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Tsarin Lab-a-a-chip tare da damar kan-site yana ba da yuwuwar saurin ganewar asali kuma yana da amfani a cikin ƙayyadaddun saitunan albarkatun inda babu kayan aikin likitanci da ƙwararrun ƙwararru.Koyaya, ƙirƙirar tsarin gwaji na kulawa wanda a lokaci guda yana da duk abubuwan da ake buƙata don rarraba ayyuka da yawa, sakin buƙatu, ingantaccen aiki, da adana dogon lokaci na reagents ya kasance babban ƙalubale.Anan mun bayyana fasahar sauya tafiye-tafiye ta lever wacce za ta iya sarrafa ruwaye ta kowace hanya, samar da daidaitattun martani da daidaituwa ga matsa lamba na iska, da kuma tsaya tsayin daka kan motsi da girgiza kwatsam.Dangane da fasahar, mun kuma bayyana ci gaban tsarin tsarin sarkar polymerase wanda ya haɗu da gabatarwar reagent, haɗawa da ayyukan amsa duk a cikin tsari ɗaya, wanda ke aiwatar da aikin "samfurin-in-amsa-fita" ga duk samfuran hanci na asibiti daga marasa lafiya 18 mura da 18 mutum controls, a cikin mai kyau concordance na fluorescence tsanani tsanani tare da daidaitattun sarkar polymerase (Pearson coefficients> 0.9).Dangane da fasahar, mun kuma bayyana ci gaban tsarin tsarin sarkar polymerase wanda ke haɗawa da gabatarwar reagent, haɗawa da ayyukan amsa duk a cikin tsari ɗaya, wanda ke cika aikin "samfurin-in-amsa-fita" ga duk samfuran hanci na asibiti daga marasa lafiya 18. tare da mura da kuma 18 mutum controls, a cikin mai kyau concordance na fluorescence tsanani tare da daidaitattun sarkar polymerase (Pearson coefficients> 0.9).Основывая стехой Технологину И ввведения смешивания ороцеспенолнение Karba - Охдоресет Кмнических отов от 18 لمациентов ст 18 пациентов от 18 пациентов и 18 отдельных контролей, в хорошем соответствии и тенсивности флуоресценци со станцертной Hotunan Пирсона> 0,9).Dangane da wannan fasaha, muna kuma bayyana ci gaban tsarin tsarin tsarin tsarin polymerase wanda ya haɗu da ayyuka na yin allura, haɗuwa, da amsawa a cikin tsari guda ɗaya, yana ba da damar samfurin amsawa ga duk samfurori na hanci na asibiti daga marasa lafiya na 18.da 18 daidaitattun sarrafawa, a cikin kyakkyawar yarjejeniya tare da daidaitaccen sarkar polymerase mai ƙarfi mai ƙarfi (ƙwararrun Pearson> 0.9).Dangane da wannan fasaha, mun kuma bayyana ci gaban tsarin tsarin sarkar polymerase wanda ke haɗa allurar reagent, haɗuwa, da ayyukan amsawa don nazarin duk samfuran hanci na asibiti daga 18 a cikin samfuran marasa lafiya na hanci. da kyau tare da daidaitaccen sarkar polymerase (ƙidar Pearson> 0.9).Dandalin da aka tsara yana ba da garantin ingantacciyar sarrafa kansa na nazarin halittu kuma ta haka zai iya haɓaka kasuwancin kewayon na'urorin gwaji na kulawa.
Cututtukan ɗan adam da suka kunno kai, kamar cutar ta COVID-19 ta 2020 wacce ta yi sanadiyyar mutuwar miliyoyin mutane, suna yin babbar barazana ga lafiyar duniya da wayewar ɗan adam1.Tun da wuri, saurin gano cututtuka daidai yana da mahimmanci don sarrafa yaduwar ƙwayar cuta da inganta sakamakon jiyya.Ainihin yanayin yanayin bincike wanda ya dogara da dakunan gwaje-gwaje na tsakiya inda ake aika samfuran gwaji zuwa asibitoci ko asibitocin bincike da kwararru ke gudanarwa a halin yanzu yana hana damar kusan mutane biliyan 5.8 a duk duniya, musamman waɗanda ke rayuwa cikin ƙayyadaddun tsarin albarkatu.inda aka samu karancin kayan aikin likitanci masu tsada da kwararrun kwararru.likitoci 2. Don haka, akwai buƙatar gaggawa don haɓaka tsarin tsarin lab-on-a-chip mara tsada da mai amfani tare da damar gwajin kulawa (POCT) wanda zai iya ba wa likitocin da ke da bayanan bincike na lokaci don yin yanke shawara na ganewar asali. .da magani 3.
Jagororin Hukumar Lafiya ta Duniya (WHO) sun bayyana cewa kyakkyawan POCT ya kamata ya zama mai araha, mai sauƙin amfani (mai sauƙin amfani tare da ƙaramin horo), daidai (kauce wa ƙiyayyar ƙarya ko tabbataccen ƙirƙira), mai sauri kuma abin dogaro (ba da kyawawan kaddarorin maimaitawa), da mai iya bayarwa (mai ikon adana dogon lokaci kuma a shirye yake don masu amfani da ƙarshensa)4.Don saduwa da waɗannan buƙatun, tsarin POCT dole ne ya samar da fasalulluka masu zuwa: m allurai don rage sa hannun hannu, a kan-buƙata saki zuwa sikelin reagent sufuri don ingantacciyar sakamakon gwaji, da ingantaccen aiki don jure girgizar muhalli.A halin yanzu, na'urar POCT da aka fi amfani da ita ita ce tsiri mai gudana ta gefe5,6 wanda ya ƙunshi yadudduka da yawa na membran nitrocellulose mai ƙarfi waɗanda ke tura ɗan ƙaramin adadin samfurin gaba, suna amsawa tare da reagents da aka riga aka yi amfani da su ta ƙarfin capillary.Kodayake suna da fa'idar ƙarancin farashi, sauƙin amfani, da sakamako mai sauri, na'urorin POCT na tushen tsiri za a iya amfani da su kawai don gwaje-gwajen halittu (misali, gwajin glucose7,8 da gwajin ciki9,10) ba tare da buƙatar nazarin matakai da yawa ba.halayen (misali loading na mahara reagents, hadawa, multiplexing).Bugu da kari, sojojin da ke sarrafa motsin ruwa (watau sojojin capillary) ba sa samar da daidaito mai kyau, musamman tsakanin batches, wanda ke haifar da rashin haɓakawa11 da yin maɗaurin raɗaɗi na gefe da farko masu amfani don ganowa mai kyau12,13.
Ƙarfafa ƙarfin masana'antu a micro- da nanoscale sun haifar da dama don haɓaka na'urorin POCT na microfluidic don ma'auni14,15,16,17.Ta hanyar daidaita kaddarorin masu dubawa 18, 19 da lissafi na tashoshi 20, 21, 22, ana iya sarrafa ƙarfin ƙarfin capillary da yawan kwararar waɗannan na'urori.Koyaya, amincin su, musamman don ruwa mai jika sosai, ya kasance ba a yarda da shi ba saboda rashin daidaiton masana'anta, lahani na kayan aiki, da azanci ga girgizar muhalli.Bugu da ƙari, tun lokacin da aka halicci motsi na capillary a mahaɗin ruwa-gas, ba za a iya gabatar da ƙarin kwarara ba, musamman bayan cika tashar microfluidic da ruwa.Don haka, don gano ƙarin rikitarwa, matakai da yawa na allurar samfurin dole ne a yi24,25.
Daga cikin na'urorin microfluidic, na'urorin microfluidic na centrifugal a halin yanzu suna ɗaya daga cikin mafi kyawun mafita don POCT26,27.Tsarin tuƙi yana da fa'ida domin ana iya sarrafa ƙarfin tuƙi ta hanyar daidaita saurin juyawa.Koyaya, rashin lahani shine ƙarfin centrifugal koyaushe yana karkata zuwa gefen waje na na'urar, yana sa wahalar aiwatar da halayen matakai da yawa da ake buƙata don ƙarin hadaddun bincike.Ko da yake ƙarin ƙarfin tuƙi (misali capillaries 28, 29 da sauran su 30, 31, 32, 33, 34, 35) ban da ƙarfin centrifugal ana gabatar da su don multifunctional dosing, canja wurin ruwa da ba a zata ba na iya faruwa saboda waɗannan ƙarin runduna gabaɗaya umarni ne. Girman ƙasa da ƙarfin centrifugal, yana mai da su tasiri kawai akan ƙananan jeri na aiki ko ba samuwa akan buƙata tare da sakin ruwa.Haɗa manipulations na pneumatic cikin microfluidics na centrifugal kamar hanyoyin centrifugal kinetic 36, 37, 38, hanyoyin thermopneumatic 39 da hanyoyin pneumatic masu aiki 40 sun tabbatar da zama madadin kyawawa.Tare da tsarin counterfugodynamic, ƙarin rami da haɗin microchannels an haɗa su cikin na'urar don aikin waje da na ciki, kodayake ingancin aikin sa (a cikin kewayon daga 75% zuwa 90%) ya dogara sosai akan adadin hawan keke da danko. na ruwa.A cikin hanyar thermopneumatic, membrane na latex da ɗakin canja wurin ruwa an ƙera su musamman don rufe ko sake buɗe mashigar lokacin da ƙarar iskan da aka kama ta yi zafi ko sanyaya.Koyaya, saitin dumama/ sanyaya yana gabatar da matsalolin amsa jinkirin kuma yana iyakance amfani dashi a cikin gwaje-gwajen zafin jiki (misali, haɓaka sarkar polymerase (PCR)).Tare da tsarin pneumatic mai aiki, ana samun sakin buƙatu da motsi na ciki ta hanyar aikace-aikacen lokaci guda na ingantacciyar matsi da daidaitattun juzu'i na juyawa ta manyan injuna masu sauri.Akwai wasu hanyoyin da suka yi nasara ta amfani da masu aikin pneumatic kawai (matsi mai kyau 41, 42 ko matsa lamba mara kyau 43) da ƙirar bawul ɗin da aka rufe.Ta hanyar jujjuya matsa lamba a cikin ɗakin pneumatic, ruwan yana jujjuya gaba ta hanyar peristaltically, kuma rufaffiyar bawul ɗin da aka saba yana hana dawowar ruwa saboda peristalsis, don haka fahimtar hadaddun ayyukan ruwa.Koyaya, a halin yanzu akwai iyakataccen adadin fasahar microfluidic waɗanda zasu iya yin hadaddun ayyukan ruwa a cikin na'urar POCT guda ɗaya, gami da rarraba ayyuka da yawa, sakin buƙatu, ingantaccen aiki, adana dogon lokaci, sarrafa ruwa mai ƙarfi, da kuma masana'anta masu tsada.Duk a lokaci guda.Rashin aikin ayyuka masu matakai da yawa na iya zama ɗaya daga cikin dalilan da ya sa wasu samfuran POCT kaɗan ne kawai na kasuwanci kamar Cepheid, Binx, Visby, Cobas Liat, da Rhonda aka samu nasarar gabatar da su a buɗe kasuwa zuwa yau.
A cikin wannan takarda, muna ba da shawarar mai kunnawa microfluidic pneumatic bisa tushen fasahar canza launin kore (FAST).FAST yana haɗa duk mahimman kaddarorin a lokaci guda don ɗimbin kewayon reagents daga microliters zuwa milliliters.FAST ya ƙunshi membranes na roba, levers da tubalan.Ba tare da aikace-aikacen matsa lamba na iska ba, membranes, levers da tubalan za a iya rufe su sosai kuma ana iya adana ruwa a ciki na dogon lokaci.Lokacin da aka yi amfani da matsi mai dacewa da daidaitawa zuwa tsayin lever, diaphragm yana faɗaɗa kuma yana tura lever zuwa wurin budewa, yana barin ruwa ya wuce.Wannan yana ba da damar ƙididdige yawan ayyuka na ruwa a cikin kascade, lokaci guda, jere ko zaɓi.
Mun haɓaka tsarin PCR ta amfani da FAST don samar da amsa-a-samfurin sakamakon gano mura A da B ƙwayoyin cuta (IAV da IBV).Mun sami ƙaramin iyaka na ganowa (LOD) na kwafi/mL 102, ƙididdigar mu ta multix ta nuna ƙayyadaddun ƙayyadaddun IAV da IBV kuma an ba da izinin cutar cututtukan mura.Sakamakon gwaji na asibiti ta amfani da samfurin swab na hanci daga marasa lafiya 18 da 18 masu lafiya suna nuna kyakkyawar daidaituwa a cikin ƙarfin haske tare da daidaitattun RT-PCR (Pearson coefficients> 0.9).Sakamakon gwaji na asibiti ta amfani da samfurin swab na hanci daga marasa lafiya 18 da 18 masu lafiya suna nuna kyakkyawar daidaituwa a cikin ƙarfin haske tare da daidaitattun RT-PCR (Pearson coefficients> 0.9).Результаты клинических испытаний спользованием образца мазка соответствие интенсивности флуоресценци стандартной ОТ-ПЦР (koэффициенты Пирсона> 0,9).Sakamakon gwaje-gwaje na asibiti ta amfani da samfurin swab na hanci daga marasa lafiya 18 da 18 masu lafiya suna nuna kyakkyawar yarjejeniya tsakanin ƙarfin haske na daidaitattun RT-PCR (Pearson's coefficients> 0.9).0.9) ............................................ Результаты клинических испытаний с использованием соответствие между интенсивностью флуоресценции стандартной ОТ-ПЦР (kоэффициент Пирсона > 0,9).Sakamakon gwaje-gwaje na asibiti ta amfani da samfurin swab na hanci daga marasa lafiya 18 da 18 masu lafiya sun nuna kyakkyawar yarjejeniya tsakanin ƙarfin haske da daidaitattun RT-PCR (Pearson's coefficient> 0.9).Ƙididdigar farashin kayan na'urar FAST-POCT kusan dalar Amurka 1 ne (Ƙarin Teburin 1) kuma ana iya ƙara ragewa ta amfani da manyan hanyoyin kera (misali gyare-gyaren allura).A zahiri, na'urorin POCT na tushen FAST suna da duk mahimman abubuwan da WHO ta umarta kuma sun dace da sabbin hanyoyin gwajin sinadarai kamar hawan keke na jini na plasma44, immunoassays-free immunoassays45 da nanobody functionalization tests46 waɗanda sune kashin bayan tsarin POCT.yiwuwa.
A kan fig.1a yana nuna tsarin dandalin FAST-POCT, wanda ya ƙunshi ɗakunan ruwa guda huɗu: ɗakin ajiya na farko, ɗakin hadawa, ɗakin amsawa, da ɗakin sharar gida.Makullin sarrafa kwararar ruwa shine ƙirar FAST (wanda ya ƙunshi membranes na roba, levers da tubalan) waɗanda ke cikin ɗakin da aka riga aka adana da ɗakin hadawa.A matsayin hanyar da aka kunna ta huhu, ƙirar FAST tana ba da daidaitaccen sarrafa ruwan ruwa, gami da rufewa/buɗe sauyawa, madaidaicin allurai, sakin ruwa akan buƙatu, ingantaccen aiki (misali, rashin kula da girgizar muhalli), da adana dogon lokaci.Dandalin FAST-POCT ya ƙunshi nau'i huɗu: Layer na baya, Layer na fim na roba, Layer na fim ɗin filastik, da murfin murfin, kamar yadda aka nuna a cikin babban ra'ayi a cikin siffa 1b (wanda kuma aka nuna dalla-dalla a Ƙarin Figures S1 da S2 ).Duk tashoshi da ɗakunan jigilar ruwa (kamar ajiyar ajiya da ɗakuna masu amsawa) an saka su a cikin PLA (polylactic acid) abubuwan da ke jere daga 0.2 mm (banshi mafi ƙarancin) zuwa 5 mm lokacin farin ciki.Kayan fim ɗin na roba shine PDMS mai kauri 300 µm wanda ke faɗaɗa cikin sauƙi lokacin da ake amfani da matsa lamba ta iska saboda “kauri mai kauri” da ƙarancin elasticity (kimanin 2.25 MPa47).Fim ɗin fim ɗin polyethylene an yi shi da polyethylene terephthalate (PET) tare da kauri na 100 µm don kare fim ɗin roba daga nakasar da ta wuce kima saboda matsin iska.Daidai da ɗakuna, madaidaicin Layer yana da levers da aka haɗa zuwa murfin murfin (wanda aka yi da PLA) ta hinges don sarrafa kwararar ruwa.Fim ɗin na roba an manne shi zuwa layin baya ta amfani da tef ɗin manne mai gefe biyu (ARseal 90880) kuma an rufe shi da fim ɗin filastik.An haɗu da yadudduka uku a kan ma'auni ta amfani da ƙirar T-clip a cikin murfin murfin.T-clamp yana da tazara tsakanin ƙafafu biyu.Lokacin da aka saka faifan bidiyo a cikin ramin, ƙafafu biyun sun dan lanƙwasa kaɗan, sannan su koma yanayinsu na asali kuma suka daure murfin da baya yayin da suke wucewa ta cikin ramin (Ƙarin Hoton S1).Sannan ana harhada yadudduka huɗu ta hanyar amfani da masu haɗawa.
Tsarin tsari na dandamali wanda ke kwatanta sassa daban-daban na aiki da fasalulluka na FAST.b Girman zane na dandalin FAST-POCT.c Hoton dandamalin da ke kusa da kwata kwata kwata kwata na dalar Amurka.
Ana nuna tsarin aiki na dandalin FAST-POCT a cikin Hoto na 2. Mahimman abubuwan da aka haɗa su ne tubalan a kan tushe na tushe da kuma hinges a kan murfin murfin, wanda ya haifar da tsangwama lokacin da aka haɗa nau'i hudu ta hanyar amfani da siffar T. .Lokacin da ba a yi amfani da matsa lamba na iska ba (fig. 2a), tsangwama mai dacewa yana haifar da hinge don lankwasa da lalacewa, kuma ana amfani da ƙarfin rufewa ta hanyar lever don danna fim ɗin roba a kan toshe, kuma an bayyana ruwa a cikin rami na hatimi. a matsayin jihar da aka rufe.Ya kamata a lura cewa a cikin wannan jiha, an lanƙwasa lever a waje, kamar yadda aka nuna a gefen gefen a cikin Fig. 2a.Lokacin da aka ba da iska (Fig. 2b), membrane na roba yana faɗaɗa waje zuwa murfin kuma yana tura lever sama, don haka buɗe rata tsakanin lefa da toshe don ruwa ya gudana zuwa ɗakin na gaba, wanda aka bayyana a matsayin buɗaɗɗen yanayi. .Bayan sakin iska, lever zai iya komawa zuwa matsayinsa na asali kuma ya kasance da ƙarfi saboda elasticity na hinge.Ana gabatar da bidiyon motsin lefa a cikin ƙarin fim ɗin S1.
A. Tsarin tsari da hotuna lokacin rufewa.Idan babu matsa lamba, lever yana danna membrane a kan toshe, kuma an rufe ruwa.b A cikin yanayi mai kyau.Lokacin da aka matsa lamba, membrane yana faɗaɗa kuma yana tura lever sama, don haka tashar ta buɗe kuma ruwa zai iya gudana.c Ƙayyade girman halayen matsi mai mahimmanci.Girman halayen sun haɗa da tsayin lefa (L), nisa tsakanin maɗauri da hinge (l) da kaurin firar lever (t).Fs shine ƙarfin daɗaɗɗa a madaidaicin ma'aunin B. q shine nauyin da aka rarraba iri ɗaya akan lefa.Tx* yana wakiltar juzu'in da aka ƙera ta lever mai hinged.Matsakaicin matsa lamba shine matsa lamba da ake buƙata don ɗaga lever da sa ruwa ya gudana.d Sakamakon nazari da gwaji na dangantaka tsakanin matsi mai mahimmanci da girman kashi.n = 6 gwaje-gwaje masu zaman kansu an yi su kuma an nuna bayanai azaman ± daidaitaccen karkatacciyar hanya.Ana gabatar da ɗanyen bayanai azaman fayilolin bayanan ɗanyen.
An ƙirƙiri samfurin nazari bisa ka'idar katako don nazarin dogaro da matsananciyar matsa lamba Pc wanda rata ta buɗe akan sigogin geometric (alal misali, L shine tsayin lefa, l shine nisa tsakanin toshe da hinge, S shine lefa Yankin lamba tare da ruwa t shine kauri na fitowar lever, kamar yadda aka nuna a cikin siffa 2c).Kamar yadda cikakken bayani a cikin Ƙarin Bayanan kula da Ƙarin Hoto S3, ratar yana buɗewa lokacin da \({P}_{c}\ge \frac{2{F}_{s}l}{SL}\), inda Fs shine magudanar ruwa. ({T}_{x}^{\ast}(={F}_{s}l)\) don kawar da dakarun da ke da alaƙa da tsangwama da kuma haifar da tanƙwara.Amsar gwaji da samfurin nazari suna nuna kyakkyawar yarjejeniya (Fig. 2d), yana nuna cewa matsananciyar matsa lamba Pc yana ƙaruwa tare da ƙara t / l da rage L, wanda aka kwatanta da sauƙi ta hanyar ƙirar katako na gargajiya, watau karfin juyi yana ƙaruwa tare da t / ɗagawa. .Don haka, nazarin ka'idar mu ya nuna a fili cewa za a iya sarrafa matsa lamba mai mahimmanci ta hanyar daidaita tsayin lever L da t / l rabo, wanda ke ba da muhimmiyar mahimmanci don tsara tsarin dandalin FAST-POCT.
Dandalin FAST-POCT yana ba da kayan aiki da yawa (wanda aka nuna a cikin Hoto 3a tare da shigarwa da gwaji), wanda shine mafi mahimmancin fasalin POCT mai nasara, inda ruwa zai iya gudana a kowace hanya kuma a kowane tsari (cascade, lokaci guda, jere) ko zaɓin multichannel. rarrabawa .- aikin dosing.A kan fig.3a(i) yana nuna yanayin juzu'i wanda ɗakuna biyu ko sama da haka ke ruɓe ta amfani da tubalan don raba raƙuman raɗaɗi daban-daban da lever don sarrafa buɗaɗɗen jihohi da rufaffiyar.Lokacin da aka matsa lamba, ruwan yana gudana daga sama zuwa ƙasa a cikin hanyar da ba ta dace ba.Ya kamata a lura cewa za a iya cika ɗakunan kascade da sinadarai masu jika ko busassun sinadarai irin su lyophilized powders.A cikin gwaji a cikin siffa 3a (i), jan tawada daga ɗakin sama yana gudana tare da shuɗi mai launin shuɗi (Copper sulfate) zuwa cikin ɗaki na biyu kuma ya juya shuɗi mai duhu lokacin da ya isa ɗakin ƙasa.Hakanan yana nuna matsi na sarrafa ruwan da ake zuƙowa.Hakazalika, lokacin da aka haɗa lefa ɗaya zuwa ɗakuna biyu, ya zama yanayin allura a lokaci ɗaya, kamar yadda aka nuna a fig.3a(ii), wanda za'a iya rarraba ruwa daidai gwargwado akan ɗakuna biyu ko fiye lokacin da aka matsa lamba.Tun da matsa lamba mai mahimmanci ya dogara da tsawon lever, za a iya daidaita tsawon lever don cimma tsarin allura na jere kamar yadda aka nuna a cikin fig.3 (iii).An haɗa dogon lever (tare da matsananciyar matsa lamba Pc_long) zuwa ɗakin B kuma an haɗa ɗan gajeren lever (tare da matsananciyar matsa lamba Pc_short> Pc_long) zuwa ɗakin A. Kamar yadda matsa lamba P1 (Pc_long <P1 <Pc_short) aka yi amfani da shi, kawai ruwa a cikin ja. zai iya gudana zuwa ɗakin B kuma lokacin da aka ƙara matsa lamba zuwa P2 (> Pc_short), ruwan shuɗi zai iya gudana zuwa ɗakin A. Wannan tsarin allura na yau da kullum ya shafi ruwa daban-daban da ke canjawa zuwa ɗakunan da ke da alaƙa a jere, wanda ke da mahimmanci ga POCT mai nasara. na'urar.An haɗa dogon lever (tare da matsananciyar matsa lamba Pc_long) zuwa ɗakin B kuma an haɗa ɗan gajeren lever (tare da matsananciyar matsa lamba Pc_short> Pc_long) zuwa ɗakin A. Kamar yadda matsa lamba P1 (Pc_long
Misali na ayyuka da yawa, watau (i) cascading, (ii) lokaci guda, (iii) jere, da (iv) zaɓen aiki.Hanyoyi suna wakiltar tafiyar aiki da sigogin waɗannan hanyoyin rarraba guda huɗu.b Sakamakon gwaje-gwajen ajiya na dogon lokaci a cikin ruwa mai lalacewa da ethanol.n = 5 gwaje-gwaje masu zaman kansu an yi su kuma an nuna bayanai azaman ± sd c.Gwajin kwanciyar hankali lokacin da na'urar FAST da na'urar bawul (CV) ke cikin (i) a tsaye da (ii) jihohi masu girgiza.(iii) Volume vs. lokaci don FAST da na'urorin CV a mitoci daban-daban na kusurwoyi.d Buga sakamakon gwaji akan buƙatar (i) na'urar FAST da (ii) na'urar CV.(iii) Dangantaka tsakanin ƙara da lokaci don FAST da na'urorin CV ta amfani da yanayin matsa lamba.Duk sandunan sikelin, 1 cm.Ana ba da albarkatun ɗanyen bayanai azaman fayilolin ɗanyen bayanai.
Dogon lokaci ajiya na reagents wani muhimmin fasali ne na na'urar POCT mai nasara wanda zai ba da damar ma'aikatan da ba a horar da su ba don ɗaukar reagents da yawa.Duk da yake yawancin fasahohi sun nuna yuwuwar su don adanawa na dogon lokaci (misali, 35 microdispensers, fakitin blister 48, da fakitin sandar 49), ana buƙatar ɗaki mai karɓa don ɗaukar fakitin, wanda ke haɓaka farashi da rikitarwa;Bugu da ƙari, waɗannan hanyoyin ajiya ba sa ba da izinin bayarwa akan buƙatu kuma suna haifar da ɓarna na reagents saboda ragowar da ke cikin marufi.An tabbatar da iyawar ajiya na dogon lokaci ta hanyar gudanar da gwajin rayuwa mai sauri ta amfani da kayan aikin PMMA na CNC saboda ƙarancin ƙarancinsa da juriya ga iskar gas (Ƙarin Hoto S5).Na'urar gwajin ta cika da ruwa da aka lalata (ruwan da aka ɗora) da 70% ethanol (mai yin sinadarai masu canzawa) a 65 ° C na kwanaki 9.Dukansu ruwan da aka cire da kuma ethanol an adana su ta amfani da foil na aluminum don toshe shiga daga sama.Ƙididdigar Arrhenius da kuzarin kunnawa shigar da aka ruwaito a cikin wallafe-wallafe50,51 an yi amfani da su don lissafin daidai lokacin.A kan fig.3b yana nuna matsakaicin sakamakon asarar nauyi don samfuran 5 da aka adana a 65 ° C na kwanaki 9, daidai da 0.30% don ruwan da aka lalata da 0.72% don 70% ethanol sama da shekaru 2 a 23 ° C.
A kan fig.3c yana nuna gwajin girgiza.Tunda bawul ɗin capillary (CV) shine mafi mashahuri hanyar sarrafa ruwa tsakanin na'urorin POCT28,29 data kasance, an yi amfani da na'urar CV mai faɗi 300 µm da zurfin 200 µm don kwatanta.Ana iya ganin cewa lokacin da na'urorin biyu suka tsaya a tsaye, ruwan da ke cikin dandalin FAST-POCT yana rufewa kuma ruwan da ke cikin na'urar CV yana kullewa saboda fadada tashar kwatsam, wanda ke rage karfin karfin.Duk da haka, yayin da mitar kusurwar maƙarƙashiya na orbital vibrator ke ƙaruwa, ruwan da ke cikin dandalin FAST-POCT ya kasance a rufe, amma ruwan da ke cikin na'urar CV yana gudana zuwa cikin ƙananan ɗakin (duba kuma Ƙarin Fim S3).Wannan yana nuna cewa madaidaitan hinges na dandalin FAST-POCT na iya amfani da ƙarfin injina mai ƙarfi zuwa ƙirar don rufe ruwan da ke cikin ɗakin.Koyaya, a cikin na'urorin CV, ana riƙe ruwa saboda ma'auni tsakanin ƙaƙƙarfan, iska, da matakan ruwa, haifar da rashin kwanciyar hankali, da rawar jiki na iya ɓata ma'auni kuma haifar da halayen kwararar da ba a zata ba.Amfanin dandalin FAST-POCT shine cewa yana samar da ingantaccen aiki kuma yana guje wa kasawa a gaban girgizar da ke faruwa a lokacin bayarwa da aiki.
Wani muhimmin fasalin dandali na FAST-POCT shine sakin da ake buƙata, wanda shine mabuɗin mahimmanci don ƙididdige ƙididdiga.A kan fig.3d yana kwatanta sakin da ake buƙata na dandalin FAST-POCT da na'urar CV.Daga fig.3d (iii) mun ga cewa na'urar FAST tana amsawa da sauri zuwa siginar matsa lamba.Lokacin da aka yi amfani da matsa lamba a kan dandalin FAST-POCT, ruwan yana gudana, lokacin da aka saki matsa lamba, nan da nan ya tsaya (Fig. 3d (i)).Za'a iya bayyana wannan aikin ta hanyar saurin dawowar ƙwanƙwasa na hinge, wanda ke danna lever baya a kan toshe, rufe ɗakin.Koyaya, ruwa ya ci gaba da gudana a cikin na'urar CV, a ƙarshe yana haifar da ƙarar ruwan da ba zato ba tsammani na kusan 100 µl bayan an saki matsin lamba (Hoto 3d(ii) da Ƙarin Fim S4).Ana iya bayyana wannan ta bacewar tasirin tasirin capillary akan cikakken jika na CV bayan allurar farko.
Ƙarfin sarrafa ruwa na sãɓãwar launukansa mai laushi da danko a cikin na'ura ɗaya ya kasance ƙalubale ga aikace-aikacen POCT.Rashin ruwa mara kyau na iya haifar da leaks ko wasu halayen kwararar da ba zato ba tsammani a cikin tashoshi, kuma ana buƙatar kayan aiki irin su vortex mixers, centrifuges da filters sau da yawa don shirya ruwa mai danko 52 .Mun gwada dangantakar dake tsakanin matsa lamba mai mahimmanci da kaddarorin ruwa (tare da kewayon wettability da danko).Ana nuna sakamakon a Table 1 da Bidiyo S5.Ana iya ganin cewa za'a iya rufe ruwa na ruwa daban-daban da danko a cikin ɗakin, kuma lokacin da aka yi amfani da matsa lamba, har ma da ruwa mai danko har zuwa 5500 cP za a iya canjawa wuri zuwa ɗakin da ke kusa, yana sa ya yiwu a gano samfurori tare da high. danko (watau sputum, samfurin danko sosai da ake amfani da shi don gano cututtukan numfashi).
Ta hanyar haɗa na'urorin rarraba kayan aiki masu yawa na sama, ana iya haɓaka kewayon na'urorin POCT masu fa'ida da yawa.An nuna misali a cikin Hoto 1. Gidan yana ƙunshe da ɗakin ajiya na farko, ɗakin hadawa, ɗakin amsawa da ɗakin sharar gida.Za'a iya adana reagents a cikin ɗakin da aka riga aka adana na tsawon lokaci mai tsawo sannan a fitar da su cikin ɗakin hadawa.Tare da matsi da ya dace, da gauraye reactants za a iya selectively canjawa wuri zuwa sharar gida dakin ko dauki dauki.
Saboda gano PCR shine ma'aunin zinare don gano ƙwayoyin cuta kamar H1N1 da COVID-19 kuma ya ƙunshi matakan amsawa da yawa, mun yi amfani da dandalin FAST-POCT don gano PCR azaman aikace-aikace.A kan fig.4 yana nuna tsarin gwajin PCR ta amfani da dandalin FAST-POCT.Na farko, an bututun reagent na eluting, Magnetic microbead reagent, maganin wankin A, da maganin wankin W a cikin ɗakunan ajiya na farko E, M, W1 da W2, bi da bi.Ana nuna matakan tallan RNA a cikin fig.4a kuma sune kamar haka: (1) lokacin da aka yi amfani da matsa lamba P1 (= 0.26 mashaya), samfurin yana motsawa cikin ɗakin M kuma an fitar da shi a cikin ɗakin haɗuwa.(2) Matsin iska P2 (= 0.12 mashaya) ana kawota ta tashar jiragen ruwa A da aka haɗa zuwa kasan ɗakin hadawa.Ko da yake da dama hanyoyin hadawa sun nuna yuwuwarsu wajen hada ruwa akan dandamalin POCT (misali hadawar maciji 53, hadawa bazuwar 54 da hadawa batch 55), ingancin hadawarsu da ingancinsu har yanzu basu gamsar ba.Yana ɗaukar hanyar haɗa kumfa, wanda aka shigar da iska a cikin kasan ɗakin hadawa don ƙirƙirar kumfa a cikin ruwa, bayan haka maƙarƙashiya mai ƙarfi zai iya cimma cikakkiyar haɗuwa cikin daƙiƙa.An gudanar da gwaje-gwajen haɗewar kumfa kuma an gabatar da sakamakon a Ƙarin Hoto S6.Ana iya ganin cewa lokacin da aka yi amfani da matsi na 0.10, cikakken haɗuwa yana ɗaukar kimanin daƙiƙa 8.Ta hanyar ƙara matsa lamba zuwa mashaya 0.20, ana samun cikakkiyar haɗuwa a cikin kusan daƙiƙa 2.Ana gabatar da hanyoyin ƙididdige ingancin hadawa a cikin sashin hanyoyin.(3) Yi amfani da magnetin rubidium don cire beads, sannan danna P3 (= 0.17 mashaya) ta tashar P don matsar da reagents zuwa cikin sharar gida.A kan fig.4b,c yana nuna matakan wankewa don cire ƙazanta daga samfurin kamar haka: (1) Ana fitar da maganin wanki A daga ɗakin W1 a cikin ɗakin haɗuwa da matsa lamba P1.(2) Sannan a yi aikin hada kumfa.(3) Ana canja wurin maganin wankin A zuwa ɗakin sharar gida, kuma ana fitar da microbeads a cikin ɗakin haɗuwa ta hanyar maganadisu.Wankewa W (Fig. 4c) yayi kama da wankan A (Fig. 4b).Ya kamata a lura cewa kowane mataki na wankewa A da W an yi sau biyu.Hoto na 4d yana nuna matakan haɓakawa don cire RNA daga beads;Matakan gabatarwa da haɓakawa iri ɗaya ne da tallan RNA da matakan wankewa da aka bayyana a sama.Yayin da ake tura reagents na haɓakawa zuwa ɗakin amsawar PCR a ƙarƙashin matsin P3 da P4 (= 0.23 mashaya), matsananciyar matsananciyar an kai don rufe hannun ɗakin amsawar PCR.Hakazalika, matsa lamba P4 kuma yana taimakawa wajen rufe hanyar zuwa ɗakin sharar gida.Don haka, an rarraba duk reagents a ko'ina a tsakanin ɗakunan amsawar PCR guda huɗu don fara halayen PCR mai yawa.An gabatar da tsarin da ke sama a cikin Ƙarin Fim ɗin S6.
A cikin matakin adsorption na RNA, ana gabatar da samfurin a cikin mashigar M kuma a yi masa allura cikin ɗakin hadawa tare da maganin bead da aka adana a baya.Bayan haɗuwa da cire granules, ana rarraba reagents a cikin ɗakin sharar gida.b da c wanke matakan, gabatar da nau'ikan kayan wankewa da aka riga aka adana a cikin ɗakin hadawa, sannan bayan haɗuwa da cire beads, canja wurin reagents zuwa ɗakin sharar gida.d Matakin hasashe: Bayan gabatar da reagents na elution, haɗewa da cire katako, ana tura reagents zuwa ɗakin amsawar PCR.Hanyoyi suna nuna tsarin aiki da sigogi masu alaƙa na matakai daban-daban.Matsi shine matsin lamba da ake yi ta ɗaiɗaikun ɗakuna.Ƙarar ita ce ƙarar ruwa a cikin ɗakin hadawa.Duk sandunan sikelin suna da 1 cm.Ana ba da albarkatun ɗanyen bayanai azaman fayilolin ɗanyen bayanai.
An gudanar da tsarin gwajin PCR kuma Ƙarin Hoto S7 yana ba da bayanan bayanan zafi ciki har da mintuna 20 na lokacin jujjuyawar lokaci da mintuna 60 na lokacin hawan keke (95 da 60 ° C), tare da zagayowar thermal ɗaya shine 90 s (Ƙarin Fim ɗin S7)..FAST-POCT yana buƙatar ƙasa da lokaci don kammala zagayowar thermal ɗaya (daƙiƙa 90) fiye da na al'ada RT-PCR (daƙiƙa 180 don zagayowar thermal ɗaya).Ana iya bayyana wannan ta wurin babban yanki zuwa girman rabo da ƙarancin ƙarancin zafi na ɗakin amsawar micro-PCR.Fuskar ɗakin shine 96.6 mm2 kuma ƙarar ɗakin shine 25 mm3, yana yin ƙasa zuwa girman girman kusan 3.86.Kamar yadda aka gani a Ƙarin Hoto S10, yankin gwajin PCR na dandalinmu yana da tsagi a kan bangon baya, yana mai da ƙasan ɗakin PCR 200 µm kauri.Ana manne da kushin roba mai zafin zafi zuwa saman dumama mai kula da zafin jiki, yana tabbatar da kusancin bayan akwatin gwaji.Wannan yana rage rashin ƙarfi na thermal na dandamali kuma yana inganta haɓakar dumama / sanyaya.A lokacin hawan keken zafi, paraffin ɗin da ke cikin dandali yana narkewa kuma yana gudana cikin ɗakin amsawar PCR, yana aiki azaman mai ɗaukar hoto don hana ƙawancewar reagent da gurɓatar muhalli (duba Ƙarin Fim ɗin S8).
Duk hanyoyin gano PCR da aka kwatanta a sama an yi su da sarrafa kansu ta amfani da na'urar da aka yi FAST-POCT na al'ada, wanda ya ƙunshi na'ura mai sarrafa matsa lamba, na'urar cirewar maganadisu, na'urar sarrafa zafin jiki, da na'urar kama siginar kyalli da na'urar sarrafawa.Na lura, mun yi amfani da dandalin FAST-POCT don warewar RNA sannan muka yi amfani da samfuran RNA da aka fitar don halayen PCR ta amfani da tsarin FAST-POCT da tsarin PCR na tebur don kwatantawa.Sakamakon ya kusan iri ɗaya kamar yadda aka nuna a Ƙarin Hoto S8.Mai aiki yana yin aiki mai sauƙi: gabatar da samfurin a cikin ɗakin M kuma ya shigar da dandamali a cikin kayan aiki.Ana samun sakamakon gwajin ƙima a cikin kusan mintuna 82.Ana iya samun cikakken bayani game da kayan aikin FAST-POCT a ƙarin adadi.C9, C10 da C11.
Mura da mura A (IAV), B (IBV), C (ICV) da D (IDV) ƙwayoyin cuta ke haifar da shi wani lamari ne da ya zama ruwan dare gama duniya.Daga cikin waɗannan, IAV da IBV ne ke da alhakin mafi munin lokuta da annoba na yanayi, suna kamuwa da 5-15% na yawan mutanen duniya, suna haifar da mummunan yanayi na 3-5 miliyan kuma suna haifar da mutuwar 290,000-650,000 a kowace shekara.Cututtukan numfashi56,57.Farkon ganewar asali na IAV da IB yana da mahimmanci don rage cututtuka da nauyin tattalin arziki mai alaƙa.Daga cikin hanyoyin bincike da ake samu, ana ɗaukar juyar da juzu'i na sarkar polymerase (RT-PCR) mafi mahimmanci, ƙayyadaddun, kuma daidai (> 99%)58,59.Daga cikin hanyoyin bincike da ake samu, ana ɗaukar juyar da juzu'i na sarkar polymerase (RT-PCR) mafi mahimmanci, ƙayyadaddun, kuma daidai (> 99%)58,59.Среди доступных диагностических методов полимеразная цепная реакция с обратной транскриптазой тельной, специфичной и точной (> 99%)58,59.Daga cikin hanyoyin bincike da ake samu, ana ɗaukar juyar da transcriptase polymerase chain reaction (RT-PCR) mafi mahimmanci, ƙayyadaddun kuma daidai (> 99%)58,59. Из доступных диагностических методов полимеразная цепная реакция льной, специфичной и точной (>99%)58,59.Daga cikin hanyoyin bincike da ake da su, ana ɗaukar juyar da juzu'i na sarkar polymerase (RT-PCR) mafi mahimmanci, ƙayyadaddun kuma daidai (> 99%)58,59.Koyaya, hanyoyin RT-PCR na al'ada suna buƙatar maimaita bututu, haɗawa, rarrabawa da canja wurin ruwa, iyakance amfani da su ta ƙwararru a cikin iyakantattun saitunan albarkatu.Anan, an yi amfani da dandalin FAST-POCT don gano PCR na IAV da IBV, bi da bi, don samun ƙananan iyakokin gano su (LOD).Bugu da ƙari, IAV da IBV sun kasance masu yawa don nuna bambanci tsakanin nau'i-nau'i daban-daban a cikin nau'i-nau'i, suna samar da dandamali mai ban sha'awa don nazarin kwayoyin halitta da kuma ikon magance cutar daidai.
A kan fig.5a yana nuna sakamakon gwajin HAV PCR ta amfani da 150 µl na RNA mai tsaftataccen hoto azaman samfuri.A kan fig.5a (i) ya nuna cewa a cikin nauyin HAV na kwafin 106 / ml, ƙarfin haske (ΔRn) zai iya kaiwa 0.830, kuma lokacin da aka rage maida hankali zuwa 102 kofe / ml, ΔRn na iya kaiwa 0.365, wanda ya dace da mafi girma fiye da haka. na rukunin mara kyau mara kyau (0.002), kusan sau 100 mafi girma.Don ƙididdigewa dangane da gwaje-gwaje masu zaman kansu guda shida, an samar da madaidaicin daidaitawar layi tsakanin tattarawar log da madaidaicin zagayowar (Ct) na IAV (Fig. 5a (ii)), R2 = 0.993, kama daga 102-106 kwafi / mL.Sakamakon yana cikin kyakkyawar yarjejeniya tare da hanyoyin RT-PCR na al'ada.A kan fig.5a (iii) yana nuna hotunan kyalli na sakamakon gwaji bayan zagayowar 40 na dandalin FAST-POCT.Mun gano cewa dandalin FAST-POCT na iya gano HAV a matsayin ƙasa da 102 kofe/mL.Koyaya, hanyar gargajiya ba ta da ƙimar Ct a kwafin 102/mL, yana mai da shi LOD na kusan kwafi 103/mL.Mun yi hasashe cewa hakan na iya kasancewa saboda babban inganci na hada kumfa.An yi gwajin gwajin PCR akan IAV RNA mai tsafta don kimanta hanyoyin hadawa daban-daban, gami da hada-hadar girgiza (hanyar hadawa iri daya kamar yadda ake yi a cikin aikin RT-PCR na al'ada), hadawar vial (wannan hanyar, 3 s a mashaya 0.12) kuma babu hadawa azaman ƙungiyar kulawa. ..Ana iya samun sakamakon a Ƙarin Hoto S12.Ana iya ganin cewa a mafi girma RNA maida hankali (106 kofe / ml), da Ct dabi'u na daban-daban hadawa hanyoyin kusan iri daya ne da kumfa hadawa.Lokacin da maida hankali na RNA ya ragu zuwa kwafin 102/mL, mahaɗin girgizawa da sarrafawa ba su da ƙimar Ct, yayin da hanyar haɗin kumfa har yanzu tana ba da ƙimar Ct na 36.9, wanda ke ƙasa da madaidaicin Ct na 38. Sakamakon ya nuna babban halayen haɗuwa. vesicles, wanda kuma aka nuna a cikin wasu wallafe-wallafen, wanda kuma zai iya bayyana dalilin da yasa hankalin FAST-POCT dandamali ya dan kadan fiye da RT-PCR na al'ada.A kan fig.5b yana nuna sakamakon binciken PCR na samfuran IBV RNA da aka tsarkake daga 101 zuwa 106 kwafi/ml.Sakamakon ya yi kama da gwajin IAV, samun R2 = 0.994 da LOD na kwafi 102/mL.
Binciken PCR na cutar mura A (IAV) tare da ƙididdigar IAV daga 106 zuwa 101 kwafi / ml ta amfani da buffer TE azaman iko mara kyau (NC).(i) Matsakaicin kyalli na ainihin lokacin.(ii) Madaidaicin daidaitawa na layi tsakanin logarithmic IAV RNA maida hankali da madaidaicin zagayowar (Ct) don FAST da hanyoyin gwaji na al'ada.(iii) Hoton IAV FAST-POCT mai kyalli bayan zagayowar 40.b, Gano PCR na cutar mura B (IBV) tare da (i) bakan haske na ainihi.(ii) Madaidaicin daidaitawa na layi da (iii) Hoton mai kyalli na FAST-POCT IBV bayan zagayowar 40.Ƙananan iyaka na ganowa (LOD) don IAV da IBV ta amfani da dandalin FAST-POCT shine 102 kofi / mL, wanda ya fi ƙasa da hanyoyin al'ada (103 kwafi / mL).c Sakamakon gwajin Multiplex na IAV da IBV.An yi amfani da GAPDH azaman kulawa mai kyau kuma an yi amfani da buffer TE azaman iko mara kyau don hana yiwuwar gurɓatawa da haɓaka bayanan baya.Za a iya bambance nau'ikan samfurin guda huɗu: (1) Gapdh-kawai Badayoyin kuɗi ("IAV-- / IBV-");(2) kamuwa da cutar IAV ("IAV+/IBV-") tare da IAV da GAPDH;(3) kamuwa da cutar IBV ("IAV-/IBV+") tare da IBV da GAPDH;(4) Cutar IAV/IBV ("IAV+/IBV+") tare da IAV, IBV da GAPDH.Layin da aka dige yana wakiltar layin kofa.n = 6 an yi gwaje-gwaje masu zaman kansu na ilimin halitta, ana nuna bayanai azaman ± daidaitaccen karkacewa.Ana gabatar da ɗanyen bayanai azaman fayilolin ɗanyen bayanai.
A kan fig.5c yana nuna sakamakon gwajin da yawa na IAV/IBV.Anan, an yi amfani da lysate na ƙwayar cuta azaman samfurin samfurin a madadin RNA mai tsafta, kuma an ƙara abubuwa guda huɗu don IAV, IBV, GAPDH (mai sarrafawa mai kyau) da TE buffer (ikon mara kyau) zuwa ɗakuna huɗu daban-daban na dandalin FAST-POCT.Ana amfani da sarrafawa mai kyau da mara kyau anan don hana yuwuwar gurɓatawa da haɓaka bayanan baya.An raba gwaje-gwajen zuwa kungiyoyi hudu: (1) samfurori marasa kyau na GAPDH ("IAV-/IBV-");(2) Cutar IAV ("IAV+/IBV-") tare da IAV da GAPDH;(3) IBV-.kamuwa da cuta (“IAV-”) -/IBV+”) IBV da GAPDH;(4) IAV/IBV ("IAV+/IBV+") kamuwa da cuta tare da IAV, IBV da GAPDH.A kan fig.5c ya nuna cewa lokacin da aka yi amfani da samfurori mara kyau, ƙarfin ƙarfin ΔRn na ɗakin kulawa mai kyau shine 0.860, kuma ΔRn na IAV da IBV sun kasance kama da mummunan iko (0.002).Don ƙungiyoyin IAV +/IBV-, IAV-/IBV + da IAV +/ IBV +, kyamarorin IAV/GAPDH, IBV/GAPDH da IAV/IBV/GAPDH sun nuna tsananin haske, bi da bi, yayin da sauran kyamarori har ma sun nuna ƙarfin haske a bango. matakin 40 bayan hawan keke na thermal.Daga gwaje-gwajen da ke sama, dandali na FAST-POCT ya nuna ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun bayanai kuma ya ba mu damar yin nau'in ƙwayoyin cuta daban-daban a lokaci guda.
Don tabbatar da cancantar asibiti na FAST-POCT, mun gwada samfurori na asibiti na 36 (samfurin swab na hanci) daga marasa lafiya na IB (n = 18) da kuma masu sarrafa IB (n = 18) (Hoto 6a).An gabatar da bayanin marasa lafiya a cikin ƙarin Teburin 3. An tabbatar da matsayin kamuwa da cutar ta IB da kansa kuma an amince da ka'idar binciken daga Asibitin Haɗin Kan Jami'ar Zhejiang (Hangzhou, Zhejiang).An raba kowane samfurin marasa lafiya zuwa kashi biyu.An sarrafa ɗaya ta hanyar amfani da FAST-POCT, ɗayan kuma an sarrafa ta ta amfani da tsarin PCR na tebur (SLAN-96P, China).Dukkanin gwaje-gwajen suna amfani da kayan aikin tsarkakewa da ganowa iri ɗaya.A kan fig.6b yana nuna sakamakon FAST-POCT da PCR na juyi na al'ada (RT-PCR).Mun kwatanta ƙarfin haske (FAST-POCT) tare da -log2 (Ct), inda Ct shine madaidaicin zagayowar don RT-PCR na al'ada.An yi yarjejeniya mai kyau tsakanin hanyoyin biyu.FAST-POCT da RT-PCR sun nuna kyakkyawar alaƙa mai ƙarfi tare da ƙimar ƙimar Pearson (r) na 0.90 (Hoto 6b).Daga nan mun tantance daidaiton bincike na FAST-POCT.An ba da rarrabuwa mai ƙarfi na fluorescence (FL) don samfurori masu kyau da marasa kyau a matsayin ma'auni na nazari mai zaman kansa (Fig. 6c).Ma'auni na FL sun kasance mafi girma a cikin marasa lafiya na IB fiye da masu sarrafawa (**** P = 3.31 × 10-19; T-test mai wutsiya biyu) (Fig. 6d).Na gaba, IBV mai karɓar aiki halaye (ROC) an ƙulla ƙira.Mun gano cewa daidaiton bincike yana da kyau sosai, tare da yanki a ƙarƙashin madaidaicin 1 (Fig. 6e).Lura cewa saboda odar abin rufe fuska na tilas a China saboda COVID-19 har zuwa 2020, ba mu gano marasa lafiya da IBD ba, don haka duk ingantattun samfuran asibiti (watau samfuran swab na hanci) na IBV ne kawai.
Tsarin nazarin asibiti.Jimlar samfurori na 36, ciki har da samfurori na marasa lafiya na 18 da 18 marasa lafiya marasa mura, an bincika su ta amfani da dandalin FAST-POCT da RT-PCR na al'ada.b Tantance daidaiton nazari tsakanin FAST-POCT PCR da RT-PCR na al'ada.Sakamakon ya kasance daidai daidai (Pearson r = 0.90).c matakan ƙarfin Fluorescence a cikin marasa lafiya na 18 IB da sarrafawa 18.d A cikin marasa lafiya na IB (+), ƙimar FL sun fi girma fiye da a cikin ƙungiyar kulawa (-) (**** P = 3.31 × 10-19; t-test mai wutsiya biyu; n = 36).Ga kowane filin murabba'i, alamar baƙar fata a tsakiya tana wakiltar tsaka-tsaki, kuma ƙasa da layin saman akwatin suna wakiltar kashi 25 da 75, bi da bi.Wasikar ta ƙara zuwa mafi ƙanƙanta da matsakaicin wuraren bayanai, waɗanda ba a la'akari da su ba.e ROC curve.Layin dige-dige d yana wakiltar ƙimar kofa da aka kiyasta daga binciken ROC.AUC na IBV shine 1. Raw data ana bayar da su azaman fayilolin bayanai danye.
A cikin wannan labarin, muna gabatar da FAST, wanda ke da halayen da ake buƙata don kyakkyawar POCT.Fa'idodin fasahar mu sun haɗa da: (1) Matsakaicin allurai (cascade, lokaci guda, jeri da zaɓi), saki akan buƙata (sauri da daidaituwa na matsa lamba) da ingantaccen aiki (vibration a digiri 150) (2) ajiya na dogon lokaci (shekaru 2 na gwajin hanzari, asarar nauyi game da 0.3%);(3) ikon yin aiki tare da ruwa mai yawa tare da nau'i mai yawa na wettability da danko (danko har zuwa 5500 cP);(4) Tattalin Arziki (Kimanin farashin kayan na'urar FAST-POCT PCR kusan dalar Amurka $1).Ta hanyar haɗa masu rarraba kayan aiki da yawa, an nuna kuma an yi amfani da tsarin dandali na FAST-POCT don gano PCR na mura A da B.FAST-POCT yana ɗaukar mintuna 82 kawai.Gwaje-gwaje na asibiti tare da samfuran swab na hanci na 36 sun nuna kyakkyawan daidaituwa a cikin ƙarfin haske tare da daidaitaccen RT-PCR (Pearson coefficients> 0.9).Gwaje-gwaje na asibiti tare da samfuran swab na hanci na 36 sun nuna kyakkyawan daidaituwa a cikin ƙarfin haske tare da daidaitaccen RT-PCR (Pearson coefficients> 0.9).Клинические тесты 36 образцами мазков из носа показалы ЦР (kоэффициенты Пирсона > 0,9).Gwaje-gwaje na asibiti tare da samfuran 36 na swabs na hanci sun nuna kyakkyawar yarjejeniya tare da tsananin haske na daidaitaccen RT-PCR (ƙirar Pearson> 0.9) .RT-PCR Клинические испытания 36 образцов мазков из показали ПЦР (kоэффициент Пирсона > 0,9).Gwajin gwaje-gwaje na asibiti na samfuran swab na hanci 36 sun nuna kyakkyawar yarjejeniya na ƙarfin haske tare da daidaitaccen RT-PCR (ƙirar Pearson> 0.9).A cikin layi daya da wannan aikin, hanyoyi daban-daban masu tasowa na kwayoyin halitta (misali, hawan keke na plasma, gwajin immunoassays marasa ƙarfi, da ƙididdigar aikin nanobody) sun nuna yuwuwar su a cikin POCT.Koyaya, saboda rashin cikakken haɗin gwiwa da ingantaccen dandamali na POCT, waɗannan hanyoyin ba makawa suna buƙatar hanyoyin aiwatarwa daban (misali, RNA ware44, incubation45 da washing46), waɗanda ke ƙara haɓaka aikin na yanzu tare da waɗannan hanyoyin don aiwatar da ayyukan POCT na ci gaba tare da. sigogin da ake buƙata.aikin fitar-a-amsa-fitarwa.A cikin wannan aikin, kodayake fam ɗin iska da aka yi amfani da shi don kunna bawul ɗin FAST yana da ƙananan isa don haɗawa a cikin kayan aikin benchtop (Fig. S9, S10), har yanzu yana cinye iko mai mahimmanci kuma yana haifar da amo.A ka'ida, ana iya maye gurbin ƙaramin nau'i na famfuna na pneumatic ta wasu hanyoyi, kamar amfani da ƙarfin lantarki ko kunna yatsa.Ƙarin haɓakawa na iya haɗawa, alal misali, kits ɗin daidaitawa don daban-daban da takamaiman ƙididdigar ƙwayoyin cuta, ta amfani da sababbin hanyoyin ganowa waɗanda basa buƙatar tsarin dumama/ sanyaya, don haka samar da dandamali na POCT mara amfani don aikace-aikacen PCR.Mun yi imanin cewa ganin cewa dandalin FAST yana ba da hanyar sarrafa ruwa, mun yi imanin cewa fasahar FAST da aka tsara tana ba da damar ƙirƙirar dandamali na gama gari ba kawai don gwajin ƙwayoyin cuta ba, har ma don kula da muhalli, gwajin ingancin abinci, kayan aiki da haɗin magunguna. ..
Kwamitin da'a na asibitin farko na jami'ar Zhejiang (IIT20220330B) ya amince da tarawa da amfani da samfuran hancin ɗan adam.An tattara samfuran swab na hanci 36, wanda ya haɗa da manya 16 <shekaru 30, manya 7> 40 shekaru, da maza 19, mata 17.An tattara samfuran swab na hanci 36, wanda ya haɗa da manya 16
FAST tushe da murfi an yi su da polylactic acid (PLA) kuma buga ta Ender 3 Pro 3D firinta (Shenzhen Transcend 3D Technology Co., Ltd.).An sayi tef mai gefe biyu daga Adhesives Research, Inc. Model 90880. An siyi fim ɗin PET mai kauri 100 µm daga McMaster-Carr.Dukansu manne da na PET an yanke su ta amfani da silhouette Cameo 2 cutter daga Silhouette America, Inc. An yi fim ɗin na roba da kayan PDMS ta hanyar yin allura.Da farko, an yanke firam ɗin PET mai kauri 200 µm ta amfani da tsarin laser kuma an manne shi zuwa takardar PMMA mai kauri 3 mm ta amfani da tef ɗin manne mai fuska biyu 100 µm.An zuba precursor na PDMS (Sylgard 184; Sashe na A: Sashe na B = 10: 1, Dow Corning) a cikin ƙirar kuma an yi amfani da sandar gilashi don cire PDMS da yawa.Bayan warkewa a 70 ° C. na 3 hours, 300 μm lokacin farin ciki fim din PDMS za a iya kwasfa daga mold.
Hotuna don rarrabawa iri-iri, buƙatu akan buƙatu da ingantaccen aiki ana ɗaukar su tare da kyamara mai sauri (Sony AX700 1000 fps).An siyi shaker orbital da aka yi amfani da shi a gwajin dogaro daga SCILOGEX (SCI-O180).Ana haifar da matsa lamba ta iska ta hanyar kwampreso na iska, kuma ana amfani da ma'aunin madaidaicin madaidaicin dijital da yawa don daidaita ƙimar matsa lamba.Tsarin gwajin halayen kwarara shine kamar haka.An shigar da ƙayyadadden adadin ruwa a cikin na'urar gwajin kuma an yi amfani da kyamara mai sauri don yin rikodin halayen kwarara.Har yanzu an ɗauki hotuna daga bidiyo na halin kwarara a ƙayyadaddun lokaci, kuma sauran yankin an ƙididdige su ta hanyar amfani da software na Image-Pro Plus, wanda aka ninka ta zurfin kamara don ƙididdige ƙarar.Ana iya samun cikakkun bayanai na tsarin gwajin ɗabi'a a Ƙarin Hoto S4.
Allurar 50µl na microbeads da 100 µl na ruwan da aka lalatar da su a cikin na'urar hadawa ta vial.An ɗauki hotuna masu haɗaɗɗiya tare da kyamara mai sauri kowane daƙiƙa 0.1 a matsi na mashaya 0.1, mashaya 0.15 da mashaya 0.2.Za'a iya samun bayanan Pixel yayin aikin haɗakarwa daga waɗannan hotuna ta amfani da software na sarrafa hoto (Photoshop CS6).Kuma za'a iya samun nasarar haɗakarwa tare da ma'auni na 53 mai zuwa.
inda M shine haɓakar hadawa, N shine jimillar adadin samfurin pixels, kuma ci da \(\bar{c}\) sune abubuwan da aka daidaita kuma ana sa ran daidaitawa.Ƙimar haɗawa ta bambanta daga 0 (0%, ba a gauraye ba) zuwa 1 (100%, cikakke gauraye).Ana nuna sakamakon a Ƙarin Hoto S6.
Kit ɗin RT-PCR na ainihi don IAV da IBV, gami da samfuran IAV da IBV RNA (cat. no. RR-0051-02/RR-0052-02, Liferiver, China), Tris-EDTA buffer (TE buffer no. B541019 , Sangon Biotech, kasar Sin), Kyatin Tsabtace RNA Mai Kyau (Sashe na No. Z-ME-0010, Liferiver, China) da GAPDH Magani (Sashe na M591101, Sangon Biotech, China) suna kasuwanci.Kit ɗin tsarkakewa na RNA ya haɗa da buffer mai ɗaure, wanki A, wanki W, eluent, microbeads na maganadisu, da mai ɗaukar acrylic.IAV da IBV kayan aikin RT-PCR na ainihi sun haɗa da haɗin gano PCR nucleic acid na IFVA da enzyme RT-PCR.Ƙara 6 µl na AcrylCarrier da 20 µl na ƙawancen maganadisu zuwa 500 µl na maganin buffer mai ɗaure, girgiza da kyau sannan a shirya maganin kwalliya.Ƙara 21 ml na ethanol don wanke A da W, girgiza sosai don samun mafita na wanke A da W, bi da bi.Bayan haka, 18 µl na cakuda PCR mai kyalli tare da IFVA nucleic acid da 1 µl na enzyme RT-PCR an ƙara su zuwa 1 µl na maganin TE, girgiza kuma an sanya su cikin dakika da yawa, suna samun 20 µl na IAV da IBV primers.
Bi hanyar tsarkakewar RNA mai zuwa: (1) tallan RNA.Pipette 526 µl na maganin pellet a cikin bututun centrifuge 1.5 ml kuma ƙara 150 µl na samfurin, sannan girgiza bututun sama da ƙasa sau 10 da hannu.Canja wurin 676 µl na cakuda zuwa ginshiƙan alaƙa da centrifuge a 1.88 x 104 g na daƙiƙa 60.Ana zubar da magudanan ruwa na gaba.(2) Matakin farko na wanka.Ƙara 500 µl na maganin wankin A zuwa ginshiƙin alaƙa, centrifuge a 1.88 x 104 g na s 40, kuma jefar da maganin da aka kashe.An maimaita wannan aikin wankin sau biyu.(3) mataki na biyu na wanka.Ƙara 500 µl na maganin wankin W zuwa ginshiƙin alaƙa, centrifuge a 1.88 × 104 g na 15 s kuma jefar da maganin da aka kashe.An maimaita wannan aikin wankin sau biyu.(4) Haske.Ƙara 200 µl na eluate zuwa ginshiƙin alaƙa da centrifuge a 1.88 x 104 g na 2 min.(5) RT-PCR: An allurar da eluate a cikin 20 μl na maganin farko a cikin bututun PCR, sannan an sanya bututun a cikin na'urar gwajin PCR na ainihi (SLAN-96P) don aiwatar da tsarin RT-PCR.Duk aikin ganowa yana ɗaukar kusan mintuna 140 (minti 20 don tsarkakewar RNA da mintuna 120 don gano PCR).
526 µl na maganin bead, 1000 µl na maganin wankin A, 1000 µl na maganin wanke W, 200 µl na eluate da 20 µl na maganin farko an ƙara da farko kuma an adana su a cikin ɗakunan M, W1, W2, E da ɗakunan gano PCR.Taron dandamali.Sa'an nan, 150 µl na samfurin da aka pipetted a cikin dakin M da FAST-POCT dandamali da aka saka a cikin gwajin kayan aiki da aka nuna a Ƙarin Hoto S9.Bayan kamar mintuna 82, an sami sakamakon gwajin.
Sai dai in an lura da haka, ana gabatar da duk sakamakon gwajin azaman ma'anar ± SD bayan mafi ƙarancin maimaitawa shida ta amfani da dandalin FAST-POCT kawai da samfuran masu zaman kansu na ilimin halitta.Ba a cire bayanai daga binciken ba.Gwaje-gwajen ba bazuwar ba ne.Masu binciken ba su makantar da ayyukan rukuni yayin gwajin.
Don ƙarin bayani game da ƙira na nazari, duba Ƙididdigar Rahoton Bincike mai alaƙa da wannan labarin.
Ana samun bayanan da ke goyan bayan sakamakon wannan binciken a cikin Ƙarin Bayani.Wannan labarin yana ba da bayanan asali.
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