Amasu okuxilonga endabuko okuthola izifo ezithathelwanayo adinga ukusetshenziswa kwezinsimbi ze-benchtop ezingafanele ukuhlolwa kwe-point-of-care (POCT).I-microfluidics esafufusa iwubuchwepheshe obuncane kakhulu, obuzenzakalelayo, futhi obuhlanganisiwe obungaba enye indlela yezindlela zendabuko zokuxilonga endaweni esheshayo, engabizi kakhulu, nenembile.Izindlela zokuxilonga zamangqamuzana zisetshenziswa kakhulu kumishini ye-microfluidic njengendlela ephumelela kakhulu yokuthola i-pathogen.Lokhu kubuyekezwa kufingqa intuthuko yakamuva ekuxilongeni kwamangqamuzana okususelwe ku-microfluidic kwezifo ezithathelwanayo ngokombono wezemfundo nowezimboni.Okokuqala, sichaza ukucutshungulwa okujwayelekile kwe-on-chip kwama-nucleic acid, okufaka isampula yokwelashwa kwangaphambili, ukukhulisa, nokufunda isignali.Izici, izinzuzo kanye nokubi kwezinhlobo ezine zamapulatifomu e-microfluidic bese kuqhathaniswa.Okulandelayo, sizoxoxa ngokusetshenziswa kwezivivinyo zedijithali zokuthola inani eliphelele lama-nucleic acid.Womabili amadivaysi okuxilonga amangqamuzana enziwe nge-microfluidic yakamuva kanye nakamuva afingqiwe njengobufakazi besimo samanje semakethe.Okokugcina, siphakamisa izinkomba zesikhathi esizayo zokuxilongwa kwe-microfluidic yezifo ezithathelwanayo.
Izifo ezithathelwanayo zibangelwa amagciwane, okuhlanganisa amagciwane, amagciwane, nama-parasites, asatshalaliswa emhlabeni wonke.Ngokungafani nezinye izifo, amagciwane atheleleka ngokushesha futhi asakazeke phakathi kwabantu nezilwane ezifuywayo ngokujova, umoya kanye nemithombo yamanzi [1].Ukuvimbela izifo ezithathelwanayo kubalulekile njengesinyathelo sezempilo yomphakathi.Amasu amathathu ayinhloko okulwa nezifo ezithathelwanayo: (1) ukulawula umthombo wamagciwane;(2) ukuphazamiseka kwendlela yokudlulisa;(3) ukuvikelwa kwezibalo zabantu abathintekayo.Phakathi kwamasu ayinhloko, ukulawulwa komthombo wokutheleleka kubhekwa njengesu elibaluleke kakhulu ngenxa yokulula kwayo kanye nezindleko eziphansi.Ukuxilongwa okusheshayo, ukuhlukaniswa, kanye nokwelashwa kwabantu abathelelekile kubalulekile, kudinga amasu okuxilonga asheshayo, abucayi, nanembile [2].Ukuxilongwa kwamanje kwezifo ezithathelwanayo ngokuvamile kuhlanganisa ukuhlolwa komtholampilo okusekelwe kuzimpawu nezimpawu kanye nezifundo zaselabhorethri ezifana nesiko lamangqamuzana nokuxilongwa kwamangqamuzana, okudinga izisebenzi eziqeqeshiwe, izinqubo ezidinga abasebenzi abaningi, kanye nemishini yokuhlola ebizayo [3, 4].Ukuvimbela ukuqubuka kwezifo ezithathelwanayo kudinga ukuxilongwa kwendawo ngokushesha, okungabizi, nokunembile, ikakhulukazi ezindaweni ezinomkhawulo wezinsiza lapho izifo ezithathelwanayo zivamile futhi zinzima [5], kanye nokwelashwa ehlane noma enkundleni yempi, lapho izimo eziphuthumayo zingalindelekile..ukunakekelwa kwezokwelapha kulinganiselwe [6].Kulo mongo, i-microfluidics ubuchwepheshe obuhlanganisa ubuchwepheshe bezinhlelo ze-microelectromechanical, i-nanotechnology, noma isayensi yezinto zokwakha ukuze kusetshenziswe uketshezi olunembile [7,8,9,10], oluhlinzeka ngamathuba amasha okutholwa kwendawo yokunakekelwa (POCT).) izifo ezithathelwanayo ngaphandle kwezibhedlela namalabhorethri.Uma kuqhathaniswa nokuxilonga okudla isikhathi okuvamile, ubuchwepheshe be-microfluidic bunikeza isampula nokonga kwezindleko zokuxilongwa kwamangqamuzana ngesikhathi sokuqubuka kwezifo.Ukusabalala komhlaba wonke kwesifo i-coronavirus 2019 (COVID-19) kubangelwe yi-acute acute kupumua i-coronavirus 2 (SARS-CoV-2), ngakho-ke ukubaluleka kwe-microfluidics ekuvimbeleni nasekulawuleni ubhubhane okufika ngesikhathi kuyagcizelelwa [11, 12 , 13].Ngokungafani nokuxilongwa kwendabuko, i-microfluidic POCT isebenzisa amadivaysi amancane aphathwayo asukela kubahlaziyi be-benchtop kuya emicu emincane yokuhlola eseceleni ukuze ihlolwe eduze kwephuzu lesampula [14].Lokhu kuhlola kufaka ukulungiselela isampula okulula noma okungekho, ukukhulisa isignali ngokushesha, nokufundwa kwesignali ebucayi okuholela esikhathini esifushane nemiphumela enembile phakathi nemizuzu.Ukutholakala nokukhiqizwa ngobuningi kwamathuluzi okunakekelwa kwezempilo asekelwe ku-microfluidic kunwebise izicelo zabo zokuxilonga ezingabizi futhi eziqondile ngaphandle kwesibhedlela, eduze kwesiguli, ngisho nasekhaya.
Phakathi kwamasu akhona okuxilonga izifo ezithathelwanayo, ukuxilongwa kwamangqamuzana kungenye yezinto ezibucayi kakhulu [15, 16].Ngaphezu kwalokho, ukuxilonga kwamangqamuzana kuvame ukusetshenziswa njengezinga legolide lokutholwa okuqhubekayo kwe-COVID-19, okuvumela ukutholwa okuqondile kwezifunda eziqondene negciwane ze-RNA noma i-DNA ngaphambi kokuqala kokuphendula kwamasosha omzimba [17, 18].Ekubuyekezweni kwamanje, sethula intuthuko yakamuva ezinqubweni zokuxilonga zamangqamuzana ezisekelwe ku-microfluidics zezifo ezithathelwanayo, kusukela kumbono wezemfundo kuya emibonweni yesikhathi esizayo yezimboni (Fig. 1).Sizoqala ngezinyathelo ezintathu ezibalulekile ekutholeni i-nucleic acid: isampula ye-on-chip yokwelashwa kwangaphambili, i-nucleic acid amplification, nokufunda isignali.Sibe sesiqhathanisa izinhlobo ezahlukene zamapulatifomu e-microfluidic nesakhiwo nomsebenzi wawo, okubonisa izici eziyingqayizivele (amandla nobuthakathaka).Ukutholwa kwe-nucleic acid yedijithali kuphinde kuxoxwe ngakho futhi kunikezwe njengesibonelo sobuchwepheshe besizukulwane sesithathu sokuthola inani eliphelele lama-molecule e-pathogen.Ngaphezu kwalokho, kuzokwethulwa amadivaysi ambalwa ajwayelekile nakamuva e-POCT azokhombisa isimo samanje semakethe ye-microfluidic POCT yokuxilongwa kwamangqamuzana.Sizoxoxa futhi sichaze umbono wethu wezicelo zesikhathi esizayo.
Amamojula ama-microfluidic chips okutholwa kwe-nucleic acid angahlukaniswa abe izigaba ezintathu (isampula, ukuqashelwa, nokusayina) ngokuya ngemisebenzi yawo [19].Phakathi kwalawa mamojula, imojula yesampula ikakhulukazi ibona isampula ye-lysis kanye nokukhishwa kwe-nucleic acid.Imojula yenzwa ilawula ngokuyinhloko ukuguqulwa nokukhulisa izimpawu ze-nucleic acid.Imojula yokusayina ithola isignali eguquliwe futhi icutshungulwe yimojuli yokuzwa.Ngokusekelwe kunqubo yokuthola ama-nucleic acid ku-chip, sizofingqa ama-chip ahlukahlukene angakwazi ukubona umsebenzi "wokufaka nokukhiphayo".
Isinyathelo sokuqala sokutholwa kwe-nucleic acid isizinda se-nucleic acid, okungukuthi ukuhlukanisa i-nucleic acid eqondiwe kusampula yoqobo.Ukukhishwa kwe-Nucleic acid kwenziwa ukuze kuhlanzwe ama-nucleic acid avela kwezinye izinto ezingcolile zamangqamuzana, kuqinisekiswe ubuqotho besakhiwo esiyinhloko sama-nucleic acid molecule, nokwandisa imiphumela.Ukukhishwa kwe-Nucleic acid kudinga isampula edingekayo ye-lysis kanye ne-nucleic acid capture, ikhwalithi nokusebenza kahle kwayo okunomthelela omkhulu ocwaningweni nasemiphumeleni yokuxilonga.Noma yimiphi imiphumela emibi ecashile phakathi nokukhipha ingase inciphise ukutholwa okwengeziwe.Isibonelo, izindlela ze-polymerase chain reaction (PCR) nezindlela ze-loop isothermal amplification (LAMP) zivinjelwa ezinye izincibilikisi eziphilayo ezisele njenge-ethanol ne-isopropanol kuma-nucleic acid isolation reagents [20].Ukukhishwa koketshezi oluwuketshezi kanye nokukhishwa kwesigaba esiqinile kuyindlela ethandwa kakhulu yokuhlukanisa ama-nucleic acid [21], nokho, ukukhishwa koketshezi oluwuketshezi ku-chip kulinganiselwe kakhulu, njengoba ama-reagents asetshenziswa ekukhipheni uketshezi oluwuketshezi abangela ukugqwala kwama-chips amaningi ama-microfluidic. .Lapha, sigqamisa izindlela zokukhipha isigaba esiqinile esekwe ku-microarray futhi siqhathanise izinzuzo nezinkinga zazo.
I-Silicon i-substrate material ehambisana ne-nucleic acid ngenxa ye-biocompatibility yayo, ukuzinza, kanye nokuguqulwa kalula [22].Okubalulekile, uma ilungiswa nge-silica noma ezinye izinto, le nhlanganisela ibonisa izakhiwo zokukhangisa ama-nucleic acid ane-nucleic ekhokhiswa kabi ngaphansi kwe-pH ephansi, izimo zikasawoti ophakeme kuyilapho i-pH ephezulu, izixazululo zikasawoti ophansi.Ngokusekelwe kulesi simo, kungenzeka ukuhlanza i-nucleic acid.
Izinhlobo ezihlukahlukene zezinto ezisekelwe ku-silica zisetshenziselwe ukukhishwa kwe-nucleic acid kuma-microfluidics, njengobuhlalu be-silica, ama-powders, izihlungi ze-microfiber, nama-silica membranes [23, 24, 25, 26].Ngokuya ngezakhiwo zezinto ezibonakalayo, izinto ezisekelwe ku-silicon zingasetshenziswa kuma-microcircuits ngezindlela ezahlukene.Isibonelo, ama-silica granules, ama-powder, nama-nanofilters okuhweba angavele afakwe ezimbotsheni noma eziteshini ezincane zama-microfluidic chips futhi asize ukukhipha ama-nucleic acid kumasampula [27, 28, 29].Ulwelwesi lwe-silica olulungiswe phezulu lungasetshenziswa futhi ukuhlanza ngokushesha i-DNA kumagciwane ngezindleko eziphansi.Ngokwesibonelo, Wang et al.[30] Ngokuhlanganisa ukusabela kokukhulisa i-denaturing ne-vesicle-mediated chain exchange ne-silica membranes embozwe nge-chitosan oligosaccharides, kwethulwa uhlelo oluphathekayo oluguquguqukayo oluthole ngempumelelo amayunithi okwenza ikholoni ye-102–108.(CFU)/ml Vibrio parahaemolyticus., futhi ubukhona begciwane babubonakala kalula.UPowell et al.[31] Ama-microarray asuselwa ku-silicon abe esesetshenziselwa ukuthola igciwane le-hepatitis C (HCV), igciwane le-human immunodeficiency (HIV), igciwane leZika, kanye ne-human papillomavirus kanye nokusakazeka okuzenzakalelayo, lapho kwakhiwe khona i-1.3 μl microreactor ehlukumezayo ukuze ibambe amagciwane e-RNA.futhi enze in situ amplification.Ngaphezu kwalezi zindlela, ama-microcolumns e-silica ashintshwe phezulu nawo adlala indima ebalulekile ekukhishweni kwe-nucleic acid, njengoba i-geometry nezakhiwo zezinto eziguqukayo zandisa kakhulu ukusebenza kahle kokukhipha.U-Chen et al.[32] uhlongoze inkundla ye-microfluidic yokuhlukaniswa kwe-RNA egxilile ephansi ngokusekelwe kumakholomu amancane e-silicon ehlanganiswe ne-amino.Le divayisi ye-microfluidic ihlanganisa uhlu lwama-micropillar angu-0.25 cm2 ku-silicon substrate ukuze kuzuzwe ukusebenza kahle okuphakeme kokukhipha ngokusebenzisa indawo ephezulu ukuya kumklamo wesilinganiso sevolumu.Inzuzo yalo mklamo ukuthi idivayisi ye-microfluidic ingafinyelela ku-95% ukusebenza kahle kokukhipha i-nucleic acid.Lawa maqhinga asekelwe ku-silicon akhombisa ukubaluleka kokuhlukanisa ngokushesha ama-nucleic acid ngezindleko eziphansi.Ngokuhlanganiswa nama-microfluidic chips, amasu okukhipha asuselwa ku-silicon awakwazi nje ukukhulisa ukusebenza kahle kokutholwa kwe-nucleic acid, kodwa futhi enze lula ukwenziwa kwe-miniaturization nokuhlanganiswa kwamadivayisi okuhlaziya [20].
Izindlela zokuhlukanisa kazibuthe zisebenzisa izinhlayiya kazibuthe ukuze zihlukanise ama-nucleic acid phambi kwendawo yangaphandle kazibuthe.Izinhlayiya kazibuthe ezivame ukusetshenziswa zifaka i-Fe3O4 noma i-γ-Fe2O3 izinhlayiya kazibuthe ezihlanganiswe ne-silica, amino ne-carboxyl [33,34,35,36].Isici esihlukanisayo sezinhlayiya kazibuthe uma kuqhathaniswa nezindlela ze-SPE ezisekelwe ku-silicon kulula ukukhohlisa nokulawula ngozibuthe bangaphandle.
Kusetshenziswa ukusebenzisana kwe-electrostatic phakathi kwe-nucleic acid ne-silica, ngaphansi kwezimo zikasawoti omningi ne-pH ephansi, ama-nucleic acid akhangiswa phezu kwezinhlayiya zikazibuthe eziboshwe nge-silica, kuyilapho ngaphansi kwezimo zosawoti ophansi kanye ne-pH ephezulu, ama-molecule angawashwa. futhi..Ubuhlalu kazibuthe obufakwe ngesilika bukwenza kube nokwenzeka ukukhipha i-DNA kumasampula evolumu enkulu (400 μL) kusetshenziswa ukunyakaza okulawulwa kazibuthe [37].Njengomboniso, uRodriguez-Mateos et al.[38] wasebenzisa ozibuthe abakwaziyo ukulawula ukudluliswa kobuhlalu kazibuthe emakamelweni ahlukene.Ngokusekelwe ezinhlayiyeni zikazibuthe ezinamekwe nge-silica, amakhophi angu-470/mL we-SARS-CoV-2 genomic RNA angakhishwa kumasampula wamanzi angcolile ukuze kutholwe ukubhalwa kabusha kwe-LAMP (RT-LAMP) futhi impendulo ingafundwa lingakapheli ihora elingu-1.iso lenyama (Fig. 2a).
Amadivayisi asuselwe ezintweni ezinozibuthe nezimbotshana.Umdwebo womqondo wedivayisi ye-microfluidic ye-IFAST RT-LAMP yokutholwa kwe-SARS-CoV-2 RNA (ithathwe kokuthi [38]).b Idivayisi encane ye-Centrifugal ye-dSPE ye-buccal swab nucleic acid (ethathwe ku- [39]).c Isampula yokugxilisa ingqondo eyakhelwe ngaphakathi esebenzisa ikhadi le-FTA® (ithathwe kokuthi [50]).d Iphepha lesihlungi se-Fusion 5 elilungiswe nge-chitosan (lithathwe ku- [51]).I-SARS-CoV-2 i-acute acute respiratory syndrome 2, i-RT-LAMP reverse transcription loop mediated isothermal amplification, abalingani bezobuchwepheshe be-FTA finders, i-NA nucleic acid
Izinhlayiya kazibuthe ezishajwe kahle zilungele ukunamathisela umgogodla we-phosphate we-nucleic acid.Esigabeni esithile sikasawoti, amaqembu e-phosphate ane-nucleic acid angakhokhiswa kahle ebusweni bezinhlayiya eziyinhlanganisela kazibuthe.Ngakho-ke, ama-nanoparticles kazibuthe anendawo elukhuni kanye nokuminyana okuphezulu kwamaqembu amino athuthukiswa ukuze kukhishwe ama-nucleic acid.Ngemuva kokuhlukaniswa kazibuthe nokuvinjwa, ama-nanoparticles kazibuthe kanye ne-DNA complexes ingasetshenziswa ngokuqondile ku-PCR, eqeda isidingo sokuhlanzwa okuyinkimbinkimbi nokuthatha isikhathi kanye nokusebenza kwe-lution [35].Ama-nanoparticles kazibuthe ahlanganiswe namaqembu e-carboxyl angalungile nawo asetshenziselwe ukuhlukanisa ama-nucleic acid akhangiswa ezindaweni ezisezingeni eliphakeme ze-polyethylene glycol nezixazululo ze-sodium chloride [36].Ngalobu buhlalu obuzibuthe obuguquliwe, ukukhishwa kwe-DNA kuhambisana nokukhulisa i-amplification okulandelayo.Dignan et al.[39] ichaze inkundla ye-centrifugal microfluidic ezenzakalelayo nephathekayo ye-nucleic acid pretreatment, evumela izisebenzi ezingezona ezobuchwepheshe ukuthi ziyisebenzise esizeni.Ukwengeza, ukuhambisana kwe-DNA ehlukanisiwe ne-LAMP, indlela efaneleke kahle yokuhlaziywa kwe-nucleic acid ye-point-of-care, iphinde ibonise izidingo zemishini encane kanye nokufaneleka kokuhlolwa kwe-colorimetric (Fig. 2b).
Izindlela zobuhlalu obuzibuthe zinikeza ithuba lokukhishwa okuzenzakalelayo, okunye kwakho okutholakala kuzikhipheli ze-nucleic acid ezizenzakalelayo [KingFisher;I-ThermoFisher (Waltham, MA, USA), QIAcube® HT;CapitalBio (Beijing, China) kanye Biomek®;Beckman (Miami, USA).), Florida, USA)].Izinzuzo zokuhlanganisa ubuhlalu obukazibuthe nama-microfluidics zingasetshenziselwa ukukhipha okuzenzakalelayo okusebenzayo kwama-nucleic acid, okungase kube namandla okuthuthukisa ukuthuthukiswa kokuxilongwa kwamangqamuzana;kodwa-ke, inhlanganisela yobuhlalu kazibuthe obunama-microfluidics isancike kakhulu ezinhlelweni zokulawula eziyinkimbinkimbi zokuguqulwa okunembile kobuhlalu kazibuthe, okuchaza ukuduma kwemikhiqizo yezentengiselwano ukuthi inkulu futhi iyabiza, okukhawulela ukusetshenziswa okuqhubekayo kobuhlalu obuzibuthe ku-POCT.
Izinto eziningana ezinama-porous ezifana nezihlungi ze-nitrocellulose eziguquliwe, amakhadi e-Finders Technology Associates (FTA), amaphepha okuhlunga asekelwe ku-polyethersulfone, nezinto eziboshwe nge-glycan nazo zisetshenziselwe ukutholwa kwe-nucleic acid [40, 41, 42, 43, 44].Izinto ezinezintambo ezinezimbobo ezinjengephepha elinemicu zaqale zasetshenziswa ukuhlukanisa i-DNA ngokuhlanganisa amangqamuzana e-DNA anemicu emide ngemicu.Izimbotshana ezincane ziholela ekuvinjelweni okuqinile ngokomzimba kwama-molecule e-DNA, okuthinta kahle ukukhishwa kwe-DNA.Ngenxa yobukhulu obuhlukahlukene be-pore bephepha le-fibrous, ukusebenza kahle kokukhipha akukwazi ukuhlangabezana nezidingo zokukhulisa i-DNA [45, 46].Ikhadi le-FTA liyiphepha lokuhlunga lezentengiselwano elisetshenziswa emkhakheni wezokwelapha ze-forensic futhi lisetshenziswa kabanzi kwezinye izindawo zokuxilonga amangqamuzana.Ngokusetshenziswa kwephepha lesihlungi se-cellulose elifakwe ngamakhemikhali ahlukahlukene ukuze limise ulwelwesi lwamaseli kusampula, i-DNA ekhishiwe ivikelwa ekonakalisweni kuze kufike eminyakeni emi-2.Muva nje, iphepha le-cellulose elifakiwe lenziwe ukuze kutholakale amangqamuzana amagciwane ahlukahlukene, okuhlanganisa i-SARS-CoV-2, i-leishmaniasis, kanye nomalaleveva [47,48,49].I-HIV ku-plasma ehlukanisiwe i-lysed ngokuqondile, futhi i-nucleic acid ye-viral icebile ku-membrane yokugeleza kwe-FTA® eyakhelwe ku-concentrator, okuvumela ukukhiqizwa okuphumelelayo kwe-nucleic acid [50] (Fig. 2c).Inkinga enkulu ngokutholwa kwe-nucleic acid kusetshenziswa amakhadi e-FTA ukuthi amakhemikhali afana ne-guanidine ne-isopropanol avimbela ukusabela okulandelayo kwe-amplification.Ukuze sixazulule le nkinga, senze iphepha lokuhlunga le-Fusion 5-chitosan-modified chitosan, elihlanganisa izinzuzo zakho kokubili kokuhlangana ngokomzimba kwama-molecule e-DNA kanye nephepha lokuhlunga i-fibrous, kanye nokukhangisa kwe-electrostatic ye-DNA kumakhompiyutha aguqulwe i-chitosan ukuze kutholakale ukukhishwa kwe-nucleic acid esebenza kahle kakhulu. ..imicu yokuhlunga [51] (Fig. 2d).Ngokufanayo, uZhu et al.[52] ubonise indlela ye-PCR elungiswe nge-chitosan esekelwe ohlelweni lwe-in situ capillary microfluidic lokuhlukaniswa ngokushesha nokutholwa kwegciwane leZika i-RNA.Ama-Nucleic acid angakhangiswa/akhishwe endaweni exubile ye-lysate/PCR, ngokulandelana, ngokusekelwe kusici sokuvula/ukuvala se-chitosan.on and off”, isabela ku-pH.
Njengoba kushiwo ngenhla, lawa maqhinga ahlanganisa izinzuzo zezinto ezihlukahlukene zesigaba esiqinile futhi andise ukusebenza kahle kokukhipha i-nucleic acid kuma-microfluidics.Ekusetshenzisweni okungokoqobo, ukusetshenziswa kwalezi zinto ngobuningi akunamali, futhi ukwelashwa okufanele kwendawo noma ukuguqulwa kwendawo yezinto ezivamile ngalezi zinto kungagcina nomsebenzi wazo.Ngakho-ke, kukholelwa ukuthi ukuqaliswa kwalezi zindlela ngemuva kocwaningo lokuhlola kunganciphisa izindleko.
Ukuhlolwa kwe-Nucleic acid ezisekelweni ze-microfluidic kuvame ukusebenzisa amasampula amancane amavolumu (< 100 µl), ngakho-ke kudinga ukukhuliswa kwama-nucleic acid aqondiwe ngama-probe athile ukuze aguqulelwe kusignali elungele ukutholwa komfula (i-optical, ugesi, kanye nozibuthe) [53, 54]. Ukuhlolwa kwe-Nucleic acid ezisekelweni ze-microfluidic kuvame ukusebenzisa amasampula amancane amavolumu (< 100 µl), ngakho-ke kudinga ukukhuliswa kwama-nucleic acid aqondiwe ngama-probe athile ukuze aguqulelwe kusignali elungele ukutholwa komfula (i-optical, ugesi, kanye nozibuthe) [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]. Uma kuhlolwa ama-nucleic acid kuzingxenyekazi ze-microfluidic, amasampula amancane amavolumu (<100 µL) avame ukusetshenziswa, ngakho-ke ukukhuliswa kwama-nucleic acid aqondiwe anama-probe akhethekile kuyadingeka ukuze kuguqulwe ibe isignali elungele ukutholwa okulandelayo (i-optical, ugesi, kanye nozibuthe) [53, 54]微流控 平台 上 的 核酸 检测 通常 使用 小样本量 (<100 μl), ].微流控 平台 上 的 核酸 检测 使用 小样本量 ((<100 μl), ]. 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]]. Ukutholwa kwama-nucleic acid kumapulatifomu e-microfluidic kuvame ukusebenzisa amasampula amancane amavolumu (<100 μl), okudinga ukukhuliswa kwama-nucleic acid aqondiwe anama-probe akhethekile ukuze awaguqule abe amasignali ukuze atholwe ngokulandelayo (i-optical, i-electrical, ne-magnetic) [53, 54]] .Ukukhulisa i-Nucleic acid kuma-microfluidics nakho kungasheshisa ukusabela, kuthuthukise imikhawulo yokuthola, kunciphise izidingo zesampula, futhi kuthuthukise ukunemba kokutholwa [55, 56].Eminyakeni yamuva nje, ngokutholwa kokutholwa okusheshayo nokunembile, izindlela ezihlukahlukene zokukhulisa i-nucleic acid ziye zasetshenziswa kuma-microfluidics, okuhlanganisa i-PCR nokunye ukusabela kwe-isothermal amplification.Lesi sigaba sizofingqa izindlela zokutholwa kwe-nucleic acid ngokusekelwe kumasistimu we-microfluidic.
I-PCR ilingisa inqubo yokuphindaphinda kwe-DNA yento ephilayo, ithiyori yayo echazwe ngokuningiliziwe kwenye indawo futhi ngeke kuxoxwe ngayo lapha.I-PCR ingakhulisa inani elincane kakhulu le-DNA/RNA eqondiwe ngenani elicacile, okwenza i-PCR ibe ithuluzi elinamandla lokutholwa ngokushesha kwama-nucleic acid.Emashumini eminyaka amuva nje, amadivaysi amaningi aphathwayo e-microfluidic ahlome nge-PCR ama-thermal cycling systems athuthukisiwe ukuze ahlangabezane nezidingo zokuxilonga ukunakekelwa kwe-point-of-care [57, 58].I-on-chip PCR ingahlukaniswa ngezinhlobo ezine (ukugeleza okuvamile, okuqhubekayo, ukushintshwa kwendawo, kanye ne-PCR ye-convective) ngokuvumelana nezindlela ezahlukene zokulawula izinga lokushisa [59].Ngokwesibonelo, Gee et al.[60] uthuthukise indlela eqondile yokuhlehla yokuloba kwe-PCR (RT-qPCR) kuplathifomu yabo ye-microfluidic yokuthola i-multiplex ye-SARS-CoV-2, amagciwane omkhuhlane A no-B kumasampuli e-swab yomphimbo (Fig. 3a) .Park et al.[61] wakha i-chip elula yokuhlaziya i-pathogen ngokuhlanganisa i-PCR yefilimu emincane, ama-electrodes, kanye ne-polydimethylsiloxane-based microfluidic module esebenza ngomunwe.Kodwa-ke, yomibili imisebenzi ihlanganisa ukushiyeka okuvamile kwe-PCR evamile.I-PCR idinga ukuhamba ngebhayisikili okushisayo, okukhawulela ukwenziwa kancane kancane kwedivayisi kanye nokunciphisa isikhathi sokuhlola.
Ukuthuthukiswa kokugeleza okuqhubekayo okususelwa ku-microfluidic kanye ne-PCR eshintshwayo emkhathini kubalulekile ukuze kubhekwane nalolu daba.Isebenzisa ishaneli ende yenyoka noma ishaneli emfushane eqondile, ukugeleza okuqhubekayo kwe-PCR kunganikeza ukukhuliswa okusheshayo ngokujikeleza ama-reagents ngenkuthalo ezindaweni ezintathu zokushisa kuqala ngepompo ye-off-chip.Lokhu kusebenza kugwema ngempumelelo isigaba soshintsho phakathi kwamazinga okushisa ahlukene okusabela futhi ngaleyo ndlela kunciphisa kakhulu isikhathi sokuhlola [62] (Fig. 3b).Kolunye ucwaningo olwenziwe nguJung et al.[63] wenze isiphakamiso sokuhlaziywa kofuzo okusha kwe-rotary PCR ehlanganisa izici ze-PCR engaguquki negelezayo ye-ultrafast kanye ne-multiplex reverse transcription PCR (Fig. 3c).Ukuze uthole i-nucleic acid amplification, i-microchip ye-PCR izozungeziswa ngamabhulokhi amathathu okushisa emazingeni okushisa ahlukene: 1. I-Denaturation block 94°C, 2. I-Annealing block ku-58°C, 3. Ibhulokhi yokunweba ku-72°C.
Ukusetshenziswa kwe-PCR kuma-microfluidics.Ukumelwa okuhleliwe kwe-dirRT-qPCR kuplathifomu ye-microfluidic (ithathwe kokuthi [60]).b Ukumelwa okuhleliwe kokugeleza okuqhubekayo kwe-PCR microarray okusekelwe kusiteshi senyoka (kuthathwe kokuthi [62]).c Ukumelwa okuhleliwe kwe-rotary PCR genetic analyzer, ehlanganisa i-microchip, amabhlokhi amathathu okushisa kanye ne-stepper motor (ethathwe ku- [63]).d Umdwebo we-PCR ye-thermoconvection ene-centrifugation nokusetha (ithathwe ku- [64]).I-DirRT-qPCR, i-quantitative reverse transcription ye-polymerase chain reaction eqondile
Isebenzisa ama-capillary nama-loops noma amapuleti amancanyana, i-convection PCR ingakhulisa ngokushesha ama-nucleic acid nge-convection yemvelo yamahhala eshisayo ngaphandle kwesidingo sepompo yangaphandle.Isibonelo, iplathifomu ye-cyclic olefin polymer microfluidic yathuthukiswa esiteji sokushisa esijikelezayo esakhiwe esisebenzisa ibhayisikili elishisayo eline-centrifugation ku-PCR loop microchannel [64] (Fig. 3d).Isixazululo sokusabela siqhutshwa yi-convection eshisayo, eshintshana ngokuqhubekayo izinga lokushisa eliphezulu neliphansi ku-microchannel enesakhiwo se-annular.Yonke inqubo yokukhulisa ingaqedwa ngemizuzu eyi-10 ngomkhawulo wokutholwa ongu-70.5 pg/channel.
Njengoba kulindelekile, i-PCR esheshayo iyithuluzi elinamandla lokuhlanganiswa ngokugcwele kwesampula-impendulo yokuxilonga amasistimu okuhlaziya ama-multiplex.I-Rapid PCR inciphisa kakhulu isikhathi esidingekayo ukuze kutholwe i-SARS-CoV-2, enomthelela ekulawuleni ngempumelelo ubhubhane lwe-COVID-19.
I-PCR idinga i-thermal cycler eyinkimbinkimbi engafanele i-POCT.Muva nje, amasu okukhulisa i-isothermal asetshenziswe kuma-microfluidics, kufaka phakathi kodwa angagcini nje ku-LAMP, i-recombinase polymerase amplification (RPA), nokukhulisa ngokusekelwe ekulandeleni kwe-nucleic acid [65,66,67,68].Ngalawa masu, ama-nucleic acid akhuliswa ezingeni lokushisa elingaguquki, okwenza kube lula ukudalwa kwezindleko eziphansi, amadivaysi e-POCT aphathekayo azwela kakhulu okuxilongwa kwamangqamuzana.
Ukuhlolwa kwe-LAMP okusekelwe ku-microfluidics ephezulu kuvumela ukutholwa okuningi kwezifo ezithathelwanayo [42, 69, 70, 71].Ngokuhambisana nohlelo lwe-centrifugal microfluidic, i-LAMP ingaqhubeka nokwenza lula ukutholwa kwe-nucleic acid [69, 72, 73, 74, 75].I-slip-and-react SlipChip yenzelwe ukutholwa okubonakalayo kwamabhaktheriya amaningi afanayo kusetshenziswa i-LAMP [76] (Fig. 4a).Lapho usebenzisa i-LAMP eyenziwe kahle ekuhlolweni, isilinganiso sesignali somsindo we-fluorescence sasicishe sibe siphindwe ka-5, futhi umkhawulo wokutholwa ufinyelele kumakhophi angu-7.2/μl we-genomic DNA. Ngaphezu kwalokho, ukuba khona kwezifo ezinhlanu ezivamile zebhaktheriya zokugaya ukudla, okuhlanganisa i-Bacillus cereus, i-Escherichia coli, i-Salmonella enterica, i-Vibrio fluvialis kanye ne-Vibrio parahaemolyticus, yabonwa ngokusekelwe endleleni engu-< 60 min. Ngaphezu kwalokho, ukuba khona kwezifo ezinhlanu ezivamile zebhaktheriya zokugaya ukudla, okuhlanganisa i-Bacillus cereus, i-Escherichia coli, i-Salmonella enterica, i-Vibrio fluvialis kanye ne-Vibrio parahaemolyticus, yabonwa ngokusekelwe endleleni engu-< 60 min.Ngaphezu kwalokho, ukuba khona kwezifo ezinhlanu ezivamile zebhaktheriya zomgudu wokugaya ukudla, okuhlanganisa i-Bacillus cereus, i-Escherichia coli, i-Salmonella enterica, i-Vibrio fluvialis ne-Vibrio parahaemolyticus, kwabonakala kusetshenziswa le ndlela ngaphansi kwemizuzu engama-60.O, 基于基于此外, 基于基于 方法 方法 在 在 在 在 内 视化 视化 五 种 的 的 的 的细菌病, 包括包括, 大弧菌 菌 弧菌弧菌 弧菌 弧菌 弧菌 弧菌 弧菌 弧菌 HIPNgaphezu kwalokho, ukuba khona kwamagciwane amahlanu avamile e-bacterial gastrointestinal, okuhlanganisa i-Bacillus cereus, i-Escherichia coli, i-Salmonella enterica, i-Vibrio fluvius, ne-Vibrio parahaemolyticus, yabonwa ngeso lengqondo kusetshenziswa le ndlela ngaphansi kwemizuzu engama-60.
Izinzuzo ze-LAMP kuma-microfluidics zihlanganisa, phakathi kokunye, ukuphendula okusheshayo nokutholwa okuncane.Kodwa-ke, ngenxa yezinga lokushisa lokusabela (elingaba ngu-70°C), ama-aerosol akhiqizwa nakanjani phakathi ne-LAMP, okuholela esilinganisweni esiphezulu esingamanga.Ukucaciswa kwe-assay, ukwakheka kwe-primer, nokulawulwa kwezinga lokushisa nakho kudinga ukuthuthukiswa kwe-LAMP.Ngaphezu kwalokho, amadizayini ama-chip asebenzisa ukutholwa okuqondiwe okuningi ku-chip eyodwa anenani elikhulu futhi kufanele athuthukiswe.Ngaphezu kwalokho, i-LAMP ifanele ukutholwa kwezinhloso eziningi okuhlanganiswe ku-chip eyodwa, okubaluleke kakhulu, kodwa kusenendawo enkulu yokuthuthukiswa.
Izinga eliphezulu elingamanga le-LAMP lingancishiswa ngokwengxenye nge-RPA, njengoba izinga lokushisa eliphansi lokusabela (~37 °C) liphumela ezinkingeni ezimbalwa zokuhwamuka [77].Kuhlelo lwe-RPA, ama-primers amabili aphikisanayo aqala ukuhlanganiswa kwe-DNA ngokubophezela ku-recombinase futhi ukukhulisa kungaqedwa phakathi kwemizuzu eyi-10 [78,79,80,81].Ngakho-ke, yonke inqubo ye-RPA ishesha kakhulu kune-PCR noma i-LAMP.Eminyakeni yamuva, ubuchwepheshe be-microfluidic buye baboniswa ukuthuthukisa isivinini nokunemba kwe-RPA [82,83,84].Ngokwesibonelo, uLiu et al.[85] ithuthukise i-microfluidic edidiyelwe ye-lateral flow polymerase recombinase amplification assay ukuze kutholwe ngokushesha nokuzwelayo kwe-SARS-CoV-2 ngokuhlanganisa okulotshiweyo okuhlanekezelwe i-RPA (RT-RPA) kanye nesistimu yokutholwa komugqa wokuhlola ukugeleza kwendawo yonke.ohlelweni olulodwa lwe-microfluidic.Umfanekiso 4b).Umkhawulo wokutholwa yikhophi engu-1/µl noma ikhophi/isampula engu-30, futhi ukutholwa kungaqedwa cishe emizuzwini engama-30.Kong et al.benze idivayisi ye-microfluidic egqokekayo.[86] wasebenzisa izinga lokushisa lomzimba kanye nesistimu yokuthola i-fluorescence esekelwe kumakhalekhukhwini ukuthola ngokushesha nangokuqondile i-HIV-1 DNA isebenzisa i-RPA (Umfanekiso 4c).Ukuhlolwa kwe-RPA okugqokekayo kuthola amakhophi angu-100/mL okulandelana okuqondiwe phakathi nemizuzu engu-24, okubonisa amandla amakhulu okuxilonga ngokushesha kwezinsana ezine-HIV-1 kuzilungiselelo ezinomkhawulo wezinsiza.
I-Isothermal amplification in point-of-care testing (POCT).Ukuthuthukiswa kanye nokukhiqizwa kwe-spin ne-reaction SlipChip.Ngemuva kokushisela i-plasma, ama-chips aphezulu naphansi ahlanganiswa nesethi yamantongomane ukuze enze i-chip yokugcina (ethathwe ku- [76]).b I-Schematic yesistimu ye-MI-IF-RPA yokutholwa kwe-COVID-19 (ithathwe kokuthi [85]).c Isikimu sokuhlolwa kwe-RPA okugqokekayo ukuze kutholwe ngokushesha i-HIV-1 DNA (ethathwe kokuthi [86]).I-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-flubite-IFD Recombinase Emitting Diode ye-LED, i-Microflubite-IFD Recombite-IF-I-IF-I-IF Emincane Ukukhulisa
I-RPA esekelwe ku-Microfluidic ithuthuka ngokushesha, nokho, izindleko zokwenziwa kwe-chip kanye nokusetshenziswa kokusabela ziphezulu kakhulu futhi kufanele zehliswe ukuze kwandiswe ukutholakala kwalobu buchwepheshe.Ngaphezu kwalokho, ukuzwela okuphezulu kwe-RPA kungathinta ukukhuliswa kwemikhiqizo engaqondile, ikakhulukazi lapho kukhona ukungcola.Le mikhawulo ingase ibe nomthelela ekusetshenzisweni kwe-RPA ezinhlelweni ezincane ze-microfluidic futhi ifanele ukwenziwa ngcono okwengeziwe.Iziqalo eziklanywe kahle kanye nama-probe okuhlosiwe okuhlukahlukene nazo ziyadingeka ukuze kuthuthukiswe ukuba nokwenzeka kwamasu ama-microfluidic asekelwe ku-RPA ku-POCT.
I-Cas13 ne-Cas12a inekhono lokuhlukanisa ngokungahleliwe ama-nucleic acid futhi ngaleyo ndlela ingathuthukiswa njengamathuluzi okuthola nokuxilonga.I-Cas13 ne-Cas12a zenziwe zisebenze lapho zibophezela ukuze ziqondise i-DNA noma i-RNA, ngokulandelana.Uma isicushiwe, iphrotheni iqala ukuhlukanisa amanye ama-nucleic acid aseduze, okuthi ngemva kwalokho umhlahlandlela we-RNAs oqondise ku-pathogen-specific nucleic acids akwazi ukuhlukanisa ama-fluorescent probe acinyiwe futhi akhulule i-fluorescence.Ngokusekelwe kulo mbono, uKellner et al.[87] ithuthukise indlela esekelwe ku-Cas13 [Specific High-sensitivity Enzymatic Reporter UnLOCKING (SHERLOCK)], kanye no-Broughton et al.[88] wenze enye indlela esekelwe ku-Cas12a [I-CRISPR Trans Reporter eqondise ku-DNA endonuclease (DTECR)].
Eminyakeni yamuva nje, kuvele izindlela ezahlukahlukene zokutholwa kwama-nucleic acid asuselwa ku-CRISPR [89, 90].Izindlela ezijwayelekile ezisekelwe ku-CRISPR zivame ukuchitha isikhathi futhi zisebenza kanzima ngenxa yezinqubo eziningi ezihlanganisa ukukhishwa kwe-nucleic acid, ukukhulisa nokutholwa kwe-CRISPR.Ukuchayeka koketshezi emoyeni kungase kwenyuse amathuba okuba nemiphumela engelona iqiniso.Ngokunikezwa okungenhla, amasistimu asekelwe ku-CRISPR adinga kakhulu ukwenziwa kahle.
Iplathifomu ye-microfluidic elawulwa umoya womoya engenza ukuhlaziya okungu-24 ngokuhambisana yenzelwe izinhlelo zokusebenza zokuthola i-CRISPR-Cas12a ne-CRISPR-Cas13a [91].Uhlelo lufakwe idivayisi yokuthola i-fluorescence edlula i-nucleic acid amplification futhi ithole ngokuzenzakalelayo amasampula e-femtomolar DNA kanye ne-RNA.U-Chen et al.[92] ukukhulisa kabusha okuhlanganisiwe kwe-recombinase ngohlelo lwe-CRISPR-Cas12a ku-centrifugal microfluidics (Fig. 5a).Lo msebenzi unqoba ubunzima bokuhlanganisa lezi zinqubo ezimbili ngoba i-Cas12a ingakwazi ukugaya i-DNA yesithunywa futhi ivimbele inqubo yokukhulisa.Ngaphezu kwalokho, u-Chen et al.[92] ngaphezu kwalokho igcine ngaphambilini ama-reagents okusabela kusilawuli se-centrifugal microfluidic ukuqedela ngokuzenzakalelayo yonke inqubo.Komunye umsebenzi, uSilva et al.[93] uthuthukise indlela yokuxilonga ngaphandle kokukhulisa i-CRISPR/Cas12a kanye ne-smartphone ukuze ithole i-SARS-CoV-2 (Fig. 5b).Lokhu kuhlolwa, okwaziwa ngokuthi uhlelo lwamahhala lokukhulisa iselula olusekelwe kumakhalekhukhwini, luhlanganisa i-enzyme encike ku-CRISPR/Cas esekelwe ekuboneni ngeso kwe-smartphone kwamasignali ebhamuza akhiqizwe yi-catalase eziteshini ezincane ze-microfluidic.Ukutholwa okuzwelayo kwamakhophi angaphansi kwama-50/µl we-nucleic acid ngaphandle kokukhulisa kusengaphambili, yonke inqubo ukusuka kumjovo wesampula ukuya ekufundweni kwesignali kuthatha imizuzu engama-71 kuphela.
Izindlela zokuthola i-Nucleic acid ezisekelwe ku-CRISPR.I-Centrifugal POCT yokuxilonga okudidiyelwe kwamangqamuzana okusekelwe ku-CRISPR (kuthathwe kokuthi [92]).b Ukuthuthukiswa kokuhlolwa kwe-CASCADE kokuhlaziywa okusekelwe kumakhalekhukhwini we-SARS-CoV-2 (kuthathwe kokuthi [93]).I-RAA recombinase amplification, i-PAM eseduze ne-protospacer motif, i-CRISPR ehlanganisa ukuphindaphinda okufushane kwe-palindromic ngezikhathi ezithile, uhlelo lwe-CASCADE ngaphandle kokukhulisa umakhalekhukhwini ngama-enzyme ancike ku-CRISPR/CAS, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride EDC
Njengesinyathelo sokugcina ekutholweni kwe-nucleic acid, ukutholwa kwesignali kubonisa ngokuqondile imiphumela yokuxilonga futhi kuyisici esibalulekile ekuthuthukisweni kwe-POCT ephumelelayo, ebucayi, nenembile.Izimpawu zingafundwa kusetshenziswa izindlela ezahlukahlukene ezifana namasu we-fluorescent, electrochemical, colorimetric kanye namagnetic.Kulesi sigaba, sichaza isizathu sendlela ngayinye futhi siqhathanise ukuxilonga kwamangqamuzana ezifo ezithathelwanayo kuma-microfluidics.
Amasu asekelwe ku-Fluorescence asetshenziswa kabanzi ekuxilongweni kwe-POCT kwezifo ezithathelwanayo ngenxa yezinzuzo zabo eziphawulekayo zokuzwela okuhle kakhulu, izindleko eziphansi, ukusebenza kalula, nokuhlaziywa kwendawo yokunakekelwa [94, 95].Lawa maqhinga asebenzisa ama-fluorophore anelebuli afana nodayi be-fluorescent nama-nanomaterials ukuze akhe isignali etholakalayo (ukuthuthukiswa kwe-fluorescence noma ukucisha).Lokhu kutholakala kuphakamisa ukuthi amasu asekelwe ku-fluorescence angahlukaniswa abe ilebula eqondile ye-fluorescent, i-signal-on, kanye nokutholwa kwe-fluorescent yokuvala isignali [96].Ukutholwa kwelebula ye-fluorescent eqondile kusebenzisa amalebula e-fluorescent akhethekile ukulebula ama-ligand athile akhiqiza inani elithile le-fluorescence lapho eboshelwe kuthagethi ngokukhetha.Ngokutholwa kwe-fluorescence esekelwe kusignali, ikhwalithi yesiginali ye-fluorescent ihlobene kahle nobukhulu bentshisekelo.Amandla e-Fluorescence awanakwa uma kungekho okuqondiwe futhi kuyabonakala uma inani elanele lethagethi likhona.Ngokuphambene, ukushuba kwe-fluorescence okutholwe yi-fluorescence "i-signal-off" ilingana ngokuphambene nenani lethagethi, iqala ifinyelela inani eliphakeme futhi iyehla kancane kancane njengoba ithagethi ikhuliswa.Isibonelo, kusetshenziswa indlela ye-CRISPR-Cas13a encike kuthagethi ye-trans-cleavage, u-Tian et al.[97] uthuthukise isu lokuqaphela inoveli ukuthola ama-RNA adlula ukuloba okuphambene ngokuqondile (Fig. 6a).Lapho kubophezeleka kuma-RNA ahambisanayo okuhlosiwe, inkimbinkimbi ye-CRISPR-Cas13-RNA ingenziwa isebenze, icuphe ukuqhekeka kwe-transcollateral yi-RNA yentatheli engaqondile.Intatheli enelebuli enokukhanya okukhanyayo [i-fluorophore (F)] icinywa isicishi (Q) singaguquki kanye nama-fluoresces lapho iklezwa inkimbinkimbi ecushiwe.
Inzuzo yokutholwa kwe-electrochemical isivinini sokutholwa okuphezulu, ukukhiqiza okulula, izindleko eziphansi, ukuthwala okulula nokulawula okuzenzakalelayo.Kuyindlela yokuhlaziya enamandla yezinhlelo zokusebenza ze-POCT.Ngokusekelwe ku-graphene field-effect transistors Gao et al.[98] ithuthukise i-nanobiosensor yokuthola i-multiplex ye-antigen yesifo se-Lyme kusuka kubhaktheriya ye-Borrelia burgdorferi enomkhawulo wokuthola we-2 pg/mL (Fig. 6b).
Ukuhlolwa kombala kusetshenziswe ezinhlelweni ze-POCT, kuhlomula ezinhlelweni zokuphatheka, izindleko eziphansi, ukulungiselela kalula, nokufunda okubukwayo.Ukutholwa kombala kungasebenzisa i-oxidation ye-peroxidase noma i-peroxidase-like nanomaterials, ukuhlanganiswa kwama-nanomaterials, kanye nokwengezwa kodayi abayinkomba ukuze kuguqulwe ulwazi mayelana nokuba khona kwama-nucleic acid ahlosiwe enze izinguquko zombala ezibonakalayo [99, 100, 101].Ngokuphawulekayo, ama-nanoparticles egolide asetshenziswa kabanzi ekuthuthukisweni kwamasu e-colorimetric, futhi ngenxa yekhono lawo lokushintsha umbala osheshayo nobalulekile, kunesithakazelo esikhulayo ekuthuthukisweni kwamapulatifomu e-POCT colorimetric for in situ ukuxilongwa kwezifo ezithathelwanayo [102].Ngedivayisi ehlanganisiwe ye-centrifugal microfluidic [103], amagciwane atholakala ekudleni kumasampula obisi angcolile angatholakala ngokuzenzakalelayo ezingeni lamangqamuzana e-bacterial 10, futhi imiphumela ingafundwa ngokubukeka phakathi kwemizuzu ye-65 (Fig. 6c).
Izindlela zokuzwa kazibuthe zingathola ngokunembile izihlaziyi zisebenzisa izinto zikazibuthe, futhi kube nentshisekelo enkulu ezinhlelweni ze-POCT emashumini eminyaka akamuva.Izindlela zokuzwa kazibuthe zinezinzuzo ezithile ezihlukile njengezisetshenziswa zikazibuthe ezibiza kancane kunezingxenye ezibizayo ze-optical.Kodwa-ke, ukusetshenziswa kwendawo kazibuthe kuthuthukisa ukusebenza kahle kokutholwa futhi kunciphisa isikhathi sokulungiselela isampula [104].Ukwengeza, imiphumela yokuhlola kazibuthe ibonisa ukucaciswa okuphezulu, ukuzwela, kanye nesilinganiso esiphezulu sesignali-kuya-nomsindo ngenxa yesignali engabalulekile yangemuva kazibuthe yamasampuli ebhayoloji [105].Sharma et al.ihlanganise umhubhe kazibuthe osuselwe ku-biosensor endaweni yesikhulumi se-microchip ephathekayo.[106] ukuthola i-multiplex yamagciwane (Fig. 6d).Ama-Biosensors athola ngokuzwela ama-nucleic acid e-subnanomolar ahlukaniswe namagciwane.
Indlela ejwayelekile yokuthola isignali.Umqondo wokutholwa kwe-hyperlocalized ye-Cas13a (ithathwe ku- [97]).b I-Graphene nanobiosensor FET ihlanganiswe ne-Lyme GroES scFv (ithathwe kokuthi [98]).c Izinkomba ze-Colorimetric zokutholwa kwe-multiplex amagciwane atholakala ekudleni ku-centrifugal microfluidic chip: amasampula No. 1 kanye No. 3 anamagciwane aqondiwe, kanye namasampuli No. 2, No. .d I-Biosensor esekelwe ekuhlanganeni komhubhe kazibuthe, okuhlanganisa iplathifomu, isikhulisi esivimbayo esakhelwe ngaphakathi, iyunithi yokulawula, kanye nokunikezwa kwamandla okukhiqiza/ukuthola isignali (kuthathwe kokuthi [106]).I-GFET Graphene FET, Escherichia coli, Escherichia coli, Salmonella typhimurium, Vibrio parahaemolyticus, Vibrio parahaemolyticus, Listeria monocytogenes, PC PC, PDMS Dimethicone, PMMA polymethyl methacrylate
Naphezu kwezici ezinhle kakhulu zezindlela zokuhlonza ezingenhla, zisenobubi.Lezi zindlela ziqhathaniswa (ithebula 1), kufaka phakathi ezinye izinhlelo zokusebenza ezinemininingwane (okuhle nokubi).
Ngokuthuthukiswa kwe-microfluidics, izinhlelo ze-microelectromechanical, i-nanotechnology kanye nesayensi yezinto zokwakha, ukusetshenziswa kwe-microfluidic chips ukuze kutholakale izifo ezithathelwanayo kuqhubekela phambili [55,96,107,108].Ukukhohliswa okunembile kwemishini emincane noketshezi kunomthelela ekunembeni kokuxilonga kanye nokusebenza kahle kwezindleko.Ngakho-ke, ukuze kuthuthukiswe okwengeziwe, kuye kwenziwa imizamo yokuthuthukisa nokuthuthukisa ama-chips, okuholela kuma-microfluidic chips ahlukahlukene anezakhiwo nemisebenzi ehlukene.Lapha sethula kafushane izinhlobo ezimbalwa ezijwayelekile zamapulatifomu e-microfluidic futhi siqhathanise izici zawo (okuhle nokubi).Ngaphezu kwalokho, izibonelo eziningi ezibalwe ngezansi zigxile kakhulu ekulweni ne-SARS-CoV-2.
Ama-LOCC ayizinhlelo zokuhlaziya eziyinkimbinkimbi ezivame kakhulu ezincane futhi ukusebenza kwazo kwenziwa nge-miniaturized kakhulu, kuhlanganiswe, ku-automated futhi kufaniswe kusuka kumjovo wesampula nokulungiswa, ukulawulwa kokugeleza nokutholwa koketshezi [109, 110].Uketshezi lusetshenziswa ngejiyomethri eklanywe ngokucophelela kanye nokusebenzisana kwemiphumela eminingi yomzimba efana ne-pressure gradients, isenzo se-capillary, i-electrodynamics, izinkundla kazibuthe namagagasi e-acoustic [111].I-LOCC ibonisa izinzuzo ezinhle kakhulu ekuhlolweni kokusebenza okuphezulu nokutholwa okuningi, ngesivinini sokuhlaziya okusheshayo, usayizi omncane wesampula, ukusetshenziswa kwamandla okuphansi, nokuphatha okuphezulu nokusebenza kahle;kodwa-ke, izisetshenziswa ze-LOCC zintekenteke kakhulu, futhi ziyakhiqiza, ziyapakishwa, futhi ziyasebenzisana.Kodwa-ke, ukuphindaphinda nokusebenzisa kabusha kubhekana nobunzima obukhulu [96].Uma kuqhathaniswa nezinye izinkundla, i-LOCC inezinzuzo eziyingqayizivele mayelana nokwehlukahlukana okukhulu kwezicelo kanye nokuhambisana okungcono kakhulu kobuchwepheshe, kodwa ukonakala kwayo nakho kusobala, okungukuthi ubunkimbinkimbi obuphezulu nokuphindaphindeka okubi.Ukuncika kumaphampu angaphandle, avame ukuba yinqwaba futhi abize, kuphinde kukhawulele ukusetshenziswa kwawo ku-POCT.
Ngesikhathi sokuqubuka kwe-COVID-19, i-LOCC yathola ukunakwa okukhulu.Ngesikhathi esifanayo, kukhona ama-chips amasha amaningana ahlanganisa ubuchwepheshe obuningana.Isibonelo, ama-smartphones manje asesetshenziswa kabanzi njengamadivayisi okuhlaziya aphathwayo futhi anamandla amakhulu okuhlanganiswa kwe-LOCC.Ilanga et al.[21] wenze i-microfluidic chip evumela ukulandelana kwe-nucleic acid ethize ye-multiplexing yamagciwane amahlanu, okuhlanganisa i-SARS-CoV-2, esebenzisa i-LAMP futhi yawahlaziya kusetshenziswa i-smartphone phakathi nehora elingu-1 ngemva kokuphela kokusabela.Njengesinye isibonelo, uSundah et al.[112] udale ukushintshwa kwamangqamuzana [i-catalytic amplification by molecular transition state switch (CATCH)] ukuze kutholwe ngokuqondile nokuzwelayo kwezinhloso ze-SARS-CoV-2 RNA kusetshenziswa ama-smartphone. I-CATCH ihambisana ne-LOCC ephathekayo futhi ifinyelela ukusebenza okuphezulu (cishe amakhophi angu-8 e-RNA/μl; < 1 h ekamelweni lokushisa) [112]. I-CATCH ihambisana ne-LOCC ephathekayo futhi ifinyelela ukusebenza okuphezulu (cishe amakhophi angu-8 e-RNA/μl; < 1 h ekamelweni lokushisa) [112]. CATCH совместим с портативным LOCC и обеспечивает превосходную производительность (примерно 8 копий РНК/мкл; < 1 ч при компечи112]. I-CATCH ihambisana ne-LOCC ephathekayo futhi inikeza umphumela omuhle kakhulu (cishe amakhophi angu-8 e-RNA/µl; < 1 h ekamelweni lokushisa) [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]. I-CATCH совместим с портативными LOCC futhi обладает превосходной производительностью (примерно 8 копий РНК/мкл; < 1 часа при комнат11 тем].) I-CATCH ihambisana nama-LOCC aphathekayo futhi isebenza kahle kakhulu (cishe amakhophi angu-8 e-RNA/µl; < ihora elingu-1 ekamelweni lokushisa) [112].Ngaphezu kwalokho, amadivayisi e-LOCC okuxilongwa kwamangqamuzana aphinde asebenzise amandla athile okushayela afana ne-vacuum, ukunwebeka, nezinkambu zikagesi.Kang et al.[113] ibonise i-PCR yesikhathi sangempela, eshesha kakhulu ye-nanoplasma-on-a-chip yokuxilonga ngokushesha nangokwenani kwe-COVID-19 emkhakheni kusetshenziswa i-vacuum plasmonic liquid chip ye-PCR.Li et al.[114] wabe esethuthukisa i-microfluidic chip edonsa kanzima eyenza ukuthi kutholakale i-COVID-19.Inkundla isebenzisa isistimu yokukhulisa i-RT-LAMP ukuze inqume ukuthi isampula lilungile yini noma libi.Kamuva, uRamachandran et al.[I-115] izuze ama-gradients enkundla kagesi afanelekile kusetshenziswa i-isotachophoresis (ITP), indlela yokugxila ye-ion ekhethiwe esetshenziswe kuma-microfluidics.Nge-ITP, i-RNA ehlosiwe evela kumasampula e-nasopharyngeal swab angahlanzwa ngokuzenzakalelayo.Khona-ke uRamachandran et al.[115] Ukuhlanganisa lokhu kuhlanzwa kwe-ITP ne-LAMP ethuthukisiwe ye-ITP kanye nezivivinyo ze-CRISPR kutholwe i-SARS-CoV-2 ku-swab yomuntu ye-nasopharyngeal kanye nezibonelo zomtholampilo cishe imizuzu engama-35.Ngaphezu kwalokho, imibono emisha ihlale ivela.Jadhav et al.[116] wenze isiphakamiso sohlelo lokuxilonga olusekelwe ku-Raman spectroscopy ethuthukisiwe ngokuhlanganiswe nedivayisi encane equkethe ama-nanotubes egolide/ahlanganiswe ngesiliva abheke phezulu noma ama-electrospun micro/nanotubes alahlwayo.Iziteshi ezincane zesihlungi esakhelwe ngaphakathi esenziwe nge-membrane ziyalahlwa.Idivayisi ikhangisa amagciwane avela oketshezini oluhlukahlukene lomzimba/ukukhishwa njengamathe, i-nasopharynx nezinyembezi.Ngakho-ke, i-virus titer iyiningi futhi igciwane lingabonakala ngokunembile ngesiginesha ye-Raman.
I-LOAD iyinkundla ye-microfluidic eyi-centrifugal lapho zonke izinqubo zilawulwa yiphrothokholi yefrikhwensi ezungezisa i-microstructured substrate [110].Idivayisi ye-LOAD ibonakala ngokusebenzisa amandla e-centrifugal njengamandla okushayela abalulekile.Uketshezi luphinde lube ngaphansi kwamandla e-capillary, Euler kanye namaCoriolis.Kusetshenziswa idivayisi ye-centrifuge, ukuhlaziya kwenziwa ekusebenzeni koketshezi okuqhubekayo ukusuka endaweni eyi-radial ukuya endaweni yangaphandle, okuqeda isidingo samashubhu angaphandle angeziwe, amaphampu, ama-actuator, nama-valve asebenzayo.Ngamafuphi, indlela eyodwa yokulawula yenza umsebenzi ube lula.Amandla asebenza oketshezini esiteshini esifanayo se-microfluidic ebangeni elifanayo ukusuka esikhungweni somthwalo ayalingana, okwenza kube nokwenzeka ukuphinda isakhiwo sesiteshi.Ngakho-ke, imishini ye-LOAD ilula futhi iyonga kakhulu ukuklama nokukhiqiza kunemishini evamile ye-LOCC, kuyilapho ukusabela kuzimele futhi kuhambisana;kodwa-ke, ngenxa yamandla aphezulu emishini yemishini ye-centrifugal, impahla ye-chip etholakalayo ilinganiselwe futhi amavolumu amancane anzima.emotweni.Ngesikhathi esifanayo, amadivayisi amaningi we-LOAD aklanyelwe ukusetshenziswa okukodwa kuphela, okubiza kakhulu ukutholwa kwenani elikhulu [96, 117, 118, 119].
Emashumini eminyaka amuva nje, i-LOAD, ebhekwa njengenye yemishini ye-microfluidic ethembisa kakhulu, ithole ukunakwa okukhulu kubacwaningi nabakhiqizi.Ngakho-ke, i-LOAD isithole ukwamukelwa okubanzi futhi isetshenziselwe ukuxilonga amangqamuzana amagciwane athathelwanayo [120, 121, 122, 123, 124], ikakhulukazi ngesikhathi sokugqashuka kwe-COVID-19.Isibonelo, ekupheleni kuka-2020, uJi et al.[60] ibonise ukuhlolwa kwe-RT-qPCR okuqondile kokutholwa okuhambisanayo okusheshayo nokuzenzakalelayo kwe-SARS-CoV-2 kanye nokutheleleka komkhuhlane A no-B kumasampula e-swab yomphimbo.Bese u-Xiong et al.[74] wethule iplathifomu ehlanganisiwe ye-discoid microfluidic ehlanganisiwe ye-LAMP ukuze kutholakale ngokushesha, okunembile, futhi ngesikhathi esisodwa ama-coronavirus okuphefumula abantu ayisikhombisa, okuhlanganisa i-SARS-CoV-2, phakathi nemizuzu engama-40.Ekuqaleni kuka-2021, u-de Oliveira et al.[73] ibonise i-polystyrene toner centrifugal microfluidic chip, esetshenziswa mathupha nge-rotator yomunwe, ukuze kuxilongwe amangqamuzana e-RT-LAMP e-COVID-19.Ngokulandelayo, uDignan et al.[39] wethule i-microdevice ephathekayo ephathekayo ye-centrifuge yokuhlanza i-SARS-CoV-2 RNA ngokuqondile ezigabeni ze-buccal swab.UMedved et al.[53] wenze isiphakamiso sohlelo lokusampula lwe-aerosol olusemgqeni lwe-SARS-CoV-2 olunevolumu encane ezungezayo ye-microfluidic fluorescent chip enomkhawulo wokutholwa wamakhophi ayi-10/μL kanye nomkhawulo omncane womjikelezo wemizuzu eyi-15.Suarez et al.[75] muva nje kubika ukuthuthukiswa kwesikhulumi se-modular centrifugal microfluidic esididiyelwe sokutholwa okuqondile kwe-SARS-CoV-2 RNA kumasampula e-nasopharyngeal swab angasebenzi kusetshenziswa i-LAMP.Lezi zibonelo zibonisa izinzuzo ezinkulu nesithembiso se-LOAD ekuxilongweni kwamangqamuzana e-COVID-19.
Ngo-1945 u-Muller noClegg [125] baqale bethula amashaneli amancane ephepheni besebenzisa isihlungi sephepha nopharafini.Ngo-2007, iqembu le-Whitesides [126] lakha isiteji sokuqala sephepha esisebenzayo sokuhlola amaprotheni noshukela.Iphepha seliyi-substrate ekahle yama-microfluidics.Iphepha linezinto ezingokwemvelo ezifana ne-hydrophilicity kanye nesakhiwo se-porous, i-biocompatibility enhle kakhulu, isisindo esilula, ukuguquguquka, ukugoqa, izindleko eziphansi, ukusetshenziswa kalula nokulula.Ama-µPAD akudala aqukethe izakhiwo ze-hydrophilic/hydrophobic ezakhelwe phezu kwama-substrates ephepha.Ngokuya ngesakhiwo esinezinhlangothi ezintathu, ama-μPAD angahlukaniswa abe ama-dimensional amabili (2D) kanye nama-3-dimensional (3D) μPAD.Ama-2D µPAD akhiqizwa ngokwakha imingcele ye-hydrophobic ukuze akhe amashaneli amancane, kuyilapho i-3D µPADs ngokuvamile enziwa ngezitaki zezendlalelo zephepha le-2D microfluidic, ngezinye izikhathi ngokugoqa iphepha, izindlela zokushelela, amashaneli avulekile, nokuphrinta kwe-3D [96].Uketshezi olunamanzi noma lwebhayoloji ku-μPAD lulawulwa ngokuyinhloko ngamandla e-capillary ngaphandle komthombo wamandla wangaphandle, okusiza ukugcinwa kwangaphambili kwama-reagents, ukuphatha isampula, nokutholwa kwe-multiplex.Kodwa-ke, ukulawula ukugeleza okunembile kanye nokutholwa kwe-multiplex kuthikazwa isivinini esinganele sokuthola, ukuzwela, nokusebenziseka kabusha [96, 127, 128, 129, 130].
Njengeplatifomu ye-microfluidic engavamile, i-μPAD iye yakhuthazwa kabanzi futhi yathuthukiswa ukuze kuhlolwe amangqamuzana ezifo ezithathelwanayo ezifana ne-HCV, i-HIV, ne-SARS-CoV-2 [131, 132].Ngokutholwa okukhethiwe nokuzwelayo kwe-HCV, uTengam et al.[133] ithuthukise inoveli biosensor esekelwe ephepheni le-fluorescent isebenzisa i-nucleic acid probe ecaciswe kakhulu esekelwe ku-pyrrolidinyl peptide.Ama-Nucleic acid awanyakaziswa ngokuhlanganyela ephepheni le-cellulose eline-oxidized ngokunciphisa i-alkylation phakathi kwamaqembu e-amino namaqembu e-aldehyde, futhi ukutholwa kusekelwe ku-fluorescence.Lezi zimpawu zingafundwa igajethi eyenziwe ngokukhethekile enekhamera ephathekayo ye-fluorescent ehlanganiswe nekhamera yeselula.Kamuva, uLu et al.[134] uklame i-electrode eguquguqukayo esekelwe ephepheni esekelwe ku-nickel/igolide nanoparticles/carbon nanotubes/polyvinyl alcohol organometallic framework composite ukuze kutholwe i-HIV target by hybridization kusetshenziswa i-methylene blue njengesibonisi se-DNA redox.Muva nje, uChowdury et al.[135] wethule umklamo weplathifomu ecatshangelwayo yokuhlolwa kwendawo yokunakekelwa kwe-µPAD kusetshenziswa amathe esiguli aluhlaza kuhlanganiswe ne-LAMP kanye nobuchwepheshe bezithombe obuphathekayo bokutholwa kohlaziyo lwe-COVID-19.
Ukuhlolwa kokugeleza kwe-lateral kuqondisa uketshezi ngamandla e-capillary futhi kulawula ukunyakaza koketshezi ngokubamanzi nezici zama-substrates anezimbotshana noma anesakhiwo esincane.Imishini yokugeleza yangemuva ihlanganisa isampula, i-conjugate, incubator kanye nokutholwa, namaphedi amuncayo.Ama-nucleic acid ku-LFA abona ama-binders athile agcinwe ngaphambili endaweni ebophayo futhi abophe njengama-complexes.Njengoba uketshezi ludlula ku-incubation kanye namapuleti okubona, izinkimbinkimbi zithathwa ama-molecule abamba atholakala emigqeni yokuhlola nokulawula, ebonisa imiphumela engafundwa ngqo ngeso lenyama.Ngokuvamile, i-LFA ingaqedwa emizuzwini engu-2-15, eshesha kunokutholwa kwendabuko.Ngenxa yomshini okhethekile, i-LFA idinga ukusebenza okumbalwa futhi ayidingi imishini eyengeziwe, okuyenza isebenziseke kakhulu.Kulula ukwenza kanye nokwenza okuncane, futhi izindleko zama-substrates asekelwe ephepheni ziphansi.Kodwa-ke, isetshenziselwa kuphela ukuhlaziywa kwekhwalithi, futhi ukutholwa komthamo kunzima kakhulu, futhi ikhono lokuphindaphinda nokuphuma kulinganiselwe kakhulu, futhi i-nucleic acid eyodwa kuphela eyanele ingatholwa ngesikhathi [96,110,127].
Nakuba izinhlelo eziningi ze-LFA zigxile ekuhloleni ama-immunoassays, ukusetshenziswa kwe-LFA kokuxilongwa kwamangqamuzana kuma-microfluidic chips nakho kuyasebenza futhi kuyathandwa [136].Endabeni yegciwane le-hepatitis B, i-HIV kanye ne-SARS-CoV-2 LFA Gong et al.[137] wenze isiphakamiso seplathifomu ye-nanoparticle LFA eguqukayo ephezulu futhi yabonisa ukuguquguquka kwalesi sigcawu esincane nesiphathekayo ngokutholwa okubucayi nokulinganisa kokuhlosiwe okuningi njenge-HBV nucleic acid.Ngaphezu kwalokho, uFu et al.[138] ibonise inoveli ye-LFA esekelwe ku-spectroscopy ye-Raman ethuthukisiwe yokuhlaziywa kobuningi be-HIV-1 DNA ekugxilweni okuphansi.Ukutholwa okusheshayo nokuzwelayo kwe-SARS-CoV-2, uLiu et al.[85] ithuthukise ukuhlaziywa kwe-microfluidic-integrated RPA lateral flow ngokuhlanganisa i-RT-RPA kanye nesistimu yokutholwa kokugeleza kwe-universal lateral ibe uhlelo olulodwa lwe-microfluidic.
Ukusetshenziswa kwamapulatifomu e-microfluidic ahlukahlukene kuyehluka kuye ngezifundo ezithile, zisebenzisa ngokugcwele amakhono kanye nezinzuzo zamapulatifomu.Ngamavalve, amaphampu namapayipi athengekayo, i-LOCC iyinkundla ebanzi kakhulu yokuhlukahluka kwezicelo nokusebenzisana negumbi elikhulu kakhulu lokuthuthukiswa.Ngakho-ke, sithemba futhi sincoma ukuthi izifundo ezintsha kakhulu zenziwe e-LOCC njengomzamo wokuqala nokuthi izimo zithuthukiswe.Ukwengeza, izindlela ezisebenza kahle nezinembayo kulindeleke ukuthi zitholakale futhi zisetshenziswe ohlelweni.I-LOAD idlula kahle kakhulu ekulawuleni okunembile koketshezi oluvela kumadivayisi akhona e-LOCC futhi ibonisa izinzuzo eziyingqayizivele kumadrayivu awodwa ngamandla aphakathi nendawo ngaphandle kwesidingo samadrayivu angaphandle, kuyilapho izimpendulo ezifanayo zingahlukaniswa futhi zivumelaniswe.Ngakho-ke, esikhathini esizayo, i-LOAD izoba inkundla ye-microfluidic eyinhloko enemisebenzi encane eyenziwa ngesandla kanye nobuchwepheshe obuvuthiwe nobuzenzakalelayo.Inkundla ye-µPAD ihlanganisa izinzuzo ze-LOCC nezinto ezisekelwe ephepheni ngezindleko eziphansi, ukuxilonga ukusetshenziswa okukodwa.Ngakho-ke, ukuthuthukiswa kwesikhathi esizayo kufanele kugxile kubuchwepheshe obulula nobuzinze kahle.Ngaphezu kwalokho, i-LFA ifaneleka kahle ukutholwa kwamehlo enqunu, ithembisa ukunciphisa ukusetshenziswa kwesampula nokusheshisa ukutholwa.Ukuqhathaniswa kwenkundla okunemininingwane kukhonjisiwe kuThebula 2.
Ukuhlaziywa kwedijithali kuhlukanisa isampula kuma-microreactors amaningi, ngayinye equkethe inani elicacile lama-molecule aqondiwe [139, 140].Ukuhlolwa kwedijithali kunikeza izinzuzo ezibalulekile zokwenza ubuningi obuphelele ngokwenza izinkulungwane zokuhlola kwamakhemikhali e-biochemical ngesikhathi esisodwa futhi ngakunye kumakhompathimenti esikali se-micron esikhundleni sesigaba esiqhubekayo.Uma kuqhathaniswa nama-microfluidis endabuko, ukusabela kwegumbi kunganciphisa umthamo wesampula, kwandise ukusebenza kahle kokusabela, futhi kuhlanganiswe kalula nezinye izindlela zokuhlaziya ngaphandle kwesidingo samashaneli, amaphampu, ama-valve, kanye nemiklamo ehlangene [141, 142, 143, 144, 145, 146, 147] .Izindlela ezimbili ezilandelayo zisetshenziswa ekuhlolweni kwedijithali ukuze kuzuzwe ukuhlukaniswa okufanayo nokunembile kwezixazululo, okuhlanganisa ama-reagents namasampula afana namaseli, ama-nucleic acid, nezinye izinhlayiya noma ama-molecule: (1) i-emulsion yehla exhaphaza ukungazinzi kwe-liquid interface;(2) ukuhlukaniswa kwe-array kwenziwa yizingqinamba zejometri zedivayisi.Ngendlela yokuqala, amaconsi aqukethe ama-reagents namasampuli kuma-microchannels angadalwa ngezindlela ezingenzi lutho ezifana ne-co-current, crossflow, flow focusing, emulsification esiteji, i-microchannel emulsification, kanye ne-membrane ngokusebenzisa amandla e-viscous shear kanye ne-emulsification ngokushintsha kwesiteshi.ukwakhiwa kwasendaweni [143, 145, 146, 148, 149] noma ukusebenzisa izindlela ezisebenzayo [150, 151], ezethula amandla engeziwe ngokulawula kagesi, kazibuthe, ezishisayo kanye nemishini.Ngendlela yokugcina, ukufana kwevolumu yoketshezi engcono kakhulu emakamelweni e-microfluidic kwabelwana ngokugcina izakhiwo zendawo ezinobukhulu obufanayo, njengama-micropits kanye ne-surface arrays [152,153,154].Ngokuphawulekayo, amaconsi ayizingxenye zokugeleza ezinkulu ezingaphinda zenziwe futhi zisetshenziswe ku-electrode array ngokusekelwe ku-digital microfluidics (DMF).I-electrowetting of dielectrics ingenye yezinkolelo-mbono ze-DMF ezifundwe kahle kakhulu, njengoba i-electrowetting ye-dielectrics ivumela ukushintshwa okunembile kwamaconsi ngamanye, ukulawula ukuma kwezimpawu zikagesi eziwuketshezi kanye ne-asymmetric ezidlula ezinhlangothini ezahlukene [141, 144].Imisebenzi eyinhloko enamaconsi ku-DMF ihlanganisa ukuhlunga, ukuhlukanisa, nokuhlanganisa [151, 155, 156], engasetshenziswa emikhakheni ehlukahlukene yokuhlaziya, ikakhulukazi ekutholeni amangqamuzana [157, 158, 159].
Ukutholwa kwe-nucleic acid yedijithali ubuchwepheshe bokuxilonga amangqamuzana esizukulwane sesithathu obulandela i-PCR evamile kanye ne-PCR yesikhathi sangempela somthamo (qPCR), ngokuhambisana nokulandelana komphumela ophezulu kanye ne-liquid biopsy.Emashumini amabili eminyaka edlule, ama-nucleic acid edijithali athuthuke ngokushesha emkhakheni wokuxilongwa kwamangqamuzana amagciwane athathelwanayo [160, 161, 162].Ukulinganisa okuphelele kokutholwa kwe-nucleic acid yedijithali kuqala ngokupakisha amasampula nama-reagents ezingxenyeni ezingazodwana ukuze kuqinisekiswe ukuthi ukulandelana okuhlosiwe ngakunye kunethuba elifanayo lokungena endaweni ngayinye ngayinye.Ngokombono, isigaba ngasinye singanikezwa ukulandelana okuqondiwe okuningi, noma kungase kungabi khona isistimu ezimele ye-microreaction.Ngezindlela ezihlukahlukene zokuzwa ezichazwe ngenhla, amagumbi anama-microbial target alandelanayo akhiqiza amasignali ngaphezu komkhawulo othile angabonwa ngeso lenyama noma ngomshini futhi alebulwe ngokuthi phozithivu, kuyilapho amanye amagumbi akhiqiza amasignali ngaphansi komkhawulo alebulwe ngokuthi positive. .ezinegethivu, ezenza isignali yesigaba ngasinye ibe i-boolean.Ngakho-ke, ngokubala inani lamakhompathi adaliwe kanye nezinga lemiphumela emihle ngemva kokusabela, amakhophi okuqala amasampula okuhlola angafaniswa kusetshenziswa ifomula yokusabalalisa ye-Poisson ngaphandle kwesidingo sejika elijwayelekile, elidingekayo ekuhlaziyweni kobuningi okujwayelekile okunjalo. njenge qPCR.[163] Uma kuqhathaniswa nezindlela zokuxilonga zamangqamuzana ezivamile, ukutholwa kwe-nucleic acid yedijithali kunezinga eliphakeme lokuzenzakalela, isivinini sokuhlaziya esiphezulu nokuzwela, ama-reagents ambalwa, ukungcoliswa okuncane, nokuklama nokukhiqiza okulula.Ngenxa yalezi zizathu, ukusetshenziswa kwezivivinyo zedijithali, ikakhulukazi izindlela ezisekelwe phansi, zokuxilongwa kwamangqamuzana, okuhlanganisa amasu okukhulisa kanye nezindlela zokufunda amasignali, kufundwe kahle ngesikhathi sokugqashuka okubucayi kwe-SARS-CoV-2.Ngokwesibonelo, Yin et al.[164] izindlela ezihlanganisiwe ze-droplet digital kanye ne-PCR esheshayo ukuthola izakhi zofuzo ze-ORF1ab, N, ne-RNase P ku-SARS-CoV-2 ku-microfluidic chip.Ngokuphawulekayo, isistimu ikwazile ukukhomba isignali enhle phakathi kwemizuzwana ye-115, esheshayo kune-PCR evamile, ebonisa ukusebenza kwayo ekutholeni iphuzu lokunakekelwa (Umfanekiso 7a).Dong et al.[165], Hlwanyela et al.[157], Chen et al.[166] kanye no-Alteri et al.[167] iphinde yasebenzisa i-droplet digital PCR (ddPCR) ukuze kutholwe i-SARS-CoV-2 kusistimu ye-microfluidic enemiphumela emihle.Ukuze uthuthukise izinga lokutholwa, u-Shen et al.[168] izuze ukuthwebula kwe-chip okusekelwe ku-ddPCR ngamasekhondi ayi-15 ngaphandle kokusebenzisa amasu okuthunga isithombe, ukusheshisa inqubo yobuchwepheshe ye-ddPCR kusuka elebhu kuya ekusetshenzisweni.Akuzona kuphela izindlela zokukhulisa ezishisayo ezifana ne-PCR, kodwa nezindlela zokukhulisa i-isothermal zisetshenziselwa ukwenza izimo zokusabela zibe lula kanye nokusabela okusheshayo.Lu et al.[71] ithuthukise i-SlipChip ukuze ihlaziye amaconsi, ekwazi ukukhiqiza amaconsi anosayizi abahlukahlukene ngokuminyana okuphezulu esinyathelweni esisodwa futhi ilinganise ama-nucleic acid akwaSARS-CoV-2 kusetshenziswa i-digital LAMP (Figure 7b).Njengobuchwepheshe obuvela ngokushesha, i-CRISPR ingadlala indima ebalulekile ekutholweni kwe-nucleic acid yedijithali ngokusebenzisa i-imaging colorimetric elula ngaphandle kwesidingo samabala engeziwe e-nucleic acid.U-Ackerman nabanye.ithuthukise ukusabela kwe-matrix okuhlanganisiwe kokuhlolwa kwe-multiplex kwama-nucleic acid.[158] ithole amagciwane angu-169 ahlobene nabantu, okuhlanganisa i-SARS-CoV-2, kumaconsi aqukethe ama-reagents okutholwa kwe-nucleic acid asekelwe ku-CRISPR-Cas13 ekuhlolweni kwe-microwell (Figure 7c).Ngaphezu kwalokho, i-isothermal amplification kanye nobuchwepheshe be-CRISPR bungasetshenziswa ohlelweni olufanayo ukuhlanganisa izinzuzo zombili.Park et al.[169] Isivivinyo sedijithali se-CRISPR/Cas12a sathuthukiswa ku-chip yezentengiselwano ye-microfluidic ukuze kutholwe i-SARS-CoV-2 ekhishiwe futhi ebulawe ukushisa ngokusekelwe ku-RT-RPA yesiteji esisodwa ngokutholwa kwesiginali kuya ngemuva okufushane nangaphezulu. isilinganiso sesikhathi., ububanzi obubanzi obuguquguqukayo kanye nokuzwela okungcono (Fig. 7d).Ezinye izincazelo zalezi zibonelo zinikezwe kuThebula lesi-3.
Ipulatifomu yedijithali ejwayelekile yokuthola i-nucleic acid.a Ukugeleza komsebenzi okusheshayo kwe-PCR yedijithali kuqukethe izinyathelo ezine ezibalulekile: ukulungiswa kwesampula, ukusatshalaliswa kwengxube yokusabela, inqubo yokukhulisa, kanye nokulinganisa okuqondisiwe (kuthathwe ku- [164]).b I-Schematic ebonisa ukuhlaziywa kwamaconsi e-SlipChip ukuze akheke amaconsi lapho kuminyene khona (kuthathwe ku-[71]).c CARMEN-Cas Umdwebo wokugeleza komsebenzi13 (uthathwe kokuthi [158]).d Uhlolojikelele lokutholwa kwegciwane ledijithali elithuthukisiwe nge-CRISPR/Cas ebhodweni elilodwa (kuthathwe kokuthi [169]).I-W/O yamanzi-kuwoyela, i-polydimethylsiloxane PDMS, i-PCR polymerase chain reaction, iqoqo ledatha ye-DAQ, okuphuma kokuhlanganisiwe kwe-PID, i-CARMEN combinatorial matrix ukusabela kokuhlolwa kwe-multiplex nucleic acid, i-SARS-CoV-2, isifo esibucayi sokuphefumula, i-coronavirus 2 , I-RT Ukukhulisa i-reverse transcriptase recombinase polymerase-RPA, isignali ye-S/B ngemuva
Isikhathi sokuthumela: Sep-15-2022
