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Lab-on-a-chip masisitimu ane pa-saiti zvikwanisiro zvinopa mukana wekukurumidza uye wakarurama kuongororwa uye anobatsira mune zviwanikwa-zvinodzora zvigadziriso uko biomedical michina uye vakadzidziswa nyanzvi havawanikwe.Nekudaro, kugadzira poindi-ye-kutarisira yekuyedza sisitimu iyo panguva imwe chete ine zvese zvinodiwa zveakawanda-inoshanda kugovera, pane-inoda kuburitswa, kuita kwakavimbika, uye kuchengetwa kwenguva refu kwemareagents kunoramba kuri dambudziko guru.Pano isu tinotsanangura lever-actuated micro travel switch tekinoroji inogona kushandura zvinwiwa chero munzira, inopa chaiyo uye yakaenzana mhinduro kune yakashandiswa mhepo yekumanikidza, uye ramba wakagadzikana uchipokana nekukasira mafambiro uye vibrations.Zvichienderana netekinoroji, isu tinotsanangurawo kuvandudzwa kwepolymerase chain reaction system iyo inobatanidza reagent sumo, kusanganisa uye kuita mabasa ese muchiitiko chimwe chete, chinogonesa "sample-in-answer-out" kuita kune ese ekiriniki nasal samples kubva kuvarwere gumi nevasere vane. Influenza uye 18 munhu anodzora, mune yakanaka concordance yefluorescence intensity ine yakajairwa polymerase chain reaction (Pearson coefficients> 0.9).Zvichibva pane tekinoroji, isu tinotsanangurawo kuvandudzwa kwepolymerase chain reaction system inobatanidza reagent sumo, kusanganisa uye kuita mabasa ese muchirongwa chimwe, chinogonesa "sample-in-answer-out" kuita kune ese ekiriniki nasal samples kubva kuvarwere gumi nevasere. neInfluenza uye 18 zvidzoreso zvemunhu, mune yakanaka concordance yefluorescence intensity ine yakajairwa polymerase chain reaction (Pearson coefficients> 0.9).Основываясь на этой технологии, мы также описываем разработку системы полимеразной цепной реакции, которая объединяет фунцияет фунцияет фунцияет фуединяет фуединяет фуединяет фуединяет фуединяет фунция реакции в одном процессе, что обеспечивает выполнение «образец-в-ответ-выход» для всех клинических образцов образцов из 18 сположение uye 18 dzidziso dzakatsauka, dzakatsauka, dzakatarwa, dzakatarwa, dzakatarwa, dzakatarwa, dzakatarwa, dzakaremara.Kubva pane tekinoroji iyi, isu tinotsanangurawo kuvandudzwa kwepolymerase chain reaction system inosanganisa mabasa ekubaya, kusanganisa, uye kuita mune imwechete maitiro, zvichiita kuti sampuli-mu-mhinduro-kunze kune ese ekiriniki nasal specimens kubva kuvarwere gumi nemasere.uye 18 kudzora kwega, mukubvumirana kwakanaka neyakajairwa polymerase chain reaction fluorescence intensity (Pearson's coefficients> 0.9).Tichibva pane tekinoroji iyi, tinotsanangurawo kugadzirwa kwepolymerase chain reaction system inobatanidza reagent jekiseni, kusanganisa, uye maitiro ekuita kuongorora ese ekiriniki nasal specimens kubva ku18 mu-sample mumhino dzemurwere. zvakanaka neyakajairwa polymerase chain reaction (Pearson's coefficient> 0.9).Ipuratifomu yakarongwa inovimbisa yakavimbika otomatiki yekuongorora biomedical uye nekudaro inogona kukurumidzira kushambadzira kweakasiyana-e-yekutarisira-yekuongorora michina.
Zvirwere zvevanhu zviri kusimukira, zvakaita sedenda ra2020 COVID-19 iro rakatora hupenyu hwemamirioni avanhu, zvinoisa njodzi kuhutano hwepasirese uye budiriro yevanhu1.Kutanga, nekukurumidza uye kwakaringana kuonekwa kwezvirwere kwakakosha kudzora kupararira kwehutachiona uye kugadzirisa mhedzisiro yekurapa.Iyo yakakosha diagnostic ecosystem yakavakirwa pakati pemaLab apo masampula ebvunzo anotumirwa kuzvipatara kana makiriniki ekuongorora uye anotungamirwa nenyanzvi ari kurambidza kupinda kwevanhu vangangosvika mabhiriyoni 5.8 pasi rese, kunyanya avo vanogara munzvimbo dzakamanikidzwa zviwanikwa.uko kune kushomeka kwemidziyo inodhura yebiomedical uye nyanzvi dzinokwanisa.makiriniki 2. Nokudaro, pane chido chekukurumidzira kuumba isingadhuri uye inoshandiswa-inoshamwaridzana lab-on-a-chip system ine-point-of-care test (POCT) inogona kupa varapi ruzivo rwekuongorora panguva yakakodzera kuti vaite zvisarudzo zvine ruzivo. .uye kurapwa 3.
Nhungamiro yeWorld Health Organisation (WHO) inotaura kuti POCT yakanakira inofanirwa kutengeka, mushandisi-ane hushamwari (nyore kushandisa nekudzidziswa kushoma), akarurama (kudzivisa manyepo enhema kana manyepo), nekukurumidza uye akavimbika (kupa zvakanaka kudzokorora zvivakwa), uye inotakurika (inokwanisa kuchengetedza kwenguva refu uye inowanikwa nyore kune vashandisi vekupedzisira)4.Kuzadzisa izvi zvinodiwa, maPOCT masisitimu anofanirwa kupa anotevera maficha: anochinja-chinja dosing kudzikisa kupindira kwemanyorero, pane-inoda kuburitswa kuyera reagent yekufambisa kune chaiyo bvunzo mhinduro, uye kuita kwakavimbika kumirisana nekudengenyeka kwezvakatipoteredza.Parizvino, iyo inonyanya kushandiswa POCT mudziyo ndeye lateral kuyerera strip5,6 ine akati wandei akaturikidzana eporous nitrocellulose membranes anosundira shoma shoma yemuenzaniso mberi, ichiita ne pre-immobilized reagents ne capillary force.Kunyangwe ivo vaine mukana wemutengo wakaderera, nyore kushandisa, uye nekukurumidza mhedzisiro, inoyerera strip-based POCT zvishandiso zvinogona kushandiswa chete kune biological bvunzo (semuenzaniso, glucose bvunzo7,8 uye nhumbu bvunzo9,10) pasina anoda akawanda-nhanho ongororo.maitiro (semuenzaniso kurodha akawanda reagents, kusanganisa, kuwanda).Mukuwedzera, masimba ekufambisa anodzora kufamba kwemvura (kureva, capillary forces) haapi hutano hwakanaka, kunyanya pakati pemabheti, zvichiita kuti urombo husina kunaka kuberekwa11 uye kuita lateral flow mabhendi anonyanya kubatsira pakuona zvakanaka12,13.
Kuwedzera hunyanzvi hwekugadzira paiyo micro- uye nanoscale yakagadzira mikana yekuvandudza microfluidic POCT zvishandiso zvekuyera zviyero14,15,16,17.Nekugadzirisa zvinhu zveiyo interface 18, 19 uye geometry yezviteshi 20, 21, 22, simba recapillary uye kuyerera kwezvishandiso izvi zvinogona kudzorwa.Nekudaro, kuvimbika kwavo, kunyanya kune zvinwiwa zvakanyorova zvakanyanya, kunoramba kusingagamuchirwe nekuda kwekugadzira zvisirizvo, hurema hwezvinhu, uye kunzwa kune kwezvakatipoteredza vibrations.Uye zvakare, sezvo kuyerera kwecapillary kunogadzirwa pane yemvura-gasi interface, hapana kumwe kuyerera kunogona kuunzwa, kunyanya mushure mekuzadza iyo microfluidic chiteshi nemvura.Naizvozvo, kuti zvionekwe zvakanyanya kuoma, matanho akati wandei ejekiseni remuenzaniso anofanira kuitwa24,25.
Pakati pemidziyo yemicrofluidic, centrifugal microfluidic zvishandiso parizvino ndiyo imwe yemhinduro dzakanakisa dzePOCT26,27.Kutyaira kwaro kunobatsira pakuti simba rekutyaira rinogona kudzorwa nekugadzirisa kumhanya kwekutenderera.Zvisineyi, chakashata ndechekuti simba recentrifugal rinogara rakanangidzirwa kumucheto kwekunze kwechigadzirwa, zvichiita kuti zviome kuita maitiro akawanda-nhanho anodiwa pakuongorora kwakaoma.Kunyangwe mamwe masimba ekutyaira (semuenzaniso capillaries 28, 29 uye mamwe akawanda 30, 31, 32, 33, 34, 35) mukuwedzera kune iyo centrifugal simba inounzwa kune yakawanda-functional dosing, kusingatarisirwe kufambisa kwemvura kunogona kuitika nekuti aya ekuwedzera masimba anowanzo mirairo. yehukuru hwakaderera pane centrifugal force, zvichiita kuti dzishande chete pamusoro pezvidiki zvekushandisa kana kusawanikwa pakudiwa nekuburitswa kwemvura.Kubatanidza kushandiswa kwepneumatic mu centrifugal microfluidics senge centrifugal kinetic nzira 36, 37, 38, thermopneumatic nzira 39 uye inoshanda pneumatic nzira 40 yakaratidza kuva imwe nzira inoyevedza.Neine counterfugodynamic nzira, imwe mhango uye inobatanidza microchannels inosanganiswa muchishandiso chezvese kunze uye mukati chiito, kunyange zvazvo kupomba kwayo kushanda zvakanaka (kubva pa75% kusvika 90%) kunoenderana zvakanyanya nehuwandu hwekupomba uye viscosity. yemvura.Mune iyo thermopneumatic nzira, iyo latex membrane uye fluid yekuchinjisa kamuri yakanyatso gadzirwa kuti iname kana kuvhurazve inopinza kana iyo yakavharirwa mhepo inodziya kana kutonhodzwa.Nekudaro, iyo yekudziya/kutonhora setup inouyisa matambudziko ekupindura anononoka uye inomisa kushandiswa kwayo mune thermosensitive assays (semuenzaniso, polymerase chain reaction (PCR) amplification).Nemaitiro anoshanda epneumatic, pane-inoda kusunungurwa uye kufamba kwemukati kunowanikwa kuburikidza nekushandisa panguva imwe chete yekumanikidza kwakanaka uye yakanyatsoenderana nekutenderera kumhanyisa nemamota akakwira-speed.Kune dzimwe nzira dzakabudirira dzinoshandisa chete pneumatic actuators (positive pressure 41, 42 kana negative pressure 43) uye kazhinji yakavharwa mavharuvhu magadzirirwo.Nekutevedzana kushandisa kudzvanywa mukamuri yepneumatic, iyo mvura inopombirwa kumberi peristaltically, uye iyo inowanzovharwa vharuvhu inodzivirira kuyerera kwemvura nekuda kweperistalsis, nekudaro inoona yakaoma mvura mashandiro.Nekudaro, pari zvino pane mashoma mashoma eiyo microfluidic tekinoroji inogona kuita yakaoma mashandiro emvura mune imwechete POCT mudziyo, kusanganisira yakawanda-inoshanda kugovera, pane-inoda kuburitswa, kuita kwakavimbika, kuchengetedza kwenguva refu, kubata kwepamusoro-viscosity zvinwiwa, uye kugadzirwa kusingadhuri.Zvose panguva imwe chete.Kushaikwa kweakawanda-nhanho mashandiro ekushanda kunogona zvakare kuve chimwe chezvikonzero nei zvishoma chete zvekutengesa zvePOCT zvigadzirwa seCepheid, Binx, Visby, Cobas Liat, uye Rhonda zvakaunzwa zvakabudirira pamusika wakavhurika kusvika parizvino.
Mubepa rino, isu tinokurudzira pneumatic microfluidic actuator yakavakirwa pagirinhi mhete micro switch tekinoroji (FAST).FAST inobatanidza zvinhu zvose zvinodiwa panguva imwe chete kune zvakasiyana-siyana zve reagents kubva ku microliters kusvika ku milliliters.FAST ine elastic membranes, levers uye zvivharo.Pasina kushandiswa kwemhepo, membrane, levers uye zvivharo zvinogona kuvharwa zvakasimba uye mvura mukati inogona kuchengetwa kwenguva yakareba.Kana kudzvinyirirwa kwakakodzera kuchiiswa uye kugadziridzwa kusvika pakureba kwechitsigiro, diaphragm inowedzera uye inosundira chibatiso panzvimbo yakavhurika, ichibvumira mvura kuti ipfuure.Izvi zvinobvumira multifunctional metering yezvinwiwa mune cascade, panguva imwe chete, inotevedzana kana yekusarudza maitiro.
Isu takagadzira PCR sisitimu tichishandisa FAST kugadzira mhinduro-mu-sample mhedzisiro yekuonekwa kwefuruwenza A uye B mavhairasi (IAV neIBV).Takawana muganho wakaderera wekuona (LOD) we102 makopi / mL, yedu multiplex assay yakaratidza chaiyo yeIAV neIBV uye yakabvumira furuwenza virus pathotyping.Mhedzisiro yekuongororwa kwekiriniki inoshandisa sampu yemhino kubva kuvarwere gumi nevasere uye vanhu gumi nevasere vane hutano vanoratidza yakanaka concordance mufluorescence intensity ine yakajairwa RT-PCR (Pearson coefficients> 0.9).Mhedzisiro yekuongororwa kwekiriniki inoshandisa sampu yemhino kubva kuvarwere gumi nevasere uye vanhu gumi nevasere vane hutano vanoratidza yakanaka concordance mufluorescence intensity ine yakajairwa RT-PCR (Pearson coefficients> 0.9).Результаты клинических испытаний с использованием образца мазка из носа от 18 пациентов и 18 здоровых лиц показывает хостовит хосистовит изпользование сценции стандартной ОТ-ПЦР (коэфффициенты Пирсона > 0,9).Mhedzisiro yemakiriniki ekuedzwa vachishandisa nasal swab sample kubva kuvarwere gumi nevasere uye gumi nevasere vanhu vane hutano vanoratidza kubvumirana kwakanaka pakati pe fluorescence intensity yeyakajairwa RT-PCR (Pearson's coefficients> 0.9).0.9................ Результаты клинических испытаний с использованием образцов назальных мазков от 18 пациентов и 18 здоровых лиц показать совиных песни ю флуоресценции и стандартной ОТ-ПЦР (коэфффициент Пирсона > 0,9).Mhedzisiro yemakiriniki ekuedzwa vachishandisa nasal swab samples kubva kuvarwere gumi nevasere uye gumi nevasere vanhu vane hutano vakaratidza kubvumirana kwakanaka pakati pefluorescence intensity uye yakajairwa RT-PCR (Pearson's coefficient> 0.9).Mutengo wezvinhu unofungidzirwa weFAST-POCT mudziyo ungangoita US$1 (Supplementary Table 1) uye unogona kudzikiswazve nekushandisa nzira huru dzekugadzira (eg jekiseni molding).Muchokwadi, FAST-yakavakirwa POCT zvishandiso zvine ese anodiwa maficha akarairwa neWHO uye anoenderana neatsva biochemical nzira dzekuongorora dzakadai seplasma thermal cycling44, amplification-yemahara immunoassays45 uye nanobody functionalization bvunzo46 inova musana wePOCT masisitimu.mukana.
Pamusoro pemuonde.1a inoratidza chimiro cheFAST-POCT papuratifomu, iyo ine makamuri mana emvura: kamuri yekutanga-yekuchengetera, kamuri yekusanganisa, kamuri yekuita, uye kamuri yekurasa.Kiyi yekudzora kuyerera kwemvura ndeye FAST dhizaini (inosanganisira elastic membranes, levers uye zvivharo) iri mu pre-yekuchengetedza kamuri uye yekusanganisa kamuri.Senzira yepneumatically actuated, iyo FAST dhizaini inopa chaiyo kuyerera kwemvura kuyerera, kusanganisira yakavharwa / yakavhurika switching, versatile dosing, pane-inoda kuburitswa kwemvura, kushanda kwakavimbika (semuenzaniso, kusanzwa kune kudengenyeka kwezvakatipoteredza), uye kuchengetedza kwenguva refu.Iyo FAST-POCT platform ine zvidimbu zvina: chidimbu chekutsigira, elastic film layer, plastiki film layer, uye chivharo chekuvhara, sezvinoratidzwa mukuona kwakakura muFig. 1b (inoratidzwawo zvakadzama muSupplementary Figures S1 uye S2 )Zvose zviteshi nemakamuri ekutakura emvura (zvakadai se-pre-storage uye reaction chambers) zvakaiswa muPLA (polylactic acid) substrates kubva pa 0.2 mm (thinnest part) kusvika 5 mm gobvu.Iyo elastic firimu zvinhu ndeye 300 µm gobvu PDMS iyo inokura nyore nyore kana mhepo yakamanikidzwa ikaiswa nekuda kwe "kutetepa ukobvu" uye yakaderera modulus yekunyungudika (inenge 2.25 MPa47).Iyo polyethylene film layer yakagadzirwa ne polyethylene terephthalate (PET) ine ukobvu hwe 100 µm kudzivirira iyo elastic firimu kubva pakunyanya deformation nekuda kwemhepo.Zvichienderana nemakamuri, iyo substrate layer ine marevhisi akabatana nevhavha layer (yakagadzirwa nePLA) nemahinji kudzora kuyerera kwemvura.Iyo elastic firimu yakanamirwa kune yekumashure layer ichishandisa kaviri-sided adhesive tepi (ARseal 90880) uye yakavharwa nepurasitiki firimu.Matanho matatu akaunganidzwa pane substrate uchishandisa T-clip dhizaini muvhavha layer.T-clamp ine mukaha pakati pemakumbo maviri.Iyo clip payakapinzwa mugoro, makumbo maviri akakotama zvishoma, ndokudzokera kune yavo yepakutanga uye zvakasimba akasunga chivharo uye nekutsigira sezvavaipfuura nepamugero (Supplementary Fig. S1).Iwo mana akaturikidzana anobva aunganidzwa pachishandiswa maconnectors.
Schematic dhizaini yepuratifomu inoratidza akasiyana siyana anoshanda makamuri uye maficha eFAST.b Dhiagiramu yakawedzera yeFAST-POCT papuratifomu.c Mufananidzo wepuratifomu padivi pemari yemadhora ekuUS.
Nzira yekushanda yeFAST-POCT platform inoratidzwa muMufananidzo 2. Zvikamu zvakakosha zvivharo panheyo yepasi uye hinges pavhavha yevhavha, iyo inoguma nekupindira kwekupindira apo zvidimbu zvina zvaunganidzwa uchishandisa T-shape. .Kana pasina simba remhepo rinoshandiswa (fig. 2a), kupindira kwakakodzera kunoita kuti hinge ikotame uye iparare, uye simba rekuvhara rinoshandiswa kuburikidza nelever kudzvanya firimu re elastic pakatarisana ne block, uye mvura iri muvharo yechisimbiso inotsanangurwa. senyika yakasimbiswa.Zvinofanira kucherechedzwa kuti muhurumende iyi, lever yakakotama kunze, sezvinoratidzwa mudivi rekuona muFig. 2a.Kana mhepo ichipihwa (Fig. 2b), elastic membrane inokwira nekunze yakananga kuchivharo uye inosundira chibatiso kumusoro, nokudaro ichivhura mukaha pakati pechivharo nechivharo chekuti mvura ipinde mukamuri rinotevera, rinotsanangurwa senzvimbo yakavhurika. .Mushure mekusunungurwa kwemhepo yemhepo, lever inogona kudzokera kunzvimbo yayo yepakutanga uye inoramba yakasimba nekuda kwekureruka kwehinge.Mavhidhiyo emafambiro emakumbo anoratidzwa mune yekuwedzera bhaisikopo S1.
A. Schematic diagram nemifananidzo kana yakavharwa.Mukusavapo kwekumanikidzwa, lever inodzvanya membrane inopesana ne block, uye mvura yakavharwa.b Mumamiriro akanaka.Kana kudzvinyirirwa kunoshandiswa, membrane inowedzera uye inosundira lever kumusoro, saka chiteshi chinovhura uye mvura inogona kuyerera.c Sarudza hunhu hwehukuru hwekumanikidza kwakakosha.Hunhu hunosanganisira kureba kwerevheti (L), chinhambwe chiri pakati pemusevedzero nehinge (l) uye ukobvu hwerevheti protrusion (t).Fs isimba rekubatanidza pa throttle point B. q ndiwo mutoro wakagovaniswa zvakafanana parevheti.Tx* inomiririra torque yakagadzirwa nehinged lever.Kudzvanyirira kwakanyanya ndiko kudzvanywa kunodiwa kusimudza lever uye kuita kuti mvura ibude.d Theoretical uye yekuedza mhedzisiro yehukama pakati pekumanikidza kwakakosha uye saizi yechinhu.n = 6 yakazvimirira kuyedza yakaitwa uye data inoratidzwa se ± yakajairwa kutsauka.Raw data inounzwa seyakagadzirwa data mafaira.
Iyo yekuongorora modhi yakavakirwa padzidziso yedanda yakagadziridzwa kuti iongorore kutsamira kweiyo yakakosha kumanikidza Pc painovhura gap pa geometric parameters (semuenzaniso, L ndiyo kureba kwechibatiso, l chinhambwe pakati pechivharo uye hinge, S is the lever Nzvimbo yekubatanidza ine mvura t ndiyo hupamhi hwehutachiona hwehutachiona, sezvinoratidzwa mumufananidzo 2c).Sezvinotsanangurwa muSupplementary Notes uye Supplementary Figure S3, mukaha unovhura apo \({P}_{c}\ge \frac{2{F}_{s}l}{SL}\), apo Fs iri torque. \ ({T}_{x}^{\ast}(={F}_{s}l)\) kubvisa masimba ane chekuita nekukanganisika fit uye kuita kuti hinge ikotame.Mhinduro yekuedza uye muenzaniso wekuongorora unoratidza kubvumirana kwakanaka (Fig. 2d), zvichiratidza kuti kudzvinyirira kwakanyanya kwePc kunowedzera nekuwedzera t / l uye kuderera kweL, iyo inotsanangurwa nyore nyore ne classical beam model, kureva kuti torque inowedzera ne t / Lift. .Nokudaro, kuongorora kwedu kwezvinyorwa kunoratidza zvakajeka kuti kudzvinyirirwa kwakanyanya kunogona kudzorwa zvakanaka nekugadzirisa urefu hwerevheti L uye t / l ratio, iyo inopa hwaro hunokosha hwekugadzirwa kweFAST-POCT platform.
Iyo FAST-POCT chikuva inopa multifunctional dispensing (inoratidzwa muMufananidzo 3a ine inset uye kuedza), inova inonyanya kukosha yePOCT yakabudirira, apo mvura inogona kuyerera chero nzira uye mune chero kurongeka (cascade, panguva imwe chete, sequential) kana yakasarudzwa multichannel. kugovera .- dosing basa.Pamusoro pemuonde.3a(i) inoratidza cascaded dosing mode umo makamuri maviri kana anopfuura anokandwa achishandisa mabhuroko kuparadzanisa akasiyana reactants uye lever yekudzora yakavhurika uye yakavharwa nyika.Kana dzvinyiriro ikaiswa, mvura inoyerera ichibva kumusoro ichienda kukamuri rezasi nenzira inoyerera.Izvo zvinofanirwa kucherechedzwa kuti makamuri ekukanda anogona kuzadzwa nemakemikari akanyorova kana makemikari akaoma akadai se lyophilized powders.Mukuedza muFig. 3a (i), inki tsvuku kubva muimba yepamusoro inoyerera pamwe chete nehupfu hwebhuruu (copper sulfate) mukamuri yechipiri uye inoshandura bhuruu yakasviba kana ichisvika muimba yepasi.Inoratidzawo kudzora kudzvanywa kwemvura iri kupombwa.Saizvozvo, kana imwe lever yakabatana kumakamuri maviri, inova iyo panguva imwe chete jekiseni modhi, sezvakaratidzwa muonde.3a(ii), umo iyo mvura inogona kugoverwa zvakafanana pamusoro pemakamuri maviri kana anopfuura kana kudzvanywa kuchishandiswa.Sezvo kudzvinyirirwa kwakakosha kunoenderana nehurefu hwechivharo, kureba kwechivharo kunogona kugadziriswa kuti uwane sequential injection pattern sezvinoratidzwa muonde.3a(iii).A refu lever (ine yakakosha pressure Pc_long) yakabatana nechamber B uye short lever (ine critical pressure Pc_short > Pc_long) yakabatana nechamber A. As pressure P1 (Pc_long <P1 <Pc_short) yakashandiswa, chete mvura iri mutsvuku. inogona kuyerera ichienda kukamuri B uye kana kudzvanywa kwakawedzera kusvika kuP2 (> Pc_pfupi), mvura yebhuruu inogona kuyerera ichienda kukamuri A. Iyi sequential jekiseni modhi inoshanda kune dzakasiyana zvinwiwa zvichiendeswa kumakamuri ayo ane hukama munhevedzano, izvo zvakakosha kuti ibudirire POCT. mudziyo.A refu lever (ine yakakosha pressure Pc_long) yakabatana nechamber B uye short lever (ine critical pressure Pc_short > Pc_long) yakabatana nechamber A. As pressure P1 (Pc_long
a Kuenzanisira yemabasa akawanda, kureva (i) cascading, (ii) panguva imwe chete, (iii) sequential, uye (iv) kusarudza basa.Iwo macurves anomiririra mafambiro ebasa uye maparameter eaya mana ekugovera modhi.b Mhedzisiro yenguva refu yekuchengetedza miedzo mumvura yakasvibiswa uye ethanol.n = 5 yakazvimirira miedzo yakaitwa uye data inoratidzwa se ± sd c.Kugadzikana bvunzo kuratidzira apo FAST mudziyo uye capillary valve (CV) mudziyo waiva mu (i) static uye (ii) vibrating states.(iii) Vhoriyamu vs. nguva yeFAST neCV zvishandiso pane akasiyana angular frequency.d Kushambadzwa kwemhinduro dzebvunzo pazvinodiwa zve (i) FAST mudziyo uye (ii) CV mudziyo.(iii) Hukama pakati pevhoriyamu uye nguva yeFAST neCV zvishandiso uchishandisa intermittent pressure mode.Ese mazariro, 1 cm.Raw data inopihwa sembishi data mafaira.
Kuchengetedzwa kwenguva refu kwemareagents chimwe chinhu chakakosha cheiyo yakabudirira POCT mudziyo unozobvumira vashandi vasina kudzidziswa kubata akawanda reagents.Nepo matekinoroji mazhinji aratidza kugona kwavo kuchengetedza kwenguva refu (semuenzaniso, 35 microdispensers, 48 blister packs, uye 49 stick packs), nzvimbo yakatsaurirwa yekugamuchira inodiwa kuti igadzirise iyo pasuru, iyo inowedzera mutengo uye kuoma;uyezve, idzi nzira dzekuchengetera hadzitenderi kugovera-pakuda uye zvinokonzeresa kutambisa reagents nekuda kwekusara mupakeji.Kukwanisa kwekuchengetedza kwenguva refu kwakasimbiswa nekuita kukurumidza kwehupenyu bvunzo uchishandisa CNC-yakagadzirwa PMMA zvinhu nekuda kwekuoma kwayo kushoma uye kuramba kupindira kwegasi (Supplementary Figure S5).Chigadzirwa chekuedza chakazadzwa nemvura yakasvibiswa (mvura yakasvibiswa) uye 70% ethanol (simulating volatile reagents) pa 65 ° C kwemazuva 9.Zvose zvakasvibiswa mvura uye ethanol zvakachengetwa uchishandisa aluminium foil kudzivirira kupinda kubva kumusoro.Iyo Arrhenius equation uye penetration activation energy yakataurwa mumabhuku50,51 yakashandiswa kuverenga iyo chaiyo-nguva yakaenzana.Pamusoro pemuonde.3b inoratidza avhareji yekurasikirwa kwehuremu mhedzisiro ye5 samples yakachengetwa pa65 ° C kwemazuva 9, yakaenzana ne0.30% yemvura yakasvibiswa uye 0.72% ye70% ethanol pamakore maviri pa23 ° C.
Pamusoro pemuonde.3c inoratidza bvunzo yekuzunguza.Sezvo iyo capillary valve (CV) iri iyo inonyanya kufarirwa nzira yekubata mvura pakati pemidziyo iripo yePOCT28,29, mudziyo weCV wakafara 300 µm uye 200 µm kudzika wakashandiswa kuenzanisa.Zvinogona kuonekwa kuti kana midziyo yose ikaramba yakamira, mvura iri muFAST-POCT papuratifomu inovhara uye mvura iri muCV device inovhara nekuda kwekukurumidza kuwedzera kwechiteshi, izvo zvinoderedza capillary forces.Nekudaro, sezvo iyo orbital vibrator's angular frequency inowedzera, mvura iri muFAST-POCT papuratifomu inoramba yakavharwa, asi iyo fluid iri muCV mudziyo inoyerera ichipinda mukamuri rezasi (onawo Supplementary Movie S3).Izvi zvinoratidza kuti mahinji akaremara eiyo FAST-POCT papuratifomu anogona kushandisa simba rakasimba remakanika kune module kuvhara zvakasimba mvura iri mukamuri.Nekudaro, mumidziyo yeCV, mvura inochengetwa nekuda kwechiyero pakati peiyo yakasimba, mhepo, uye mvura nhanho, kugadzira kusagadzikana, uye vibrations zvinogona kukanganisa chiyero uye kukonzera isingatarisirwe kuyerera maitiro.Kubatsira kweFAST-POCT papuratifomu ndeyokuti inopa kushanda kwakavimbika uye inodzivirira kukundikana pamberi pehupo hwekudengenyeka kunowanzoitika panguva yekusununguka uye kushanda.
Chimwe chinhu chakakosha cheFAST-POCT papuratifomu ndeye-inoda kuburitswa, chinova chinhu chakakosha chekuongorora kwehuwandu.Pamusoro pemuonde.3d inofananidza kuburitswa-kwekuda kweiyo FAST-POCT chikuva uye mudziyo weCV.Kubva muonde.3d (iii) tinoona kuti FAST device inopindura nokukurumidza kune chiratidzo chekumanikidza.Apo kudzvinyirirwa kwakashandiswa kuFAST-POCT platform, mvura yakayerera, apo kudzvinyirira kwakabudiswa, kuyerera kwakabva kwamira (Fig. 3d (i)).Ichi chiito chinogona kutsanangurwa nekukurumidza elastic kudzoka kwehinge, iyo inotsikirira lever kumashure kune block, kuvhara kamuri.Nekudaro, mvura yakaramba ichiyerera mumudziyo weCV, zvichizoguma nekuwanda kwemvura isingatarisirwe ingangoita 100 µl mushure mekumanikidzwa kuburitswa (Mufananidzo 3d(ii) uye Supplementary Movie S4).Izvi zvinogona kutsanangurwa nekutsakatika kwecapillary pinning effect pakunyorova kwakazara kweCV mushure mejekiseni rekutanga.
Iko kugona kubata zvinwiwa zvekusiyana kwekunyorova uye viscosity muchishandiso chimwe chete chinoramba chiri dambudziko kune POCT application.Kusanyorova kwemvura kunogona kutungamirira kumvura kana mamwe maitiro asingatarisirwi ekuyerera mumatanho, uye ancillary midziyo yakadai sevortex mixers, centrifuges uye mafirita zvinowanzodiwa kugadzirira zvakanyanya viscous zvinwiwa 52.Isu takaedza hukama pakati pekumanikidza kwakanyanya uye zvinwiwa zvemvura (zvine huwandu hwakasiyana hwekunyorova uye viscosity).Mhedzisiro inoratidzwa muTafura 1 uye Vhidhiyo S5.Zvinogona kuonekwa kuti zvinwiwa zvakasiyana-siyana zvekunyorova uye viscosity zvinogona kuvharirwa mukamuri, uye kana kudzvanywa kuchiiswa, kunyangwe zvinwiwa zvine viscosity inosvika 5500 cP zvinogona kuendeswa kukamuri iri padyo, zvichiita kuti zvikwanise kuona masampula ane yakakwira. viscosity (kureva sputum, muenzaniso wakajeka unoshandiswa pakuongorora zvirwere zvekufema).
Nekubatanidza iyo iri pamusoro apa yakawanda inoshanda yekuparadzira michina, dzakasiyana siyana dzeFAST-based POCT zvishandiso zvinogona kugadzirwa.Muenzaniso unoratidzwa muMufananidzo 1. Chimiro chine pre-storage chamber, musanganiswa wekamuri, kamuri yekupindura uye marara.Reagents inogona kuchengetwa mu pre-yekuchengetera mukamuri kwenguva yakawedzerwa yenguva uye yozoburitswa mukamuri yekusanganisa.Nekumanikidzwa kwakakodzera, izvo zvakasanganiswa zvinogadzirisa zvinogona kusarudzwa kuendeswa kukamuri yemarara kana mukamuri yekuita.
Nekuti PCR kucherechedzwa ndiyo chiyero chegoridhe chekuona utachiona hwakaita seH1N1 neCOVID-19 uye inosanganisira akawanda maitiro ekuita, takashandisa FAST-POCT chikuva chekuonekwa kwePCR sekushandisa.Pamusoro pemuonde.4 inoratidza PCR yekuyedza maitiro uchishandisa FAST-POCT chikuva.Chekutanga, iyo eluting reagent, magineti microbead reagent, wash mhinduro A, uye yekugezesa mhinduro W zvakaiswa pombi mumakamuri ekutanga ekuchengetedza E, M, W1 uye W2, zvichiteerana.Matanho eRNA adsorption anoratidzwa mufig.4a uye ndeaya anotevera: (1) kana kumanikidza P1 (= 0.26 bar) kunoshandiswa, sampu inopinda mukamuri M uye inoburitswa mukamuri yekusanganisa.(2) Mhepo yekumanikidza P2 (= 0.12 bar) inopihwa kuburikidza nechiteshi A chakabatana nepasi pekamuri yekusanganisa.Kunyange zvazvo nzira dzinoverengeka dzekusanganisa dzakaratidza kugona kwavo mukusanganisa zvinwiwa pamapuratifomu ePOCT (semuenzaniso kusanganisa nyoka 53, kusarongeka kusanganisa 54 uye batch kusanganisa 55), kusanganisa kwavo kugona uye kubudirira hazvisati zvichigutsikana.Iyo inotora nzira yekusanganisa bubble, umo mhepo inounzwa muzasi mekamuri yekusanganisa kuti igadzire mabhubhu mumvura, mushure meiyo vortex ine simba inogona kuwana kusanganisa kwakazara mukati memasekondi.Bubble kusanganisa kuyedza kwakaitwa uye mhedzisiro inoratidzwa muSupplementary Figure S6.Zvinogona kuonekwa kuti kana kudzvinyirirwa kwe0.10 bar kunoshandiswa, kusanganiswa kwakakwana kunotora anenge 8 seconds.Nekuwedzera kumanikidza kune 0.20 bar, kusanganisa kwakazara kunowanikwa mukati memasekondi maviri.Nzira dzekuverenga kusanganisa kusanganisa dzinoratidzwa muchikamu cheNzira.(3) Shandisa magineti yerubidium kubvisa mabhero, wobva wadzvanya P3 (= 0.17 bar) kuburikidza nechiteshi chengarava P kuti ufambise mareagents mukamuri yemarara.Pamusoro pemuonde.4b,c inoratidza matanho ekugezesa kubvisa tsvina kubva mumuenzaniso sezvinotevera: (1) Mushonga wekugeza A kubva mukamuri W1 inobudiswa mukamuri yekumanikidza P1.(2) Wobva waita iyo bubble kusanganisa maitiro.(3) Iyo yekugezesa mhinduro A inoendeswa kune tsvina yemvura mukamuri, uye ma microbead ari mukamuri yekusanganisa anoburitswa kunze nemagineti.Kushambidza W (Fig. 4c) yakanga yakafanana nekugeza A (Fig. 4b).Izvo zvinofanirwa kucherechedzwa kuti imwe neimwe yekugeza nhanho A uye W yakaitwa kaviri.Mufananidzo 4d unoratidza nhanho dze elution kuti elute iyo RNA kubva kumabhedhi;iyo elution uye kusanganisa sumo matanho akafanana neRNA adsorption uye kugeza matanho anotsanangurwa pamusoro.Sezvo elution reagents inotamirwa muPCR reaction chamber pasi pekumanikidzwa P3 uye P4 (= 0.23 bar), iyo yakakosha kudzvanywa inosvika kusimbisa ruoko rwePCR reaction chamber.Saizvozvo, iyo P4 kudzvanywa inobatsirawo kuvhara ndima kuenda kukamuri yemarara.Nekudaro, ese elution reagents akagovaniswa zvakaenzana pakati pemakamuri mana ePCR maitiro ekutanga maitiro akawanda ePCR.Maitiro ari pamusoro anoratidzwa muSupplementary Movie S6.
Mune iyo RNA adsorption nhanho, sampu inounzwa muinopinza M uye inopinzwa mukamuri yekusanganisa pamwe neyakachengeterwa bead mhinduro.Mushure mekusanganisa uye kubvisa granules, ma reagents anogoverwa mukamuri yemarara.b uye c nhanho dzekugeza, suma mareagents akasiyana-siyana akafanochengeterwa mukamuri yekusanganisa, uye mushure mekusanganisa nekubvisa mabhero, endesa mareagents kukamuri yemarara.d Elution nhanho: Mushure mekuzivisa iyo elution reagents, kusanganisa uye kuburitsa bead, mareagents anoendeswa kuPCR reaction chamber.Iwo macurves anoratidza mafambiro ebasa uye zvinoenderana nematanho ematanho akasiyana.Dzvinyiriro idzvinyiriro inoitwa kuburikidza nemakamuri ega ega.Vhoriyamu ihuwandu hwemvura mukamuri yekusanganisa.Ese mazariro ane 1 cm.Raw data inopihwa sembishi data mafaira.
Nzira yekuongorora PCR yakaitwa uye Supplementary Figure S7 inopa mafaera ekupisa anosanganisira maminetsi makumi maviri enguva yekudzokesa yakanyorwa uye maminetsi makumi matanhatu enguva yekupisa bhasikoro (95 uye 60 °C), paine kutenderera kumwe kwekupisa kuri makumi mapfumbamwe (Supplementary Movie S7)..FAST-POCT inoda nguva shoma yekupedzisa imwe thermal cycle (90 seconds) pane yakajairwa RT-PCR (180 seconds yeimwe thermal cycle).Izvi zvinogona kutsanangurwa nepamusoro penzvimbo kusvika kuhuwandu hwehuwandu uye yakaderera inertia yekupisa kweiyo micro-PCR reaction chamber.Iyo yekamuri yepamusoro i96.6 mm2 uye vhoriyamu yekamuri iri 25 mm3, ichiita iyo pamusoro kune vhoriyamu reshiyo inosvika 3.86.Sezvinoonekwa muSupplementary Figure S10, iyo PCR bvunzo nzvimbo yepuratifomu yedu ine groove pane yekuseri, ichiita pasi pePCR kamuri 200 µm gobvu.Iyo thermally conductive elastic pad inosungirirwa kune inodziya pamusoro peiyo tembiricha controller, kuve nechokwadi chekubata kwakasimba nekuseri kwebhokisi rekuyedza.Izvi zvinoderedza inertia yekupisa kwepuratifomu uye inovandudza kupisa / kutonhora kushanda zvakanaka.Munguva yekupisa bhasikoro, parafini yakaiswa mupuratifomu inonyunguduka uye inoyerera ichipinda muPCR reaction chamber, ichiita sechisimbiso kudzivirira reagent evaporation uye kusvibiswa kwezvakatipoteredza (ona Supplementary Movie S8).
Yese PCR yekuona maitiro anotsanangurwa pamusoro aive otomatiki akazara achishandisa tsika yakagadzirwa FAST-POCT chiridzwa, chinosanganisira yakarongwa dhizaini yekudzora unit, magnetic extraction unit, tembiricha control unit, uye fluorescent chiratidzo chekutora uye kugadzirisa unit.Zvecherechedzo, isu takashandisa iyo FAST-POCT chikuva cheRNA yekuzviparadzanisa nevamwe ndokuzoshandisa yakatorwa RNA samples yePCR maitiro tichishandisa iyo FAST-POCT system uye desktop PCR system yekuenzanisa.Mhedzisiro yacho yaive yakafanana neyakaratidzwa muSupplementary Figure S8.Mushandi anoita basa rakareruka: anosuma sample muM-chamber uye anoisa chikuva muchiridzwa.Quantitative test results inowanikwa mukati memaminitsi makumi masere nemaviri.Ruzivo rwakadzama nezve FAST-POCT maturusi anogona kuwanikwa mune yekuwedzera nhamba.C9, C10 uye C11.
Furuwenza inokonzerwa nefuruwenza A (IAV), B (IBV), C (ICV) uye D (IDV) mavhairasi chinhu chinowanzoitika pasi rose.Pakati peizvi, IAV neIBV ndiyo inokonzera nyaya dzakanyanya kuoma uye zvirwere zvemwaka, zvichitapura 5-15% yehuwandu hwepasi rose, zvichikonzera 3-5 miriyoni zviitiko zvakakomba uye zvichikonzera 290,000-650,000 kufa pagore.Zvirwere zvekufema56,57.Kuongororwa kwekutanga kweIAV neIB kwakakosha kuderedza kurwara uye mutoro wehupfumi wakabatana.Pakati pemaitiro anowanikwa ekuongorora, iyo reverse transcriptase polymerase chain reaction (RT-PCR) inoonekwa seyakanyanya kujeka, yakananga, uye yakarurama (> 99%) 58,59.Pakati pemaitiro anowanikwa ekuongorora, iyo reverse transcriptase polymerase chain reaction (RT-PCR) inoonekwa seyakanyanya kujeka, yakananga, uye yakarurama (> 99%) 58,59.Среди доступных диагностических методов полимеразная цепная реакция с обратной транскриптазой (ОТ-ПЦР) считается наийболеин > 99%)58,59.Pakati penzira dziripo dzekuongorora, reverse transcriptase polymerase chain reaction (RT-PCR) inoonekwa seyakanyanya kunzwisiswa, yakananga uye yakarurama (> 99%) 58,59. Из доступных диагностических методов полимеразная цепная реакция с обратной транскриптазой (ОТ-ПЦР) считается наийболее чулье, песни 99%)58,59.Panzira dziripo dzekuongorora, reverse transcriptase polymerase chain reaction (RT-PCR) inoonekwa seyakanyanya kunzwisiswa, yakananga uye yakarurama (> 99%) 58,59.Nekudaro, nzira dzechinyakare dzeRT-PCR dzinoda kudzokororwa pombi, kusanganisa, kugovera uye kuendesa mvura, kudzikisira kushandiswa kwavo nenyanzvi mune zvigadziriso-zvishoma zvigadziriso.Pano, iyo FAST-POCT papuratifomu yakashandiswa paPCR yekuona IAV neIBV, zvichiteerana, kuwana yavo yakaderera muganho wekuonekwa (LOD).Pamusoro pezvo, IAV neIBV zvakave zvakapetwa kuti zvisarura pakati pemhando dzakasiyana dzemarudzi, zvichipa chikuva chinovimbisa chekuongorora genetic uye kugona kubata chirwere ichi nemazvo.
Pamusoro pemuonde.5a inoratidza mhedzisiro yekuongororwa kweHAV PCR uchishandisa 150 µl yeyakacheneswa hutachiona RNA semuenzaniso.Pamusoro pemuonde.5a (i) inoratidza kuti paHAV yehuwandu hwe106 makopi / ml, iyo fluorescence intensity (ΔRn) inogona kusvika ku0.830, uye kana iyo yakaderedzwa kusvika ku102 makopi / ml, ΔRn inogona kusvika ku0.365, iyo inofananidzwa nepamusoro kupfuura iyo. yeboka risina chinhu rinodzora (0.002), rinenge 100 nguva dzakakwirira.Pakuverengera kwakavakirwa pazviyedzo zvitanhatu zvakazvimiririra, mutsara wekugadzirisa curve wakagadzirwa pakati pelog concentration uye cycle cycle (Ct) yeIAV (Fig. 5a (ii)), R2 = 0.993, kubva ku102-106 makopi / mL.mibairo iri muchibvumirano chakanaka neyakajairwa RT-PCR nzira.Pamusoro pemuonde.5a(iii) inoratidza mifananidzo yefluorescent yemhedzisiro yebvunzo mushure me40 kutenderera kweFAST-POCT papuratifomu.Takaona kuti FAST-POCT papuratifomu inogona kuona HAV yakaderera se102 makopi/mL.Zvisinei, nzira yechinyakare haina kukosha kweCt pa102 makopi/mL, zvichiita kuti ive LOD yeanenge 103 makopi/mL.Isu takafungidzira kuti izvi zvinogona kunge zviri nekuda kwekushanda kwakanyanya kwekusanganiswa kwebubble.PCR bvunzo dzekuyedza dzakaitwa pane yakacheneswa IAV RNA yekuongorora dzakasiyana nzira dzekusanganisa, kusanganisira kusanganisa kusanganisa (yakafanana nzira yekusanganisa seyakajairwa RT-PCR mashandiro), vial kusanganisa (iyi nzira, 3 s pa 0.12 bar) uye hapana kusanganisa seboka rinodzora. ..Mhedzisiro yacho inogona kuwanikwa muSupplementary Figure S12.Zvinogona kuoneka kuti paRNA yakakwira (106 makopi/mL), iyo Ct tsika dzemhando dzakasiyana dzekusanganisa dzakada kufanana neyekusanganisa bubble.Apo RNA concentration yakaderera kusvika ku102 makopi / mL, shake mix uye kutonga kwakanga kusina Ct values, nepo bubble mix nzira ichiri kupa Ct kukosha kwe36.9, iyo yaiva pasi peCt threshold ye 38. Zvigumisiro zvinoratidza hukuru hwekusanganiswa hunhu. vesicles, izvo zvakaratidzwawo mune mamwe mabhuku, izvo zvinogona zvakare kutsanangura kuti sei kunzwisiswa kweFAST-POCT papuratifomu kwakakwira zvishoma pane yakajairwa RT-PCR.Pamusoro pemuonde.5b inoratidza mhedzisiro yePCR kuongororwa kweakacheneswa IBV RNA samples kubva ku101 kusvika ku106 makopi / ml.Mhedzisiro yacho yaive yakafanana neyeIAV bvunzo, kuwana R2 = 0.994 uye LOD ye102 makopi/mL.
ongororo yePCR yefuruwenza A virus (IAV) ine IAV concentrations kubva pa106 kusvika ku101 makopi/mL uchishandisa TE buffer senge negative control (NC).(i) Nguva chaiyo fluorescence curve.(ii) Linear calibration curve pakati pe logarithmic IAV RNA concentration uye kutenderera chikumbaridzo (Ct) yeFAST uye yakajairwa nzira dzekuyedza.(iii) IAV FAST-POCT fluorescent mufananidzo mushure me40 kutenderera.b, PCR kuonekwa kwefuruwenza B virus (IBV) ne (i) real-time fluorescence spectrum.(ii) Linear calibration curve uye (iii) FAST-POCT IBV fluorescence mufananidzo mushure me40 kutenderera.Muganho wakaderera wekuona (LOD) weIAV neIBV uchishandisa FAST-POCT chikuva chaive 102 makopi/mL, iyo yakaderera pane yakajairika nzira (103 copies/mL).c Multiplex bvunzo mhinduro dzeIAV neIBV.GAPDH yakashandiswa seyakanaka kudzora uye TE buffer yakashandiswa seyakashata kutonga kudzivirira kusvibiswa kunogona kuitika uye kukwidziridzwa kwemashure.Mhando ina dzakasiyana dzemuenzaniso dzinogona kusiyaniswa: (1) GAPDH-chete sampuli dzisina kunaka ("IAV-/IBV-");(2) IAV utachiona ("IAV +/IBV-") neIAV neGAPDH;(3) IBV utachiona (“IAV-/IBV +”) neIBV neGAPDH;(4) IAV/IBV utachiona (“IAV+/IBV+”) neIAV, IBV neGAPDH.Mutsetse une doti unomiririra mutsara wechikumbaridzo.n = 6 biologically yakazvimiririra kuyedza yakaitwa, data inoratidzwa se ± yakajairwa kutsauka.Raw data inounzwa seyakagadzirwa data mafaira.
Pamusoro pemuonde.5c inoratidza mhedzisiro yekuwanda kwebvunzo yeIAV/IBV.Pano, virus lysate yakashandiswa semuenzaniso wemhinduro panzvimbo yeRNA yakacheneswa, uye mana ekutanga eIAV, IBV, GAPDH (positive control) uye TE buffer (negative control) akawedzerwa kumakamuri mana ekuita akasiyana eFAST-POCT papuratifomu.Positive and negative controls inoshandiswa pano kudzivirira kusvibiswa kunogona kuitika uye kusimudzira kumashure.Miedzo yakakamurwa kuva mapoka mana: (1) GAPDH-negative samples ("IAV-/IBV-");(2) IAV-infected ("IAV +/IBV-") inopesana neIAV neGAPDH;(3) IBV-.utachiona (“IAV-”) -/IBV+”) IBV neGAPDH;(4) IAV/IBV (“IAV+/IBV+”) utachiona neIAV, IBV uye GAPDH.Pamusoro pemuonde.5c inoratidza kuti apo sampuli dzisina kunaka dzakashandiswa, iyo fluorescence intensity ΔRn yekamuri yekutonga yakanaka yaiva 0.860, uye ΔRn yeIAV neIBV yakanga yakafanana nekutonga kusina kunaka (0.002).Kune iyo IAV+/IBV-, IAV-/IBV+ uye IAV+/IBV+ mapoka, iyo IAV/GAPDH, IBV/GAPDH uye IAV/IBV/GAPDH makamera airatidza kusimba kwefluorescence, zvichiteerana, nepo mamwe makamera aitoratidza kusimba kwefluorescence kumashure. nhanho ye40 mushure mekupisa bhasikoro.Kubva pamiyedzo iri pamusoro, iyo FAST-POCT papuratifomu yakaratidza yakasarudzika uye yakatibvumira kuti panguva imwe chete pathotype dzakasiyana dzefuruwenza mavhairasi.
Kuti tisimbise kushandiswa kwekliniki yeFAST-POCT, takaedza 36 kliniki mienzaniso (nose swab specimens) kubva kune IB varwere (n = 18) uye vasiri-IB controls (n = 18) (Mufananidzo 6a).Ruzivo rwemurwere runoratidzwa muSupplementary Table 3. IB chirwere chetachiona chakasimbiswa zvakasununguka uye chirongwa chekudzidza chakagamuchirwa neZhejiang University First Affiliated Hospital (Hangzhou, Zhejiang).Muenzaniso wega wega wevarwere wakakamurwa kuva mapoka maviri.Imwe yakagadziriswa ichishandisa FAST-POCT uye imwe yacho yakagadziriswa uchishandisa desktop PCR system (SLAN-96P, China).Mayedzo ese ari maviri anoshandisa zvakafanana kucheneswa uye kuona kits.Pamusoro pemuonde.6b inoratidza mhedzisiro yeFAST-POCT uye yakajairwa reverse transcription PCR (RT-PCR).Isu takaenzanisa iyo fluorescence intensity (FAST-POCT) ne -log2 (Ct), uko Ct ndiyo inotenderera chiyero cheyakajairika RT-PCR.Pakanga pane kubvumirana kwakanaka pakati penzira mbiri idzi.FAST-POCT uye RT-PCR yakaratidza kuwirirana kwakasimba kwakanaka nePearson's ratio (r) kukosha kwe0.90 (Mufananidzo 6b).Takazoongorora kurongeka kwekuongorora kweFAST-POCT.Fluorescence intensity (FL) kugoverwa kwemaitiro akanaka uye asina kunaka akapiwa seyero yakazvimirira yekuongorora (Fig. 6c).FL tsika dzaive dzakakwirira zvakanyanya muvarwere veIB kupfuura mukutonga (**** P = 3.31 × 10-19; maviri-tailed t-test) (Fig. 6d).Tevere, IBV inogamuchira maitiro ekushandisa (ROC) macurves akarongwa.Takaona kuti kurongeka kwekuongorora kwaiva kwakanaka kwazvo, ine nzvimbo iri pasi pemutsara we1 (Fig. 6e).Ndokumbira utarise kuti nekuda kwekurongeka kwemask kuChina nekuda kweCCIDID-19 kubva muna 2020, hatisati taziva varwere vane IBD, saka ese akanaka ekiriniki emuenzaniso (kureva, nasal swab specimens) aive eIBV chete.
Chidzidzo chekiriniki dhizaini.Huwandu hwemasamples makumi matatu nematanhatu, kusanganisira gumi nemasere evarwere uye 18 asiri efuruwenza controls, akaongororwa pachishandiswa FAST-POCT chikuva uye yakajairika RT-PCR.b Ongorora kuenderana kwekuongorora pakati peFAST-POCT PCR uye yakajairwa RT-PCR.Mhedzisiro yacho yakabatana zvakanaka (Pearson r = 0.90).c Fluorescence intensity mazinga mu18 IB varwere uye gumi nemasere ekudzora.d Muvarwere veIB (+), FL maitiro aive akakwira zvakanyanya kupfuura muboka rekutonga (-) (**** P = 3.31 × 10-19; maviri-tailed t-test; n = 36).Pasikweya yega yega, mucherechedzo mutema uri pakati unomiririra wepakati, uye mitsetse yepasi neyepamusoro yebhokisi inomiririra 25th uye 75th percentiles, zvichiteerana.Ndebvu dzinowedzera kusvika kune shoma uye yakanyanya data mapoinzi, ayo asingatariswe ekunze.e ROC curve.Mutsetse une doti d unomiririra kukosha kwechikumbaridzo kunofungidzirwa kubva mukuongorora kweROC.Iyo AUC yeIBV ndeye 1. Raw data inopiwa semafaira e data mbishi.
Muchikamu chino, tinopa FAST, ine hunhu hunodiwa kune yakanakira POCT.Zvakanakira tekinoroji yedu zvinosanganisira: (1) Versatile dosing (cascade, panguva imwe chete, sequential uye yakasarudzika), kusunungurwa pane zvinodiwa (kukurumidza uye kuenzanirana kusunungurwa kwekumanikidzwa kwakaiswa) uye kushanda kwakavimbika (vibration pa150 degrees) (2) kuchengetedza kwenguva refu. (2 makore ekukurumidza kuyedzwa, kurasikirwa nehuremu nezve 0.3%);(3) kugona kushanda nezvinwiwa zvine huwandu hwakasiyana hwekunyorova uye viscosity (viscosity kusvika ku5500 cP);(4) Economical (Inofungidzirwa mutengo weiyo FAST-POCT PCR mudziyo ingangoita US$1).Nekubatanidza madhizaini akawanda, yakasanganiswa FAST-POCT chikuva chePCR yekuona mavhairasi efuruwenza A uye B yakaratidzwa uye yakashandiswa.FAST-POCT inongotora 82 maminetsi.Miedzo yekiriniki ine 36 nasal swab samples yakaratidza yakanaka concordance mufluorescence intensity ine yakajairwa RT-PCR (Pearson coefficients> 0.9).Miedzo yekiriniki ine 36 nasal swab samples yakaratidza yakanaka concordance mufluorescence intensity ine yakajairwa RT-PCR (Pearson coefficients> 0.9).Клинические тесты с 36 образцами мазков из носа показали хорошее соответствие интенсивности флуоресценции стандартной ОТ-ПЦенция ОТ-ПЦенция ОТ-ПЦенция ОТ-ПЦ (коэртый ОТ-ПЦРе.Miedzo yemakiriniki ine 36 samples dzenasal swabs dzakaratidza kubvumirana kwakanaka nefluorescence intensity yeyakajairwa RT-PCR (Pearson's coefficients> 0.9).RT-PCR Клинические испытания 36 образцов мазков из носа показали хорошее совпадение интенсивности флуоресценции со стандартной ОТ-эфиценции стандартной ОТ-эфинценции стандартной ОТ-ПфинцийKuongororwa kwekiriniki kwemakumi matatu nenhanhatu swab specimens kwakaratidza kubvumirana kwakanaka kwefluorescence kusimba neyakajairwa RT-PCR (Pearson's coefficient> 0.9).Zvichienderana nebasa iri, nzira dzakasiyana-siyana dziri kubuda dzebiochemical (semuenzaniso, plasma thermal cycling, amplification-free immunoassays, uye nanobody functionalization assays) dzakaratidza kugona kwavo muPOCT.Nekudaro, nekuda kwekushaikwa kweiyo yakazara yakazara uye yakasimba yePOCT chikuva, nzira idzi dzinoda zvakapatsanurwa pre-processing maitiro (eg, RNA isolation44, incubation45 uye washing46), iyo inowedzera kuzadzisa basa razvino nemaitiro aya ekuita epamberi POCT mabasa ne. iyo inodiwa parameters.tora-in-response-output performance.Mune basa iri, kunyange zvazvo pombi yemhepo inoshandiswa kugadzirisa FAST valve idiki zvakakwana kuti ibatanidzwe mubenchtop chiridzwa (Fig. S9, S10), ichiri kushandisa simba guru uye inoita ruzha.Mumusimboti, diki fomu factor pneumatic pombi inogona kutsiviwa nedzimwe nzira, sekushandiswa kwesimba remagetsi kana munwe actuation.Kumwe kuvandudzwa kunogona kusanganisira, semuenzaniso, kugadzirisa kits kune akasiyana uye chaiwo biochemical assays, uchishandisa nzira nyowani dzekuona dzisingade kudziyisa / kutonhora masisitimu, nekudaro ichipa chishandiso-isina POCT chikuva chePCR application.Isu tinotenda kuti zvakapihwa kuti FAST chikuva chinopa nzira yekushandisa zvinwiwa, isu tinotenda kuti yakatsanangurwa FAST tekinoroji inopa mukana wekugadzira yakajairika chikuva kwete chekuongorora biomedical chete, asiwo chekutarisa nharaunda, kuongororwa kwemhando yezvokudya, zvinhu uye kugadzirwa kwezvinodhaka. ..
Kuunganidzwa uye kushandiswa kwezviyereso zvemhino zvemunhu zvakabvumidzwa neEthics Committee yeZhejiang University First Affiliated Hospital (IIT20220330B).36 nasal swab samples dzakaunganidzwa, dzinosanganisira 16 vakuru vane makore makumi matatu ekuberekwa, 7 vakuru> 40 makore, uye 19 varume, 17 vakadzi.36 nasal swab samples dzakaunganidzwa, dzinosanganisira 16 vakuru vane makore makumi matatu ekuberekwa, 7 vakuru> 40 makore, uye 19 varume, 17 vakadzi.Было собрано 36 образцов мазков из носа, в которых приняли участие 16 взрослых < 30 лет, 7 взрослых старше 40 лемун17 инге 19Zviyero makumi matatu nenhanhatu zvenasal swab zvakatorwa kubva kuvakuru gumi nevatanhatu vane makore makumi matatu ekuberekwa, 7 vakuru vane makore 40 okuberekwa, 19 varume ne17 vakadzi..Demographic data inoratidzwa muSupplementary Table 3. Mvumo ine ruzivo yakawanikwa kubva kune vose vatori vechikamu.Vese vatori vechikamu vaifungidzirwa kuti vane furuwenza uye vakaongororwa vachizvidira pasina muripo.
Iyo FAST base uye chivharo chakagadzirwa ne polylactic acid (PLA) uye yakadhindwa neEnder 3 Pro 3D printer (Shenzhen Transcend 3D Technology Co., Ltd.).Tepi ine mativi maviri yakatengwa kubva kuAdhesives Research, Inc. Model 90880. PET firimu rinorema 100 µm rakatengwa kubva kuMcMaster-Carr.Zvose zvakanamatira uye PET firimu zvakatemwa uchishandisa Silhouette Cameo 2 cutter kubva kuSilhouette America, Inc. Iyo elastic film inogadzirwa nePDMS zvinhu nekuumba jekiseni.Chekutanga, 200 µm gobvu PET furemu yakachekwa pachishandiswa laser system uye yakanamirwa kune 3 mm gobvu PMMA sheet uchishandisa 100 µm kaviri-sided adhesive tepi.Iyo PDMS precursor (Sylgard 184; Chikamu A: Chikamu B = 10: 1, Dow Corning) yakabva yadirwa muforoma uye tsvimbo yegirazi yakashandiswa kubvisa yakawandisa PDMS.Mushure mekurapa pa 70 ° C. kwemaawa matatu, iyo 300 μm gobvu yePDMS firimu inogona kupepetwa kubva muforoma.
Mapikicha ekugovera kwakasiyana-siyana, pa-inoda kushambadza uye kuita kwakavimbika kunotorwa nekamera inomhanya kwazvo (Sony AX700 1000 fps).Iyo orbital shaker yakashandiswa muyedzo yekuvimbika yakatengwa kubva kuSCILOGEX (SCI-O180).Iyo mhepo yekumanikidza inogadzirwa nemhepo compressor, uye akati wandei edhijitari precision pressure regulators anoshandiswa kugadzirisa kukosha kwekumanikidza.Iyo yekuyerera maitiro ekuyedza maitiro ndeaya anotevera.Huwandu hwakafanotemerwa hwemvura hwakaiswa muyedzo mudziyo uye kamera yekumhanyisa yakashandiswa kurekodha mafambiro ekuyerera.Zvichiri mifananidzo zvakabva zvatorwa kubva kumavhidhiyo ekuyerera maitiro panguva dzakatarwa, uye nzvimbo yakasara yakaverengerwa uchishandisa Image-Pro Plus software, iyo yakazowedzerwa nekudzika kwekamera kuverenga vhoriyamu.Ruzivo rwekuyerera maitiro ekuyedza system inogona kuwanikwa muSupplementary Figure S4.
Jekiseni 50 µl ye microbeads uye 100 µl yemvura yakasvibiswa muvharo yekusanganisa mudziyo.Mapikicha akasanganiswa ekuita akatorwa nekamera yekumhanya kwese kwese 0.1 masekonzi pakumanikidzwa kwe0.1 bar, 0.15 bar uye 0.2 bar.Ruzivo rwePixel panguva yekusanganisa maitiro anogona kuwanikwa kubva pamifananidzo iyi uchishandisa mafoto ekugadzirisa software (Photoshop CS6).Uye kusanganisa kugona kunogona kuwanikwa neinotevera Equation 53.
uko M ndiko kugona kusanganisa, N ndiyo nhamba yese yemapikisesi emuenzaniso, uye ci uye \(\ bar{c}\) ndiwo akajairwa uye anotarisirwa akajairwa.Kusanganisa kushanda zvakanaka kunobva ku0 (0%, isina kusanganiswa) kusvika ku1 (100%, yakasanganiswa zvizere).Migumisiro inoratidzwa muSupplementary Figure S6.
Real-time RT-PCR kit yeIAV neIBV, kusanganisira IAV neIBV RNA samples (katsi no. RR-0051-02/RR-0052-02, Liferiver, China), Tris-EDTA buffer (TE buffer no. B541019 , Sangon Biotech, China), Positive Control RNA Purification Kit (Chikamu No. Z-ME-0010, Liferiver, China) uye GAPDH Solution (Chikamu No. M591101, Sangon Biotech, China) inotengeswa.Iyo RNA yekuchenesa kit inosanganisira inosunga buffer, kugeza A, kugeza W, eluent, magnetic microbeads, uye acrylic mutakuri.IAV uye IBV chaiyo-nguva RT-PCR kits inosanganisira IFVA nucleic acid PCR yekuona musanganiswa uye RT-PCR enzyme.Wedzera 6 µl yeAcrylCarrier uye 20 µl yemagineti mabedhi kusvika pa500 µl yekusunga buffer solution, zunza zvakanaka wozogadzira mushonga webead.Wedzera 21 ml ye ethanol kushambidza A uye W, zunza zvakanaka kuti uwane mhinduro dzekugezesa A uye W, zvichiteerana.Zvadaro, 18 µl yefluorescent PCR musanganiswa neIFVA nucleic acid uye 1 µl yeRT-PCR enzyme yakawedzerwa kune 1 µl yeTE mhinduro, yakazununguswa uye centrifuged kwemasekonzi akati wandei, kuwana 20 µl yeIAV uye IBV yekutanga.
Tevedza nzira inotevera yekuchenesa RNA: (1) RNA adsorption.Pipette 526 µl yemushonga wepellet muchubhu ye1.5 ml centrifuge wowedzera 150 µl yesampuli, wozunza chubhu nemaoko kumusoro nekudzika kagumi.Chinja 676 µl yemusanganiswa kune affinity column uye centrifuge pa 1.88 x 104 g kwemasekonzi makumi matanhatu.Madhini anotevera anobva araswa.(2) Nhanho yekutanga yekugeza.Wedzera 500 µl yewash solution A kune affinity column, centrifuge pa 1.88 x 104 g kwema 40 s, uye rasa mhinduro yakashandiswa.Iyi nzira yekugeza yakadzokororwa kaviri.(3) chikamu chechipiri chekugeza.Wedzera 500 µl yewash solution W kune affinity column, centrifuge pa 1.88 × 104 g kwema 15 s uye bvisa mhinduro inoshandiswa.Iyi nzira yekugeza yakadzokororwa kaviri.(4) Elution.Wedzera 200 µl ye eluate kune affinity column uye centrifuge pa 1.88 x 104 g kwemaminetsi maviri.(5) RT-PCR: Iyo eluate yakaiswa mu20 μl yeprimer solution muPCR chubhu, ipapo chubhu yakaiswa mune chaiyo-nguva PCR test appatus (SLAN-96P) kuita iyo RT-PCR maitiro.Iyo yese yekuona maitiro inotora angangoita 140 maminetsi (20 maminetsi ekucheneswa kweRNA uye 120 maminetsi ekuonekwa kwePCR).
526 µl yemushonga webead, 1000 µl yewash solution A, 1000 µl yewash solution W, 200 µl of eluate uye 20 µl yeprimer solution yakatanga yawedzerwa nekuchengetwa mumakamuri M, W1, W2, E uye PCR.Platform assembly.Zvadaro, 150 µl yemuenzaniso yakapinzwa mukamuri M uye FAST-POCT chikuva chakaiswa muchiridzwa chekuyedza chinoratidzwa muSupplementary Figure S9.Mushure memaminitsi makumi masere nemaviri, mhinduro dzebvunzo dzakawanikwa.
Kunze kwekunge zvaonekwa neimwe nzira, mhinduro dzese dzebvunzo dzinounzwa sezvinoreva ± SD mushure mezvishoma zvitanhatu zvinodzokorora uchishandisa chete FAST-POCT papuratifomu uye masampuli akazvimirira ega.Hapana data yakabviswa kubva pakuongorora.Miedzo haina kungoitika.Vatsvakurudzi vakanga vasina mapofu kumapoka emabasa panguva yekuedza.
Kuti uwane rumwe ruzivo nezve dhizaini yekudzidza, ona Nature Research Report abstract yakabatana nechinyorwa ichi.
Mashoko anotsigira zvakabuda muchidzidzo ichi anowanikwa muSupplementary Information.Ichi chinyorwa chinopa data rekutanga.
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