwaye ayiyi kuxoxwa apha.I-PCR inokwandisa inani elincinci kakhulu le-DNA / i-RNA ekujoliswe kuyo kwizinga elicacileyo, okwenza i-PCR ibe sisixhobo esinamandla sokufunyanwa ngokukhawuleza kwe-nucleic acids.Kumashumi eminyaka akutshanje, izixhobo ezininzi eziphathwayo ze-microfluidic ezixhotyiswe ngeenkqubo zebhayisikile ezishushu ze-PCR ziye zaphuhliswa ukuze zihlangabezane neemfuno zoxilongo lwe-point-of-care diagnostics [57, 58].I-PCR ye-on-chip inokwahlulwa ibe ziindidi ezine (ukuhamba okuqhelekileyo, okuqhubekayo, ukutshintshelwa kwendawo, kunye ne-PCR ye-convective) ngokweendlela ezahlukeneyo zokulawula ubushushu [59].Ngokomzekelo, uGee et al.[60] iphuhlise indlela yoshicilelo oluthe ngqo lwe-PCR (RT-qPCR) kwiqonga labo le-microfluidic lokufunyanwa kwe-multiplex ye-SARS-CoV-2, iintsholongwane zomkhuhlane A kunye no-B kwiisampulu ze-swab yomqala (Fig. 3a) .Park et al.[61] yakha i-chip yokuhlalutya i-pathogen elula ngokudibanisa ifilimu encinci ye-PCR, i-electrodes, kunye ne-polydimethylsiloxane-based based microfluidic module.Nangona kunjalo, yomibini imisebenzi iquka iintsilelo eziqhelekileyo zePCR eqhelekileyo.I-PCR ifuna ibhayisekile eshushu, enqanda ukuphuculwa kwesixhobo esincinci kunye nexesha elincitshisiweyo lovavanyo.
Ukuphuhliswa kokuhamba okuqhubekayo okusekelwe kwi-microfluidic kunye ne-space-switched PCR kubalulekile ukulungisa lo mba.Ukusebenzisa umjelo omde wenyoka okanye umjelo omfutshane othe tye, i-PCR ehambayo eqhubekayo inokubonelela ngokukhawuleza kokukhulisa ngokujikeleza ngokusebenzayo ii-reagents kwiindawo ezintathu zokushisa kwangaphambili kunye nepompo ye-off-chip.Lo msebenzi uthintela ngempumelelo isigaba sokutshintsha phakathi kwamaqondo okushisa ahlukeneyo kwaye ngaloo ndlela unciphisa kakhulu ixesha lokuvavanya [62] (Umfanekiso 3b).Kwesinye isifundo nguJung et al.[63] icetywe i-rotary PCR genetic analyzer entsha edibanisa iimpawu ze-PCR ezisisigxina kunye nokuhamba kwe-ultrafast kunye ne-multiplex ye-reverse transcription PCR (Umfanekiso 3c).Ukwandisa i-nucleic acid, i-microchip ye-PCR iya kujikelezwa ngeebhloko ezintathu zokufudumeza kwiqondo lokushisa elihlukeneyo: 1. Ibhloko ye-Denaturation 94 ° C, 2. I-Annealing block kwi-58 ° C, 3. Ibhloko yokwandisa kwi-72 ° C.
Ukusetyenziswa kwe-PCR kwi-microfluidics.Ukumelwa okucwangcisiweyo kwe-dirRT-qPCR kwiqonga le-microfluidic (ithathwe kwi- [60]).b Ukumelwa okucwangcisiweyo kwenkqubo eqhubekayo yePCR microarray esekelwe kumjelo wenyoka (ithathwe kwi- [62]).c Umelo olucwangcisiweyo lwe-rotary PCR yomhlalutyi wemfuza, ebandakanya i-microchip, iibhloko ezintathu zokufudumeza kunye ne-motor stepper (ethathwe kwi- [63]).d Umzobo we-PCR ye-thermoconvection kunye ne-centrifugation kunye nokusekwa (ithathwe kwi- [64]).I-DirRT-qPCR, ubungakanani obuthe ngqo bereverse transcription polymerase chain reaction
Ukusebenzisa i-capillaries kunye ne-loops okanye iipleyiti ezinqabileyo, i-PCR ye-convection inokwandisa ngokukhawuleza i-nucleic acids nge-convection ye-thermal yamahhala yendalo ngaphandle kwesidingo sempompo yangaphandle.Ngokomzekelo, i-cyclic olefin polymer iplatifomu ye-microfluidic yaphuhliswa kwinqanaba lokufudumala elijikelezayo elisebenzisa ibhayisikili ye-thermal nge-centrifugation kwi-PCR loop microchannel [64] (Umfanekiso 3d).Isisombululo sokuphendula siqhutyelwa yi-convection ye-thermal, eqhubekayo itshintshisa ukushisa okuphezulu kunye nokuphantsi kwi-microchannel enesakhiwo se-annular.Yonke inkqubo yokukhulisa ingagqitywa kwimizuzu eyi-10 kunye nomda wokufumanisa we-70.5 pg / channel.
Njengoko kulindelekile, i-PCR ekhawulezayo sisixhobo esinamandla sokudibanisa ngokupheleleyo isampuli-impendulo ye-molecular diagnostic and multiplex analysis systems.I-PCR ekhawulezileyo yehlisa kakhulu ixesha elifunekayo lokubona i-SARS-CoV-2, enegalelo kulawulo olusebenzayo lwesibetho se-COVID-19.
I-PCR idinga i-thermal cycler enzima engafanelekanga kwi-POCT.Ngoku kutshanje, iindlela ze-isothermal amplification zisetyenziswe kwi-microfluidics, kubandakanywa kodwa ingaphelelanga kwi-LAMP, i-recombinase polymerase amplification (RPA), kunye nokukhulisa ngokusekelwe kwi-nucleic acid sequences [65,66,67,68].Ngobu buchule, i-nucleic acids ikhuliswa kwiqondo lokushisa elingaguqukiyo, iququzelela ukudalwa kweendleko eziphantsi, izixhobo eziphathwayo ze-POCT eziphathwayo zokuxilongwa kweemolekyuli.
I-high-throughput microfluidics-based-LAMP assays ivumela ukufunyanwa kwezifo ezosulelayo ezininzi [42, 69, 70, 71].Ngokudibanisa nenkqubo ye-centrifugal ye-microfluidic, i-LAMP inokuqhubela phambili ukuququzelela ukuzenzekelayo kokufunyanwa kwe-nucleic acid [69, 72, 73, 74, 75].I-spin-and-react SlipChip yaphuhliswa ukukhangela okubonakalayo kwiibhaktheriya ezininzi ezihambelanayo usebenzisa i-LAMP [76] (umzobo 4a).Xa usebenzisa i-LAMP ephuculweyo kwi-assay, i-fluorescence signal-to-noise ratio yayimalunga ne-5-fold, kwaye umda wokufumanisa ufikelele kwiikopi ze-7.2 / μl ze-genomic DNA. Ngaphezu koko, ubukho be-bacterial pathogens ye-digestive emihlanu eqhelekileyo, kuquka i-Bacillus cereus, i-Escherichia coli, i-Salmonella enterica, i-Vibrio fluvialis kunye ne-Vibrio parahaemolyticus, yabonwa ngokusekelwe kwindlela <60 min. Ngaphezu koko, ubukho be-bacterial pathogens ye-digestive emihlanu eqhelekileyo, kuquka i-Bacillus cereus, i-Escherichia coli, i-Salmonella enterica, i-Vibrio fluvialis kunye ne-Vibrio parahaemolyticus, yabonwa ngokusekelwe kwindlela <60 min.Ngaphezu koko, ubukho be-bacterial pathogens eqhelekileyo ye-digestive tract, kuquka i-Bacillus cereus, i-Escherichia coli, i-Salmonella enterica, i-Vibrio fluvialis kunye ne-Vibrio parahaemolyticus, yabonwa ngokusebenzisa le ndlela ngaphantsi kwemizuzu engama-60.此外，基于该方法在<60分钟内可视化了五种常见消化道细菌病原体的存在，包括蜡状芽孢杆菌、大肠杆菌、肠沙门氏菌、河流弧菌和副溶血性弧菌。此外, 基于 该 在 在 视化 了 了 五 种 细菌病 的 的 的 的 的 的 的, 包括 芽孢杆菌, 大 肠杆菌, 大 肠杆菌 菌, 肠 肠杆菌 菌, 弧菌 氏 菌 性 性 性 性 ... 弧菌 弧菌 弧菌 弧菌弧菌 弧菌 弧菌 弧菌 弧菌 弧菌 弧菌 HIPUkongezelela, ubukho bezilwanyana eziqhelekileyo ze-bacterial gastrointestinal pathogens, kuquka i-Bacillus cereus, i-Escherichia coli, i-Salmonella enterica, i-Vibrio fluvius, kunye ne-Vibrio parahaemolyticus, yabonwa ngokusebenzisa le ndlela ngaphantsi kwemizuzu engama-60.
Izibonelelo ze-LAMP kwi-microfluidics zibandakanya, phakathi kwezinye, ukuphendula ngokukhawuleza kunye nokufunyanwa kwe-miniaturized.Nangona kunjalo, ngenxa yobushushu bokusabela (malunga ne-70 ° C), ii-aerosols ngokuqinisekileyo ziveliswa ngexesha le-LAMP, okukhokelela kwizinga eliphezulu lobuxoki.Ukuchaneka kwe-Assay, uyilo lwe-primer, kunye nolawulo lobushushu nalo kufuneka lulungiselelwe i-LAMP.Ukongeza, uyilo lwetshiphu oluphumeza ukuchongwa okujoliswe kuko okuninzi kwitshiphu enye luxabiso elikhulu kwaye kufuneka luphuhliswe.Ukongeza, i-LAMP ifanelekile ukufumanisa iinjongo ezininzi ezidityanisiweyo kwi-chip enye, ebaluleke kakhulu, kodwa kusekho indawo eninzi yophuhliso.
Izinga eliphezulu lobuxoki eliqinisekileyo le-LAMP lingancitshiswa ngokuyinxenye nge-RPA, njengoko iqondo lobushushu elisezantsi ngokwentelekiso (~37 °C) liphumela kwiingxaki ezimbalwa zomphunga [77].Kwinkqubo ye-RPA, ii-primers ezimbini ezichaseneyo ziqala i-DNA synthesis ngokubophelela kwi-recombinase kunye nokukhulisa ingagqitywa kwimizuzu eyi-10 [78,79,80,81].Ngoko ke, yonke inkqubo ye-RPA ikhawuleza kakhulu kune-PCR okanye i-LAMP.Kwiminyaka yakutshanje, iteknoloji ye-microfluidic iboniswe ukuqhubela phambili ukuphucula isantya kunye nokuchaneka kwe-RPA [82,83,84].Umzekelo, uLiu et al.[85] iphuhlise i-microfluidic edityanisiweyo ye-lateral flow polymerase recombinase assay yeamplification ukufunyaniswa okukhawulezileyo nokunovakalelo kwe-SARS-CoV-2 ngokudibanisa i-RPA eshicilelweyo ebuyela umva (RT-RPA) kunye nenkqubo yokukhangela umcu wokuqukuqela jikelele jikelele.kwinkqubo enye microfluidic.Umfanekiso 4b).Umda wokuchongwa yikopi enye/µl okanye iikopi ezingama-30/isampulu, kwaye ubhaqo lungagqitywa malunga nemizuzu engama-30.Kong et al.baphuhlise isixhobo esinxitywayo semicrofluidic.[86] esetyenzisiweyo ubushushu bomzimba kunye neselfowuni-based fluorescence inkqubo yokufumanisa fluorescence ngokukhawuleza nangokuthe ngqo HIV-1 DNA usebenzisa RPA (Figure 4c).Uvavanyo olunxitywayo lwe-RPA lufumanisa iikopi ezili-100/mL zolandelelwano ekujoliswe kulo kwimizuzu engama-24, ebonisa amandla amakhulu okuxilongwa ngokukhawuleza kweentsana ezosulelwe yi-HIV-1 kwiindawo ezilinganiselweyo.
I-Isothermal amplification kwi-point-of-care testing (POCT).Uphuhliso kunye nokuveliswa kwe-spin kunye ne-reaction SlipChip.Emva kwe-plasma welding, iichips eziphezulu kunye nezantsi zahlanganiswa kunye neseti yamantongomane ukwenza i-chip yokugqibela (ithathwe kwi- [76]).b Uyilo lwenkqubo ye-MI-IF-RPA yokufunyaniswa kwe-COVID-19 (ithatyathwe kwi- [85]).c Schematic yovavanyo lwe-RPA olunxitywayo lokubona ngokukhawuleza i-HIV-1 DNA (ithatyathwe kwi- [86]).SE Salmonella enterica, VF Vibrio fluvius, VP Vibrio parahaemolyticus, BC Bacillus cereus, EC Escherichia coli, FAM carboxyfluorescein, human immunodeficiency virus HIV, RPA recombinase polymerase amplification, LED light emitting diode, MI-Flurow-IF-Idayidi ye-Microfluores Ephakathi kwe-Microfluore-IF-IF-IF-I-microfluorescein Ukwandisa
I-RPA esekelwe kwi-Microfluidic ikhula ngokukhawuleza, nangona kunjalo, ixabiso lokwenziwa kwe-chip kunye nokusetyenziswa kokusabela liphezulu kakhulu kwaye kufuneka lincitshiswe ukwandisa ukufumaneka kobu buchwepheshe.Ukongeza, uvakalelo oluphezulu lwe-RPA lunokuchaphazela ukunyuswa kweemveliso ezingezizo ezodwa, ngakumbi xa kukho ungcoliseko.Le mida inokuchaphazela ukusetyenziswa kwe-RPA kwiinkqubo ze-microfluidic kwaye ifanele ukuphuculwa ngakumbi.Iiprayimari eziyilwe kakuhle kunye neeprobe kwiithagethi ezahlukeneyo nazo ziyafuneka ukuze kuphuculwe ukuba nokwenzeka kwe-RPA-based microfluidic izicwangciso kwi-POCT.
I-Cas13 kunye ne-Cas12a inamandla okuqhawula ngokungaqhelekanga i-nucleic acids kwaye ngoko inokuphuhliswa njengezixhobo zokufumanisa kunye nokuxilonga.I-Cas13 kunye ne-Cas12a zivuliwe xa zibophelela kwi-DNA okanye i-RNA, ngokulandelelanayo.Nje ukuba isebenze, iprotheni iqala ukucanda ezinye ii-nucleic acids ezikufutshane, emva koko isikhokelo se-RNAs esijolise kwi-pathogen-specific nucleic acids sinokucama iiprobes ezicinyiweyo ze-fluorescent kwaye zikhuphe i-fluorescence.Ngokusekelwe kule thiyori, uKellner et al.[87] iphuhlise indlela ye-Cas13-based [Specific High-sensitivity Enzymatic Reporter UnLOCKING (SHERLOCK)], kunye noBroughton et al.[88] iphuhlise enye indlela esekelwe kwi-Cas12a [i-CRISPR Trans Reporter ejolise kwi-DNA endonuclease (DTECR)].
Kwiminyaka yakutshanje, iindlela ezahlukeneyo zokufunyanwa kwe-nucleic acids esekwe kwi-CRISPR ziye zavela [89, 90].Iindlela eziqhelekileyo ezisekelwe kwi-CRISPR zihlala zichitha ixesha kwaye zisebenza ngamandla ngenxa yeenkqubo ezininzi ezibandakanya i-nucleic acid extraction, ukukhulisa kunye nokufunyanwa kwe-CRISPR.Ukuba sesichengeni solwelo emoyeni kunokunyusa amathuba okuba neziphumo ezingezizo ezingalunganga.Ukunika oku ngasentla, iinkqubo ezisekwe kwi-CRISPR zifuna kakhulu ukulungiswa.
I-platform ye-microfluidic elawulwa nge-pneumatic enokwenza uhlalutyo lwe-24 ngokuhambelanayo iphuhliswe kwi-CRISPR-Cas12a kunye ne-CRISPR-Cas13a izicelo zokufumanisa [91].Inkqubo ixhotywe ngesixhobo sokufumanisa i-fluorescence edlula i-nucleic acid amplification kwaye ibone ngokuzenzekelayo i-femtomolar DNA kunye neesampuli ze-RNA.UChen et al.[92] ukuhlanganiswa kwe-recombinase amplification kunye ne-CRISPR-Cas12a inkqubo kwi-centrifugal microfluidics (Umfanekiso 5a).Lo msebenzi unqoba ubunzima bokudibanisa ezi nkqubo zimbini kuba i-Cas12a inokugaya i-DNA yesithunywa kwaye inqanda inkqubo yokukhulisa.Ukongeza, uChen et al.[92] ukongeza kwangaphambili i-reagents yokusabela kwi-centrifugal microfluidic control ukugqiba ngokuzenzekelayo yonke inkqubo.Komnye umsebenzi, uSilva et al.[I-93] iphuhlise indlela yokuxilonga ngaphandle kwe-CRISPR / Cas12a yokukhulisa kunye ne-smartphone ukuze ibone i-SARS-CoV-2 (umzobo 5b).Olu vavanyo, olubizwa ngokuba yi-cell phone-based amplification-free system, lubandakanya i-enzyme ye-CRISPR / i-Cas-exhomekeke kwi-smartphone ebonakalayo ye-catalase-generated bubble signals kwi-microfluidic channels.Ukufunyaniswa okubuthathaka kweekopi ezingaphantsi kwe-50 / µl ye-nucleic acid ngaphandle kokwandiswa kwangaphambili, yonke inkqubo ukusuka kwinaliti yesampuli ukuya ekufundeni umqondiso kuthatha imizuzu engama-71 kuphela.
Iindlela zokubona i-Nucleic acid esekelwe kwi-CRISPR.I-POCT ye-Centrifugal yokuxilongwa kwe-molecular edibeneyo esekelwe kwi-CRISPR (ithathwe kwi- [92]).b Uphuhliso lovavanyo lweCASCADE lohlalutyo olusekwe kwi-smartphone ye-SARS-CoV-2 (elungiswe ukusuka [93]).I-RAA recombinase amplification, i-PAM ekufutshane ne-protospacer motif, i-CRISPR edibeneyo yokuphindaphinda i-palindromic emfutshane ngamaxesha aqhelekileyo, inkqubo ye-CASCADE ngaphandle kwe-cell phone amplification kunye ne-CRISPR / i-CAS-exhomekeke kwi-enzymes, i-1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride EDC
Njengesinyathelo sokugqibela ekubonweni kwe-nucleic acid, ukufumanisa umqondiso kubonakalisa ngokuthe ngqo iziphumo zokuxilonga kwaye yinto ebalulekileyo ekuphuhliseni i-POCT esebenzayo, enobucayi kunye nechanekileyo.Iimpawu zinokufundwa kusetyenziswa iindlela ezahlukeneyo ezifana ne-fluorescent, i-electrochemical, i-colorimetric kunye ne-magnetic strategy.Kweli candelo, sichaza ingqiqo yendlela nganye kwaye sithelekise i-molecular diagnostics yezifo ezosulelayo kwi-microfluidics.
Izicwangciso ezisekelwe kwi-Fluorescence zisetyenziswa ngokubanzi kwi-POCT yokuxilongwa kwezifo ezithathelwanayo ngenxa yeenzuzo zabo eziphawulekayo zovelwano olubalaseleyo, ixabiso eliphantsi, ukulula ukusebenza, kunye nohlalutyo lwe-point-of-care [94, 95].Ezi zicwangciso zisebenzisa i-fluorophores ebhalwe njenge-fluorescent dyes kunye nama-nanomaterials ukwenza uphawu olubonakalayo (ukuphucula i-fluorescence okanye ukucima).Oku kufunyaniswayo kuphakamisa ukuba izicwangciso ezisekelwe kwi-fluorescence zinokwahlulwa zibe zilebhile ezithe ngqo ze-fluorescent, i-signal-on, kunye nokufunyanwa kwe-signal-off fluorescent [96].Ukufunyanwa kweleyibhile ye-fluorescent ngokuthe ngqo kusebenzisa iileyibhile ezikhethekileyo ze-fluorescent ukubeka iilebhile ezithile ezivelisa umlinganiselo othile we-fluorescence xa ubophelelwe ngokukhethiweyo kwithagethi.Ukufumana umqondiso we-fluorescence, umgangatho womqondiso we-fluorescent uhambelana ngokufanelekileyo nobukhulu bomdla.I-Fluorescence intensity ayihoywa ngokungabikho kwethagethi kwaye ibonakala xa isixa esaneleyo sethagethi ikhona.Ngokuchasene noko, ubunzulu be-fluorescence efunyenwe yi-"signal-off" fluorescence ihambelana ngokuphambeneyo nomyinge wethagethi, ekuqaleni ifikelela kwixabiso eliphezulu kwaye iyancipha ngokuthe ngcembe njengoko ithagethi isanda.Ngokomzekelo, usebenzisa i-CRISPR-Cas13a exhomekeke ekujoliswe kuyo kwi-trans-cleavage mechanism, i-Tian et al.[97] iphuhlise isicwangciso senoveli yokuqaphela i-RNAs edlula ushicilelo olujikelezayo ngokuthe ngqo (Umfanekiso 6a).Emva kokuzibophelela kwii-RNA ezijoliswe kuzo, i-CRISPR-Cas13-RNA i-complex inokuthi isebenze, ibangele i-transcollateral cleavage yi-RNAs yentatheli engabonakaliyo.Intatheli ebhalwe ngefluorescent [i-fluorophore (F)] icinywa sisicimi (Q) singaguquki kunye ne-fluoresces xa sicandwa yi-complex esebenzayo.
Inzuzo yokufumanisa i-electrochemical isantya esiphezulu sokufumanisa, ukuveliswa okulula, ixabiso eliphantsi, ukuthwala lula kunye nokulawula ngokuzenzekelayo.Yindlela enamandla yohlalutyo lwezicelo ze-POCT.Ngokusekwe kwigraphene field-effect transistors Gao et al.[98] iphuhlise i-nanobiosensor yokufumanisa i-multiplex ye-antigen ye-Lyme yesifo esivela kwi-Borrelia burgdorferi ibhaktheriya kunye nomda wokufumanisa we-2 pg / mL (Fig. 6b).
Uvavanyo lwe-Colorimetric lusetyenziswe kwizicelo ze-POCT, ezixhamla kwiinzuzo zokuphatheka, iindleko eziphantsi, ukulungiswa lula, kunye nokufunda okubonakalayo.Ukufunyanwa kombala kunokusebenzisa i-oxidation ye-peroxidase okanye i-peroxidase-like nanomaterials, ukuhlanganiswa kwama-nanomaterials, kunye nokongezwa kwedayi yesalathisi ukuguqula ulwazi malunga nobukho be-nucleic acids ekujoliswe kuyo kwiinguqu zombala ezibonakalayo [99, 100, 101].Ngokucacileyo, i-nanoparticles yegolide isetyenziswa ngokubanzi kuphuhliso lwezicwangciso zemibala, kwaye ngenxa yokukwazi ukukhuthaza utshintsho olukhawulezayo nolubalulekileyo lwemibala, kukho umdla okhulayo kuphuhliso lwamaqonga emibala ye-POCT yokuxilongwa kwezifo ezosulelayo [102].Ngesixhobo esihlanganisiweyo se-centrifugal microfluidic [103], iipathogens zokutya kwiisampuli zobisi ezingcolileyo zingabonwa ngokuzenzekelayo kwinqanaba leeseli zebhaktheriya ze-10, kwaye iziphumo zingafundwa ngokubonakalayo ngaphakathi kwemizuzu ye-65 (umzobo 6c).
Ubuchwephesha bokuva imagnethi bunokubona ngokuchanekileyo abahlalutyi besebenzisa imathiriyeli yamagnetic, kwaye kubekho umdla obalulekileyo kwizicelo ze-POCT kumashumi eminyaka akutshanje.Ubuchwephesha bokuva imagnethi bunoncedo olulodwa olulodwa njengemathiriyeli yemagnethi enexabiso eliphantsi kunezixhobo zamehlo ezibizayo.Nangona kunjalo, ukusetyenziswa kwendawo yamagnetic kuphucula ukusebenza kakuhle kunye nokunciphisa ixesha lokulungiselela isampuli [104].Ukongeza, iziphumo zokuphonononga kazibuthe zibonisa ukuchaneka okuphezulu, ubuntununtunu, kunye nomlinganiselo ophezulu wesignali-kwingxolo ngenxa yomqondiso ongabalulekanga ongasemva wemagnethi weesampulu zebhayoloji [105].Sharma et al.idityaniswe i-junction yemagnethi yetonela esekwe kwi-biosensor kwiqonga le-microchip ephathekayo.[106] ukufumanisa i-multiplex ye-pathogens (Umfanekiso 6d).I-Biosensor ibona ngononophelo i-subnanomolar nucleic acids ekwanti kwi-pathogens.
Indlela yokubona umqondiso oqhelekileyo.Ingqikelelo yokufumanisa i-hyperlocalized ye-Cas13a (iguqulelwe kwi- [97]).b IGraphene nanobiosensor FET indibaniselwano neLyme GroES scFv (ithatyathwe kwi [98]).c Izibonakaliso zemibala yokufumanisa i-multiplex ye-pathogens yokutya kwi-centrifugal microfluidic chip: I-No. .d I-Biosensor esekelwe kwi-junction ye-magnetic tunnel junction, kubandakanywa iqonga, i-amplifier yokuthintela eyakhelweyo, iyunithi yokulawula, kunye nokunikezelwa kwamandla okuvelisa umqondiso / ukufunyanwa (kulungiswe kwi- [106]).IGFET Graphene FET, Escherichia coli, Escherichia coli, Salmonella typhimurium, Vibrio parahaemolyticus, Vibrio parahaemolyticus, Listeria monocytogenes, PC PC, PDMS Dimethicone, PMMA polymethyl methacrylate
Nangona iimpawu ezibalaseleyo zezi ndlela zokubona zingentla, zisenezingeloncedo.Ezi ndlela zifaniswa (itheyibhile 1), kubandakanywa ezinye izicelo ezineenkcukacha (iinzuzo kunye neengxaki).
Ngokuphuhliswa kwe-microfluidics, iinkqubo ze-microelectromechanical, i-nanotechnology kunye nesayensi yezinto eziphathekayo, ukusetyenziswa kwee-chips ze-microfluidic zokubona izifo ezithathelwanayo zihlala ziqhubela phambili [55,96,107,108].Ukuphathwa ngokuchanekileyo kwezixhobo ezincinci kunye nolwelo kunegalelo ekuchanekeni koxilongo kunye nokusebenza kakuhle kweendleko.Ke ngoko, uphuhliso olongezelelweyo, iinzame zenziwe zokwandisa kunye nokuphucula iitshiphusi, okukhokelela kwiichips ezahlukeneyo ze-microfluidic ezinezakhiwo ezahlukeneyo kunye nemisebenzi.Apha sazisa ngokufutshane iintlobo ezininzi eziqhelekileyo zamaqonga e-microfluidic kwaye sithelekise iimpawu zabo (iinzuzo kunye neengxaki).Ukongeza, uninzi lwemizekelo edweliswe ngezantsi ijolise ikakhulu ekulweni i-SARS-CoV-2.
Ii-LOCC zezona zixhaphakileyo iinkqubo zokuhlalutya ezintsonkothileyo ezincinci kwaye imisebenzi yazo incinci, idityaniswe, izenzekelayo kwaye ifaniswe ukusuka kwinaliti yesampulu kunye nokulungiselela, ukulawula ukuhamba kunye nokubonwa kolwelo [109, 110].Ulwelo lulawulwa ngobuchule bejometri eyilwe ngononophelo kunye nokusebenzisana kweziphumo ezininzi zomzimba ezinje ngemitha yoxinzelelo, inyathelo le-capillary, i-electrodynamics, imagnethi kunye namaza e-acoustic [111].I-LOCC ibonisa iingenelo ezigqwesileyo kwi-high-throughput screening kunye nokufumanisa ezininzi, kunye nesantya sokuhlalutya ngokukhawuleza, ubungakanani besampulu encinci, ukusetyenziswa kwamandla aphantsi, kunye nolawulo oluphezulu kunye nokusebenza kakuhle;nangona kunjalo, izixhobo zeLOCC zibuthathaka kakhulu, kwaye zenza, ukupakishwa, kunye nokunxibelelana.Nangona kunjalo, ukuphindaphinda kunye nokusebenzisa kwakhona kujongana nobunzima obukhulu [96].Xa kuthelekiswa namanye amaqonga, iLOCC ineengenelo ezizodwa malunga neyona ndlela iphezulu yeyantlukwano kunye nokuhambelana kobuchwepheshe obugqwesileyo, kodwa izinto ezingeloncedo zayo zikwacacile, ezizezi, ukuntsonkotha okuphezulu kunye nokuphindaphindeka okubi.Ukuxhomekeka kwiimpompo zangaphandle, ezihlala zininzi kwaye zibiza, zinciphisa ngakumbi ukusetyenziswa kwazo kwi-POCT.
Ngexesha lokuqhambuka kwe-COVID-19, iLOCC yafumana ingqwalasela enkulu.Kwangaxeshanye, kukho iitshiphusi ezintsha ezininzi ezidibanisa itekhnoloji ezininzi.Umzekelo, ii-smartphones ngoku zisetyenziswa ngokubanzi njengezixhobo zokuhlalutya eziphathekayo kwaye zinamandla amakhulu okudityaniswa kwe-LOCC.Sun et al.[21] yenze i-chip ye-microfluidic evumela ukuphindaphinda ukulandelelana kwe-nucleic acid ethile ye-pathogens ezintlanu, kubandakanya i-SARS-CoV-2, isebenzisa i-LAMP kwaye yahlalutya kusetyenziswa i-smartphone kwiyure enye emva kokuphela kokusabela.Njengomnye umzekelo, uSundah et al.[112] wenze i-molecular switch [catalytic amplification by molecular transition state switch (CATCH)] yokuchongwa ngokuthe ngqo nokunovakalelo lweethagethi ze-SARS-CoV-2 RNA kusetyenziswa ii-smartphones. I-CATCH iyahambelana ne-LOCC ephathekayo kwaye ifezekisa ukusebenza okuphezulu (malunga neekopi ze-8 ze-RNA / μl; < 1 h kwiqondo lobushushu begumbi) [112]. I-CATCH iyahambelana ne-LOCC ephathekayo kwaye ifezekisa ukusebenza okuphezulu (malunga neekopi ze-8 ze-RNA / μl; < 1 h kwiqondo lobushushu begumbi) [112]. Ukubamba совместим с портативным LOCC kunye обеспечивает превосходную производительность (примерно 8 копий РНК/мкл; < 1 ч при компелой12]. I-CATCH iyahambelana ne-LOCC ephathekayo kwaye ibonelela ngokugqwesa kokuphumelela (malunga neekopi ezi-8 ze-RNA/µl; < 1 h kwiqondo lobushushu begumbi) [112]. CATCH 与便携式LOCC 兼容并具有卓越的性能（大约8 RNA 拷贝/μl；室温下<1 小时）[112]. CATCH 与便携式LOCC 兼容并具有卓越的性能（大约8 RNA 拷贝/μl；室温下<1 小时）[112]. CATCH совместим с портативными LOCC и обладает превосходной производительностью (примерно 8 копий РНК/мкл; < 1 часа при комнат121 тем) I-CATCH iyahambelana neeLOCC eziphathwayo kwaye inomsebenzi oncomekayo (malunga neekopi ezisi-8 zeRNA/µl; < 1 iyure kwiqondo lobushushu begumbi) [112].Ukongeza, izixhobo zeLOCC zoxilongo lwemolekyuli zisebenzisa amandla athile okuqhuba anje ngevacuum, ukolula, kunye neendawo zombane.Kang et al.[113] ubonise i-PCR yexesha lokwenyani, i-ultra-fast nanoplasma-on-a-chip yokufunyaniswa ngokukhawuleza nangobungakanani be-COVID-19 ebaleni kusetyenziswa itshiphu ye-PCR yolwelo lwe-vacuum.Li et al.[114] emva koko uphuhlise i-microfluidic chip eyolula eyenze ukuba kufunyaniswe i-COVID-19.Iqonga lisebenzisa inkqubo yokukhulisa i-RT-LAMP ukugqiba ukuba isampuli ifanelekile okanye imbi.Emva koko, uRamachandran et al.[I-115] iphumelele i-gradients yendawo yombane efanelekileyo usebenzisa i-isotachophoresis (ITP), i-ion ekhethiweyo yokugxininisa ubuchule obuphunyezwe kwi-microfluidics.Nge-ITP, i-RNA ekujoliswe kuyo kwiisampuli ze-nasopharyngeal swab eziluhlaza zinokucocwa ngokuzenzekelayo.Emva koko uRamachandran et al.[115] Ukudibanisa oku kuhlanjululwa kwe-ITP kunye ne-ITP-ephuculweyo ye-LAMP kunye novavanyo lwe-CRISPR luchonge i-SARS-CoV-2 kwi-swab ye-nasopharyngeal yabantu kunye neesampuli zeklinikhi malunga nemizuzu engama-35.Ukongezelela, iingcamango ezintsha zihlala zivela.Jadhav et al.[116] icebise isikimu sokuxilonga esisekwe kumphezulu ophuculweyo weRaman spectroscopy ngokudityaniswa nesixhobo semicrofluidic esiqulathe ngokuthe nkqo igolide/isilivere egqunywe ngekhabhoni nanotubes okanye i-electrospun micro/nanotubes elahlayo.I-Membrane-isebenzayo i-microchannels eyakhelwe-ngaphakathi yokucoca iyalahlwa.Isixhobo sibhengeza iintsholongwane ezivela kulwelo olwahlukeneyo lomzimba/ukukhutshulwa okunje ngamathe, i-nasopharynx kunye neenyembezi.Ke, i-virus titer ininzi kwaye intsholongwane inokuchongwa ngokuchanekileyo ngumsayino weRaman.
I-LOAD yiplatifomu ye-centrifugal microfluidic apho zonke iinkqubo zilawulwa yi-protocol frequency ejikeleza i-microstructured substrate [110].Isixhobo se-LOAD sibonakaliswa ngokusebenzisa amandla e-centrifugal njengamandla okuqhuba abalulekileyo.Ulwelo lukwaxhomekeke kwi-capillary, i-Euler kunye nemikhosi yeCoriolis.Ukusebenzisa isixhobo se-centrifuge, uhlalutyo lwenziwa ekusebenzeni kolwelo oluqhubekayo ukusuka kwi-radial ukuya kwindawo yangaphandle, ukuphelisa imfuno ye-tubing yangaphandle eyongezelelweyo, iimpompo, ii-actuator, kunye neevalvu ezisebenzayo.Ngamafutshane, indlela yokulawula enye yenza lula ukusebenza.Imikhosi esebenza kulwelo kwijelo le-microfluidic efanayo kumgama ofanayo ukusuka kwiziko lomthwalo liyalingana, okwenza kube lula ukuphinda isakhiwo setshaneli.Ngaloo ndlela, izixhobo ze-LOAD zilula kwaye zinoqoqosho ngakumbi ukuyila kunye nokuveliswa kunezixhobo eziqhelekileyo ze-LOCC, ngelixa iimpendulo zizimeleyo kwaye zihambelana;nangona kunjalo, ngenxa yamandla omatshini aphezulu ezixhobo ze-centrifugal, imathiriyeli ye-chip ekhoyo ilinganiselwe kwaye imiqulu encinci inzima.kwimoto.Ngexesha elifanayo, izixhobo ezininzi ze-LOAD zenzelwe ukusetyenziswa okukodwa kuphela, okuxabisa kakhulu ukufumanisa ubukhulu obukhulu [96, 117, 118, 119].
Kumashumi eminyaka akutshanje, i-LOAD, ethathwa njengenye yezona zixhobo zithembisayo ze-microfluidic, ifumene ingqalelo enkulu kubaphandi kunye nabavelisi.Ke, i-LOAD ifumene ukwamkelwa okubanzi kwaye isetyenziselwe ukuxilongwa kweemolekyuli zeepathogens ezosulelayo [120, 121, 122, 123, 124], ngakumbi ngexesha lokuqhambuka kwe-COVID-19.Umzekelo, ekupheleni kuka-2020, uJi et al.[60] ubonise uvavanyo oluthe ngqo lwe-RT-qPCR lokufunyaniswa okukhawulezileyo nokuzenzekela okufanayo kwe-SARS-CoV-2 kunye nosulelo lomkhuhlane A kunye no-B kwisampulu zomqala.Emva koko Xiong et al.[74] thaca iqonga elidityanisiweyo le-LAMP le-discoid microfluidic ngokukhawuleza, echanekileyo, kwaye ngaxeshanye ukubhaqwa kwe-coronavirus yabantu ezisixhenxe zokuphefumla, kubandakanya i-SARS-CoV-2, kwimizuzu engama-40.Ekuqaleni kuka-2021, de Oliveira et al.[73] wabonisa i-polystyrene toner centrifugal microfluidic chip, eqhutywa ngesandla ngencam yokujikeleza, ye-RT-LAMP yokuxilongwa kweemolekyuli ze-COVID-19.Emva koko, uDignan et al.[39] thaca isixhobo esiphathwayo se-centrifuge microdevice yokucoca i-SARS-CoV-2 RNA ngokuthe ngqo kumacandelo e-buccal swab.UMedved et al.[53] icebise ngenkqubo yesampulu ye-aerosol engaphakathi kwe-SARS-CoV-2 enomthamo omncinci ojikelezayo we-microfluidic fluorescent chip enomlinganiselo wokubhaqwa kweekopi ezili-10/μL kunye nomyinge womjikelo omncinane wemizuzu eli-15.Suarez et al.[75] kutshanje uxele uphuhliso lweqonga elidityanisiweyo le-centrifugal microfluidic lokuchongwa ngokuthe ngqo kwe-SARS-CoV-2 RNA kwiisampuli ze-nasopharyngeal swab ezingasebenziyo zisebenzisa i-LAMP.Le mizekelo ibonisa iingenelo ezinkulu kunye nesithembiso se-LOAD kuxilongo lwemolekyuli ye-COVID-19.
Ngo-1945 uMuller kunye noClegg [125] babonisa kuqala amajelo e-microfluidic ephepheni besebenzisa iphepha lokucoca kunye neparafini.Kwi-2007, iqela le-Whitesides [126] lenze iqonga lephepha lokuqala elisebenzayo kwiprotheyini kunye novavanyo lwe-glucose.Iphepha liye laba yi-substrate efanelekileyo ye-microfluidics.Iphepha lineempawu zendalo ezifana ne-hydrophilicity kunye ne-porous structure, i-biocompatibility egqwesileyo, ubunzima bokukhanya, ukuguquguquka, ukugoqa, ixabiso eliphantsi, ukusetyenziswa lula kunye nokulula.Ii-Classical µPADs ziquka i-hydrophilic/hydrophobic structures ezakhelwe phezu kwesubstrates zephepha.Ngokuxhomekeke kwisakhiwo esine-dimensional, i-μPADs inokwahlulwa ibe yi-dimensional (2D) kunye ne-three-dimensional (3D) μPADs.I-2D µPADs ziveliswa ngokwenza imida ye-hydrophobic ukwenza i-microfluidic channels, ngelixa i-3D µPADs ziqhele ukwenziwa ukusuka kwi-stacks ye-2D microfluidic paper, ngamanye amaxesha ngokusongwa kwephepha, ubuchule bokutyibilika, iziteshi ezivulekileyo, kunye noshicilelo lwe-3D [96].Ulwelo olunamanzi okanye lwebhayoloji kwi-μPAD lulawulwa ngokuyintloko ngamandla e-capillary ngaphandle komthombo wamandla wangaphandle, ukuququzelela ukugcinwa kwangaphambili kwee-reagents, ukuphathwa kwesampulu, kunye nokufunyanwa kwe-multiplex.Nangona kunjalo, ulawulo oluchanekileyo lokuhamba kunye nokuchongwa kwe-multiplex kuthintelwa yisantya esinganelanga sokufumanisa, uvakalelo, kunye nokusebenziseka kwakhona [96, 127, 128, 129, 130].
Njengeqonga le-microfluidic elingaqhelekanga, i-μPAD iye yakhuthazwa ngokubanzi kwaye yaphuhliswa ukuxilongwa kwe-molecular yezifo ezosulelayo ezifana ne-HCV, i-HIV, kunye ne-SARS-CoV-2 [131, 132].Ukuchongwa okukhethiweyo kunye nobuthathaka be-HCV, uTengam et al.[133] iphuhlise inoveli biosensor esekwe kwiphepha le-fluorescent usebenzisa i-nucleic acid probe ethile esekelwe kwi-pyrrolidinyl peptide.I-Nucleic acids i-covalently immobilised kwiphepha le-cellulose elifakwe i-oxid ngokunciphisa i-alkylation phakathi kwamaqela e-amino kunye namaqela e-aldehyde, kwaye ukufunyanwa kusekelwe kwi-fluorescence.Ezi zibonakaliso zinokufundwa yigajethi eyenziwe ngokukodwa kunye nekhamera ephathekayo ye-fluorescent ngokudibanisa nekhamera yeselula.Emva koko, uLu et al.[134] yenzelwe i-electrode eguquguqukayo esekwe kwiphepha esekelwe kwi-nickel / i-nanoparticles yegolide / i-carbon nanotubes / i-polyvinyl alcohol organometallic framework composites ye-HIV ekufumaneni i-target target nge-DNA hybridization usebenzisa i-methylene blue njengesalathisi se-DNA redox.Kutshanje, uChowdury et al.[135] thaca uyilo lweqonga locingelo lovavanyo lwendawo yokhathalelo µPAD kusetyenziswa amathe esigulane ekrwada zidityaniswe neLAMP kunye neteknoloji yomfanekiso ophathwayo ukubhaqwa kohlalutyi lwe-COVID-19.
Iimvavanyo ze-lateral flow zikhokela ulwelo ngamandla e-capillary kunye nokulawula ukuhamba kwamanzi ngokumanzisa kunye neempawu ze-porous or microstructured substrates.Izixhobo ezihamba ngasecaleni zibandakanya isampuli, i-conjugate, i-incubator kunye nokufumanisa, kunye neepads ezifunxayo.Iimolekyuli ze-nucleic acid kwi-LFA ziqaphela izibophelelo ezithile ezigcinwe kwangaphambili kwindawo yokubopha kwaye zibophe njengezinto eziyinkimbinkimbi.Njengoko i-liquid idlula kwi-incubation kunye neeplate zokubona, ii-complexes zithathwa ngama-molecule wokubamba abekwe kwimigca yokuvavanya kunye nokulawula, ebonisa iziphumo ezinokufundwa ngokuthe ngqo kwiso lenyama.Ngokuqhelekileyo, i-LFA inokugqitywa kwi-2-15 imizuzu, ngokukhawuleza kunokufumanisa kwendabuko.Ngenxa yomatshini okhethekileyo, i-LFA idinga imisebenzi embalwa kwaye ayifuni izixhobo ezongezelelweyo, nto leyo eyenza ukuba isebenziseke kakhulu.Kulula ukwenza kunye nokwenza i-miniaturize, kwaye iindleko ze-substrates ezisekelwe ephepheni ziphantsi.Nangona kunjalo, isetyenziselwa kuphela uhlalutyo lwekhwalithi, kwaye ukufumanisa ubungakanani kunzima kakhulu, kwaye ukukwazi ukuphindaphinda kunye nokugqithisa kuncinci kakhulu, kwaye inye kuphela i-nucleic acid eyaneleyo inokufunyanwa ngexesha [96,110,127].
Nangona uninzi lwezicelo ze-LFA zijolise kwi-immunoassays, ukusetyenziswa kwe-LFA yokuxilongwa kweemolekyuli kwi-chips microfluidic iyasebenza kwaye iyathandwa [136].Kwimeko yentsholongwane ye-hepatitis B, i-HIV kunye ne-SARS-CoV-2 LFA Gong et al.[137] icebise i-platform ye-nanoparticle ye-LFA ephezulu yokuguqulwa kwaye ibonise ukuguquguquka kweli qonga elincinci kunye ne-portable ngokufumanisa ubuthathaka kunye nobuninzi beethagethi ezininzi ezifana ne-HBV nucleic acid.Ukongeza, uFu et al.[138] ibonise inoveli ye-LFA esekelwe kwi-Raman spectroscopy ephuculweyo yomgangatho wohlalutyo lobuninzi be-HIV-1 DNA kwiindawo eziphantsi.Ukufunyaniswa okukhawulezileyo nobuthathaka kwe-SARS-CoV-2, uLiu et al.[85] iphuhlise i-microfluidic-integrated RPA lateral flow analysis ngokudibanisa i-RT-RPA kunye ne-universal lateral flow flow system kwi-single microfluidic system.
Ukusetyenziswa kwamaqonga ahlukeneyo e-microfluidic kuyahluka ngokuxhomekeke kwizifundo ezithile, ukuthatha inzuzo epheleleyo yezakhono kunye neenzuzo zamaqonga.Ngeevalvu ezifikelelekayo, iimpompo kunye needucts, iLOCC lelona qonga libanzi lolwahlulo lwezicelo kunye nokusebenzisana kunye negumbi elikhulu lophuhliso.Ke ngoko, sinethemba kwaye sicebisa ukuba olona phando lutsha luqhutywe eLOCC njengelinge lokuqala kwaye iimeko ziphuculwe.Ukongeza, iindlela ezisebenzayo nezichanekileyo zilindeleke ukuba kufunyanwe kwaye zisetyenziswe kwinkqubo.I-LOAD igqwesa kulawulo oluchanekileyo lwamanzi avela kwizixhobo ezikhoyo ze-LOCC kwaye ibonisa inzuzo eyodwa kwi-drive enye ngamandla e-centrifugal ngaphandle kwesidingo sokuqhuba kwangaphandle, ngelixa iimpendulo ezihambelanayo zinokuhlukana kwaye zihambelane.Ke, kwixa elizayo, i-LOAD iya kuba yeyona ndawo iphambili ye-microfluidic iqonga elinomsebenzi omncinci wezandla kunye nobuchwephesha obuqolileyo kunye nobuchwephesha.Iqonga le-µPAD lidibanisa uncedo lwe-LOCC kunye nemathiriyeli esekwe ephepheni ngexabiso eliphantsi, ukuxilongwa kokusetyenziswa kanye.Ngoko ke, uphuhliso lwexesha elizayo kufuneka lugxininise kwiiteknoloji ezifanelekileyo kunye nezisekwe kakuhle.Ukongezelela, i-LFA ifaneleke kakuhle ukubonwa kwamehlo e-naked, ithembisa ukunciphisa ukusetyenziswa kwesampuli kunye nokukhawuleza ukufumanisa.Uthelekiso oluneenkcukacha lweqonga luboniswe kwiThebhile yesi-2.
Uhlalutyo lwedijithali lwahlula isampuli kwii-microreactors ezininzi, nganye kuzo iqulethe inani elicacileyo leemolekyuli ekujoliswe kuzo [139, 140].Uvavanyo lwedijithali lubonelela ngoncedo olubalulekileyo lokwenza umyinge opheleleyo ngokwenza amawaka eemvavanyo zebhayokhemikhali ezinxuseneyo ngaxeshanye kunye nomntu ngamnye kumacandelo esikali se-micron kunesigaba esiqhubekayo.Xa kuthelekiswa ne-microfluidics yendabuko, ukuphendula kwe-compartment kunokunciphisa umthamo wesampuli, ukwandisa ukusebenza kakuhle kokuphendula, kwaye zidityaniswe lula kunye nezinye iindlela zokuhlalutya ngaphandle kwesidingo semijelo, iimpompo, iivalve, kunye noyilo oludibeneyo [141, 142, 143, 144, 145, 146, 147] .Ezi ndlela zimbini zilandelayo zisetyenziswa kwiimvavanyo zedijithali ukufezekisa ukwahlula okufanayo kunye nokuchanekileyo kwezisombululo, kubandakanywa i-reagents kunye neesampuli ezifana neeseli, i-nucleic acids, kunye nezinye iincinci okanye ii-molecule: (1) i-emulsions yokulahla i-emulsions isebenzisa ukungazinzi kwe-liquid interface;(2) ulwahlulo lwe-array luqhutywa yimiqobo yejometri yesixhobo.Kwindlela yokuqala, amaconsi aqulethe i-reagents kunye neesampuli kwii-microchannels zinokudalwa ngeendlela ezingabonakaliyo ezifana ne-co-current, crossflow, flowing focus, emulsification staged, i-microchannel emulsification, kunye ne-membrane ngokusebenzisa i-viscous shear forces kunye ne-emulsification kunye noshintsho lwesiteshi.indawo yokuhlala [143, 145, 146, 148, 149] okanye ukusebenzisa iindlela ezisebenzayo [150, 151], ezizisa amandla ongezelelweyo ngolawulo lombane, umbane, i-thermal kunye nomatshini.Kwindlela yokugqibela, ukufana komthamo we-fluid best kumagumbi e-microfluidic kwabelwana ngokugcina izakhiwo zendawo ezinobungakanani obufanayo, njenge-micropits kunye ne-surface arrays [152,153,154].Ngokucacileyo, amathontsi ngamacandelo okuqukuqela aphambili anokuthi aveliswe kwaye aguqulwe kuluhlu lwe-electrode esekwe kwi-digital microfluidics (DMF).I-electrowetting ye-dielectrics yenye yezona thiyori ze-DMF ezifundwe kakhulu, ekubeni i-electrowetting ye-dielectrics ivumela ukuguqulwa okuchanekileyo kwamathontsi ngamanye, ukulawula imilo yolwelo kunye nemiqondiso yombane ye-asymmetric edlula kumacala ahlukeneyo [141, 144].Imisebenzi ephambili kunye namaconsi kwi-DMF ibandakanya ukuhlenga, ukuhlukana, kunye nokudibanisa [151, 155, 156], enokuthi isetyenziswe kwiinkalo ezahlukeneyo zokuhlalutya, ngokukodwa ekuboneni i-molecular [157, 158, 159].
Ukubhaqwa kwe-nucleic acid yeDijithali sisizukulwana sesithathu sokuxilonga itekhnoloji yemolekyuli elandela i-PCR eqhelekileyo kunye ne-PCR yexesha lokwenyani lobungakanani (qPCR), ngokuhambelana nolandelelwano oluphezulu kunye ne-biopsy yolwelo.Kwiminyaka emibini edlulileyo, i-digital nucleic acids iphuhliswe ngokukhawuleza kwintsimi yokuxilongwa kwee-molecular of pathogens ezosulelayo [160, 161, 162].Ubungakanani obupheleleyo bokufunyanwa kwe-nucleic acid yedijithali iqala ngokupakisha iisampulu kunye nee-reagents kwiikhompatimenti zomntu ngamnye ukuqinisekisa ukuba ulandelelwano ngalunye olujoliswe kuyo lunethuba elifanayo lokungena kwikhompatimenti nganye.Ngokwethiyori, icandelo ngalinye linokwabelwa ulandelelwano lweethagethi ezininzi, okanye kungabikho sistim ezimeleyo ye-microreaction.Ngokusebenzisa iindlela ezahlukeneyo zokuvamvo ezichazwe ngasentla, iikhompatimenti ezine-microbial target sequences ezivelisa imiqondiso ngaphezu komda othile zinokubonwa ngeliso lenyama okanye ngomatshini kwaye zibhalwe njengezilungileyo, ngelixa ezinye iikhompatimenti ezivelisa imiqondiso engaphantsi komgubasi zibhalwe njengezilungileyo. .Ezingalunganga, ezenza umqondiso wecandelo ngalinye ube yi boolean.Ke, ngokubala inani lamacandelo adalwe kunye nesantya seziphumo ezilungileyo emva kokusabela, iikopi zoqobo zeesampulu zovavanyo zinokudityaniswa kusetyenziswa iPoisson ifomula yokuhambisa ngaphandle kwemfuneko yegophe eliqhelekileyo, elifunekayo kuhlalutyo lwesiqhelo lobungakanani obunjalo. njenge qPCR.[163] Xa kuthelekiswa neendlela zemveli zokuxilonga iimolekyuli, ukufumanisa i-nucleic acid yedijithali kunezinga eliphezulu lokuzenzekelayo, isantya esiphezulu sokuhlalutya kunye novakalelo, ii-reagents ezimbalwa, ukungcoliseka okuncinci, kunye noyilo olulula kunye nokuveliswa.Ngezi zizathu, ukusetyenziswa kovavanyo lwedijithali, ngakumbi iindlela ezisezantsi, kuxilongo lweemolekyuli, ukudibanisa ukukhulisa kunye nobuchule bokufunda umqondiso, kufundwe kakuhle ngexesha lokuqhambuka okubalulekileyo kwe-SARS-CoV-2.Ngokomzekelo, uYin et al.[164] idijithi edityanisiweyo yedijithali kunye neendlela ezikhawulezayo ze-PCR zokubona i-ORF1ab, N, kunye nofuzo lwe-RNase P kwi-SARS-CoV-2 kwi-microfluidic chip.Ngokucacileyo, inkqubo yakwazi ukuchonga isibonakaliso esilungileyo ngaphakathi kwemizuzwana ye-115, ekhawulezayo kune-PCR eqhelekileyo, ebonisa ukusebenza kwayo ekubonweni kwe-point-of-care-care (Figure 7a).UDong et al.[165], Yihlwayela et al.[157], Chen et al.[166] kunye noAlteri et al.[167] ikwafakele idroplet digital PCR (ddPCR) ukubona i-SARS-CoV-2 kwinkqubo yemicrofluidic eneziphumo ezincomekayo.Ukuphucula ngakumbi izinga lokubona, uShen et al.[168] iphumelele i-ddPCR-based imaging chip kwisithuba nje se-15 s ngaphandle kokusetyenziswa kweendlela zokuthunga umfanekiso, ukukhawulezisa inkqubo yeteknoloji ye-ddPCR ukusuka kwilebhu ukuya kwisicelo.Ayizizo kuphela iindlela zokukhulisa ubushushu ezifana ne-PCR ezisetyenziswayo, kodwa neendlela zokukhulisa i-isothermal zisetyenziselwa ukwenza lula iimeko zokusabela kunye nokuphendula ngokukhawuleza.Lu et al.[71] iphuhlise iSlipChip yohlalutyo lwamathontsi, ekwaziyo ukuvelisa amathontsi obungakanani obahlukeneyo kuxinaniso oluphezulu kwinyathelo elinye kunye nokulinganisa i-SARS-CoV-2 nucleic acids kusetyenziswa i-LAMP yedijithali (Figure 7b).Njengobuchwephesha obuvela ngokukhawuleza, i-CRISPR inokudlala indima ebalulekileyo ekubhaqweni kwe-nucleic acid yedijithali ngokusebenzisa umfanekiso ocacileyo we-colorimetric ngaphandle kwesidingo sokongeza amabala e-nucleic acid.UAckerman et al.iphuhlise ukusabela kwe-matrix edibeneyo yokuvavanya i-multiplex ye-nucleic acids.[158] yafumanisa iintsholongwane ezili-169 ezinxulumene nomntu, kubandakanywa i-SARS-CoV-2, kumathontsi aqulethe i-CRISPR-Cas13-based based nucleic acid reagents kwi-microwell assay (Figure 7c).Ukongezelela, i-isothermal amplification kunye ne-CRISPR iteknoloji ingasetyenziselwa kwinkqubo efanayo ukudibanisa izibonelelo zombini.Park et al.[169] Uvavanyo lwedijithali lwe-CRISPR/Cas12a lwaphuhliswa kwi-chip ye-microfluidic yorhwebo ukuze kufunyanwe i-SARS-CoV-2 ekhutshiweyo kunye nobushushu obubulewe ngokusekwe kwinqanaba elinye le-RT-RPA kunye nophawu olufutshane kunye oluphezulu oluya ngasemva. umlinganiselo wexesha., uluhlu olubanzi oluguquguqukayo kunye novakalelo olungcono (Umfanekiso 7d).Ezinye iinkcazo zale mizekelo zinikwe kwiThebhile yesi-3.
Iqonga ledijithali eliqhelekileyo lokufunyanwa kwe-nucleic acid.a Ukuhamba kwe-PCR yedijithali ekhawulezayo iquka amanyathelo amane abalulekileyo: ukulungiswa kwesampulu, ukuhanjiswa komxube wokusabela, inkqubo yokukhulisa, kunye nomlinganiselo ojoliswe kuwo (owenziwe kwi- [164]).b Isicwangciso esibonisa uhlalutyo lwamathontsi e-SlipChip ukwenzela ukwakheka kwamathontsi kuxinaniso oluphezulu ( luthatyathwe kwi- [71]).c CARMEN-Cas umzobo wokuhamba komsebenzi13 (ulungiswe ukusuka [158]).d Isishwankathelo sokufunyanwa kwentsholongwane yedijithali ephezulu kunye ne-CRISPR/Cas kwimbiza enye (ithathwe kwi- [169]).I-W/O yamanzi e-oyile, i-polydimethylsiloxane PDMS, i-PCR i-polymerase chain reaction, i-DAQ yokuqokelelwa kwedatha, i-PID edityanisiweyo ephuma kuyo, i-CARMEN i-combinatorial matrix reaction ye-multiplex yovavanyo lwe-nucleic acid, i-SARS-CoV-2, i-acute acute breathing syndrome, i-coronavirus 2, I-RT Ukwandiswa kwe-reverse transcriptase recombinase polymerase-RPA, isignali ye-S/B ngasemva