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Machitidwe a Lab-on-a-chip omwe ali ndi mphamvu zopezeka pamalowa amapereka mwayi wodziwa mwamsanga komanso molondola ndipo ndi othandiza pazikhazikiko zomwe zili ndi zida zomwe zida zamankhwala ndi akatswiri ophunzitsidwa bwino sapezeka.Komabe, kupanga njira yoyesera yoyezetsa yomwe nthawi imodzi imakhala ndi zofunikira zonse zoperekera ntchito zambiri, kumasulidwa kwa zofuna, ntchito yodalirika, ndi kusungidwa kwa nthawi yaitali kwa reagents kumakhalabe vuto lalikulu.Apa tikufotokoza ukadaulo wosinthira ma lever-actuated micro travel switch womwe umatha kuwongolera madzi mbali iliyonse, kupereka kuyankha molondola komanso molingana ndi kuthamanga kwa mpweya, ndikukhalabe okhazikika pakusuntha kwadzidzidzi ndi kugwedezeka.Kutengera ukadaulo, timafotokozanso za chitukuko cha polymerase unyolo kachitidwe kachitidwe kamene kamaphatikiza kuyambika kwa reagent, kusanganikirana ndikuchita ntchito zonse munjira imodzi, zomwe zimakwaniritsa magwiridwe antchito a "zitsanzo-mu-yankho-kunja" kwa zitsanzo zonse zachipatala kuchokera kwa odwala 18 omwe ali ndi vuto laukadaulo. Fuluwenza ndi 18 amazilamulira payekha, mogwirizana bwino fluorescence kwambiri ndi muyezo polymerase chain reaction (Pearson coefficients> 0.9).Kutengera ukadaulo, timafotokozanso za chitukuko cha polymerase chain reaction system yomwe imaphatikiza kuyambitsa kwa reagent, kusanganikirana ndikuchita ntchito zonse munjira imodzi, zomwe zimakwaniritsa magwiridwe antchito a "zitsanzo-mu-yankho-kunja" kwa zitsanzo zonse zachipatala za odwala 18. ndi Influenza ndi 18 zowongolera payekha, mogwirizana ndi mphamvu ya fluorescence ndi standard polymerase chain reaction (Pearson coefficients> 0.9).Основываясь на этой технологии, мы также описываем разработку системы полимеразной цепной реакции, которая объединяет фунция, которая объединяет функаивка, которая объединяет функаивка реакции в одном процессе, что обеспечивает выполнение «образец-в-ответ-выход» для всех клинических образцов образцов из 18 сположение ndi 18 отдельных контролей, хорошем соответствии интенсивности флуоресценции стандартной полимеразной цепной реакцией (0,9,9).Kutengera luso limeneli, ifenso kufotokoza chitukuko cha polymerase unyolo kachitidwe kachitidwe kaphatikizidwe ntchito jekeseni, kusakaniza, ndi anachita mu njira imodzi, kuloleza chitsanzo-mu-yankho-kunja kwa onse chipatala zitsanzo mphuno kuchokera 18 odwala fuluwenza.ndi 18 zowongolera payekhapayekha, mogwirizana ndi muyezo wa polymerase chain reaction fluorescence intensity (Pearson's coefficients> 0.9).Kutengera luso limeneli, ifenso kufotokoza chitukuko cha polymerase unyolo kachitidwe kachitidwe kaphatikizidwe jekeseni reagent, kusakaniza, ndi anachita ntchito kusanthula onse chipatala zitsanzo m'mphuno kuchokera 18 mu-chitsanzo mphuno wodwala zitsanzo.Chimfine ndi 18 amazilamulira munthu, fulorosisi mphamvu chikufanana. bwino ndi polymerase chain reaction (Pearson's coefficient> 0.9).Pulatifomu yomwe ikuyembekezeredwa imatsimikizira kudalirika kwa kusanthula kwa biomedical ndipo motero imatha kufulumizitsa kutsatsa kwa zida zingapo zoyesera.
Matenda a anthu omwe akubwera, monga mliri wa 2020 COVID-19 womwe wapha miyoyo ya anthu mamiliyoni ambiri, akuwopseza kwambiri thanzi lapadziko lonse lapansi komanso chitukuko cha anthu1.Kuzindikira koyambirira, kofulumira komanso kolondola kwa matenda ndikofunikira kuti tipewe kufalikira kwa kachilomboka ndikuwongolera zotsatira zamankhwala.Dongosolo lodziwika bwino lazachilengedwe lotengera ma lab apakati pomwe zitsanzo zoyezetsa zimatumizidwa ku zipatala kapena zipatala zomwe zimayendetsedwa ndi akatswiri pano zikulepheretsa anthu pafupifupi 5.8 biliyoni padziko lonse lapansi, makamaka omwe akukhala movutikira.kumene kuli kusowa kwa zipangizo zodula zachipatala ndi akatswiri oyenerera.azachipatala 2. Chifukwa chake, pakufunika mwachangu kukhazikitsa njira yotsika mtengo komanso yosavuta kugwiritsa ntchito lab-on-a-chip yokhala ndi kuthekera koyezetsa magazi (POCT) komwe kungathe kupatsa madokotala chidziwitso chowunikira nthawi yake kuti apange zisankho zodziwika bwino. .ndi chithandizo 3.
Malangizo a World Health Organisation (WHO) akuti POCT yabwino iyenera kukhala yotsika mtengo, yosavuta kugwiritsa ntchito (yosavuta kugwiritsa ntchito popanda maphunziro ochepa), yolondola (peŵani zoyipa zabodza kapena zonama), yachangu komanso yodalirika (perekani zinthu zabwino zobwerezabwereza), ndi zobweretsedwa (zotha kusungidwa kwa nthawi yayitali komanso kupezeka mosavuta kwa ogwiritsa ntchito)4.Kuti akwaniritse izi, machitidwe a POCT akuyenera kupereka izi: dosing yosunthika kuti muchepetse kulowererapo pamanja, kumasulidwa kofunidwa kuti muchepetse zoyendera kuti mupeze zotsatira zolondola, komanso magwiridwe antchito odalirika kuti athe kupirira kugwedezeka kwa chilengedwe.Pakalipano, chipangizo cha POCT chomwe chimagwiritsidwa ntchito kwambiri ndi lateral flow strip5,6 yomwe imakhala ndi zigawo zingapo za porous nitrocellulose nembanemba zomwe zimakankhira pang'ono pang'ono kutsogolo, zomwe zimakhudzidwa ndi pre-immobilized reagents ndi capillary force.Ngakhale ali ndi mwayi wotsika mtengo, kugwiritsa ntchito mosavuta, komanso zotsatira zachangu, zida za POCT zokhala ndi ma flow-based POCT zitha kugwiritsidwa ntchito pakuyesa kwachilengedwe (mwachitsanzo, mayeso a glucose7,8 ndi mayeso apakati9,10) osafunikira kuwunika kwamagawo angapo.zochita (mwachitsanzo, kutsitsa ma reagents angapo, kusakaniza, kuchulukitsa).Kuonjezera apo, mphamvu zoyendetsa galimoto zomwe zimayendetsa kayendetsedwe ka madzi (ie, mphamvu za capillary) sizimapereka kusinthasintha kwabwino, makamaka pakati pa magulu, zomwe zimapangitsa kuti pakhale kuberekana kosakwanira11 ndikupanga magulu othamanga kwambiri kuti azitha kuzindikira bwino12,13.
Kuthekera kowonjezereka kopanga pa micro- ndi nanoscale kwapanga mwayi wopanga zida za microfluidic POCT zoyezera kuchulukana14,15,16,17.Posintha mawonekedwe a mawonekedwe 18, 19 ndi geometry ya mayendedwe 20, 21, 22, mphamvu ya capillary ndi kuthamanga kwa zida izi zitha kuwongoleredwa.Komabe, kudalirika kwawo, makamaka pazakumwa zonyowa kwambiri, kumakhalabe kosavomerezeka chifukwa chopanga zolakwika, zolakwika zakuthupi, komanso chidwi ndi kugwedezeka kwa chilengedwe.Kuonjezera apo, popeza kutuluka kwa capillary kumapangidwira pa mawonekedwe amadzimadzi-gasi, palibe kutuluka kwina komwe kungayambitsidwe, makamaka mutatha kudzaza njira ya microfluidic ndi madzi.Chifukwa chake, kuti muzindikire zovuta kwambiri, njira zingapo za jakisoni wachitsanzo ziyenera kuchitidwa24,25.
Pakati pa zida za microfluidic, zida za centrifugal microfluidic pano ndi imodzi mwamayankho abwino kwambiri a POCT26,27.Njira yake yoyendetsera ndi yopindulitsa chifukwa mphamvu yoyendetsa galimoto imatha kuwongoleredwa ndikusintha liwiro lozungulira.Komabe, choyipa ndichakuti mphamvu ya centrifugal nthawi zonse imalunjikitsidwa kumphepete kwakunja kwa chipangizocho, zomwe zimapangitsa kuti zikhale zovuta kugwiritsa ntchito njira zambiri zomwe zimafunikira pakuwunika kovutirapo.Ngakhale mphamvu zowonjezera zoyendetsa (monga ma capillaries 28, 29 ndi ena ambiri 30, 31, 32, 33, 34, 35) kuwonjezera pa mphamvu ya centrifugal imayambitsidwa kuti igwiritsidwe ntchito mosiyanasiyana, kusamutsidwa kwamadzi kosayembekezereka kumatha kuchitikabe chifukwa mphamvu zowonjezera izi nthawi zambiri zimalamulidwa. kutsika kwambiri kuposa mphamvu yapakati, kupangitsa kuti ikhale yogwira ntchito pamagawo ang'onoang'ono ogwiritsira ntchito kapena kusapezeka pakufunika ndi kutulutsa kwamadzimadzi.Kuphatikiza ma pneumatic manipulations mu centrifugal microfluidics monga centrifugal kinetic njira 36, 37, 38, thermopneumatic njira 39 ndi yogwira pneumatic njira 40 zatsimikizira kukhala njira yokopa.Ndi njira ya counterfugodynamic, chowonjezera chowonjezera ndi ma microchannels amaphatikizidwa mu chipangizo cha kunja ndi mkati, ngakhale kuti kupopera kwake (kuchokera ku 75% mpaka 90%) kumadalira kwambiri chiwerengero cha kupopera ndi kukhuthala. cha madzi.Mu njira ya thermopneumatic, nembanemba ya latex ndi chipinda chosinthira madzimadzi chimapangidwa makamaka kuti chisindikize kapena kutsegulanso cholowera pamene mpweya wotsekeka watenthedwa kapena utakhazikika.Komabe, khwekhwe lotenthetsera/kuzirala limabweretsa mavuto oyankha pang'onopang'ono ndikuchepetsa kugwiritsidwa ntchito kwake pamayesero a thermosensitive (mwachitsanzo, polymerase chain reaction (PCR) amplification).Ndi njira yogwira mtima ya pneumatic, kumasulidwa kofunidwa ndi kuyenda kwamkati kumatheka kupyolera mukugwiritsa ntchito panthawi imodzimodziyo ya kukakamiza kwabwino komanso kufananizidwa bwino ndi liwiro lozungulira ndi ma motors othamanga kwambiri.Palinso njira zina zopambana zogwiritsira ntchito ma pneumatic actuators okha (kuthamanga kwabwino 41, 42 kapena kupanikizika koipa 43) ndi mapangidwe a valve omwe nthawi zambiri amatsekedwa.Pogwiritsira ntchito motsatizana kukakamiza mu chipinda cha pneumatic, madzi amaponyedwa kutsogolo kwa peristaltically, ndipo valavu yomwe imakhala yotsekedwa imalepheretsa kutuluka kwa madzi chifukwa cha peristalsis, motero kuzindikira ntchito zovuta zamadzimadzi.Komabe, pakali pano pali umisiri wochepa chabe wa teknoloji ya microfluidic yomwe ingathe kuchita ntchito zovuta zamadzimadzi mu chipangizo chimodzi cha POCT, kuphatikizapo kugawa kwamitundu yambiri, kumasulidwa kwa zofuna, ntchito yodalirika, kusungirako nthawi yaitali, kusamalira zakumwa zamadzimadzi kwambiri, ndi kupanga zotsika mtengo.Zonse nthawi imodzi.Kuperewera kwa ntchito zambiri zogwirira ntchito kungakhalenso chimodzi mwa zifukwa zomwe malonda ochepa a POCT monga Cepheid, Binx, Visby, Cobas Liat, ndi Rhonda adayambitsidwa bwino pamsika wotseguka mpaka pano.
Mu pepala ili, tikupangira cholumikizira cha pneumatic microfluidic chotengera ukadaulo wobiriwira wa mphete yaying'ono (FAST).FAST imaphatikiza zinthu zonse zofunika panthawi imodzimodzi pamagulu osiyanasiyana a reagents kuchokera ku microliters mpaka milliliters.FAST imakhala ndi nembanemba zotanuka, ma levers ndi midadada.Popanda kugwiritsa ntchito mphamvu ya mpweya, ma nembanemba, ma levers ndi midadada amatha kutsekedwa mwamphamvu ndipo madzi mkati mwake amatha kusungidwa kwa nthawi yayitali.Pamene kukakamiza koyenera kuikidwa ndi kusinthidwa ku utali wa lever, diaphragm imakula ndi kukankhira lever pamalo otseguka, kulola madzi kudutsa.Izi zimalola kuti madzi azigwira ntchito mosiyanasiyana m'njira zingapo, nthawi imodzi, motsatizana kapena mosankha.
Tapanga kachitidwe ka PCR pogwiritsa ntchito FAST kuti tipeze mayankho-mu-zitsanzo kuti tipeze ma virus a fuluwenza A ndi B (IAV ndi IBV).Tinapeza malire otsika (LOD) a 102 makope / mL, ma multiplex assay adawonetsa zenizeni za IAV ndi IBV ndipo amalola kuti tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo.Zotsatira zoyezetsa zachipatala pogwiritsa ntchito chitsanzo cha mphuno kuchokera kwa odwala 18 ndi anthu 18 athanzi amawonetsa kuvomerezana kwabwino kwa fluorescence intensity ndi standard RT-PCR (Pearson coefficients> 0.9).Zotsatira zoyezetsa zachipatala pogwiritsa ntchito chitsanzo cha mphuno kuchokera kwa odwala 18 ndi anthu 18 athanzi amawonetsa kuvomerezana kwabwino kwa fluorescence intensity ndi standard RT-PCR (Pearson coefficients> 0.9).Zolemba za 18 zapita ndi 18 zotsogola лиц показывает хостовыт из 18 пациентов испытания сценции стандартной ОТ-ПЦР (коэфффициенты Пирсона > 0,9).Zotsatira za mayesero azachipatala pogwiritsa ntchito chitsanzo cha mphuno kuchokera kwa odwala 18 ndi anthu 18 athanzi amasonyeza mgwirizano wabwino pakati pa mphamvu ya fluorescence ya RT-PCR yokhazikika (Pearson's coefficients> 0.9).0.9............... Результаты клинических испытаний с использованием образцов назальных мазков от 18 пациентов и 18 здоровых лиц показать достовиный песни ю флуоресценции ndi стандартной ОТ-ПЦР (коэфффициент Пирсона > 0,9).Zotsatira zamayesero azachipatala pogwiritsa ntchito zitsanzo za mphuno za odwala 18 ndi anthu 18 athanzi zidawonetsa mgwirizano wabwino pakati pa mphamvu ya fluorescence ndi RT-PCR wamba (Pearson's coefficient> 0.9).Mtengo woyerekeza wa chipangizo cha FAST-POCT ndi pafupifupi US$1 (Table Supplementary Table 1) ndipo ukhoza kuchepetsedwanso pogwiritsa ntchito njira zazikulu zopangira (monga jekeseni).M'malo mwake, zida za POCT zochokera ku FAST zili ndi zofunikira zonse zomwe WHO idalamula ndipo zimagwirizana ndi njira zatsopano zoyesera zamankhwala am'madzi am'madzi monga plasma thermal cycling44, amplification-free immunoassays45 ndi nanobody functionalization test46 yomwe ili msana wa machitidwe a POCT.kuthekera.
Pa mkuyu.1a ikuwonetsa mawonekedwe a nsanja ya FAST-POCT, yomwe ili ndi zipinda zinayi zamadzimadzi: chipinda chosungiramo zinthu zakale, chipinda chosakanikirana, chipinda chochitiramo zinthu, ndi chipinda cha zinyalala.Chinsinsi cha kuwongolera kwamadzimadzi ndi mapangidwe a FAST (opangidwa ndi nembanemba zotanuka, ma levers ndi midadada) yomwe ili m'chipinda chosungirako ndi chipinda chosanganikirana.Monga njira yogwiritsira ntchito pneumatically, mapangidwe a FAST amapereka kayendetsedwe kabwino ka madzimadzi, kuphatikizapo kutsekedwa / kutsegula, kusinthasintha kosinthasintha, kutulutsa madzi omwe akufuna, ntchito yodalirika (mwachitsanzo, kusamva kugwedezeka kwa chilengedwe), ndi kusunga kwa nthawi yaitali.Pulatifomu ya FAST-POCT ili ndi zigawo zinayi: chigawo chothandizira, filimu yowonongeka, filimu ya pulasitiki, ndi chivundikiro, monga momwe tawonetsera mu chithunzithunzi cha 1b (chikuwonetsedwanso mwatsatanetsatane mu Supplementary Figures S1 ndi S2). ).Njira zonse ndi zipinda zonyamulira zamadzimadzi (monga zosungirako zisanachitike ndi zipinda zochitirapo kanthu) zimayikidwa mu magawo a PLA (polylactic acid) kuyambira 0.2 mm (gawo locheperako) mpaka 5 mm wandiweyani.Mafilimu otanuka ndi 300 µm wandiweyani wa PDMS omwe amakula mosavuta pamene kuthamanga kwa mpweya kumagwiritsidwa ntchito chifukwa cha "kuwonda kwake" ndi kutsika kwa modulus (pafupifupi 2.25 MPa47).Filimu ya polyethylene imapangidwa ndi polyethylene terephthalate (PET) yokhala ndi makulidwe a 100 µm kuteteza filimu yotanuka kuti isawonongeke kwambiri chifukwa cha kuthamanga kwa mpweya.Mogwirizana ndi zipinda, gawo lapansili lili ndi ma levers olumikizidwa ndi chivundikiro (chopangidwa ndi PLA) ndi hinges kuwongolera kutuluka kwamadzi.Filimu yotanukayo idamata pamsana wotsatira pogwiritsa ntchito tepi yomatira mbali ziwiri (ARseal 90880) ndikukutidwa ndi filimu yapulasitiki.Zigawo zitatu zidasonkhanitsidwa pagawo laling'ono pogwiritsa ntchito kamangidwe ka T-clip pachivundikirocho.T-clamp ili ndi kusiyana pakati pa miyendo iwiri.Pamene kopanira anaikapo mu poyambira, miyendo iwiri anawerama pang'ono, ndiye anabwerera ku chikhalidwe chawo choyambirira ndi mwamphamvu womanga chivindikiro ndi kuthandizira pamene iwo anadutsa poyambira (Supplementary mkuyu. S1).Zigawo zinayizo zimasonkhanitsidwa pogwiritsa ntchito zolumikizira.
Chithunzi chojambula cha nsanja chomwe chikuwonetsa magawo osiyanasiyana ogwira ntchito ndi mawonekedwe a FAST.b Chithunzi chokulitsa cha nsanja ya FAST-POCT.c Chithunzi cha nsanja pafupi ndi kotala ya dollar yaku US.
Njira yogwirira ntchito ya nsanja ya FAST-POCT ikuwonetsedwa mu Chithunzi 2. Zigawo zazikuluzikulu ndizozitsulo zomwe zili pamtunda wapansi ndi zolembera pazitsulo zophimba, zomwe zimapangitsa kuti pakhale kusokoneza pamene zigawo zinayi zikusonkhanitsidwa pogwiritsa ntchito T-mawonekedwe. .Pamene palibe kukakamiza kwa mpweya kumagwiritsidwa ntchito (mkuyu 2a), kusokoneza kokwanira kumapangitsa kuti hinge ipindike ndi kupunduka, ndipo mphamvu yosindikizira imagwiritsidwa ntchito kupyolera muzitsulo kuti isindikize filimu yotanuka pa chipikacho, ndipo madzi omwe ali mu chisindikizo amatanthauzidwa. ngati dziko losindikizidwa.Tikumbukenso kuti m'chigawo ichi, lever akupindika kunja, monga momwe mbali view mu mkuyu 2a.Mpweya ukaperekedwa (mkuyu 2b), nembanemba yotanuka imatuluka kunja kupita ku chivundikiro ndikukankhira chotchinga mmwamba, motero imatsegula mpata pakati pa chitsulocho ndi chipika chamadzimadzi kuti chilowe mu chipinda chotsatira, chomwe chimatanthauzidwa ngati malo otseguka. .Kuthamanga kwa mpweya kumatuluka, lever imatha kubwerera kumalo ake oyambirira ndikukhalabe olimba chifukwa cha kusungunuka kwa hinge.Makanema amayendedwe a lever amawonetsedwa mu kanema wowonjezera wa S1.
A. Chithunzi chojambula ndi zithunzi zikatsekedwa.Popanda kukakamiza, lever imakanikiza nembanemba pa chipikacho, ndipo madziwo amatsekedwa.b Ali bwino.Kupanikizika kukagwiritsidwa ntchito, nembanemba imakula ndikukankhira lever mmwamba, kotero kuti njirayo imatseguka ndipo madzi amatha kuyenda.c Dziwani kukula kwake kwa kukakamiza kofunikira.Makhalidwe amaphatikizapo kutalika kwa lever (L), mtunda wapakati pa chowongolera ndi hinge (l) ndi makulidwe a lever protrusion (t).Fs ndi mphamvu yophatikizira pa throttle point B. q ndi katundu wogawidwa mofanana pa lever.Tx* imayimira torque yopangidwa ndi lever yokhala ndi hinged.Kupanikizika kwakukulu ndiko kukakamiza kofunikira kuti mukweze lever ndikupanga madzi amadzimadzi.d Zotsatira zongoyerekeza ndi zoyesera za ubale pakati pa kukakamizidwa kofunikira ndi kukula kwa chinthu.n = Zoyesera zodziyimira za 6 zidachitidwa ndipo deta ikuwonetsedwa ngati ± kupatuka kokhazikika.Data yaiwisi imaperekedwa ngati mafayilo osasinthika.
Chitsanzo chowunikira chotengera chiphunzitso cha mtengo chapangidwa kuti chiwunikire kudalira kwamphamvu kwambiri ya Pc pomwe kusiyana kumatseguka pazigawo za geometric (mwachitsanzo, L ndi kutalika kwa lever, l ndi mtunda pakati pa chipika ndi hinge, S ndi lever Malo okhudzana ndi madzi t ndi makulidwe a lever protrusion , monga momwe tawonetsera mkuyu 2c).Monga tafotokozera mu Supplementary Notes and Supplementary Figure S3, kusiyana kumatseguka pamene \({P}_{c}\ge \frac{2{F}_{s}l}{SL}\), pomwe Fs ndi torque. \ ({T}_{x}^{\ast}(={F}_{s}l)\) kuti athetse mphamvu zomwe zimagwirizanitsidwa ndi kusokoneza kokwanira ndikupangitsa kuti hinge ipinde.Mayankho oyesera ndi chitsanzo chowunikira amasonyeza mgwirizano wabwino (mkuyu 2d), kusonyeza kuti kupanikizika kwakukulu kwa Pc kumawonjezeka ndi kuwonjezeka kwa t / l ndi kuchepa kwa L, zomwe zimafotokozedwa mosavuta ndi chitsanzo chamtengo wapatali, mwachitsanzo, torque ikuwonjezeka ndi t / Lift. .Choncho, kusanthula kwathu kwachidziwitso kumasonyeza bwino kuti kupanikizika kwakukulu kungathe kuyendetsedwa bwino mwa kusintha kutalika kwa lever L ndi chiwerengero cha t / l, chomwe chimapereka maziko ofunikira pakupanga nsanja ya FAST-POCT.
Pulatifomu ya FAST-POCT imapereka kugawa kwazinthu zambiri (zowonetsedwa mu Chithunzi 3a ndi choyikapo ndi kuyesa), chomwe ndi chinthu chofunikira kwambiri cha POCT yopambana, pomwe madzi amatha kuyenda mbali iliyonse komanso mwanjira iliyonse (kutayika, munthawi yomweyo, motsatizana) kapena kusankha njira zambiri. kupereka .- ntchito ya dosing.Pa mkuyu.3a (i) ikuwonetsa njira yochepetsera momwe zipinda ziwiri kapena zingapo zimaponyedwa pogwiritsa ntchito midadada kuti alekanitse ma reactants osiyanasiyana ndi lever yowongolera malo otseguka ndi otsekedwa.Pamene kupanikizika kukugwiritsidwa ntchito, madzi amadzimadzi amayenda kuchokera kumtunda kupita ku chipinda chapansi m'njira yowonongeka.Tikumbukenso kuti kutsika zipinda akhoza kudzazidwa ndi chonyowa mankhwala kapena youma mankhwala monga lyophilized ufa.Poyesera mu Chithunzi 3a (i), inki yofiira yochokera ku chipinda chapamwamba imayenda pamodzi ndi ufa wa buluu (copper sulfate) mu chipinda chachiwiri ndikusintha buluu wakuda ikafika m'chipinda chapansi.Zimasonyezanso mphamvu yolamulira madzimadzi omwe amapopedwa.Mofananamo, lever imodzi ikalumikizidwa ndi zipinda ziwiri, imakhala njira yojambulira nthawi imodzi, monga momwe tawonetsera mkuyu.3a (ii), momwe madzi amatha kugawidwa mofanana pazipinda ziwiri kapena kuposerapo pamene kukakamizidwa kukugwiritsidwa ntchito.Popeza kupanikizika kwakukulu kumadalira kutalika kwa lever, kutalika kwa lever kungasinthidwe kuti akwaniritse ndondomeko ya jekeseni yotsatizana monga momwe tawonetsera mkuyu.3a (iii).Chingwe chachitali (chokhala ndi zovuta zovuta Pc_long) chinagwirizanitsidwa ndi chipinda B ndi chowongolera chachifupi (chokhala ndi kupanikizika kwakukulu Pc_short> Pc_long) chinagwirizanitsidwa ndi chipinda A. Monga kupanikizika kwa P1 (Pc_long <P1 <Pc_short) kunagwiritsidwa ntchito, madzi okhawo ali ofiira. imatha kuyenderera kuchipinda B ndipo kukakamiza kudawonjezeredwa mpaka P2 (> Pc_short), madzi abuluu amatha kupita kuchipinda A. Njira yojambulira yotsatizanayi imagwira ntchito ku zakumwa zosiyanasiyana zomwe zimasamutsira ku zipinda zawo motsatizana, zomwe ndizofunikira kwambiri kuti POCT ikhale yopambana. chipangizo.Chingwe chachitali (chokhala ndi zovuta zovuta Pc_long) chinagwirizanitsidwa ndi chipinda B ndi chowongolera chachifupi (chokhala ndi kupanikizika kwakukulu Pc_short> Pc_long) chinagwirizanitsidwa ndi chipinda A. Monga kupanikizika kwa P1 (Pc_long
Chithunzi cha ntchito zambiri, mwachitsanzo (i) kutsitsa, (ii) nthawi imodzi, (iii) motsatizana, ndi (iv) ntchito yosankha.Ma curve akuyimira kayendetsedwe ka ntchito ndi magawo amitundu inayi yogawa.b Zotsatira za mayesero osungira nthawi yaitali m'madzi osungunuka ndi ethanol.n = Zoyeserera zodziyimira 5 zidachitidwa ndipo deta ikuwonetsedwa ngati ± sd c.Ziwonetsero zoyesa kukhazikika pamene chipangizo cha FAST ndi capillary valve (CV) chidali mu (i) static ndi (ii) ma vibrating states.(iii) Volume vs. nthawi ya FAST ndi CV zida pamitundu yosiyanasiyana ya angular.d Kufalitsa zotsatira za mayeso pakufunika kwa (i) chipangizo cha FAST ndi (ii) chipangizo cha CV.(iii) Ubale pakati pa voliyumu ndi nthawi ya zida za FAST ndi CV pogwiritsa ntchito njira yapakati yapakati.Mipiringidzo yonse, 1 cm.Data yaiwisi imaperekedwa ngati mafayilo osasinthika.
Kusungidwa kwa nthawi yayitali kwa reagents ndi chinthu china chofunikira cha chipangizo chopambana cha POCT chomwe chidzalola ogwira ntchito osaphunzitsidwa kusamalira ma reagents angapo.Ngakhale matekinoloje ambiri awonetsa kuthekera kwawo kosungirako nthawi yayitali (mwachitsanzo, ma microdispensers 35, mapaketi 48 a matuza, ndi mapaketi a ndodo 49), chipinda chodzipatulira cholandirira chimafunika kuti chigwirizane ndi phukusi, zomwe zimawonjezera mtengo ndi zovuta;kuonjezera apo, njira zosungirazi sizimalola kugawikana pakufunidwa ndipo zimabweretsa kuwonongeka kwa ma reagents chifukwa cha zotsalira m'paketi.Kuthekera kosungirako kwa nthawi yayitali kudatsimikiziridwa poyesa moyo wofulumira pogwiritsa ntchito zida za PMMA zopangidwa ndi CNC chifukwa chakuuma kwake pang'ono komanso kukana kutulutsa mpweya (Supplementary Figure S5).Zida zoyesera zidadzazidwa ndi madzi opangidwa ndi deionized (madzi a deionized) ndi 70% ethanol (simulating reagents) pa 65 ° C kwa masiku 9.Madzi onse opangidwa ndi deionized ndi ethanol adasungidwa pogwiritsa ntchito zojambulazo za aluminiyamu kuti atseke kulowa kuchokera pamwamba.Arrhenius equation ndi mphamvu yolowera mkati yomwe inalembedwa m'mabuku50,51 anagwiritsidwa ntchito kuwerengera nthawi yeniyeni yofanana.Pa mkuyu.3b imasonyeza kulemera kwapakati pa zotsatira za 5 zosungidwa pa 65 ° C kwa masiku 9, zofanana ndi 0,30% za madzi opangidwa ndi deionized ndi 0,72% kwa 70% ethanol pazaka 2 pa 23 ° C.
Pa mkuyu.3c ikuwonetsa kuyesa kwa vibration.Popeza valavu ya capillary (CV) ndiyo njira yodziwika kwambiri yogwiritsira ntchito madzimadzi pakati pa zipangizo zomwe zilipo POCT28,29, chipangizo cha CV 300 µm m'lifupi ndi 200 µm kuya chinagwiritsidwa ntchito poyerekezera.Zitha kuwoneka kuti pamene zipangizo zonse ziwiri zimakhala zokhazikika, madzimadzi omwe ali mu FAST-POCT amasindikiza ndipo madzi a mu chipangizo cha CV amatseka chifukwa cha kuwonjezereka kwadzidzidzi kwa njirayo, yomwe imachepetsa mphamvu za capillary.Komabe, monga mafupipafupi a orbital vibrator akuwonjezeka, madzimadzi mu FAST-POCT nsanja amakhalabe osindikizidwa, koma madzimadzi mu chipangizo cha CV amalowa m'chipinda chapansi (onaninso Supplementary Movie S3).Izi zikuwonetsa kuti ma hinges opunduka a nsanja ya FAST-POCT atha kugwiritsa ntchito mphamvu yamakina amphamvu ku gawoli kuti atseke mwamphamvu madzi muchipindacho.Komabe, mu zida za CV, zamadzimadzi zimasungidwa chifukwa cha kukhazikika pakati pa magawo olimba, mpweya, ndi madzimadzi, kupanga kusakhazikika, ndi kugwedezeka kumatha kusokoneza kukhazikika ndikuyambitsa machitidwe oyenda mosayembekezereka.Ubwino wa nsanja ya FAST-POCT ndikuti imapereka magwiridwe antchito odalirika ndikupewa zolephera pamaso pa kugwedezeka komwe kumachitika nthawi yobereka ndikugwira ntchito.
Chinthu chinanso chofunikira pa nsanja ya FAST-POCT ndiyo kumasulidwa kwake komwe kumafunikira, zomwe ndizofunikira kwambiri pakuwunika kuchuluka.Pa mkuyu.3d ikufanizira kutulutsidwa kofunidwa kwa nsanja ya FAST-POCT ndi chipangizo cha CV.Kuchokera mkuyu.3d (iii) tikuwona kuti chipangizo cha FAST chimayankha mofulumira ku chizindikiro chokakamiza.Pamene kupanikizika kunagwiritsidwa ntchito pa nsanja ya FAST-POCT, madziwo amayenda, pamene kupanikizika kunatulutsidwa, kutuluka nthawi yomweyo kunasiya (mkuyu 3d (i)).Izi zitha kufotokozedwa ndikubwerera mwachangu kwa hinge, komwe kumakankhira lever kumbuyo kwa chipika, kutseka chipindacho.Komabe, madzimadzi anapitiriza kuyenda mu chipangizo cha CV, pamapeto pake kumapangitsa kuti madzi amadzimadzi achulukane pafupifupi 100 µl pambuyo potulutsa mphamvu (Chithunzi 3d(ii) ndi Supplementary Movie S4).Izi zitha kufotokozedwa ndi kutha kwa mphamvu ya capillary pinning pakunyowetsa kwathunthu kwa CV pambuyo jekeseni woyamba.
Kutha kuthana ndi zakumwa zonyowa mosiyanasiyana komanso ma viscosity pazida zomwezo zimakhalabe zovuta pamapulogalamu a POCT.Kunyowa kosauka kungayambitse kutayikira kapena khalidwe lina losayembekezereka loyenda muzitsulo, ndi zipangizo zothandizira monga vortex mixers, centrifuges ndi zosefera nthawi zambiri zimafunika kukonzekera zakumwa zowoneka bwino kwambiri 52.Tinayesa kugwirizana pakati pa kupanikizika kwakukulu ndi katundu wamadzimadzi (wokhala ndi kunyowa kosiyanasiyana ndi kukhuthala).Zotsatira zikuwonetsedwa mu Table 1 ndi Video S5.Zitha kuwonedwa kuti zakumwa za wettability zosiyanasiyana ndi mamasukidwe akayendedwe akhoza kusindikizidwa mu chipinda, ndipo pamene kuthamanga ntchito, ngakhale zakumwa ndi mamasukidwe akayendedwe kukhuthala mpaka 5500 cP akhoza anasamutsidwa ku chipinda moyandikana, kuti athe kuzindikira zitsanzo ndi mkulu. viscosity (mwachitsanzo, sputum, chitsanzo chowoneka bwino chomwe chimagwiritsidwa ntchito pozindikira matenda a kupuma).
Mwa kuphatikiza zida zogawira zomwe zili pamwambazi, zida zambiri za POCT zochokera ku FAST zitha kupangidwa.Chitsanzo chikuwonetsedwa mu Chithunzi 1. Chomeracho chili ndi chipinda chosungiramo zinthu zakale, chipinda chosakaniza, chipinda chochitiramo zinthu komanso chipinda chotaya zinyalala.Ma reagents akhoza kusungidwa mu chipinda chosungiramo chisanadze kwa nthawi yotalikirapo kenako ndikutulutsidwa mu chipinda chosanganikirana.Ndi kuthamanga koyenera, ma reactants osakanikirana amatha kusamutsidwa kuchipinda cha zinyalala kapena chipinda chochitiramo.
Chifukwa kuzindikira kwa PCR ndiye muyeso wagolide wozindikira tizilombo toyambitsa matenda monga H1N1 ndi COVID-19 ndipo kumaphatikizapo njira zingapo zochitira, tidagwiritsa ntchito nsanja ya FAST-POCT yozindikira PCR ngati ntchito.Pa mkuyu.4 ikuwonetsa kuyesa kwa PCR pogwiritsa ntchito nsanja ya FAST-POCT.Choyamba, reagent eluting, maginito microbead reagent, kusamba njira A, ndi kusamba njira W anali pipetted mu chisanadze yosungirako zipinda E, M, W1 ndi W2, motero.Magawo a RNA adsorption akuwonetsedwa mkuyu.4a ndipo ali motere: (1) pamene kukakamiza P1 (= 0.26 bar) kumagwiritsidwa ntchito, chitsanzocho chimasunthira mu chipinda cha M ndikutulutsidwa mu chipinda chosakaniza.(2) Kuthamanga kwa mpweya P2 (= 0.12 bar) kumaperekedwa kudzera pa doko A yolumikizidwa pansi pa chipinda chosakaniza.Ngakhale njira zingapo zosakanikirana zawonetsa kuthekera kwawo pakusakaniza zakumwa pamasamba a POCT (mwachitsanzo, kusakaniza kwa serpentine 53, kusakaniza mwachisawawa 54 ndi kusakaniza kwa batch 55), kusakaniza kwawo bwino komanso kugwira ntchito kwake sikuli kokwanira.Iwo utenga kuwira kusanganikirana njira, imene mpweya anayambitsa pansi pa kusanganikirana chipinda kulenga thovu mu madzi, ndiyeno wamphamvu vortex akhoza kukwaniritsa kusakaniza wathunthu mkati masekondi.Zoyeserera zosakaniza ma Bubble zidachitika ndipo zotsatira zake zimaperekedwa mu Supplementary Figure S6.Zitha kuwoneka kuti kukakamiza kwa 0,10 bar kukugwiritsidwa ntchito, kusakaniza kwathunthu kumatenga pafupifupi masekondi 8.Powonjezera kukakamiza kwa bar 0,20, kusakaniza kwathunthu kumatheka pafupifupi 2 masekondi.Njira zowerengetsera kusakaniza bwino zimaperekedwa mu gawo la Njira.(3) Gwiritsani ntchito maginito a rubidium kuti mutulutse mikanda, kenako panikizani P3 (= 0.17 bar) kudzera pa doko P kuti musunthire ma reagents muchipinda cha zinyalala.Pa mkuyu.4b,c ikuwonetsa masitepe otsuka kuti achotse zonyansa kuchokera pachitsanzo motere: (1) Njira yotsuka A kuchokera kuchipinda W1 imatulutsidwa muchipinda chophatikizira P1.(2) Kenako chitani kusakaniza kuwira.(3) Njira yotsuka A imasamutsidwa kuchipinda chamadzi otayira, ndipo ma microbead omwe ali m'chipinda chosakanikirana amatulutsidwa ndi maginito.Kusamba W (mkuyu 4c) kunali kofanana ndi kutsuka A (mkuyu 4b).Tiyenera kuzindikira kuti sitepe iliyonse yosamba A ndi W inachitidwa kawiri.Chithunzi 4d chikuwonetsa masitepe a elution kuti alute RNA kuchokera ku mikanda;masitepe oyambira ndi kusanganikirana ndi ofanana ndi kutsatsa kwa RNA ndi masitepe ochapira omwe afotokozedwa pamwambapa.Pamene ma reagents a elution amasamutsidwa mu chipinda chochitira PCR pansi pa zovuta P3 ndi P4 (= 0.23 bar), kupanikizika kwakukulu kumafikira kusindikiza mkono wa chipinda chochitira PCR.Momwemonso, kuthamanga kwa P4 kumathandizanso kusindikiza njira yopita kuchipinda cha zinyalala.Chifukwa chake, ma reagents onse a elution adagawidwa mofanana pakati pa zipinda zinayi za PCR kuti ayambitse machitidwe ambiri a PCR.Njira yomwe ili pamwambapa ikuwonetsedwa mu Supplementary Movie S6.
Mu sitepe ya RNA adsorption, chitsanzocho chimalowetsedwa mu cholowetsa M ndikulowetsa mu chipinda chosakaniza pamodzi ndi yankho la mkanda lomwe linasungidwa kale.Pambuyo posakaniza ndi kuchotsa ma granules, ma reagents amagawidwa mu chipinda cha zinyalala.b ndi c kutsuka masitepe, yambitsani zotsukira zosiyanasiyana zosungidwa kale m'chipinda chosanganikirana, ndipo mutatha kusakaniza ndikuchotsa mikandayo, tumizani ma reagents kuchipinda chamadzi otayira.d Elution sitepe: Pambuyo poyambitsa ma reagents a elution, kusakaniza ndi kuchotsa mikanda, ma reagents amasamutsidwa ku chipinda cha PCR.Ma curve akuwonetsa kayendetsedwe ka ntchito ndi magawo okhudzana ndi magawo osiyanasiyana.Kupsyinjika ndiko kupanikizika komwe kumachitika kudzera m'zipinda za munthu aliyense.Volume ndi kuchuluka kwa madzi mu chipinda chosakaniza.Mipiringidzo yonse ndi 1 cm.Data yaiwisi imaperekedwa ngati mafayilo osasinthika.
Njira yoyesera ya PCR idachitika ndipo Supplementary Figure S7 ikuwonetsa mbiri zotentha kuphatikiza mphindi 20 za nthawi yolemba mobwereza ndi mphindi 60 zanthawi yotentha yoyendetsa njinga (95 ndi 60 °C), kuzungulira kumodzi komwe kumakhala 90 s (Supplementary Movie S7)..FAST-POCT imafuna nthawi yocheperako kuti amalize kutenthetsa kumodzi (masekondi 90) kusiyana ndi RT-PCR wamba (masekondi 180 pa kutentha kumodzi).Izi zitha kufotokozedwa ndi kuchuluka kwapamwamba kwa kuchuluka kwa voliyumu komanso kutsika kwapang'onopang'ono kwapang'onopang'ono PCR reaction chamber.Chipinda chapamwamba ndi 96.6 mm2 ndipo voliyumu yachipinda ndi 25 mm3, kupangitsa kuti chiŵerengero chapamwamba chikhale pafupifupi 3.86.Monga tawonera mu Supplementary Figure S10, malo oyesera a PCR papulatifomu yathu ali ndi poyambira kumbuyo, kupangitsa pansi pachipinda cha PCR kukhala 200 µm wandiweyani.Thermally conductive zotanuka pad amamangiriridwa pa Kutentha pamwamba pa chowongolera kutentha, kuonetsetsa kukhudzana kolimba ndi kumbuyo kwa bokosi loyesera.Izi zimachepetsa kutentha kwa nsanja ndikuwongolera kutentha / kuzizira bwino.Pa njinga matenthedwe, parafini ophatikizidwa mu nsanja amasungunuka ndi kuyenda mu PCR anachita chipinda, kuchita ngati sealant kupewa reagent evaporation ndi kuipitsidwa chilengedwe (onani Supplementary Movie S8).
Njira zonse zodziwira za PCR zomwe zafotokozedwa pamwambapa zinali zongochitika zokha pogwiritsa ntchito chida chopangidwa ndi FAST-POCT, chomwe chimakhala ndi pulogalamu yowongolera kuthamanga, maginito otulutsa maginito, gawo lowongolera kutentha, ndi cholumikizira chizindikiro cha fulorosenti.Chodziwikiratu, tidagwiritsa ntchito nsanja ya FAST-POCT pakudzipatula kwa RNA kenako tidagwiritsa ntchito zitsanzo za RNA zochotsedwa pamachitidwe a PCR pogwiritsa ntchito FAST-POCT system ndi desktop PCR system poyerekeza.Zotsatira zake zinali zofanana ndi zomwe zikuwonetsedwa mu Supplementary Figure S8.Wogwira ntchitoyo amachita ntchito yosavuta: amalowetsa chitsanzo mu chipinda cha M ndikuyika nsanja mu chida.Zotsatira za mayeso ochulukira zimapezeka pafupifupi mphindi 82.Zambiri za zida za FAST-POCT zitha kupezeka muzowonjezera.C9, C10 ndi C11.
Fuluwenza yoyambitsidwa ndi ma virus a fuluwenza A (IAV), B (IBV), C (ICV) ndi D (IDV) ndizochitika padziko lonse lapansi.Mwa awa, IAV ndi IBV ndi omwe amayambitsa milandu yoopsa kwambiri komanso miliri yanyengo, yomwe imakhudza 5-15% ya anthu padziko lapansi, zomwe zimapangitsa kuti anthu 3-5 miliyoni azidwala kwambiri komanso kufa 290,000-650,000 pachaka.Matenda a kupuma56,57.Kuzindikira koyambirira kwa IAV ndi IB ndikofunikira kuti muchepetse kudwala komanso zovuta zokhudzana ndi zachuma.Mwa njira zodziwira zomwe zilipo, reverse transcriptase polymerase chain reaction (RT-PCR) imatengedwa kuti ndiyovuta kwambiri, yeniyeni, komanso yolondola (> 99%) 58,59.Mwa njira zodziwira zomwe zilipo, reverse transcriptase polymerase chain reaction (RT-PCR) imatengedwa kuti ndiyovuta kwambiri, yeniyeni, komanso yolondola (> 99%) 58,59.Среди доступных диагностических методов полимеразная цепная реакция с обратной транскриптазой (ОТ-ПЦР) считается наийболее видео > 99%)58,59.Mwa njira zodziwira zomwe zilipo, reverse transcriptase polymerase chain reaction (RT-PCR) imawonedwa ngati yovuta kwambiri, yeniyeni komanso yolondola (> 99%) 58,59. Из доступных диагностических методов полимеразная цепная реакция с обратной транскриптазой (ОТ-ПЦР) считается наийболее чулье, песни 99%)58,59.Mwa njira zodziwira zomwe zilipo, reverse transcriptase polymerase chain reaction (RT-PCR) imatengedwa ngati yovuta kwambiri, yeniyeni komanso yolondola (> 99%) 58,59.Komabe, njira zachikhalidwe za RT-PCR zimafuna kubwereza mapaipi, kusakaniza, kutulutsa ndi kusamutsa madzimadzi, kuchepetsa kugwiritsidwa ntchito kwawo ndi akatswiri pamakonzedwe opanda zida.Apa, nsanja ya FAST-POCT idagwiritsidwa ntchito pozindikira PCR ya IAV ndi IBV, motsatana, kuti apeze malire awo otsika (LOD).Kuonjezera apo, IAV ndi IBV zakhala zikuchulukirachulukira kuti zisankhe pakati pa mitundu yosiyanasiyana ya mitundu yosiyanasiyana ya zamoyo, kupereka malo odalirika owonetsera ma genetic ndi kuthekera kochiza matendawa molondola.
Pa mkuyu.5a ikuwonetsa zotsatira za kuyezetsa kwa HAV PCR pogwiritsa ntchito 150 µl ya ma virus oyeretsedwa a RNA monga chitsanzo.Pa mkuyu.5a (i) imasonyeza kuti pa HAV ndende ya 106 makope / ml, mphamvu ya fluorescence (ΔRn) imatha kufika ku 0.830, ndipo pamene ndende imachepetsedwa kukhala makope a 102 / ml, ΔRn ikhoza kufika ku 0.365, yomwe ikugwirizana ndipamwamba kuposa izo. ya gulu lopanda kanthu lowongolera (0.002), pafupifupi nthawi 100 kupitilira apo.Pakuwerengera motengera kuyeserera kodziyimira pawokha sikisi, mzere wowongolera mzere unapangidwa pakati pa chipika cha chipika ndi malo ozungulira (Ct) wa IAV (mkuyu 5a (ii)), R2 = 0.993, kuyambira 102-106 makope / mL.zotsatira zimagwirizana bwino ndi njira wamba RT-PCR.Pa mkuyu.5a (iii) imasonyeza zithunzi za fulorosenti za zotsatira zoyesa pambuyo pa maulendo a 40 a nsanja ya FAST-POCT.Tidapeza kuti nsanja ya FAST-POCT imatha kuzindikira HAV yotsika ngati makope 102/mL.Komabe, njira yachikhalidwe ilibe mtengo wa Ct pa 102 makope/mL, kupangitsa LOD kukhala pafupifupi 103 makope/mL.Tidaganiza kuti izi zitha kukhala chifukwa chakuchita bwino kwambiri kwakusakanizika kwa thovu.Kuyesa kwa PCR kunachitika pa IAV RNA yoyeretsedwa kuti iwunikire njira zosiyanasiyana zosanganikirana, kuphatikiza kusakaniza kogwedezeka (njira yosakanikirana yofananira monga momwe amachitira ochiritsira RT-PCR), kusakaniza vial (njira iyi, 3 s pa 0,12 bar) ndipo palibe kusakanikirana ngati gulu lolamulira. ..Zotsatira zitha kupezeka mu Supplementary Figure S12.Zitha kuwoneka kuti pagulu lapamwamba la RNA (makopi 106/mL), mayendedwe a Ct anjira zosiyanasiyana zosanganikirana amakhala pafupifupi ofanana ndi kusakaniza kuwira.Pamene ndondomeko ya RNA inatsikira ku 102 makope / mL, kusakaniza kugwedeza ndi kuwongolera kunalibe Ct, pamene njira yosakanikirana ya bubble inaperekabe mtengo wa Ct wa 36.9, womwe unali pansi pa Ct 38. Zotsatira zimasonyeza khalidwe losakanikirana kwambiri. ma vesicles, omwe adawonetsedwanso m'mabuku ena, omwe angafotokozenso chifukwa chake kukhudzika kwa nsanja ya FAST-POCT ndikokwera pang'ono kuposa RT-PCR wamba.Pa mkuyu.5b ikuwonetsa zotsatira za kusanthula kwa PCR kwa zitsanzo zoyeretsedwa za IBV RNA kuyambira 101 mpaka 106 makope/ml.Zotsatira zinali zofanana ndi kuyesa kwa IAV, kukwaniritsa R2 = 0.994 ndi LOD ya 102 makope / mL.
kusanthula kwa PCR kwa kachilombo ka fuluwenza A (IAV) yokhala ndi ndende ya IAV kuyambira 106 mpaka 101 makope/mL pogwiritsa ntchito TE buffer ngati control negative (NC).(i) Nthawi yeniyeni yokhotakhota ya fluorescence.(ii) Linear calibration curve pakati pa logarithmic IAV RNA concentration and cycle threshold (Ct) ya FAST ndi njira wamba zoyesera.(iii) Chithunzi cha fluorescent cha IAV FAST-POCT pambuyo pa mizungu 40.b, PCR kuzindikira kachilombo ka fuluwenza B (IBV) ndi (i) nthawi yeniyeni ya fluorescence spectrum.(ii) Linear calibration curve ndi (iii) FAST-POCT IBV fluorescence image pambuyo pa 40 cycle.Malire otsika ozindikira (LOD) a IAV ndi IBV pogwiritsa ntchito nsanja ya FAST-POCT inali 102 makope / mL, yomwe ili yotsika kusiyana ndi njira zamakono (103 makope / mL).c Zotsatira za mayeso angapo a IAV ndi IBV.GAPDH idagwiritsidwa ntchito ngati chiwongolero chabwino ndipo TE buffer idagwiritsidwa ntchito ngati chiwongolero choyipa kuti chiteteze kuipitsidwa komwe kungachitike komanso kukulitsa kumbuyo.Mitundu inayi ya zitsanzo ingasiyanitsidwe: (1) GAPDH-okha zitsanzo zoipa (“IAV-/IBV-”);(2) matenda a IAV ("IAV +/IBV-") ndi IAV ndi GAPDH;(3) Matenda a IBV ("IAV-/IBV +") ndi IBV ndi GAPDH;(4) Matenda a IAV/IBV (“IAV+/IBV+”) ndi IAV, IBV ndi GAPDH.Mzere wa madontho umayimira poyambira.n = Zoyeserera za 6 zodziyimira pawokha za biologically zidachitika, deta ikuwonetsedwa ngati ± kupatuka kokhazikika.Data yaiwisi imaperekedwa ngati mafayilo osasinthika.
Pa mkuyu.5c ikuwonetsa zotsatira za mayeso ochulukitsa a IAV/IBV.Apa, kachilombo ka lysate kanagwiritsidwa ntchito ngati chitsanzo cha yankho m'malo mwa RNA yoyeretsedwa, ndipo zoyambira zinayi za IAV, IBV, GAPDH (zowongolera zabwino) ndi TE buffer (kuwongolera koyipa) zidawonjezedwa kuzipinda zinayi zosiyana za nsanja ya FAST-POCT.Kuwongolera kwabwino ndi koyipa kumagwiritsidwa ntchito pano kuti apewe kuipitsidwa komwe kungachitike komanso kukulitsa maziko.Mayeserowa adagawidwa m'magulu anayi: (1) zitsanzo za GAPDH-negative ("IAV-/IBV-");(2) IAV-infected ("IAV +/IBV-") motsutsana ndi IAV ndi GAPDH;(3) IBV-.kachilombo (“IAV-”) -/IBV+”) IBV ndi GAPDH;(4) IAV/IBV (“IAV+/IBV+”) matenda ndi IAV, IBV ndi GAPDH.Pa mkuyu.5c ikuwonetsa kuti zitsanzo zoyipa zikagwiritsidwa ntchito, mphamvu ya fluorescence ΔRn ya chipinda chowongolera chabwino inali 0.860, ndipo ΔRn ya IAV ndi IBV inali yofanana ndi kuwongolera koyipa (0.002).Kwa magulu a IAV+/IBV-, IAV-/IBV+ ndi IAV+/IBV+, makamera a IAV/GAPDH, IBV/GAPDH ndi IAV/IBV/GAPDH amawonetsa kulimba kwa fluorescence, motsatana, pomwe makamera ena amawonetsa kulimba kwa fulorosisi kumbuyo. mlingo wa 40 pambuyo pa njinga yamoto yotentha.Kuchokera ku mayesero omwe ali pamwambawa, nsanja ya FAST-POCT inasonyeza tsatanetsatane ndipo inatilola nthawi imodzi kuti tipeze mavairasi osiyanasiyana a chimfine.
Kuti titsimikizire kugwiritsa ntchito kwachipatala kwa FAST-POCT, tinayesa zitsanzo zachipatala za 36 (zitsanzo za mphuno za mphuno) kuchokera kwa odwala IB (n=18) ndi olamulira omwe si a IB (n=18) (Chithunzi 6a).Zambiri za odwala zimaperekedwa mu Supplementary Table 3. Mkhalidwe wa matenda a IB unatsimikiziridwa paokha ndipo ndondomeko yophunzirira inavomerezedwa ndi Zhejiang University First Affiliated Hospital (Hangzhou, Zhejiang).Chitsanzo chilichonse cha odwala chinagawidwa m'magulu awiri.Imodzi idakonzedwa pogwiritsa ntchito FAST-POCT ndipo ina idakonzedwa pogwiritsa ntchito kompyuta PCR system (SLAN-96P, China).Mayesero onsewa amagwiritsa ntchito zida zofanana zoyeretsera ndi kuzindikira.Pa mkuyu.6b ikuwonetsa zotsatira za FAST-POCT ndi PCR (RT-PCR).Tidayerekeza mphamvu ya fluorescence (FAST-POCT) ndi -log2(Ct), pomwe Ct ndiye gawo lozungulira la RT-PCR wamba.Panali mgwirizano wabwino pakati pa njira ziwirizi.FAST-POCT ndi RT-PCR adawonetsa mgwirizano wabwino kwambiri ndi mtengo wa Pearson (r) wa 0.90 (Chithunzi 6b).Kenako tidawunika kulondola kwa FAST-POCT.Fluorescence intensity (FL) kugawa kwa zitsanzo zabwino ndi zoipa kunaperekedwa ngati muyeso wodziyimira pawokha (mkuyu 6c).Makhalidwe a FL anali apamwamba kwambiri mwa odwala IB kuposa momwe amawongolera (**** P = 3.31 × 10-19; t-mayeso awiri) (mkuyu 6d).Kenako, ma curve a IBV receiver (ROC) adakonzedwa.Tinapeza kuti kulondola kwa matenda kunali kwabwino kwambiri, ndi malo omwe ali pansi pa 1 (mkuyu 6e).Chonde dziwani kuti chifukwa cholamula kuyitanitsa chigoba ku China chifukwa cha COVID-19 kuyambira 2020, sitinazindikire odwala omwe ali ndi IBD, kotero kuti zitsanzo zonse zabwino zachipatala (ie, zitsanzo za swab za m'mphuno) zinali za IBV zokha.
Kapangidwe ka maphunziro azachipatala.Zitsanzo zonse za 36, kuphatikizapo zitsanzo za odwala 18 ndi 18 zowongolera zopanda chimfine, zidafufuzidwa pogwiritsa ntchito nsanja ya FAST-POCT ndi RT-PCR wamba.b Unikani kusasinthika kosanthula pakati pa FAST-POCT PCR ndi RT-PCR wamba.Zotsatira zinali zogwirizana bwino (Pearson r = 0.90).c Fluorescence intensity milingo mwa odwala 18 IB ndi 18 zowongolera.d Odwala a IB (+), FL mfundo zinali zapamwamba kwambiri kuposa gulu lolamulira (-) (****P = 3.31 × 10-19; t-mayeso awiri; n = 36).Pachiwembu chilichonse, cholembera chakuda chapakati chimayimira wapakati, ndipo mizere yapansi ndi yapamwamba ya bokosilo imayimira maperesenti a 25 ndi 75, motsatana.Ndevu zimafikira kuzinthu zochepa komanso zochulukirapo, zomwe sizimaganiziridwa kuti ndizochepa.ndi ROC curve.Mzere wa madontho d umayimira mtengo woyerekeza kuchokera pakuwunika kwa ROC.AUC ya IBV ndi 1. Data yaiwisi imaperekedwa ngati mafayilo amtundu wa data.
Munkhaniyi, tikuwonetsa FAST, yomwe ili ndi mawonekedwe ofunikira pa POCT yabwino.Ubwino waukadaulo wathu umaphatikizapo: (1) Madontho osiyanasiyana (kutsika, nthawi imodzi, zotsatizana ndi kusankha), kumasulidwa pakufunika (kutulutsa mwachangu komanso molingana ndi kukakamizidwa kogwiritsidwa ntchito) ndi magwiridwe antchito odalirika (kugwedezeka kwa madigiri 150) (2) kusungidwa kwanthawi yayitali. (zaka 2 za kuyezetsa mwachangu, kuchepa thupi pafupifupi 0.3%);(3) kuthekera kugwira ntchito ndi zakumwa zokhala ndi kunyowa komanso kukhuthala (makamakamakamaka mpaka 5500 cP);(4) Zachuma (Kuyerekeza mtengo wazinthu za chipangizo cha FAST-POCT PCR ndi pafupifupi US $ 1).Mwa kuphatikiza ma dispensers amitundu yambiri, nsanja yophatikizika ya FAST-POCT ya PCR kuzindikira ma virus a fuluwenza A ndi B idawonetsedwa ndikuyikidwa.FAST-POCT imangotenga mphindi 82.Mayesero azachipatala okhala ndi zitsanzo za 36 za nasal swab adawonetsa kuvomerezana kwamphamvu kwa fluorescence ndi standard RT-PCR (Pearson coefficients> 0.9).Mayesero azachipatala okhala ndi zitsanzo za 36 za nasal swab adawonetsa kuvomerezana kwamphamvu kwa fluorescence ndi standard RT-PCR (Pearson coefficients> 0.9).Клинические тесты с 36 образцами мазков из носа показали хорошее соответствие интенсивности флуоресценции стандартной ОТ-ПЦенций ОТ-ПЦ (коэфенценции стандартной ОТ-ПЦР,фифортной 0.Kuyeza kwachipatala ndi zitsanzo 36 za swabs za m'mphuno kunasonyeza kuvomerezana bwino ndi mphamvu ya fluorescence ya RT-PCR (Pearson's coefficients> 0.9).RT-PCR Клинические испытания 36 образцов мазков из носа показали хорошее совпадение интенсивности флуоресценции со стандартной ОТ-эфинценции стандартной ОТ-эфинций ОТ-эфинценции стандартной ОТэфенценцийKuyesa kwachipatala kwa zitsanzo za 36 za swab zamphuno kunasonyeza kuvomerezana kwamphamvu kwa fluorescence ndi RT-PCR yokhazikika (Pearson's coefficient> 0.9).Mogwirizana ndi ntchitoyi, njira zosiyanasiyana zomwe zikubwera (mwachitsanzo, kuyendetsa njinga yamoto ya plasma, ma immunoassays opanda amplification, ndi nanobody functionalization assays) awonetsa kuthekera kwawo mu POCT.Komabe, chifukwa cha kusowa kwa nsanja ya POCT yophatikizika bwino komanso yolimba, njirazi zimafunikira njira zosiyana zosinthira (mwachitsanzo, RNA isolation44, incubation45 ndi washing46), zomwe zimakwaniritsanso ntchito yomwe ilipo ndi njira izi kukhazikitsa ntchito zapamwamba za POCT ndi magawo ofunikira.kutengera-mu-kuyankha-zotuluka.Mu ntchitoyi, ngakhale kuti pampu ya mpweya yomwe imagwiritsidwa ntchito poyambitsa valavu ya FAST ndi yaying'ono yokwanira kuti igwirizane ndi chida cha benchtop (Mkuyu S9, S10), imagwiritsabe ntchito mphamvu zazikulu ndikupanga phokoso.M'malo mwake, mapampu ang'onoang'ono amtundu wa pneumatic amatha kusinthidwa ndi njira zina, monga kugwiritsa ntchito mphamvu yamagetsi yamagetsi kapena kuyambitsa chala.Zowonjezereka zowonjezereka zingaphatikizepo, mwachitsanzo, kusintha zida zowunikira zosiyana ndi zenizeni za biochemical, pogwiritsa ntchito njira zatsopano zodziwira zomwe sizikufuna kutenthetsa / kuzizira, motero zimapereka chida chopanda zida cha POCT cha mapulogalamu a PCR.Timakhulupirira kuti popeza nsanja ya FAST imapereka njira yoyendetsera zakumwa, tikukhulupirira kuti ukadaulo wa FAST womwe waperekedwa umapereka mwayi wopanga nsanja yofananira osati kungoyesa kwachilengedwe kokha, komanso kuyang'anira chilengedwe, kuyesa kwa zakudya, kaphatikizidwe kazinthu ndi mankhwala. ..
Kusonkhanitsa ndi kugwiritsa ntchito zitsanzo za swab za m'mphuno za munthu kwavomerezedwa ndi Komiti Yowona za Ethics ya Zhejiang University First Affiliated Hospital (IIT20220330B).Zitsanzo 36 za swab za m'mphuno zinasonkhanitsidwa, kuphatikizapo akuluakulu 16 ochepera zaka 30, akuluakulu 7> zaka 40, ndi amuna 19, 17 akazi.Zitsanzo 36 za swab za m'mphuno zinasonkhanitsidwa, kuphatikizapo akuluakulu 16 ochepera zaka 30, akuluakulu 7> zaka 40, ndi amuna 19, 17 akazi.Было собрано 36 образцов мазков из носа, в которых приняли участие 16 взрослых <30 лет, 7 взрослых старше 40 лет17 инге 19.Zitsanzo makumi atatu ndi zisanu ndi chimodzi za swab za m'mphuno zinatengedwa kuchokera kwa akuluakulu 16 ochepera zaka 30, akuluakulu 7 azaka zopitilira 40, amuna 19 ndi akazi 17..Deta ya chiwerengero cha anthu ikuperekedwa mu Supplementary Table 3. Chilolezo chodziwitsidwa chinapezedwa kuchokera kwa onse omwe atenga nawo mbali.Onse omwe adatenga nawo mbali adaganiziridwa kuti ali ndi chimfine ndipo adayesedwa mwakufuna kwawo popanda chipukuta misozi.
FAST base ndi chivindikiro amapangidwa ndi polylactic acid (PLA) ndi kusindikizidwa ndi Ender 3 Pro 3D printer (Shenzhen Transcend 3D Technology Co., Ltd.).Tepi ya mbali ziwiri idagulidwa ku Adhesives Research, Inc. Model 90880. Filimu ya PET yokhuthala 100 µm idagulidwa kuchokera kwa McMaster-Carr.Zonse zomatira ndi filimu ya PET zinadulidwa pogwiritsa ntchito Silhouette Cameo 2 cutter kuchokera ku Silhouette America, Inc. Filimu yotanuka imapangidwa ndi zinthu za PDMS ndi jekeseni.Choyamba, 200 µm wandiweyani PET chimango chinadulidwa pogwiritsa ntchito laser system ndikumata ku 3 mm wandiweyani pepala la PMMA pogwiritsa ntchito tepi yomatira ya mbali ziwiri ya 100 µm.Kalambulabwalo wa PDMS (Sylgard 184; Gawo A: Gawo B = 10: 1, Dow Corning) adatsanuliridwa mu nkhungu ndipo ndodo yagalasi idagwiritsidwa ntchito kuchotsa PDMS yochulukirapo.Pambuyo pochiritsa pa 70 ° C. kwa maola atatu, filimu ya 300 μm yokhuthala ya PDMS imatha kusenda nkhungu.
Zithunzi zogawanitsa mosiyanasiyana, zosindikiza zomwe mukufuna komanso magwiridwe antchito odalirika amatengedwa ndi kamera yothamanga kwambiri (Sony AX700 1000 fps).Chombo cha orbital chomwe chinagwiritsidwa ntchito poyesa kudalirika chinagulidwa kuchokera ku SCILOGEX (SCI-O180).Kuthamanga kwa mpweya kumapangidwa ndi mpweya wa compressor, ndipo zowongolera zingapo za digito zolondola zowongolera zimagwiritsidwa ntchito kusintha kuchuluka kwa kuthamanga.Njira yoyezera khalidwe lakuyenda ndi motere.Kuchulukitsidwa kwamadzimadzi kunalowetsedwa mu chipangizo choyesera ndipo kamera yothamanga kwambiri inagwiritsidwa ntchito kulemba khalidwe loyenda.Zithunzizo zidatengedwa kuchokera kumavidiyo amayendedwe oyenda nthawi zoikika, ndipo malo otsalawo adawerengedwa pogwiritsa ntchito pulogalamu ya Image-Pro Plus, yomwe idachulukitsidwa ndi kuya kwa kamera kuwerengera voliyumu.Tsatanetsatane wa njira yoyezera machitidwe oyenda imapezeka mu Supplementary Figure S4.
Imani 50 µl ya timikanda tating'onoting'ono ndi 100 µl yamadzi osakanizidwa mu chipangizo chosakaniza.Zithunzi zosakanikirana zojambulidwa zidajambulidwa ndi kamera yothamanga kwambiri masekondi aliwonse a 0.1 pazovuta za 0.1 bar, 0.15 bar ndi 0.2 bar.Zambiri za pixel panthawi yosakaniza zitha kupezeka pazithunzizi pogwiritsa ntchito pulogalamu yokonza zithunzi (Photoshop CS6).Ndipo kusakaniza bwino kumatha kutheka ndi Equation 53 yotsatirayi.
pomwe M ndiye kusakaniza bwino, N ndiye kuchuluka kwa ma pixel a zitsanzo, ndipo ci ndi \(\bar{c}\) ndizokhazikika komanso zoyembekezeka zokhazikika.Kusakaniza koyenera kumayambira 0 (0%, osasakaniza) mpaka 1 (100%, osakanizidwa kwathunthu).Zotsatira zikuwonetsedwa mu Supplementary Figure S6.
Zida zenizeni za RT-PCR za IAV ndi IBV, kuphatikiza zitsanzo za IAV ndi IBV RNA (paka no. RR-0051-02/RR-0052-02, Liferiver, China), Tris-EDTA buffer (TE buffer no. B541019 , Sangon Biotech, China), Positive Control RNA Purification Kit (Gawo No. Z-ME-0010, Liferiver, China) ndi GAPDH Solution (Gawo No. M591101, Sangon Biotech, China) ndi malonda.Zida zoyeretsera za RNA zimaphatikizapo chotchinga chomangira, kuchapa A, kuchapa W, eluent, maginito ma microbead, ndi chonyamulira cha acrylic.IAV ndi IBV zida zenizeni za RT-PCR zikuphatikizapo IFVA nucleic acid PCR kuzindikira mix ndi RT-PCR enzyme.Onjezani 6 µl ya AcrylCarrier ndi 20 µl ya mikanda ya maginito ku 500 µl ya njira yomangira yotsekera, gwedezani bwino kenaka konzani njira yothira mikanda.Onjezerani 21 ml ya ethanol kuti musambe A ndi W, gwedezani bwino kuti mupeze njira zotsuka A ndi W, motero.Kenaka, 18 µl ya fulorosenti PCR osakaniza ndi IFVA nucleic acid ndi 1 µl ya RT-PCR enzyme anawonjezeredwa ku 1 µl ya TE yankho, kugwedezeka ndi centrifuged kwa masekondi angapo, kupeza 20 µl wa IAV ndi IBV zoyambira.
Tsatirani njira zotsatirazi zoyeretsera RNA: (1) RNA adsorption.Pipette 526 µl ya mankhwala a pellet mu chubu cha 1.5 ml centrifuge ndikuwonjezera 150 µl ya zitsanzo, kenaka gwedezani chubucho mmwamba ndi pansi nthawi khumi.Tumizani 676 µl ya zosakaniza ku gawo logwirizana ndi centrifuge pa 1.88 x 104 g kwa masekondi 60.Ma drains otsatirawa amatayidwa.(2) Gawo loyamba la kuchapa.Onjezani 500 µl wa mankhwala ochapira A ku gawo la affinity, centrifuge pa 1.88 x 104 g kwa 40 s, ndikutaya yankho lomwe mwagwiritsa ntchito.Kusamba kumeneku kunabwerezedwa kawiri.(3) siteji yachiwiri ya kuchapa.Onjezani 500 µl wa njira yotsuka W ku gawo logwirizana, centrifuge pa 1.88×104 g kwa 15 s ndikutaya yankho lomwe mwagwiritsa ntchito.Kusamba kumeneku kunabwerezedwa kawiri.(4) Chidziwitso.Onjezani 200 µl wa eluate ku gawo logwirizana ndi centrifuge pa 1.88 x 104 g kwa 2 min.(5) RT-PCR: Eluate idabayidwa mu 20 μl ya yankho la primer mu chubu cha PCR, ndiye chubucho chinayikidwa mu chipangizo choyesera cha PCR (SLAN-96P) kuti akwaniritse ndondomeko ya RT-PCR.Njira yonse yodziwira imatenga pafupifupi mphindi 140 (mphindi 20 pakuyeretsa RNA ndi mphindi 120 kuti PCR izindikire).
526 µl ya yankho la mkanda, 1000 µl wa wash solution A, 1000 µl wa wash solution W, 200 µl wa eluate ndi 20 µl wa primer solution adawonjezedwa ndikusungidwa mu zipinda za M, W1, W2, E ndi PCR.Msonkhano wa nsanja.Kenako, 150 µl yachitsanzo idalowetsedwa muchipinda M ndipo nsanja ya FAST-POCT idayikidwa mu chida choyesera chomwe chikuwonetsedwa mu Supplementary Figure S9.Pambuyo pa mphindi 82, zotsatira za mayeso zidapezeka.
Pokhapokha ngati tafotokozera, zotsatira zonse zoyesa zimaperekedwa ngati ± SD patatha zosachepera zisanu ndi chimodzi pogwiritsa ntchito nsanja ya FAST-POCT yokha komanso zitsanzo zodziyimira pawokha.Palibe deta yomwe idachotsedwa pakuwunika.Zoyeserera sizichitika mwachisawawa.Ofufuzawo sanali akhungu ku ntchito zamagulu panthawi yoyesera.
Kuti mumve zambiri pamapangidwe amaphunziro, onani Nature Research Report abstract yolumikizidwa ndi nkhaniyi.
Deta yothandizira zotsatira za kafukufukuyu ikupezeka mu Zowonjezera Zowonjezera.Nkhaniyi imapereka deta yoyambirira.
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