Labarai

Dabarun bincike na al'ada don gano cututtuka masu yaduwa suna buƙatar amfani da kayan aikin benchtop waɗanda basu dace da gwajin kulawa ba (POCT).Microfluidics da ke tasowa shine ƙwanƙwasa, mai sarrafa kansa, da kuma haɗaɗɗen fasaha wanda shine yuwuwar madadin hanyoyin gargajiya don sauri, ƙarancin farashi, ingantaccen bincike akan rukunin yanar gizo.Ana amfani da hanyoyin gano ƙwayoyin ƙwayoyin cuta sosai a cikin na'urorin microfluidic azaman hanyoyin mafi inganci don gano ƙwayoyin cuta.Wannan bita yana taƙaita ci gaba na baya-bayan nan a cikin bincike-binciken ƙwayoyin cuta na tushen microfluidic daga mahangar ilimi da masana'antu.Da farko, mun bayyana nau'in sarrafa kan-chip na acid nucleic, gami da samfurin pretreatment, haɓakawa, da karatun sigina.Ana kwatanta halaye, fa'idodi da rashin amfani na nau'ikan dandamali guda huɗu na microfluidic.Na gaba, za mu tattauna yin amfani da ƙididdigar dijital don cikakken ƙididdige acid nucleic.Dukansu na gargajiya da na kwanan nan na kasuwanci na na'urorin gano ƙwayoyin ƙwayoyin cuta na microfluidic an taƙaita su azaman shaida na halin yanzu na kasuwa.A ƙarshe, muna ba da shawarar jagororin gaba don gano microfluidic na cututtukan cututtuka.
Cututtuka masu yaduwa suna haifar da cututtuka, ciki har da ƙwayoyin cuta, ƙwayoyin cuta, da ƙwayoyin cuta, waɗanda ke yaduwa a duniya.Ba kamar sauran cututtuka ba, ƙwayoyin cuta da sauri suna kamuwa da kamuwa da cuta kuma suna yaduwa tsakanin mutane da dabbobin gida ta hanyar alluran rigakafi, kafofin watsa labarai na iska da ruwa [1].Rigakafin cututtuka yana da mahimmanci a matsayin ma'aunin lafiyar jama'a.Manyan dabaru guda uku don yakar cututtuka masu yaduwa: (1) sarrafa tushen kamuwa da cuta;(2) katsewar hanyar watsawa;(3) kariya ga al'umma masu rauni.Daga cikin manyan dabarun, sarrafa tushen kamuwa da cuta ana la'akari da mafi mahimmancin dabarun saboda dacewa da ƙarancin farashi.Gaggawa ga ganewar asali, keɓewa, da kuma kula da masu kamuwa da cutar suna da mahimmanci, suna buƙatar sauri, m, da ingantattun dabarun gano cutar [2].Binciken na yau da kullun na cututtukan cututtuka yawanci yana haɗa gwajin asibiti bisa alamu da alamu da binciken dakin gwaje-gwaje kamar al'adun tantanin halitta da ƙididdigar ƙwayoyin cuta, waɗanda ke buƙatar ƙwararrun ma'aikata, hanyoyin aiki mai ƙarfi, da kayan gwaji masu tsada [3, 4].Rigakafin kamuwa da cututtuka yana buƙatar gaggawa, mara tsada, da ingantaccen ganewar asali na gida, musamman a wuraren da ba a iyakance albarkatun ƙasa ba inda cututtuka masu yaduwa suka zama ruwan dare kuma suna da tsanani [5], da kuma magani a cikin jeji ko a fagen fama, inda ba za a iya tsinkaya ga gaggawa ba..kiwon lafiya yana da iyaka [6].A cikin wannan mahallin, microfluidics fasaha ce da ta haɗu da fasahar tsarin microelectromechanical, nanotechnology, ko kimiyyar kayan don daidaitaccen magudin ruwa [7,8,9,10], yana ba da sabbin dama don gano kulawa (POCT).) masu kamuwa da cuta a wajen asibitoci da dakunan gwaje-gwaje.Idan aka kwatanta da bincike-bincike na al'ada na cin lokaci, fasahar microfluidic tana ba da samfuri da tanadin farashi don gwajin ƙwayoyin cuta yayin barkewar cututtuka.Yaduwar cutar coronavirus ta duniya 2019 (COVID-19) tana haifar da mummunan cutar sankara na numfashi coronavirus 2 (SARS-CoV-2), don haka an sake jaddada mahimmancin microfluidics don rigakafin kan lokaci da sarrafa cutar ta [11, 12] , 13].Ba kamar bincike na al'ada ba, microfluidic POCT yana amfani da ƙananan na'urori masu ɗaukuwa waɗanda suka fito daga masu nazarin benchtop zuwa ƙananan ɗigon gwaji na gefe don gwadawa kusa da wurin samfur [14].Waɗannan gwaje-gwajen sun ƙunshi sauƙi ko babu shiri na samfur, haɓaka sigina mai sauri, da karatun siginar mai hankali wanda ke haifar da ɗan gajeren lokaci da ingantaccen sakamako a cikin mintuna.Samuwar da yawan samar da kayan aikin kiwon lafiya na tushen microfluidic sun fadada aikace-aikacen bincike masu amfani da tsada da kai tsaye a wajen asibiti, kusa da mai haƙuri, har ma a gida.
Daga cikin dabarun da ake da su don gano cututtuka masu yaduwa, binciken kwayoyin halitta yana daya daga cikin mafi mahimmanci [15, 16].Bugu da kari, ana amfani da binciken kwayoyin cuta sau da yawa azaman ma'aunin zinare don ci gaba da gano COVID-19, yana ba da damar gano takamaiman yankuna na RNA ko DNA kai tsaye kafin fara amsawar rigakafi [17, 18].A cikin bita na yanzu, muna gabatar da sababbin ci gaba a cikin tsarin bincike na kwayoyin halitta na microfluidics don cututtuka masu cututtuka, daga hangen nesa na ilimi zuwa hangen nesa na masana'antu na gaba (Fig. 1).Za mu fara da matakai masu mahimmanci guda uku a cikin gano nucleic acid: samfurin pretreatment na kan-chip, haɓaka acid nucleic, da karatun sigina.Sa'an nan kuma muka kwatanta nau'ikan dandamali na microfluidic daban-daban tare da tsarin su da aikin su, suna nuna halaye na musamman (ƙarfi da rauni).Ana ci gaba da tattauna gano acid nucleic na dijital kuma an ba da shi azaman misali na fasaha na ƙarni na uku don ƙididdige adadin ƙwayoyin cuta masu kamuwa da cuta.Bugu da kari, za a gabatar da na'urorin POCT da yawa na yau da kullun da na zamani na kasuwanci don nuna halin yanzu na kasuwar POCT na microfluidic don bincikar kwayoyin halitta.Za mu kuma tattauna kuma mu bayyana hangen nesanmu don aikace-aikace na gaba.
Modules na microfluidic kwakwalwan kwamfuta don gano nucleic acid za a iya raba su zuwa rukuni uku (samfurin, ganewa, da sigina) gwargwadon ayyukansu [19].Daga cikin waɗannan nau'ikan, samfurin samfurin galibi yana gane samfurin lysis da cirewar acid nucleic.Tsarin firikwensin firikwensin yana sarrafa jujjuyawa da haɓaka siginar acid nucleic.Tsarin siginar yana gano siginar da aka canza kuma aka sarrafa ta tsarin ji.Dangane da tsarin gano acid nucleic akan guntu, za mu taƙaita nau'ikan kwakwalwan kwamfuta daban-daban waɗanda zasu iya gane aikin "shigarwa da fitarwa".
Mataki na farko na gano acid nucleic shine hakar acid nucleic, watau ware acid nucleic da aka yi niyya daga samfurin asali.Ana yin hakar acid nucleic don tsarkake acid ɗin nucleic daga sauran gurɓatattun ƙwayoyin cuta, tabbatar da amincin tsarin farko na ƙwayoyin ƙwayoyin nucleic acid, da haɓaka sakamako.Hakar acid nucleic yana buƙatar lysis na samfurin da ake buƙata da kuma kama acid nucleic, inganci da inganci wanda ke da babban tasiri akan bincike da sakamakon bincike.Duk wani illolin da hankali yayin hakar na iya iyakance ƙarin ganowa.Misali, maganin sarkar polymerase (PCR) da hanyoyin haɓaka isothermal madauki (LAMP) ana hana su ta wasu sauran kaushi na halitta kamar ethanol da isopropanol a cikin keɓancewar keɓancewar acid nucleic [20].Hakar ruwa-ruwa da tsattsauran lokaci sune mafi mashahuri hanyoyin don ware ƙwayoyin nucleic acid [21], duk da haka, hakar ruwa-ruwa akan guntu yana da iyakancewa sosai, tunda reagents da ake amfani da su wajen fitar da ruwa-ruwa suna haifar da lalata yawancin kwakwalwan kwamfuta na microfluidic. .Anan, muna haskaka hanyoyin samar da ingantaccen lokaci na tushen microarray kuma muna kwatanta fa'idodi da rashin amfanin su.
Silicon wani abu ne mai dacewa da acid nucleic saboda daidaituwarsa, kwanciyar hankali, da sauƙin gyarawa [22].Mahimmanci, lokacin da aka gyara tare da silica ko wasu kayan, wannan haɗin yana nuna kaddarorin don tallata ƙwayoyin nucleic da ba su da kyau a ƙarƙashin ƙananan pH, yanayin gishiri mai girma yayin da suke haɓaka tare da babban pH, ƙananan hanyoyin gishiri.Bisa ga wannan al'amari, yana yiwuwa a tsarkake nucleic acid.
An yi amfani da nau'i-nau'i na kayan tushen silica don haɓakar acid nucleic a cikin microfluidics, irin su silica beads, powders, microfiber filters, da silica membranes [23, 24, 25, 26].Dangane da kaddarorin kayan, ana iya amfani da kayan tushen silicon a cikin microcircuits ta hanyoyi daban-daban.Misali, silica granules, foda, da nanofilters na kasuwanci ana iya sanya su kawai a cikin pores ko microchannels na kwakwalwan microfluidic kuma suna taimakawa cire acid nucleic daga samfurori [27, 28, 29].Hakanan za'a iya amfani da membranes na silica da aka gyaggyara don tsarkake DNA cikin sauri daga ƙwayoyin cuta a farashi mai sauƙi.Alal misali, Wang et al.[30] Ta hanyar haɗa halayen haɓakawa tare da musayar sarkar tsaka-tsaki mai tsaka-tsaki tare da silica membranes wanda aka lulluɓe da chitosan oligosaccharides, an ƙaddamar da wani tsari mai ɗaukar hoto wanda ya sami nasarar gano ƙungiyoyin mallaka na 102-108.(CFU)/ml Vibrio parahaemolyticus., kuma kasancewar kwayar cutar ta kasance a bayyane.Powell et al.[31] Daga nan sai aka yi amfani da siliki-based microarrays don gano cutar hanta ta C (HCV), ƙwayar cuta ta mutum (HIV), cutar Zika, da papillomavirus na mutum da kuma yaduwa ta atomatik, inda aka samar da microreactor 1.3 μl don kama ƙwayoyin RNA.da kuma yi a wurin ƙarawa.Bugu da ƙari ga waɗannan hanyoyin, ƙananan silica microcolumns da aka gyara saman suma suna taka muhimmiyar rawa wajen fitar da acid nucleic, kamar yadda lissafin lissafi da kaddarorin kayan da ke gyare-gyare suna ƙara haɓaka haɓakar haɓaka.Chen et al.[32] ya ba da shawarar dandali na microfluidic don keɓewar RNA mai ƙarancin hankali dangane da microcolumn silicon mai rufin amino.Wannan na'urar microfluidic tana haɗe tsararrun 0.25 cm2 micropillars akan ƙaramin siliki don cimma ƙimar haɓaka mafi girma ta wurin babban yanki zuwa ƙirar ƙimar girma.Amfanin wannan ƙira shine cewa na'urar microfluidic zata iya cimma har zuwa 95% ingancin hakar nucleic acid.Waɗannan dabarun tushen silicon suna nuna ƙimar keɓewar acid nucleic cikin sauri a farashi mai sauƙi.A haɗe tare da kwakwalwan kwamfuta na microfluidic, dabarun haɓakar tushen silicon ba za su iya haɓaka haɓakar gano acid nucleic kawai ba, har ma da sauƙaƙe ƙarami da haɗakar na'urorin bincike [20].
Hanyoyin rabuwa na Magnetic suna amfani da barbashi na maganadisu don ware acid nucleic a gaban filin maganadisu na waje.Abubuwan da aka fi amfani da su na maganadisu sun haɗa da Fe3O4 ko γ-Fe2O3 ɓangarorin maganadisu masu rufi da silica, amino da carboxyl [33,34,35,36].Siffar bambance-bambancen ƙwayoyin magnetic idan aka kwatanta da hanyoyin SPE na tushen silicon shine sauƙin magudi da sarrafawa tare da maganadisu na waje.
Yin amfani da hulɗar electrostatic tsakanin nucleic acid da silica, a ƙarƙashin yanayi na gishiri mai girma da ƙananan pH, ana amfani da acid nucleic a saman silica-mai rufi na magnetic barbashi, yayin da a karkashin yanayi na low gishiri da high pH, ​​da kwayoyin za a iya wanke. sake..Silica-rufin Magnetic beads yana ba da damar cire DNA daga manyan samfuran girma (400 μL) ta amfani da motsi mai sarrafa maganadisu [37].A matsayin nuni, Rodriguez-Mateos et al.[38] An yi amfani da maganadisu mai kunnawa don sarrafa canja wurin beads na maganadisu zuwa ɗakuna daban-daban.Dangane da silica-mai rufi barbashi Magnetic, 470 kofe/ml na SARS-CoV-2 genomic RNA za a iya fitar da shi daga sharar gida samfurin ga LAMP reverse transcription gano (RT-LAMP) da amsa za a iya karanta a cikin 1 hour.ido tsirara (Fig. 2a).
Na'urori bisa la'akari da kayan maganadisu da porous.Tsarin ra'ayi na na'urar microfluidic IFAST RT-LAMP don gano SARS-CoV-2 RNA (wanda aka karɓa daga [38]).b Centrifugal micro na'urar don dSPE na buccal swab nucleic acid (wanda aka daidaita daga [39]).c Gina-hannun na'ura mai sarrafa kai ta amfani da katin FTA® (wanda aka saba daga [50]).d Fusion 5 takarda tace da aka gyara tare da chitosan (wanda aka daidaita daga [51]).SARS-CoV-2 mai tsananin ciwo na numfashi na numfashi coronavirus 2, RT-LAMP juyar da madaidaicin madaidaicin haɓakawar isothermal, abokan fasahar FTA, NA nucleic acid
Ingantattun ƙwayoyin maganadisu masu caji suna da kyau don haɗa kashin bayan phosphate na acid nucleic.A wani yanki na gishiri, ƙungiyoyin phosphate da aka caje su na acid nucleic za a iya cajin su da kyau a saman ɓangarorin magnetic composite.Saboda haka, Magnetic nanoparticles tare da m surface da kuma babban yawa na amino kungiyoyin da aka ɓullo da domin hakar na nucleic acid.Bayan rarrabuwar maganadisu da toshewa, ana iya amfani da nanoparticles na Magnetic da rukunin DNA kai tsaye a cikin PCR, wanda ke kawar da buƙatar hadaddun da ɗaukar lokaci mai tsafta da ayyukan haɓaka [35].Hakanan an yi amfani da nanoparticles na Magnetic mai rufi tare da ƙungiyoyin carboxyl mara kyau don raba acid nucleic da aka tallata akan saman a cikin babban taro polyethylene glycol da mafita sodium chloride [36].Tare da waɗannan ƙwanƙolin maganadisu da aka gyaggyarawa, hakar DNA ya dace da haɓakawa na gaba.Dignan et al.[39] ya bayyana wani dandamali mai sarrafa kansa da šaukuwa na centrifugal microfluidic don pretreatment na acid nucleic, kyale ma'aikatan da ba na fasaha ba suyi amfani da shi akan wurin.Bugu da ƙari, daidaitawar DNA da aka keɓe tare da LAMP, hanyar da ta dace da mahimmanci don nazarin kwayoyin nucleic acid, yana kara nuna ƙananan buƙatun kayan aiki da dacewa don ƙididdigar launi (Fig. 2b).
Hanyoyin katako na Magnetic suna ba da yuwuwar hakar ta atomatik, wasu daga cikinsu suna wanzuwa a cikin masu cire acid nucleic mai sarrafa kansa [KingFisher;ThermoFisher (Waltham, MA, Amurka), QIAcube® HT;CapitalBio (Beijing, China) da kuma Biomek®;Beckman (Miami, Amurka).), Florida, Amurka).Ana iya amfani da fa'idodin haɗa beads na maganadisu tare da microfluidics don ingantaccen hakar acid nucleic ta atomatik, wanda zai iya haɓaka haɓakar cututtukan ƙwayoyin cuta;duk da haka, hade da Magnetic beads tare da microfluidics har yanzu dogara sosai a kan hadaddun sarrafawa tsarin domin daidai magudi na maganadisu beads, wanda ya bayyana shahararsa na kasuwanci kayayyakin zama girma da kuma tsada, wanda iyakance kara aikace-aikace na Magnetic beads a POCT.
An kuma yi amfani da wasu abubuwa masu ƙyalli kamar gyaran gyare-gyare na nitrocellulose filters, Katunan Fasahar Fasahar Fasaha (FTA), takaddun matatun polyethersulfone, da kayan da aka yi da glycan don gano nucleic acid [40, 41, 42, 43, 44].An fara amfani da kayan ɓangarorin ƙorafi irin su takarda mai fibrous don keɓe DNA ta hanyar shigar da kwayoyin DNA masu tsayin jiki tare da zaruruwa.Ƙananan pores suna haifar da ƙaƙƙarfan ƙuntatawa ta jiki na kwayoyin DNA, wanda ke tasiri sosai akan hakar DNA.Saboda nau'ikan pore daban-daban na takarda fibrous, ingancin hakar ba zai iya biyan buƙatun haɓaka DNA ba [45, 46].Katin FTA takarda ce ta kasuwanci ta kasuwanci da ake amfani da ita a fagen ilimin likitanci kuma ana amfani da ita sosai a wasu wuraren binciken kwayoyin.Ta hanyar yin amfani da takarda tace cellulose da aka yi da sinadarai daban-daban don lalata membranes tantanin halitta a cikin samfurin, ana kiyaye DNA ɗin da aka saki daga lalacewa har zuwa shekaru 2.Kwanan nan, an samar da takarda cellulose mai ciki don gano ƙwayoyin cuta na ƙwayoyin cuta daban-daban, gami da SARS-CoV-2, leishmaniasis, da zazzabin cizon sauro [47,48,49].HIV a cikin keɓaɓɓen plasma an lysed kai tsaye, kuma kwayar nucleic acid tana haɓaka a cikin membrane FTA® da aka gina a cikin mai tattarawa, wanda ke ba da damar samar da ingantaccen acid nucleic [50] (Fig. 2c).Babban matsala tare da gano nucleic acid ta amfani da katunan FTA shine cewa sinadarai irin su guanidine da isopropanol suna hana halayen haɓakawa na gaba.Don magance wannan matsalar, mun ƙirƙiri takarda tace Fusion 5 chitosan-gyara, wanda ya haɗu da fa'idodin duka biyun interlacing ta jiki na kwayoyin DNA da takarda tace fibrous, da adsorption na electrostatic na DNA akan mahaɗan chitosan da aka gyaggyara don cimma ingantaccen haɓakar acid nucleic. ..tace zaruruwa [51] (Hoto 2d).Hakanan, Zhu et al.[52] ya nuna hanyar PCR da aka gyara na chitosan bisa tsarin microfluidic capillary in situ don keɓanta da sauri da gano ƙwayar cutar Zika RNA.Ana iya ƙara acid ɗin nucleic a cikin madaidaicin lysate/PCR mai gauraya, bisa la'akari da kayan kunnawa da kashewa na chitosan.kunnawa da kashewa", mai amsawa ga pH.
Kamar yadda aka ambata a sama, waɗannan dabarun sun haɗu da fa'idodin ingantaccen kayan lokaci daban-daban kuma suna haɓaka haɓakar haɓakar acid nucleic a cikin microfluidics.A cikin aikace-aikacen aikace-aikacen, amfani da waɗannan kayan a cikin adadi mai yawa ba shi da ƙima, kuma daidaitaccen jiyya na ƙasa ko gyare-gyare na kayan gama gari tare da waɗannan kayan kuma na iya adana aikin su.Sabili da haka, an yi imanin cewa aiwatar da waɗannan dabarun bayan nazarin gwaji na iya rage farashi.
Gwajin Nucleic acid akan dandamali na microfluidic sau da yawa yana amfani da ƙananan samfurin ƙira (<100 µl), don haka yana buƙatar haɓaka abubuwan nucleic acid ɗin da aka yi niyya tare da takamaiman bincike don juyawa zuwa siginar da ta dace don gano ƙasa (na gani, lantarki, da magnetic) [53, 54]. Gwajin Nucleic acid akan dandamali na microfluidic sau da yawa yana amfani da ƙananan samfurin ƙira (<100 µl), don haka yana buƙatar haɓaka abubuwan nucleic acid ɗin da aka yi niyya tare da takamaiman bincike don juyawa zuwa siginar da ta dace don gano ƙasa (na gani, lantarki, da magnetic) [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]. Lokacin gwajin ƙwayoyin nucleic a kan dandamali na microfluidic, ana amfani da ƙananan samfurin ƙididdiga (<100 µL) sau da yawa, don haka ana buƙatar ƙara girman acid nucleic acid tare da bincike na musamman don canza shi zuwa sigina mai dacewa don ganowa na gaba (na gani, lantarki, da magnetic) [53, 54].的平台 og 的 核酸核酸 的核酸 的使用 的核酸 的核酸), 因此 需要 使用 特定 探针 扩增目标 游核酸, 以以, 电学电学 游便于 游检测 检测 (53, 54 ].平台平台 og 的 核酸核酸 的核酸 的小样本量 的核酸), 因此 需要 特定特定), 因此, 以以 游转换游游游 下载 (53, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 54, 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]]. Gano acid nucleic akan dandamali na microfluidic yawanci yana amfani da ƙananan samfuran samfura (<100 μl), wanda ke buƙatar haɓakar acid nucleic da aka yi niyya tare da bincike na musamman don canza su zuwa sigina don ganowa na gaba (na gani, lantarki, da magnetic) [53, 54]] .Nucleic acid ƙarawa a cikin microfluidics kuma na iya hanzarta halayen, haɓaka iyakoki na ganowa, rage buƙatun samfurin, da haɓaka daidaiton ganowa [55, 56].A cikin 'yan shekarun nan, tare da ganewar sauri da ingantaccen ganewa, an yi amfani da hanyoyi daban-daban na haɓakawa na nucleic acid a cikin microfluidics, ciki har da PCR da wasu halayen haɓakawa na isothermal.Wannan sashe zai taƙaita hanyoyin gano nucleic acid bisa tsarin microfluidic.
PCR simulation ne na tsarin kwafi na DNA na kwayoyin halitta, ka'idar wacce aka bayyana dalla-dalla a wani wuri kuma ba za a yi magana a nan ba.PCR na iya haɓaka ɗan ƙaramin adadin DNA/RNA da aka yi niyya a ƙaƙƙarfan ƙima, yana mai da PCR kayan aiki mai ƙarfi don gano ƙwayoyin nucleic cikin sauri.A cikin 'yan shekarun da suka gabata, yawancin na'urorin microfluidic masu ɗaukuwa sanye da tsarin hawan keke na PCR an haɓaka su don saduwa da buƙatun ƙididdigar kulawa [57, 58].Ana iya raba PCR akan guntu zuwa nau'ikan guda huɗu (na al'ada, ci gaba da gudana, sauyawa ta sarari, da PCR mai jujjuyawa) bisa ga hanyoyin sarrafa zafin jiki daban-daban [59].Alal misali, Gee et al.[60] sun haɓaka hanyar juzu'in juzu'i na PCR (RT-qPCR) kai tsaye akan dandamalin microfluidic na kansu don ganowa da yawa na SARS-CoV-2, mura A da ƙwayoyin cuta B a cikin samfuran swab na makogwaro (Fig. 3a).Park et al.[61] ya gina guntu na bincike mai sauƙi ta hanyar haɗa fim ɗin PCR na bakin ciki, na'urori masu lantarki, da tsarin microfluidic na tushen polydimethylsiloxane mai yatsa.Koyaya, duka biyun suna aiki sun haɗa da kasawar gama gari na PCR na al'ada.PCR na buƙatar hawan keke na zafi, wanda ke iyakance ƙara ƙarancin na'urar da rage lokacin gwaji.
Haɓaka ci gaba da gudana bisa microfluidic da PCR mai canza sararin samaniya yana da mahimmanci don magance wannan batu.Yin amfani da doguwar tashar maciji ko tashoshi madaidaiciya madaidaiciya, ci gaba da gudana PCR na iya samar da haɓakawa da sauri ta hanyar zazzage reagents a cikin yankuna preheat uku tare da famfo mai kashe guntu.Wannan aikin ya sami nasarar guje wa yanayin canji tsakanin yanayin zafi daban-daban kuma don haka yana rage lokacin gwaji sosai [62] (Fig. 3b).A wani binciken da Jung et al.[63] ya ba da shawarar sabon mai nazarin kwayoyin halittar PCR na jujjuya wanda ya haɗu da halaye na ƙayyadaddun PCR da kwarara don ultrafast da multiplex reverse transcription PCR (Fig. 3c).Don ƙara girman acid nucleic, PCR microchip za a juya ta cikin tubalan dumama a yanayin zafi daban-daban: 1. Denaturation block 94°C, 2. Annealing block at 58°C, 3. Expansion block at 72°C.
Aikace-aikacen PCR a cikin microfluidics.Wakilin tsari na dirRT-qPCR akan dandamalin microfluidic (wanda aka daidaita daga [60]).b Tsarin tsari na ci gaba da gudana PCR microarray dangane da tashar maciji (wanda aka daidaita daga [62]).c Misalin tsari na mai nazarin kwayoyin halittar PCR na jujjuya, wanda ya kunshi microchip, tubalan dumama guda uku da injin stepper (wanda aka saba daga [63]).d Zane na thermoconvection PCR tare da centrifugation da saitin (wanda aka daidaita daga [64]).DirRT-qPCR, juzu'i mai jujjuya juyi kai tsaye amsa sarkar polymerase
Yin amfani da capillaries da madaukai ko ma faranti na bakin ciki, convection PCR na iya haɓaka acid ɗin nucleic da sauri ta hanyar haɓakar yanayin zafi na halitta ba tare da buƙatar famfo na waje ba.Misali, an ɓullo da dandali na cyclic olefin polymer microfluidic a kan ƙera gyare-gyaren dumamar yanayi wanda ke amfani da hawan keke na zafi tare da centrifugation a cikin microchannel madauki na PCR [64] (Fig. 3d).Maganin amsawa yana gudana ta hanyar convection na thermal, wanda ke ci gaba da musanya high da ƙananan zafin jiki a cikin microchannel tare da tsarin shekara-shekara.Za'a iya kammala dukkan tsarin haɓakawa a cikin mintuna 10 tare da iyakar ganowa na 70.5 pg/tashar.
Kamar yadda aka zata, PCR mai sauri kayan aiki ne mai ƙarfi don cikakken haɗaɗɗen samfurin amsawar ƙwayoyin cuta da tsarin bincike mai yawa.PCR mai sauri yana rage lokacin da ake buƙata don gano SARS-CoV-2, wanda ke ba da gudummawa ga ingantaccen sarrafa cutar ta COVID-19.
PCR yana buƙatar hadaddun mahaɗan mai zazzagewa wanda bai dace da POCT ba.Kwanan nan, an yi amfani da fasahar haɓakar isothermal zuwa microfluidics, ciki har da amma ba'a iyakance ga LAMP ba, recombinase polymerase amplification (RPA), da haɓakawa bisa jerin nucleic acid [65,66,67,68].Tare da waɗannan fasahohin, ƙwayoyin nucleic acid suna haɓaka a cikin yanayin zafi akai-akai, suna sauƙaƙe ƙirƙirar ƙarancin farashi, na'urorin POCT masu ɗaukar nauyi sosai don bincikar ƙwayoyin cuta.
Babban matakan samar da microfluidics na tushen LAMP yana ba da damar gano cututtuka da yawa [42, 69, 70, 71].A haɗe tare da tsarin microfluidic na centrifugal, LAMP na iya ƙara sauƙaƙe aikin gano nucleic acid [69, 72, 73, 74, 75].SlipChip mai jujjuya-da-react an haɓaka shi don gano gani na ƙwayoyin cuta masu kama da juna ta amfani da LAMP [76] (Fig. 4a).Lokacin amfani da ingantacciyar LAMP a cikin tantancewar, ƙimar sigina-zuwa-amo rabo ya kai kusan ninki 5, kuma iyakar ganowa ya kai kwafi 7.2/μl na DNA na genomic. Bugu da ƙari, kasancewar ƙwayoyin ƙwayoyin cuta na ƙwayoyin cuta guda biyar masu narkewa, ciki har da Bacillus cereus, Escherichia coli, Salmonella enterica, Vibrio fluvialis da Vibrio parahaemolyticus, an gani bisa hanyar a cikin <60 min. Bugu da ƙari, kasancewar ƙwayoyin ƙwayoyin cuta na ƙwayoyin cuta guda biyar masu narkewa, ciki har da Bacillus cereus, Escherichia coli, Salmonella enterica, Vibrio fluvialis da Vibrio parahaemolyticus, an gani bisa hanyar a cikin <60 min.Bugu da ƙari, kasancewar ƙwayoyin cuta guda biyar na ƙwayoyin cuta na ƙwayar cuta, ciki har da Bacillus cereus, Escherichia coli, Salmonella enterica, Vibrio fluvialis da Vibrio parahaemolyticus, an hango su ta hanyar amfani da wannan hanya a cikin ƙasa da minti 60.此外, 基于 该 可 <60 分钟 内 可 视化 了了 的 的 细菌病存在 的 的存在, 包括包括, 肠 菌, 河流 弧菌 和 副溶血性 弧菌.此外, 基于 该 方法 <60 分钟 内 视化 了 五 种 细菌病 的 的 的 消化道 消化道 细菌病 的 的 的 的 的 的 的 的 的 的 的 的 的 的 的 的 的 的 的 的 的 的 的 的 的 的 的 的 的 的弧菌 弧菌 弧菌 弧菌 弧菌 弧菌 弧菌 弧菌 HIPBugu da ƙari, an hango kasancewar ƙwayoyin ƙwayoyin cuta guda biyar na ƙwayoyin cuta na hanji, ciki har da Bacillus cereus, Escherichia coli, Salmonella enterica, Vibrio fluvius, da Vibrio parahaemolyticus, ta hanyar amfani da wannan hanya a cikin ƙasa da minti 60.
Fa'idodin LAMP a cikin microfluidics sun haɗa da, da sauransu, saurin amsawa da ƙarancin ganowa.Koyaya, saboda yawan zafin jiki (kimanin 70 ° C), babu makawa ana haifar da iska a lokacin LAMP, yana haifar da ƙimar inganci na ƙarya.Ƙayyadaddun ƙayyadaddun ƙididdiga, ƙira na farko, da sarrafa zafin jiki suma suna buƙatar inganta su don LAMP.Bugu da ƙari, ƙirar guntu waɗanda ke aiwatar da gano maƙasudi da yawa akan guntu ɗaya suna da ƙima sosai kuma yakamata a haɓaka su.Bugu da kari, LAMP ya dace da gano maƙasudi da yawa da aka haɗa a cikin guntu ɗaya, wanda ke da mahimmanci, amma har yanzu akwai ɗaki mai yawa don haɓakawa.
Za a iya rage girman ƙimar ƙimar LAMP tare da RPA, saboda ƙarancin zafin jiki (~ 37 ° C) yana haifar da ƙananan matsalolin ƙafewa [77].A cikin tsarin RPA, maɓalli biyu masu gaba da juna sun fara haɗin DNA ta hanyar ɗaure zuwa sake haɗawa da haɓakawa za a iya kammala su cikin mintuna 10 [78,79,80,81].Saboda haka, duk tsarin RPA yana da sauri fiye da PCR ko LAMP.A cikin 'yan shekarun nan, an nuna fasahar microfluidic don ƙara haɓaka sauri da daidaito na RPA [82,83,84].Alal misali, Liu et al.[85] ɓullo da wani microfluidic hadedde a kaikaice kwarara kwarara polymerase recombinase amplification assay ga m da kuma m ganewa na SARS-CoV-2 ta hade da juyi kwafin RPA (RT-RPA) da kuma duniya kai tsaye kwarara gwajin tsiri tsarin gano tsiri.cikin tsarin microfluidic guda ɗaya.Hoto na 4b).Iyakar ganowa shine 1 kwafi/µl ko kwafi 30/samfuri, kuma ana iya kammala ganowa cikin kusan mintuna 30.Kong et al.sun ƙera na'urar microfluidic mai sawa.[86] yayi amfani da zafin jiki da tsarin gano hasken haske mai tushen wayar hannu don gano HIV-1 DNA cikin sauri da kai tsaye ta amfani da RPA (Hoto 4c).Gwajin RPA mai sawa yana gano kwafi/ml 100 na jerin abubuwan da aka yi niyya a cikin mintuna 24, yana nuna babban yuwuwar saurin gano jarirai masu kamuwa da cutar kanjamau a cikin iyakokin iyakacin albarkatu.
Ƙaddamar da Isothermal a cikin gwajin kulawa (POCT).Haɓakawa da samar da juzu'i da amsa SlipChip.Bayan waldawar plasma, an haɗa guntu na sama da ƙasa tare da saitin goro don samar da guntu na ƙarshe (wanda aka daidaita daga [76]).b Tsarin tsarin MI-IF-RPA don gano COVID-19 (an daidaita shi daga [85]).c Tsarin gwaji na RPA mai sawa don gano HIV-1 DNA da sauri (wanda aka karɓa daga [86]).SE Salmonella enterica, VF Vibrio fluvius, VP Vibrio parahaemolyticus, BC Bacillus cereus, EC Escherichia coli, FAM carboxyfluorescein, HIV immunodeficiency cutar HIV, RPA recombinase polymerase amplification, LED haske emitting diode, MI-IF-RPA Microfluidics Laterrase-integFlower-Flower. Ƙarawa
RPA na tushen Microfluidic yana haɓaka cikin sauri, duk da haka, farashin ƙirƙira guntu da amfani da amsa ya yi yawa kuma dole ne a rage shi don ƙara samun wannan fasaha.Bugu da kari, babban hankali na RPA na iya shafar haɓaka samfuran da ba takamaiman samfuran ba, musamman a gaban gurɓatawa.Waɗannan iyakoki na iya shafar aikace-aikacen RPA a cikin tsarin microfluidic da ƙarin haɓakawa.Ana kuma buƙatar ƙwararrun ƙirar ƙira da bincike don manufa daban-daban don haɓaka yuwuwar dabarun microfluidic tushen RPA a cikin POCT.
Cas13 da Cas12a suna da ikon tsinke acid nucleic ba da gangan ba don haka ana iya haɓaka su azaman ganowa da kayan aikin bincike.Cas13 da Cas12a ana kunna su akan ɗaure zuwa DNA ko RNA, bi da bi.Da zarar an kunna, sunadaran zai fara tsinke sauran acid nucleic na kusa, bayan haka jagorar RNAs da ke niyya takamaiman acid nucleic na pathogen zai iya toshe binciken da aka kashe kuma ya saki haske.Bisa ga wannan ka'idar, Kellner et al.[87] ya haɓaka hanyar tushen Cas13 [Takamaiman High-sensitivity Enzymatic Reporter UnlockING (SHERLOCK)], da Broughton et al.[88] ya ɓullo da wata hanyar da ta danganci Cas12a [CRISPR Trans Reporter wanda ke niyya DNA endonuclease (DTECR)].
A cikin 'yan shekarun nan, hanyoyi daban-daban don gano acid nucleic dangane da CRISPR sun bayyana [89, 90].Hanyoyin tushen CRISPR na al'ada galibi suna cin lokaci da aiki mai ƙarfi saboda hanyoyin da yawa waɗanda suka haɗa da hakar acid nucleic, haɓakawa da gano CRISPR.Fitar da ruwa zuwa iska na iya ƙara damar samun sakamako mai kyau na ƙarya.Ganin abin da ke sama, tsarin tushen CRISPR yana buƙatar haɓakawa sosai.
An haɓaka dandamali na microfluidic mai sarrafa huhu wanda zai iya yin nazarin 24 a layi daya don aikace-aikacen gano CRISPR-Cas12a da CRISPR-Cas13a [91].An sanye da tsarin tare da na'urar gano haske wanda ke ƙetare haɓakar acid nucleic kuma ta atomatik gano samfuran DNA na femtomolar da RNA.Chen et al.[92] hadedde recombinase haɓakawa tare da tsarin CRISPR-Cas12a a cikin centrifugal microfluidics (Fig. 5a).Wannan aikin yana shawo kan wahalar haɗa waɗannan matakai guda biyu saboda Cas12a na iya narke DNA na manzo kuma ya hana tsarin haɓakawa.Bugu da kari, Chen et al.[92] Bugu da ƙari, an riga an adana reagents na amsawa a cikin kulawar microfluidic na centrifugal don kammala dukkan tsari ta atomatik.A cikin wani aikin, Silva et al.[93] ɓullo da hanyar bincike ba tare da CRISPR/Cas12a haɓakawa da wayar hannu don gano SARS-CoV-2 (Fig. 5b).Wannan ƙididdiga, wanda aka sani da tsarin haɓakawa na tushen wayar salula, ya haɗa da enzyme mai dogaro da CRISPR/Cas wanda ya dogara akan hangen nesa ta wayar salula na siginar kumfa mai haifar da catalase a cikin tashoshi na microfluidic.Gano mai hankali na ƙasa da kwafin 50/µl na nucleic acid ba tare da haɓakawa ba, gabaɗayan tsari daga allurar samfur zuwa karatun siginar yana ɗaukar mintuna 71 kawai.
Hanyoyin gano Nucleic acid bisa CRISPR.Centrifugal POCT don haɗaɗɗen bincike na kwayoyin halitta bisa CRISPR (wanda aka karɓa daga [92]).b Haɓaka gwajin CASCADE don nazarin tushen wayar hannu na SARS-CoV-2 (wanda aka karɓa daga [93]).RAA recombinase amplification, PAM kusa da protospacer motif, CRISPR clustered short palindromic maimaitawa a lokaci-lokaci tsarin, CASCADE tsarin ba tare da wayar salula karawa tare da CRISPR / CAS-degara enzymes, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride EDC.
A matsayin mataki na ƙarshe a cikin gano acid nucleic, gano siginar kai tsaye yana nuna sakamakon bincike kuma muhimmin abu ne a cikin haɓaka ingantaccen POCT mai inganci, mai hankali da daidaito.Ana iya karanta sigina ta amfani da hanyoyi daban-daban kamar su fluorescent, electrochemical, colorimetric da dabarun maganadisu.A cikin wannan sashe, mun bayyana ma'anar kowace hanya kuma muna kwatanta binciken kwayoyin cututtuka na cututtuka a cikin microfluidics.
Ana amfani da dabarun tushen Fluorescence don bincikar POCT na cututtukan cututtuka saboda fa'idodinsu na ban mamaki na ingantacciyar hankali, ƙarancin farashi, sauƙin aiki, da ƙididdigar kulawa [94, 95].Waɗannan dabarun suna amfani da alamar fluorophores kamar rini mai kyalli da nanomaterials don ƙirƙirar siginar da za a iya ganowa (haɓaka kyalli ko kashewa).Wannan binciken yana nuna cewa dabarun tushen kyalli za a iya raba su zuwa lakabin mai walƙiya kai tsaye, siginar kunnawa, da gano siginar mai walƙiya [96].Gano lakabin mai kyalli kai tsaye yana amfani da alamun kyalli na musamman don yiwa lakabin ligands na musamman waɗanda ke haifar da takamaiman adadin haske lokacin zaɓin ɗaure zuwa manufa.Don gano tushen sigina, ingancin siginar kyalli yana da alaƙa da girman sha'awa.Ƙarfin fluorescence ba shi da ƙima idan babu manufa kuma ana iya gano shi lokacin da isasshen adadin abin da ake nufi ya kasance.Sabanin haka, tsananin hasken walƙiya da aka gano ta hanyar walƙiya ta “sigina-kashe” yana daidaita daidai da adadin maƙasudi, da farko ya kai matsakaicin ƙima kuma a hankali yana raguwa yayin da aka haɓaka manufa.Misali, ta amfani da hanyar CRISPR-Cas13a mai dogaro da kai-manufa, Tian et al.[97] ya ɓullo da dabarar gane sabon labari don gano RNA waɗanda ke ƙetare juzu'i kai tsaye (Fig. 6a).Bayan ɗaure ga RNAs masu maƙasudi, za a iya kunna hadaddun CRISPR-Cas13-RNA, yana haifar da rarrabuwar kawuna ta RNAs marasa takamaiman.Mai ba da labari mai suna [fluorophore (F)] yana kashe shi ta hanyar quencher (Q) cikakke da kyalli lokacin da hadaddun da ke kunnawa ya fashe.
Amfanin ganowa na electrochemical shine babban saurin ganowa, samar da sauƙi, ƙananan farashi, sauƙin ɗauka da sarrafawa ta atomatik.Hanya ce ta nazari mai ƙarfi don aikace-aikacen POCT.Dangane da transistors filin graphene Gao et al.[98] ya haɓaka nanobiosensor don ganowa da yawa na cutar Lyme antigens daga kwayoyin Borrelia burgdorferi tare da iyakar ganowa na 2 pg / mL (Fig. 6b).
An yi amfani da gwaje-gwajen launi a cikin aikace-aikacen POCT, suna amfana daga fa'idodin ɗaukar hoto, ƙarancin farashi, sauƙin shiri, da karatun gani.Ganewar launi na launi na iya amfani da iskar shaka na peroxidase ko peroxidase-kamar nanomaterials, tarawar nanomaterials, da ƙari na dyes mai nuna alama don canza bayani game da kasancewar acid nucleic da aka yi niyya zuwa canje-canjen launi na bayyane [99, 100, 101].Musamman ma, ana amfani da nanoparticles na gwal a cikin haɓaka dabarun launi, kuma saboda ikon su na haifar da saurin canje-canjen launi, ana samun karuwar sha'awar ci gaban dandamalin launi na POCT don gano yanayin cututtukan cututtuka [102].Tare da na'urar microfluidic na centrifugal da aka haɗa [103], ƙwayoyin cuta na abinci a cikin gurɓataccen samfuran madara za a iya gano su ta atomatik a matakin ƙwayoyin ƙwayoyin cuta 10, kuma ana iya karanta sakamakon a gani a cikin minti 65 (Fig. 6c).
Dabarun ji na maganadisu na iya gano masu nazari daidai ta amfani da kayan maganadisu, kuma an sami sha'awar aikace-aikacen POCT a cikin 'yan shekarun nan.Dabarun tsinkayar maganadisu suna da wasu fa'idodi na musamman kamar kayan maganadisu masu arha maimakon tsadar abubuwan gani.Koyaya, yin amfani da filin maganadisu yana haɓaka haɓakar ganowa kuma yana rage lokacin shirye-shiryen samfurin [104].Bugu da ƙari, sakamakon binciken maganadisu yana nuna ƙayyadaddun ƙayyadaddun ƙayyadaddun bayanai, hankali, da babban sigina-zuwa amo saboda ƙarancin siginar bangon maganadisu na samfuran halittu [105].Sharma et al.hadedde mahadar ramin maganadisu mai tushen biosensor zuwa dandalin microchip mai ɗaukuwa.[106] don ganowa da yawa na ƙwayoyin cuta (Fig. 6d).Biosensors suna gano acid nucleic subnanomolar waɗanda ke ware daga ƙwayoyin cuta.
Hanyar gano sigina ta al'ada.Manufar gano hyperlocalized Cas13a (wanda aka daidaita daga [97]).b Graphene nanobiosensor FET a hade tare da Lyme GroES scFv (wanda aka daidaita daga [98]).c Alamun launi don ganowa da yawa na ƙwayoyin cuta na abinci a cikin guntun microfluidic na centrifugal: Na 1 da No. 3 samfurori tare da ƙwayoyin cuta, da kuma No. .d Biosensor dangane da mahaɗar rami na maganadisu, gami da dandamali, ginanniyar toshe amplifier, naúrar sarrafawa, da samar da wutar lantarki don samar da sigina/saye (wanda aka daidaita daga [106]).GFET Graphene FET, Escherichia coli, Escherichia coli, Salmonella typhimurium, Vibrio parahaemolyticus, Vibrio parahaemolyticus, Listeria monocytogenes, PC PC, PDMS Dimethicone, PMMA polymethyl methacrylate
Duk da kyawawan halaye na hanyoyin gano sama, har yanzu suna da rashin amfani.Ana kwatanta waɗannan hanyoyin (tebur 1), gami da wasu aikace-aikace tare da cikakkun bayanai (ribobi da fursunoni).
Tare da ci gaban microfluidics, tsarin microelectromechanical, nanotechnology da kimiyyar kayan aiki, amfani da kwakwalwan kwamfuta na microfluidic don gano cututtukan cututtuka yana ci gaba da ci gaba [55,96,107,108].Madaidaicin sarrafa ƙananan kayan aiki da ruwaye suna ba da gudummawa ga daidaiton bincike da ingancin farashi.Sabili da haka, don ƙarin haɓakawa, an yi ƙoƙari don ingantawa da haɓaka kwakwalwan kwamfuta, wanda ya haifar da kwakwalwan microfluidic daban-daban tare da tsari da ayyuka daban-daban.Anan zamu ɗan gabatar da nau'ikan dandamali na microfluidic da yawa kuma muna kwatanta halayensu (riba da fursunoni).Bugu da ƙari, yawancin misalan da aka jera a ƙasa sun fi mayar da hankali kan yaƙar SARS-CoV-2.
LOCCs sune tsarin nazarin hadaddun hadaddun hadaddun da aka fi sani da su kuma ayyukansu suna da ƙarancin ƙima, haɗaka, sarrafa kai da daidaitawa daga samfurin allura da shirye-shirye, sarrafa kwarara da gano ruwa [109, 110].Ana sarrafa ruwaye ta hanyar ƙira da ƙira a hankali da kuma hulɗar yawancin tasirin jiki kamar gradients matsa lamba, aikin capillary, electrodynamics, filayen maganadisu da raƙuman sauti [111].LOCC yana nuna fa'idodi masu kyau a cikin babban aikin nunawa da ganowa da yawa, tare da saurin bincike mai sauri, ƙaramin samfurin ƙima, ƙarancin wutar lantarki, da babban gudanarwa da ingantaccen aiki;duk da haka, na'urorin LOCC suna da laushi sosai, kuma suna ƙera, marufi, da haɗin kai.Koyaya, haɓakawa da sake amfani da su suna fuskantar manyan matsaloli [96].Idan aka kwatanta da sauran dandamali, LOCC yana da fa'idodi na musamman dangane da matsakaicin bambancin aikace-aikacen da mafi kyawun dacewa da fasaha, amma rashin amfanin sa kuma a bayyane yake, wato babban rikitarwa da rashin maimaitawa.Dogaro da famfo na waje, waɗanda galibi suna da girma da tsada, yana ƙara iyakance amfani da su a cikin POCT.
Yayin barkewar COVID-19, LOCC ta sami kulawa da yawa.A lokaci guda, akwai sabbin kwakwalwan kwamfuta da yawa waɗanda ke haɗa fasaha da yawa.Misali, wayoyin komai da ruwanka yanzu ana amfani da su azaman na'urorin bincike masu ɗaukar nauyi kuma suna da babban yuwuwar haɗakar LOCC.Sun et al.[21] ya ƙirƙira guntun microfluidic wanda ke ba da damar ɗimbin takamaiman jerin nucleic acid na ƙwayoyin cuta guda biyar, gami da SARS-CoV-2, ta amfani da LAMP kuma yayi nazarin su ta amfani da wayar hannu a cikin awa 1 bayan ƙarshen abin.Kamar wani misali, Sundah et al.[112] ya ƙirƙiri maɓalli na ƙwayoyin cuta [ƙarfafa haɓakawa ta hanyar sauya yanayin canjin kwayoyin halitta (CATCH)] don gano kai tsaye da tsinkayen hari na SARS-CoV-2 RNA ta amfani da wayoyin hannu. CATCH ya dace da LOCC mai ɗaukuwa kuma yana samun kyakkyawan aiki (kimanin kwafin RNA 8 / μl; <1 h a zazzabi na ɗaki) [112]. CATCH ya dace da LOCC mai ɗaukuwa kuma yana samun kyakkyawan aiki (kimanin kwafin RNA 8 / μl; <1 h a zazzabi na ɗaki) [112]. CATCH совместим с портативным LOCC и обеспечивает превосходную производительность ( примерно 8 копий Ремер 8 копий. CATCH ya dace da LOCC mai ɗaukuwa kuma yana ba da ingantaccen kayan aiki (kimanin kwafin RNA 8/µl; <1 h a zafin ɗaki) [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）[12] 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）[12] CATCH совместим с портативными LOCC и обладает превосходной производной CATCH ya dace da LOCCs masu ɗaukuwa kuma yana da kyakkyawan aiki (kimanin kwafin RNA 8/µl; <1 hour a zafin ɗaki) [112].Bugu da ƙari, na'urorin LOCC don bincikar ƙwayoyin cuta suma suna amfani da wasu ƙarfin tuƙi kamar su vacuum, stretch, da lantarki.Kang et al.[113] ya nuna ainihin lokaci, ultra-sauri nanoplasma-on-a-chip PCR don saurin ganewa da ƙididdigewa na COVID-19 a cikin filin ta amfani da guntu PCR vacuum plasmonic liquid.Li et al.[114] Daga baya ya haɓaka guntu microfluidic mai shimfiɗawa wanda ya ba da damar gano cutar ta COVID-19.Dandalin yana amfani da tsarin haɓakawa na RT-LAMP don tantance ko samfurin yana da inganci ko mara kyau.Bayan haka, Ramachandran et al.[115] ya sami daidaitattun filayen lantarki ta hanyar amfani da isotachophoresis (ITP), dabarar mayar da hankali ga ion da aka aiwatar a cikin microfluidics.Tare da ITP, RNA da aka yi niyya daga samfuran swab na nasopharyngeal za a iya tsarkake ta atomatik.Sai Ramachandran et al.[115] Haɗa wannan tsarkakewar ITP tare da ingantaccen LAMP da CRISPR assays an gano SARS-CoV-2 a cikin ɗan adam nasopharyngeal swab da samfuran asibiti a cikin kusan mintuna 35.Bugu da ƙari, sababbin ra'ayoyin suna fitowa kullum.Jadhav et al.[116] ya ba da shawarar tsarin bincike dangane da ingantaccen yanayin Raman spectroscopy a haɗe tare da na'urar microfluidic mai ƙunshe da ko dai daidaitaccen zinari/mai rufin carbon nanotubes mai rufin azurfa ko kuma microspun micro/nanotubes.Ƙirƙirar ƙaƙƙarfan ƙayyadaddun ƙayyadaddun ƙayyadaddun tashoshi masu tacewa ana iya zubarwa.Na'urar tana toshe ƙwayoyin cuta daga magudanar ruwa/fitowar jiki daban-daban kamar su yau, nasopharynx da hawaye.Don haka, adadin ƙwayoyin ƙwayoyin cuta suna da yawa kuma ana iya gano kwayar cutar daidai da sa hannun Raman.
LOAD dandamali ne na microfluidic na centrifugal wanda duk matakai ana sarrafa su ta hanyar ƙa'idar mitar da ke jujjuya siginar ƙirar microstructured [110].Ana siffanta na'urar LOAD ta amfani da ƙarfin centrifugal azaman muhimmin ƙarfin tuƙi.Liquids kuma suna ƙarƙashin ikon capillary, Euler da sojojin Coriolis.Yin amfani da na'urar centrifuge, ana yin nazari a ci gaba da aikin ruwa daga radial zuwa ciki zuwa waje, kawar da buƙatar ƙarin bututu na waje, famfo, masu kunnawa, da bawuloli masu aiki.A takaice, hanyar sarrafawa guda ɗaya tana sauƙaƙe aiki.Sojojin da ke aiki a kan ruwa a cikin tashar microfluidic guda ɗaya a daidai wannan nisa daga cibiyar kaya suna daidai, wanda ya sa ya yiwu a sake maimaita tsarin tashar.Don haka, kayan LOAD sun fi sauƙi kuma mafi tattalin arziki don ƙira da ƙira fiye da na'urorin LOCC na al'ada, yayin da halayen sun kasance masu zaman kansu da kuma daidaitawa;duk da haka, saboda babban ƙarfin injina na kayan aikin centrifugal, kayan guntu da ke samuwa yana da iyaka kuma ƙananan kundin suna da wahala.zuwa mota.A lokaci guda, yawancin na'urorin LOAD an tsara su don amfani guda ɗaya kawai, wanda ke da tsada don gano manyan sikelin [96, 117, 118, 119].
A cikin 'yan shekarun nan, LOAD, wanda aka yi la'akari da ɗayan mafi kyawun na'urorin microfluidic, ya sami kulawa mai yawa daga masu bincike da masana'antun.Don haka, LOAD ya sami karɓuwa sosai kuma an yi amfani dashi don bincikar ƙwayoyin ƙwayoyin cuta na ƙwayoyin cuta [120, 121, 122, 123, 124], musamman lokacin barkewar COVID-19.Misali, a karshen 2020, Ji et al.[60] ya nuna gwajin kai tsaye na RT-qPCR don gano saurin ganowa ta atomatik na SARS-CoV-2 da mura A da B a cikin samfuran swab na makogwaro.Sannan Xiong et al.[74] ya gabatar da wani dandali na discoid microfluidic mai haɗa LAMP don saurin, daidai, da gano cutar coronavirus na ɗan adam guda bakwai, gami da SARS-CoV-2, cikin mintuna 40.A farkon 2021, de Oliveira et al.[73] ya nuna guntu na polystyrene toner centrifugal microfluidic guntu, ana sarrafa shi da hannu tare da rotator tip, don gano ƙwayoyin ƙwayoyin cuta na RT-LAMP na COVID-19.Bayan haka, Dignan et al.[39] ya gabatar da na'ura mai ɗaukar hoto ta atomatik don tsarkakewa na SARS-CoV-2 RNA kai tsaye daga sassan swab na buccal.Medved et al.[53] ya ba da shawarar tsarin samfurin aerosol na layi na SARS-CoV-2 tare da ƙaramin ƙarar jujjuyawar guntu mai kyalli tare da iyakar ganowa na kwafi 10 / μL da ƙaramin iyakar zagayowar mintuna 15.Suarez et al.[75] kwanan nan ya ba da rahoton ci gaba da ingantaccen dandamali na microfluidic na centrifugal don gano kai tsaye na SARS-CoV-2 RNA a cikin samfuran swab na nasopharyngeal da ba a kunna zafi ba ta amfani da LAMP.Waɗannan misalan suna nuna babban fa'idodi da alƙawarin LOAD a cikin ƙididdigar ƙwayoyin cuta na COVID-19.
A cikin 1945 Muller da Clegg [125] sun fara gabatar da tashoshi na microfluidic akan takarda ta amfani da takarda tace da paraffin.A cikin 2007, ƙungiyar Whitesides [126] ta ƙirƙiri dandamalin takarda na farko don gwajin furotin da glucose.Takarda ta zama manufa mai mahimmanci don microfluidics.Takardar tana da kaddarorin da suka dace irin su hydrophilicity da tsarin porous, kyakkyawan yanayin halitta, nauyi mai sauƙi, sassauci, foldability, ƙananan farashi, sauƙin amfani da dacewa.µPADs na gargajiya sun ƙunshi sifofin hydrophilic/hydrophobic da aka gina akan madaurin takarda.Dangane da tsari mai girma uku, μPADs za a iya raba zuwa kashi biyu (2D) da uku (3D) μPADs.Ana samar da 2D µPADs ta hanyar samar da iyakokin hydrophobic don samar da tashoshi na microfluidic, yayin da 3D µPADs yawanci ana yin su ne daga tarin yadudduka na takarda microfluidic na 2D, wani lokacin ta hanyar nada takarda, dabarun zamewa, tashoshi na buɗe, da bugu na 3D [96].Ruwan ruwa mai ruwa ko na halitta akan μPAD ana sarrafa su da farko ta hanyar ƙarfin capillary ba tare da tushen wutar lantarki na waje ba, yana sauƙaƙe ajiyar preagents, sarrafa samfurin, da ganowa da yawa.Koyaya, ingantaccen sarrafa kwarara da ganowa da yawa suna fuskantar cikas ta ƙarancin saurin ganowa, hankali, da sake amfani da su [96, 127, 128, 129, 130].
A matsayin dandali na microfluidic wanda ba a saba gani ba, μPAD ya sami haɓaka sosai kuma an haɓaka shi don gano ƙwayoyin ƙwayoyin cuta na cututtukan cututtuka kamar HCV, HIV, da SARS-CoV-2 [131, 132].Don zaɓin ganowa da sanin yakamata na HCV, Tengam et al.[133] ya ɓullo da wani sabon labari biosensor dangane da takarda mai kyalli ta amfani da takamaiman bincike na acid nucleic wanda ya dogara da pyrrolidinyl peptide.Nucleic acid suna daɗaɗɗen motsi a kan takarda cellulose da aka yi da oxidized ta hanyar rage alkylation tsakanin ƙungiyoyin amino da ƙungiyoyin aldehyde, kuma ganowa ya dogara ne akan haske.Ana iya karanta waɗannan sigina ta na'urar da aka kera ta musamman tare da kyamarar kyamarori mai ɗaukar hoto tare da kyamarar wayar salula.Bayan haka, Lu et al.[134] ya ƙera na'urar lantarki mai sassauƙa ta takarda bisa nickel / zinariya nanoparticles / carbon nanotubes / polyvinyl barasa organometallic tsarin haɗe-haɗe don gano cutar HIV ta hanyar haɓakar DNA ta amfani da methylene blue azaman mai nuna alamar DNA redox.Kwanan nan, Chowdury et al.[135] ya gabatar da ƙirar dandamali mai ƙima don gwajin kulawa µPAD ta amfani da ɗanyen mai haƙuri a haɗe tare da LAMP da fasahar hoto mai ɗaukar hoto don gano COVID-19 analyte.
Gwaje-gwajen kwararar ruwa na gefe suna jagorantar ruwaye ta hanyar ƙarfin ƙarfin jiki da sarrafa motsin ruwa ta hanyar jikewa da halaye na ɓangarorin da ba su da ƙarfi ko ƙaramin tsari.Na'urorin kwarara na gefe sun ƙunshi samfuri, haɗin kai, incubator da ganowa, da faɗuwar ruwa.Kwayoyin kwayoyin acid nucleic a cikin LFA suna gane takamaiman masu ɗaure waɗanda aka riga aka adana su a wurin dauri kuma suna ɗaure azaman hadaddun.Yayin da ruwa ke wucewa ta cikin faranti da ganowa, ana kama rukunin ta hanyar kwayoyin kama da ke kan layin gwaji da sarrafawa, suna nuna sakamakon da za a iya karantawa kai tsaye zuwa ido tsirara.Yawanci, ana iya kammala LFA a cikin mintuna 2-15, wanda ya fi saurin ganowa na gargajiya.Saboda tsari na musamman, LFA yana buƙatar ƴan ayyuka kuma baya buƙatar ƙarin kayan aiki, wanda ya sa ya zama mai sauƙin amfani.Yana da sauƙi don ƙirƙira da rage girman, kuma farashin kayan aikin takarda ya ragu.Duk da haka, ana amfani da shi ne kawai don ƙididdige ƙididdiga, kuma gano ƙididdiga yana da wuyar gaske, kuma ikon yin yawa da kayan aiki yana da iyaka sosai, kuma isashen nucleic acid guda ɗaya kawai za a iya ganowa a lokaci guda [96,110,127].
Kodayake yawancin aikace-aikacen LFA suna mayar da hankali kan immunoassays, amfani da LFA don bincikar kwayoyin halitta a cikin kwakwalwan microfluidic shima yana da tasiri kuma sananne [136].A cikin yanayin cutar hanta B, HIV da SARS-CoV-2 LFA Gong et al.[137] ya ba da shawarar dandamali na nanoparticle LFA mai canzawa kuma ya nuna haɓakar wannan ƙaramin dandali mai ɗaukuwa ta hanyar gano maƙasudi da ƙididdige maƙasudi da yawa kamar HBV nucleic acid.Bugu da kari, Fu et al.[138] ya nuna wani labari LFA dangane da ingantaccen Raman spectroscopy don ƙididdigar ƙididdiga na HIV-1 DNA a ƙananan ƙira.Don ganowa cikin sauri da kulawa na SARS-CoV-2, Liu et al.[85] ɓullo da microfluidic-haɗe-haɗe-haɗe-haɗe-haɗe-haɗe-haɗe-haɗe RPA bincike kwarara ta hanyar hada RT-RPA da kuma na duniya baki ya kwarara tsarin ganewa a cikin wani microfluidic tsarin guda.
Aikace-aikacen dandamali na microfluidic daban-daban sun bambanta dangane da takamaiman karatu, suna cin cikakkiyar fa'idar iyawa da fa'idodin dandamali.Tare da bawuloli masu araha, famfo da ducts, LOCC shine mafi kyawun dandamali don bambancin aikace-aikacen da haɗin kai tare da mafi girman ɗaki don haɓakawa.Don haka, muna fata kuma muna ba da shawarar cewa a gudanar da sabon binciken a LOCC a matsayin ƙoƙari na farko kuma a inganta yanayin.Bugu da ƙari, ana sa ran za a gano hanyoyin da suka fi dacewa da kuma amfani da su a cikin tsarin.LOAD ya yi fice a daidaitaccen sarrafa ruwa daga na'urorin LOCC da ke da su kuma yana nuna fa'idodi na musamman a cikin tuƙi guda ɗaya ta hanyar centrifugal ƙarfi ba tare da buƙatar tuƙi na waje ba, yayin da amsoshi masu kama da juna za su iya bambanta da aiki tare.Don haka, a nan gaba, LOAD zai zama babban dandamali na microfluidic tare da ƙarancin ayyukan hannu da ƙarin balagagge da fasaha mai sarrafa kansa.Dandalin µPAD ya haɗu da fa'idodin LOCC da kayan tushen takarda don ƙarancin farashi, ƙididdigar amfani guda ɗaya.Don haka, ci gaban gaba ya kamata ya mayar da hankali kan ingantattun fasahohin da suka dace.Bugu da ƙari, LFA ya dace da ganewar ido tsirara, yana yin alkawarin rage yawan amfani da samfurin da kuma saurin ganowa.Ana nuna cikakken kwatancen dandamali a cikin Tebur 2.
Nazari na dijital ya raba samfurin zuwa yawancin microreactors, kowannensu yana ƙunshe da adadi mai ƙima na kwayoyin manufa [139, 140].Ƙididdiga na dijital suna ba da fa'idodi masu mahimmanci don yin cikakken ƙididdigewa ta hanyar yin dubunnan gwaje-gwajen gwaje-gwajen sinadarai masu kama da juna a lokaci guda da ɗaiɗaiku a cikin ma'aunin ma'aunin micron maimakon a cikin ci gaba da lokaci.Idan aka kwatanta da microfluidics na gargajiya, halayen ɗabi'a na iya rage girman samfurin, haɓaka haɓakar amsawa, kuma a sauƙaƙe haɗa su tare da sauran hanyoyin bincike ba tare da buƙatar tashoshi ba, famfo, bawuloli, da ƙirar ƙira [141, 142, 143, 144, 145, 146, 147].Ana amfani da waɗannan hanyoyi guda biyu masu zuwa a cikin ƙididdigar dijital don cimma daidaituwa da daidaitaccen rabuwa na mafita, gami da reagents da samfurori kamar ƙwayoyin sel, acid nucleic, da sauran ƙwayoyin cuta ko ƙwayoyin cuta: (1) sauke emulsion masu amfani da rashin zaman lafiyar ruwa;(2) Rarraba tsararru ana aiwatar da shi ta iyakokin jumlolin na'urar.A cikin hanyar farko, za a iya ƙirƙirar droplets dauke da reagents da samfurori a cikin microchannels ta hanyar m hanyoyin kamar co-current, crossflow, kwarara mayar da hankali, mataki emulsification, microchannel emulsification, da membranes ta viscous karfi karfi da emulsification tare da tashar canji.yanki [143, 145, 146, 148, 149] ko amfani da hanyoyin aiki [150, 151], wanda ke gabatar da ƙarin makamashi ta hanyar lantarki, Magnetic, thermal da sarrafa inji.A cikin tsarin na ƙarshe, mafi kyawun daidaiton ƙarar ruwa a cikin ɗakunan microfluidic ana raba su ta hanyar kiyaye tsarin sarari iri ɗaya, kamar micropits da tsararrun saman [152,153,154].Musamman ma, ɗigogi sune manyan sassan kwarara waɗanda kuma za'a iya ƙirƙira su kuma ana sarrafa su akan tsarin lantarki dangane da microfluidics na dijital (DMF).Electrowetting na dielectrics yana ɗaya daga cikin mafi kyawun ka'idodin DMF da aka yi nazari, tun da electrowetting na dielectrics yana ba da damar yin amfani da daidaitattun digo na mutum, sarrafa siffar ruwa da siginonin lantarki na asymmetric suna wucewa ta bangarori daban-daban [141, 144].Babban ayyuka tare da digo a cikin DMF sun haɗa da rarrabuwa, rarrabuwa, da haɗuwa [151, 155, 156], waɗanda za a iya amfani da su a fannoni daban-daban na bincike, musamman a gano kwayoyin halitta [157, 158, 159].
Gano acid nucleic na dijital fasaha ce ta gwajin kwayoyin halitta ta ƙarni na uku da ke bin PCR na al'ada da PCR na ainihin lokaci (qPCR), a layi daya tare da babban tsari da biopsy na ruwa.A cikin shekaru ashirin da suka gabata, ƙwayoyin nucleic acid na dijital sun haɓaka cikin sauri a fagen binciken ƙwayoyin cuta na ƙwayoyin cuta [160, 161, 162].Cikakkun ƙididdigewa na gano nucleic acid na dijital yana farawa tare da tattara samfuran da reagents cikin ɗakunan mutum ɗaya don tabbatar da cewa kowane jeri na manufa yana da yuwuwar shigar kowane ɗaki.A bisa ka'ida, kowane sashe na iya ƙila a sanya maƙasudin maƙasudi da yawa, ko kuma ƙila ba a sami tsarin microreaction mai zaman kansa ba.Ta hanyar hanyoyi daban-daban na ganewa da aka bayyana a sama, sassan da ke da jerin abubuwan da ke haifar da sigina a sama da wani kofa ana iya ganin su da ido tsirara ko ta na'ura kuma ana lakafta su a matsayin tabbatacce, yayin da sauran sassan da ke samar da sigina da ke ƙasa da bakin kofa ana lakafta su da kyau. .marasa kyau, waɗanda ke sa siginar kowane sashe ya zama boolean.Don haka, ta hanyar ƙididdige adadin ɗakunan da aka ƙirƙira da ƙimar sakamako mai kyau bayan amsawa, ana iya daidaita kwafin asali na samfuran gwajin ta amfani da tsarin rarraba Poisson ba tare da buƙatar madaidaicin lanƙwasa ba, wanda ake buƙata don ƙididdigar ƙididdiga na yau da kullun kamar haka. kamar qPCR.[163] Idan aka kwatanta da hanyoyin gano kwayoyin halitta na al'ada, gano nucleic acid na dijital yana da babban digiri na aiki da kai, saurin bincike da hankali, ƙarancin reagents, ƙarancin gurɓatawa, da ƙira da ƙira mafi sauƙi.Don waɗannan dalilai, an yi nazarin amfani da ƙididdigar dijital, musamman hanyoyin da aka saukar, don bincikar ƙwayoyin cuta, haɗa haɓakawa da dabarun karanta siginar, an yi nazari sosai yayin barkewar cutar SARS-CoV-2.Alal misali, Yin et al.[164] Haɗaɗɗen dijital da hanyoyin PCR masu sauri don gano kwayoyin halittar ORF1ab, N, da RNase P a cikin SARS-CoV-2 a cikin guntu microfluidic.Musamman ma, tsarin ya sami damar gano sigina mai kyau a cikin 115 seconds, wanda ya fi sauri fiye da PCR na al'ada, yana nuna tasirinsa a cikin ganewar kulawa (Figure 7a).Dong et al.[165], Sow et al.[157], Chen et al.[166] da Alteri et al.[167] Hakanan ana amfani da PCR dijital dijital (ddPCR) don gano SARS-CoV-2 a cikin tsarin microfluidic tare da sakamako mai ban sha'awa.Don ƙara haɓaka ƙimar ganowa, Shen et al.[168] ya sami ddPCR na tushen guntu hoto a cikin kadan kamar 15 s ba tare da amfani da dabarun dinki na hoto ba, yana hanzarta aiwatar da fasahar ddPCR daga lab zuwa aikace-aikace.Ba wai kawai hanyoyin haɓaka yanayin zafi kamar PCR ana amfani da su ba, amma ana amfani da hanyoyin haɓaka isothermal don sauƙaƙe yanayin amsawa da saurin amsawa.Lu et al.[71] ɓullo da SlipChip don nazarin droplet, mai iya haifar da ɗigon ruwa masu girma dabam a manyan yawa a mataki ɗaya da ƙididdige acid nucleic SARS-CoV-2 ta amfani da LAMP na dijital (Hoto 7b).A matsayin fasahar haɓaka da sauri, CRISPR kuma na iya taka muhimmiyar rawa a cikin gano nucleic acid ta dijital ta hanyar hoto mai dacewa ba tare da buƙatar ƙarin tabo na acid nucleic ba.Ackerman et al.ya haɓaka halayen matrix na haɗin gwiwa don kimantawa da yawa na acid nucleic.[158] an gano ƙwayoyin cuta guda 169 da ke da alaƙa da ɗan adam, gami da SARS-CoV-2, a cikin ɗigon ruwa mai ɗauke da CRISPR-Cas13 na tushen nucleic acid reagents a cikin gwajin microwell (Hoto 7c).Bugu da ƙari, haɓakawar isothermal da fasahar CRISPR za a iya amfani da su a cikin tsarin guda ɗaya don haɗa amfanin duka biyu.Park et al.[169] An haɓaka ƙimar dijital ta CRISPR/Cas12a a cikin guntun microfluidic na kasuwanci don ganowar SARS-CoV-2 da aka cire da kuma kashe zafi dangane da RT-RPA mai mataki ɗaya tare da gajeriyar sigina mafi girma zuwa ga bango. rabon lokaci., Faɗi mai ƙarfi mai ƙarfi da mafi kyawun hankali (Fig. 7d).An ba da wasu kwatancen waɗannan misalan a cikin Tebur 3.
Babban dandamali na dijital don gano nucleic acid.Matsakaicin saurin aiki na PCR na dijital ya ƙunshi matakai huɗu masu mahimmanci: shirye-shiryen samfurin, rarraba cakudawar amsawa, tsarin haɓakawa, da ƙididdige ƙididdigewa (wanda aka daidaita daga [164]).b Tsari yana nuna nazarin ɗigon SlipChip don samuwar digo a babban yawa (wanda aka daidaita daga [71]).c CARMEN-Cas zane-zane na aikin aiki13 (an daidaita shi daga [158]).d Bayanin ci gaba na gano ƙwayoyin cuta na dijital tare da CRISPR/Cas a cikin tukunya ɗaya (wanda aka karɓa daga [169]).W/O ruwa-in-man, polydimethylsiloxane PDMS, PCR polymerase sarkar dauki, DAQ tarin bayanai, PID gwargwado integral abin da ya faru, CARMEN combinatorial matrix dauki ga multiplex nucleic acid kimantawa, SARS-CoV-2, m numfashi ciwo, coronavirus 2 , RT Amplification na juyar da rubutun recombinase polymerase-RPA, siginar S/B a bango