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Sistemụ lab-on-a-chip nwere ikike dị na saịtị na-enye ikike maka nyocha ngwa ngwa na nke ziri ezi ma bara uru na ntọala ejiri akụrụngwa ebe akụrụngwa biomedical na ndị ọkachamara zụrụ azụ adịghị.Otú ọ dị, ịmepụta usoro nyocha nke nlekọta nke na-enwe ihe niile dị mkpa n'otu oge maka ịnyefe ọtụtụ ọrụ, ntọhapụ na-achọ, arụmọrụ a pụrụ ịdabere na ya, na nchekwa ogologo oge nke reagents ka bụ nnukwu ihe ịma aka.N'ebe a, anyị na-akọwa teknụzụ mgba ọkụ obere njem na-arụ ọrụ lever nke nwere ike ịchịkwa mmiri mmiri n'akụkụ ọ bụla, na-enye nzaghachi ziri ezi na nha anya maka nrụgide ikuku etinyere, ma kwụsie ike megide mmegharị mberede na ịma jijiji.Dabere na teknụzụ, anyị na-akọwakwa mmepe nke usoro mmeghachi omume nke polymerase nke na-ejikọta mmeghe reagent, ịgwakọta na mmeghachi omume na-arụ ọrụ niile n'otu usoro, nke na-arụ ọrụ “nlere-na-aza-apụ” maka ihe nlele imi ụlọ ọgwụ niile sitere na ndị ọrịa 18. Influenza na 18 onye njikwa, na ezi nkwekọ nke fluorescence ike na ọkọlọtọ polymerase chain mmeghachi omume (Pearson coefficients> 0.9).Dabere na teknụzụ, anyị na-akọwakwa mmepe nke usoro mmeghachi omume nke polymerase nke na-ejikọta mmeghe reagent, ịgwakọta na mmeghachi omume na-arụ ọrụ niile n'otu usoro, nke na-arụ ọrụ “nlere-na-aza-apụ” maka ụdị imi ụlọ ọgwụ niile sitere na ndị ọrịa 18. ya na influenza na 18 onye njikwa, na ezigbo nkwekọrịta nke fluorescence ike na ọkọlọtọ polymerase chain mmeghachi omume (Pearson coefficients> 0.9).Основ jaая ная Ну ОНЛА Нnиииииииииииииииииииииииииииииии, колиерая, кбъерая Ọzọkwa Вения реагент: рноаеспет в вдномаеваеваеваеваеваеваеваеваеваеваеваеваеваенение Икиччссиххи Изацса г гипацитов гиптп и 18 Пирсона> 0,9).Dabere na nkà na ụzụ a, anyị na-akọwakwa mmepe nke usoro mmeghachi omume nke polymerase nke na-ejikọta ọrụ nke ịgbanye, ịgwakọta, na imeghachi omume n'otu usoro, na-eme ka nlele-na-azaghachi maka ụdị imi niile nke ụlọ ọgwụ sitere na ndị ọrịa 18 influenza.yana njikwa mmadụ 18, na nkwekọrịta dị mma na ọkọlọtọ polymerase chain reaction fluorescence intensity (ọnụọgụ Pearson> 0.9).Dabere na nkà na ụzụ a, anyị na-akọwakwa mmepe nke usoro mmeghachi omume nke polymerase nke na-ejikọta reagent injection, ịgwakọta, na mmeghachi omume mmeghachi omume iji nyochaa ihe niile gbasara imi imi site na 18 in-sample nasal patient specimens.Influenza na 18 onye njikwa, fluorescence ike dakọtara. nke ọma na mmeghachi omume yinye polymerase ọkọlọtọ (ọnụọgụ Pearson> 0.9).Ikpo okwu a na-atụ aro na-ekwe nkwa na akpaaka a pụrụ ịdabere na ya nke nyocha biomedical ma si otú ahụ nwee ike mee ka azụmaahịa nke ngwaọrụ nyocha dị iche iche na-ahụ maka nlekọta anya.
Ọrịa ụmụ mmadụ na-apụta, dị ka ọrịa COVID-19 nke 2020 nke gburula ndụ ọtụtụ nde mmadụ, na-etinye nnukwu ihe egwu na ahụike ụwa na mmepeanya mmadụ1.Nchọpụta ọrịa nke mbụ, ngwa ngwa na nke ziri ezi dị oke mkpa iji chịkwaa mgbasa nke nje ma melite nsonaazụ ọgwụgwọ.Usoro gburugburu ebe obibi nchọpụta bụ isi nke dabere na ụlọ nyocha etiti ebe a na-eziga nlele ule n'ụlọ ọgwụ ma ọ bụ ụlọ ọgwụ nyocha na ndị ọkachamara na-agba ọsọ ugbu a na-egbochi ohere maka ihe fọrọ nke nta ka ọ bụrụ ijeri mmadụ 5.8 n'ụwa niile, ọkachasị ndị bi na ntọala akụrụngwa.ebe enweghi akụrụngwa biomedical dị oke ọnụ na ndị ọkachamara ruru eru.Ndị dọkịta ụlọ ọgwụ 2. Ya mere, ọ dị mkpa ngwa ngwa ịmepụta usoro nyocha ụlọ nyocha na-adịghị ọnụ na onye ọrụ nwere ike nyocha nke nlekọta (POCT) nke nwere ike inye ndị dọkịta na-ahụ maka nchọpụta n'oge iji mee mkpebi nyocha. .na ọgwụgwọ 3.
Ntuziaka Healthtù Ahụ Ike (wa (WHO) na-ekwu na POCT dị mma kwesịrị ịdị ọnụ ala, enyi na enyi (dị mfe iji na obere ọzụzụ), nke ziri ezi (zere ihe ọjọọ ụgha ma ọ bụ ihe ndị na-adịghị mma), ngwa ngwa na ntụkwasị obi (nye ezigbo ihe nrụgharị), na anapụta (nwere ike ịchekwa ogologo oge yana ọ dị ngwa ngwa maka ndị ọrụ njedebe)4.Iji mezuo ihe ndị a chọrọ, sistemu POCT ga-enwerịrị atụmatụ ndị a: ụdị doses dị iche iche iji belata ntinye aka, ntọhapụ achọrọ ka ọ bụrụ ụgbọ njem reagent maka nsonaazụ ule ziri ezi, yana arụmọrụ a pụrụ ịdabere na ya iji guzogide ịma jijiji gburugburu ebe obibi.Ugbu a, ngwaọrụ POCT a na-ejikarị eme ihe bụ eriri mpụta nke na-agbapụta5,6 nke nwere ọtụtụ oghere nke membran nitrocellulose porous na-ebuli obere ihe nlele na-aga n'ihu, na-emeghachi omume na reagents ndị a na-emegharịbeghị site na ike capillary.N'agbanyeghị na ha nwere uru nke ọnụ ala dị ala, ịdị mfe nke iji, yana nsonaazụ ngwa ngwa, ngwaọrụ POCT na-agbapụta nwere ike iji naanị maka nnwale nke ndu (dịka ọmụmaatụ, ule glucose7,8 na ule ime ime9,10) na-achọghị nyocha ọtụtụ ọkwa.mmeghachi omume (dịka nbudata nke otutu reagents, ngwakọta, multiplexing).Na mgbakwunye, ike ịnya ụgbọ ala nke na-achịkwa mmegharị mmiri (ya bụ, ndị agha capillary) anaghị enye ezigbo nguzosi ike, ọkachasị n'etiti batches, na-ebute mmeghari na-adịghị mma11 na-eme ka eriri mpụta n'akụkụ bara uru maka nchọpụta dị mma12,13.
Ike mmepụta ihe gbasaa na micro- na nanoscale ewepụtala ohere maka mmepe nke microfluidic POCT ngwaọrụ maka nha nha14,15,16,17.Site n'ịgbanwe njirimara nke interface 18, 19 na geometry nke ọwa 20, 21, 22, enwere ike ịchịkwa ike capillary na ọsọ ọsọ nke ngwaọrụ ndị a.Agbanyeghị, ntụkwasị obi ha, ọkachasị maka mmiri mmiri nwere mmiri mmiri, ka bụ ihe a na-anabataghị n'ihi ezighi ezi n'ichepụta, nrụrụ ihe, yana nghọta maka ịma jijiji gburugburu ebe obibi.Tụkwasị na nke ahụ, ebe ọ bụ na a na-emepụta ihe na-agbapụta capillary na mmiri mmiri-gas interface, ọ dịghị ihe ọzọ na-asọpụta nwere ike ịmalite, karịsịa mgbe ejiri mmiri jupụta ọwa microfluidic.Ya mere, maka nchọpụta mgbagwoju anya, a ghaghị ime ọtụtụ nzọụkwụ nke injection sample24,25.
N'ime ngwaọrụ microfluidic, ngwaọrụ centrifugal microfluidic bụ otu n'ime ngwọta kachasị mma maka POCT26,27.Usoro draịva ya bara uru n'ihi na enwere ike ijikwa ike ịnya ụgbọ ala site na ịhazigharị ọsọ ntụgharị.Otú ọ dị, ihe ọghọm ya bụ na a na-eduzi ike centrifugal mgbe niile n'akụkụ mpụta nke ngwaọrụ ahụ, na-eme ka o sie ike imejuputa mmeghachi omume ọtụtụ nzọụkwụ achọrọ maka nyocha dị mgbagwoju anya.Ọ bụ ezie na ndị ọzọ na-anya ụgbọ ala (dịka capillaries 28, 29 na ọtụtụ ndị ọzọ 30, 31, 32, 33, 34, 35) na mgbakwunye na centrifugal ike na-ewebata maka multifunctional dosing, na-atụghị anya mmiri nyefe ka nwere ike ime n'ihi na ndị a ọzọ agha na-n'ozuzu iwu. ịdị ukwuu dị ala karịa ike centrifugal, na-eme ka ha dị irè naanị n'obere obere ọrụ na-arụ ọrụ ma ọ bụ na-adịghị adị na achọrọ ya na mwepụta mmiri mmiri.Ịgbakwunye mmeghari pneumatic n'ime microfluidics centrifugal dị ka usoro centrifugal kinetic 36, 37, 38, ụzọ thermopneumatic 39 na ụzọ pneumatic na-arụ ọrụ 40 egosila na ọ bụ ihe ọzọ mara mma.Site na usoro counterfugodynamic, a na-ejikọta oghere na microchannels na-ejikọta ya na ngwaọrụ maka ma mpụga na ime ime, n'agbanyeghị na arụmọrụ mgbapụta ya (na nso 75% ruo 90%) na-adabere na ọnụ ọgụgụ nke mgbapụta mgbapụta na viscosity. nke mmiri mmiri.N'ime usoro thermopneumatic, akpụkpọ ahụ latex na ọnụ ụlọ na-ebufe mmiri ka emebere kpọmkwem iji mechie ma ọ bụ meghee ntinye ahụ mgbe ụda ikuku tọrọ atọ dị ọkụ ma ọ bụ mee ka ọ dị jụụ.Otú ọ dị, nhazi ikpo ọkụ / oyi na-ewebata nsogbu nzaghachi ngwa ngwa ma gbochie iji ya na nyocha thermosensitive (dịka ọmụmaatụ, mmeghachi omume polymerase (PCR) amplification).Site na usoro pneumatic na-arụsi ọrụ ike, a na-enweta ntọhapụ nke achọrọ na mmegharị ime site na ntinye n'otu oge nke nrụgide dị mma yana kpomkwem na-agbagharị agbagharị ọsọ site na moto dị elu.Enwere ụzọ ndị ọzọ na-aga nke ọma na-eji naanị pneumatic actuators (nrụgide dị mma 41, 42 ma ọ bụ nrụgide na-adịghị mma 43) yana atụmatụ valvụ mechiri emechi.Site n'itinye nrụgide n'otu n'otu n'ime ụlọ pneumatic, a na-ebufe mmiri ahụ n'ihu peristaltically, na valvụ a na-emechi emechi na-egbochi nlọghachi nke mmiri mmiri n'ihi peristalsis, si otú a na-achọpụta ọrụ mmiri dị mgbagwoju anya.Otú ọ dị, enwere ugbu a nanị ọnụ ọgụgụ dị nta nke teknụzụ microfluidic nke nwere ike ịrụ ọrụ mmiri dị mgbagwoju anya n'otu ngwaọrụ POCT, gụnyere inyefe ọtụtụ ọrụ, ntọhapụ achọrọ, arụmọrụ a pụrụ ịdabere na ya, nchekwa ogologo oge, njikwa mmiri mmiri dị elu. na mmepụta ihe na-efu ọnụ.Ha niile n'otu oge.Enweghị ọrụ ọrụ ọtụtụ nzọụkwụ nwekwara ike ịbụ otu n'ime ihe kpatara na ọ bụ naanị ngwaahịa POCT ole na ole azụmahịa dị ka Cepheid, Binx, Visby, Cobas Liat, na Rhonda ka ewebatala nke ọma n'ahịa mepere emepe ruo taa.
N'ime akwụkwọ a, anyị na-atụ aro ka onye na-eme ihe na-eme ka microfluidic pneumatic dabere na teknụzụ mgbanaka mgbịrịgba green (FAST).FAST na-ejikọta ihe niile dị mkpa n'otu oge maka ọtụtụ reagents sitere na microliters ruo milliliters.FAST nwere akpụkpọ anụ, levers na ngọngọ.Na-enweghị ntinye nke ikuku ikuku, enwere ike mechie membranes, levers na blocks na mmiri mmiri dị n'ime nwere ike ịchekwa ogologo oge.Mgbe a na-etinye nrụgide kwesịrị ekwesị ma gbanwee ya na ogologo nke lever, diaphragm na-agbasawanye ma na-akwanye lever ahụ na oghere oghere, na-ekwe ka mmiri na-agafe.Nke a na-enye ohere ịlele mmiri mmiri multifunctional n'otu oge, n'otu oge, usoro ma ọ bụ nhọrọ.
Anyị emepụtala usoro PCR site na iji FAST iji mepụta nzaghachi-n'ihe nlele maka nchọpụta nke nje influenza A na B (IAV na IBV).Anyị nwetara njedebe dị ala nke nchọpụta (LOD) nke 102 mbipụta/mL, nyocha anyị multiplex gosipụtara nkọwapụta maka IAV na IBV ma kwe ka nje nje nje influenza.Nsonaazụ nnwale ụlọ ọgwụ site na iji nlele imi imi sitere na ndị ọrịa 18 na ndị mmadụ 18 nwere ahụike gosipụtara ezigbo nkwenye na ike ọkụ na ọkọlọtọ RT-PCR (Pearson coefficients> 0.9).Nsonaazụ nnwale ụlọ ọgwụ site na iji nlele imi imi sitere na ndị ọrịa 18 na ndị mmadụ 18 nwere ahụike gosipụtara ezigbo nkwenye na ike ọkụ na ọkọlọtọ RT-PCR (Pearson coefficients> 0.9).Результаты клинических испытаний с использованием образ | сответствие интенсивности флуоресценции стандартной ОТ-ПЦР (mkpụrụ osisi > 0,9).Nsonaazụ nke nnwale ụlọ ọgwụ site na iji nlele imi imi sitere na ndị ọrịa 18 na ndị mmadụ 18 nwere ahụike na-egosi nkwekọrịta dị mma n'etiti ike ọkụ nke ọkọlọtọ RT-PCR (Pearson's coefficients> 0.9).0.9) ……………………………………………………………………………………. Результаты клинических испытаний с использованием 18 соответствие между интенсивностью флуоресценции стандартной ОТ-ПЦР (коэффициент Пирсона> 0,9).Nsonaazụ nke nnwale ụlọ ọgwụ site na iji ụdị swab imi sitere na ndị ọrịa 18 na ndị mmadụ 18 nwere ahụike gosipụtara ezigbo nkwekọrịta n'etiti ike ọkụ na ọkọlọtọ RT-PCR (Pearson's coefficient> 0.9).Ọnụ ego a na-akwụ maka ngwa ngwa FAST-POCT dị ihe dị ka US$1 (Table 1 nke mgbakwunye) ma enwere ike belata ya site na iji usoro nrụpụta buru ibu (dịka ịkpụzi injection).N'ezie, ngwa ngwa POCT dabere na FAST nwere njirimara niile dị mkpa nke WHO nyere iwu ma kwekọọ na ụzọ nnwale biochemical ọhụrụ dị ka ịgba ígwè ikuku plasma thermal44, amplification-free immunoassays45 na nanobody functionalization tests46 bụ ọkpụkpụ azụ nke sistemụ POCT.ekwe omume.
Na fig.1a na-egosi nhazi nke ikpo okwu FAST-POCT, nke nwere ọnụ ụlọ mmiri anọ: ebe a na-edebe ebe nchekwa, ụlọ ngwakọta, ụlọ mmeghachi omume, na ụlọ mkpofu.Isi ihe na-eme ka njikwa mmiri na-asọ asọ bụ FAST imewe (nke nwere membranes na-agbanwe agbanwe, levers na blocks) dị n'ime ụlọ a na-ebu ụzọ echekwa na ọnụ ụlọ agwakọta.Dị ka usoro a na-emegharị pneumatically, FAST imewe na-enye njikwa nrịba mmiri ziri ezi, gụnyere mgbanaka mechiri emechi / mepere emepe, ngbanwe dị iche iche, mwepụta mmiri achọrọ, ọrụ a pụrụ ịdabere na ya (dịka ọmụmaatụ, enweghị mmetụta na ịma jijiji gburugburu), yana nchekwa ogologo oge.Ngwa ngwa FAST-POCT nwere akwa anọ: oyi akwa nkwado, ihe nkiri na-agbanwe agbanwe, ihe nkiri ihe nkiri plastik na mkpuchi mkpuchi, dị ka egosiri na nlele abawanyela na fig. 1b (nke egosiri n'ụzọ zuru ezu na S1 na S2). ).Ọwa niile na ebe a na-ebufe mmiri (dị ka ebe a na-ebuga mbụ na ụlọ mmeghachi omume) bụ agbakwunyere na PLA (polylactic acid) mkpụrụ sitere na 0.2 mm (akụkụ kacha mkpa) ruo 5 mm gbara ọkpụrụkpụ.Ihe nkiri na-agbanwe agbanwe bụ 300 µm gbara ọkpụrụkpụ PDMS nke na-agbasa ngwa ngwa mgbe a na-etinye nrụgide ikuku n'ihi "oke ọkpụrụkpụ" ya na modul dị ala nke elasticity (ihe dịka 2.25 MPa47).Ihe nkiri polyethylene bụ nke polyethylene terephthalate (PET) nwere ọkpụrụkpụ nke 100 µm iji chebe ihe nkiri na-agbanwe agbanwe site na nrụrụ oke n'ihi nrụgide ikuku.N'ikwekọ na ọnụ ụlọ ahụ, oyi akwa nke mkpụrụ osisi nwere levers jikọtara na mkpuchi mkpuchi (nke PLA) site na hinges iji chịkwaa mmiri mmiri.A na-ejikọta ihe nkiri na-agbanwe agbanwe na oyi akwa nkwado site na iji teepu nrapado nwere akụkụ abụọ (ARseal 90880) ma kpuchie ya na ihe nkiri plastik.A na-agbakọta akwa atọ n'elu ihe eji eme ihe T-clip na oyi akwa mkpuchi.T-mwekota nwere oghere n'etiti ụkwụ abụọ.Mgbe etinyere clip ahụ n'ime oghere ahụ, ụkwụ abụọ ahụ na-ehulata ntakịrị, wee laghachi na ọnọdụ mbụ ha wee kechie mkpuchi na nkwado ka ha na-agafe n'okporo ụzọ ahụ (Mgbakwunye Fig. S1).A na-ejikọta akwa anọ ahụ site na iji njikọ.
Eserese atụmatụ nke ikpo okwu na-egosi akụkụ dị iche iche na-arụ ọrụ yana atụmatụ FAST.b Eserese nke ikpo okwu FAST-POCT.c Foto nke ikpo okwu na-esote otu mkpụrụ ego US nkeji iri na ise.
E gosipụtara usoro na-arụ ọrụ nke ikpo okwu FAST-POCT na Figure 2. Ihe ndị bụ isi bụ ihe mgbochi dị na oyi akwa na mkpuchi na mkpuchi mkpuchi, nke na-eme ka ntinye nnyonye anya mgbe a na-ejikọta okpukpu anọ site na iji T-ụdị. .Mgbe a na-etinyeghị nrụgide ikuku (fig. 2a), nnyonye anya dabara na-eme ka mgbanaka ahụ na-ehulata ma gbanwee, a na-etinyekwa ike mkpuchi site na lever iji pịa ihe nkiri na-agbanwe agbanwe megide ngọngọ ahụ, a na-akọwakwa mmiri mmiri dị na oghere akara. dị ka akara akara.Ekwesiri iburu n'uche na na steeti a, a na-ehulata eriri ahụ n'èzí, dị ka e gosiri n'akụkụ anya na Fig. 2a.Mgbe a na-enye ikuku (Fig 2b), akpụkpọ ahụ na-agbanwe agbanwe na-agbasapụ n'èzí gaa n'ihu mkpuchi ma na-akwali lever elu, si otú a na-emepe oghere n'etiti lever na ngọngọ maka mmiri na-asọba n'ime ụlọ na-esote, bụ nke a kọwara dị ka oghere oghere. .Mgbe ntọhapụ nke ikuku ikuku, lever nwere ike ịlaghachi n'ọnọdụ mbụ ya ma nọgide na-ejidesi ike n'ihi ngbanwe nke hinge.E gosipụtara vidiyo nke mmegharị lever na ihe nkiri mgbakwunye S1.
A. Eserese eserese na foto mgbe emechiri ya.Na enweghị nrụgide, lever na-agbanye akpụkpọ ahụ na ngọngọ ahụ, a na-emechikwa mmiri mmiri.b N'ọnọdụ dị mma.Mgbe a na-etinye nrụgide, akpụkpọ ahụ na-agbasawanye ma na-akwali lever elu, ya mere ọwa ahụ ga-emepe na mmiri nwere ike ịgbapụta.c Chọpụta nha njirimara nke nrụgide dị oke egwu.Akụkụ njirimara gụnyere ogologo nke lever (L), anya dị n'etiti ihe mmịfe na mgbanaka (l) na ọkpụrụkpụ nke protrusion lever (t).Fs bụ ike mkpirisi na throttle point B. q bụ ibu ekesara n'otu n'otu na lever.Tx* na-anọchi anya ike ike nke ihe nleba higed mebere.Nrụgide dị oke mkpa bụ nrụgide achọrọ iji bulie lever ma mee ka mmiri na-asọpụta.d Nsonaazụ nyocha na nnwale nke mmekọrịta dị n'etiti nrụgide dị oke egwu na nha mmewere.n = 6 nyocha onwe ha ka emere ma gosipụta data dị ka ± ọkọlọtọ ọkọlọtọ.A na-ewepụta data raw dị ka faịlụ data raw.
Emepụtala ihe nlere anya dabere na tiori beam iji nyochaa ndabere nke oke nrụgide Pc nke ọdịiche ahụ na-emepe na paramita geometric (dịka ọmụmaatụ, L bụ ogologo nke lever, l bụ ebe dị anya n'etiti ngọngọ na hinge, S bụ lever Mpaghara kọntaktị na mmiri mmiri t bụ ọkpụrụkpụ nke protrusion lever, dị ka e gosiri na 2c).Dị ka nkọwa zuru ezu na Ndenye mgbakwunye na ihe atụ S3, oghere ahụ ga-emepe mgbe \({P}_{c}\ge \frac{2{F}_{s}l}{SL}\), ebe Fs bụ ihe mgbawa. \ ({T}_{x}^{\ast}(={F}_{s}l)\) iji kpochapụ ike ndị metụtara nnyonye anya dabara ma mee ka mgbanaka ahụ gbadaa.Nzaghachi nnwale na ụdị nyocha na-egosi nkwekọrịta dị mma (Fig 2d), na-egosi na nrụgide dị oke egwu Pc na-abawanye na t / l na-ebelata L, nke a na-akọwa ngwa ngwa site na ụdị ọkụ ọkụ oge gboo, ya bụ na torque na-abawanye na t / Ebuli. .Ya mere, nyocha usoro anyị na-egosi n'ụzọ doro anya na nrụgide dị oke egwu nwere ike ịchịkwa nke ọma site n'ịgbanwe ogologo ogologo L na t / l, nke na-enye ihe ndabere dị mkpa maka nhazi nke ikpo okwu FAST-POCT.
Ngwa ngwa FAST-POCT na-enye mgbasa ozi multifunctional (gosipụtara na Figure 3a na inset na nnwale), nke bụ akụkụ kachasị mkpa nke POCT na-aga nke ọma, ebe mmiri nwere ike na-asọba n'akụkụ ọ bụla na n'usoro ọ bụla (cascade, simultaneous, sequential) ma ọ bụ multichannel nhọrọ. nkesa .- arụ ọrụ dosing.Na fig.3a(i) na-egosi ọnọdụ dosing cascaded nke a na-agbanye ụlọ abụọ ma ọ bụ karịa site na iji blocks iji kewapụta ihe dị iche iche na-emeghachi omume na lever iji chịkwaa steeti mepere emepe na mechiri emechi.Mgbe etinyere nrụgide, mmiri mmiri na-esi na elu gaa n'ọnụ ụlọ dị ala n'ụzọ na-adịghị mma.Ekwesiri ighota na ime ulo nke cascade nwere ike juputara na kemịkalụ mmiri ma ọ bụ kemịkalụ akọrọ dị ka ntụ ntụ lyophilized.N'ime nnwale dị na Fig. 3a (i), ink na-acha uhie uhie si n'elu ụlọ na-asọba yana ntụ ntụ na-acha anụnụ anụnụ (ọla kọpa sulfate) n'ime ụlọ nke abụọ wee gbanwee ọchịchịrị na-acha anụnụ anụnụ mgbe ọ rutere n'ọnụ ụlọ dị ala.Ọ na-egosikwa nrụgide nchịkwa maka mmiri a na-agbapụta.N'otu aka ahụ, mgbe ejikọrọ otu lever n'ime ụlọ abụọ, ọ na-aghọ ụdị ịgba ọgwụ n'otu oge, dị ka egosiri na fig.3a (ii), nke enwere ike kesaa mmiri mmiri n'otu ọnụ ụlọ abụọ ma ọ bụ karịa mgbe etinyere nrụgide.Ebe ọ bụ na nrụgide dị oke egwu na-adabere n'ogologo nke lever ahụ, ogologo nke lever nwere ike gbanwee iji nweta usoro injection usoro dị ka egosiri na fig.3a (iii).A na-ejikọta ogologo lever (nke nwere nrụgide dị egwu Pc_long) na ụlọ B na obere lever (na nrụgide siri ike Pc_short> Pc_long) jikọtara ya na ụlọ A. Dị ka nrụgide P1 (Pc_long <P1 <Pc_short) etinyere, naanị mmiri mmiri na-acha uhie uhie. nwere ike ịgbaba n'ime ụlọ B na mgbe nrụgide ahụ na-abawanye na P2 (> PC_short), mmiri mmiri na-acha anụnụ anụnụ nwere ike ịbanye n'ime ụlọ A. Usoro injection usoro a na-emetụta mmiri dị iche iche na-ebufe n'ime ụlọ ndị metụtara ya n'usoro, nke dị oké mkpa maka POCT na-aga nke ọma. ngwaọrụ.A na-ejikọta ogologo lever (nke nwere nrụgide dị egwu Pc_long) na ụlọ B na obere lever (na nrụgide siri ike Pc_short> Pc_long) jikọtara ya na ụlọ A. Dị ka nrụgide P1 (Pc_long
ihe atụ nke ọrụ multifunctional, ntụgharị (i) cascading, (ii) otu oge, (iii) usoro, na (iv) ọrụ nhọrọ.Ọkpụkpụ ndị ahụ na-anọchi anya usoro ọrụ na parampat nke ụdị nkesa anọ a.b Nsonaazụ nke ule nchekwa ogologo oge na mmiri deionized na ethanol.n = 5 nyocha onwe ha ka emere ma gosi data dị ka ± sd c.Ngosipụta nkwụsi ike mgbe ngwaọrụ FAST na ngwaọrụ capillary valve (CV) nọ na (i) static na (ii) steeti na-ama jijiji.(iii) Mpịakọta vs. oge maka ngwa ngwa FAST na CV n'ọtụtụ akụkụ akụkụ dị iche iche.d Mbipụta nsonaazụ ule na-achọ (i) Ngwa ngwa ngwa na (ii) ngwaọrụ CV.(iii) Mmekọrịta dị n'etiti olu na oge maka ngwa ngwa FAST na CV na-eji ọnọdụ nrụgide oge.Ogwe ọnụ ọgụgụ niile, 1 cm.A na-enye data raw dị ka faịlụ data raw.
Ogologo oge nchekwa nke reagents bụ akụkụ ọzọ dị mkpa nke ngwaọrụ POCT na-aga nke ọma nke ga-ekwe ka ndị ọrụ na-azụghị azụ nwee ike ijikwa ọtụtụ reagents.Ọ bụ ezie na ọtụtụ teknụzụ egosila ikike ha maka nchekwa ogologo oge (dịka ọmụmaatụ, 35 microdispensers, 48 blister packs, na 49 stick packs), a na-achọ ụlọ nnabata raara onwe ya nye iji nabata ngwugwu ahụ, nke na-abawanye ọnụ ahịa na mgbagwoju anya;Ọzọkwa, usoro nchekwa ndị a anaghị ekwe ka a na-ekesa ihe a na-achọsi ike ma na-ebute mbibi nke reagents n'ihi ihe fọdụrụ na nkwakọ ngwaahịa.A kwadoro ikike nchekwa ogologo oge site n'ịme ule ndụ ngwa ngwa site na iji CNC-machined PMMA ihe n'ihi ntakịrị ike ya na iguzogide ikuku gas (Ngbakwunye S5).Ngwa nnwale ahụ juputara na mmiri deionized (mmiri deionized) na 70% ethanol (na-eme ka reagents na-agbanwe agbanwe) na 65ºC maka ụbọchị 9.A na-echekwa ma mmiri deionized na ethanol site na iji foil aluminum iji gbochie ịnweta site n'elu.Eji Arrhenius equation na penetration activation nke akọwara n'ime akwụkwọ 50,51 ka ejiri gbakọọ otu oge.Na fig.3b na-egosi nkezi nsonaazụ ọnwụ maka ihe nlele 5 echekwara na 65 ° C maka ụbọchị 9, ya na 0.30% maka mmiri deionized na 0.72% maka 70% ethanol karịa afọ 2 na 23 Celsius.
Na fig.3c na-egosi ule ịma jijiji.Ebe ọ bụ na valvụ capillary (CV) bụ usoro eji eji mmiri eme ihe kachasị ewu ewu n'etiti ngwaọrụ POCT28,29 dị ugbu a, a na-eji ngwaọrụ CV 300 µm n'obosara na 200 µm miri maka ntụnyere.Enwere ike ịhụ na mgbe ngwaọrụ abụọ ahụ nọgidere na-eguzosi ike, mmiri mmiri dị na FAST-POCT ikpo okwu na-emechi na mmiri dị na ngwaọrụ CV n'ihi mgbasawanye na mberede nke ọwa, nke na-ebelata ike capillary.Otú ọ dị, ka akụkụ akụkụ nke orbital vibrator na-abawanye, mmiri dị n'elu ikpo okwu FAST-POCT na-anọgide na-emechi, mana mmiri dị na ngwaọrụ CV na-abanye n'ime ụlọ dị ala (leekwa ihe nkiri S3 mgbakwunye).Nke a na-egosi na nrụrụ nrụrụ nke ikpo okwu FAST-POCT nwere ike itinye ike nrụzi siri ike na modul iji mechie mmiri mmiri dị n'ime ụlọ ahụ.Otú ọ dị, na ngwaọrụ CV, a na-edobe mmiri mmiri n'ihi nguzozi n'etiti usoro siri ike, ikuku, na mmiri mmiri, na-emepụta enweghị ike, na ịma jijiji nwere ike imebi nguzozi ahụ ma mee ka omume na-asọpụta na-atụghị anya ya.Uru nke ikpo okwu FAST-POCT bụ na ọ na-enye ọrụ a pụrụ ịdabere na ya ma na-ezere ọdịda n'ihu mkpọtụ nke na-emekarị n'oge nnyefe na ọrụ.
Akụkụ ọzọ dị mkpa nke ikpo okwu FAST-POCT bụ ntọhapụ ya na-achọ, nke bụ isi ihe achọrọ maka nyocha ọnụọgụ.Na fig.3d tụlere ntọhapụ nke ngwa FAST-POCT na ngwaọrụ CV.Site na fig.3d (iii) anyị na-ahụ na ngwaọrụ FAST na-anabata ngwa ngwa na mgbama nrụgide.Mgbe a na-etinye nrụgide n'elu ikpo okwu FAST-POCT, mmiri na-asọpụta, mgbe a na-ahapụ nrụgide ahụ, mmiri ahụ kwụsịrị ozugbo (Fig 3d (i)).Enwere ike ịkọwa ihe omume a site na nlọghachi ngwa ngwa na-agbanwe agbanwe nke hinge, nke na-agbanye eriri azụ na ngọngọ ahụ, mechie ụlọ ahụ.Agbanyeghị, mmiri gara n'ihu na-asọba na ngwaọrụ CV, n'ikpeazụ bute oke mmiri a na-atụghị anya ya nke ihe dịka 100 µl ka ewepụtara nrụgide (Njirimara 3d(ii) na ihe nkiri mgbakwunye S4).Enwere ike ịkọwa nke a site na mwepu nke mmetụta pinning capillary n'elu mmiri zuru oke nke CV mgbe ịgba ọgwụ mgbochi mbụ gasịrị.
Ikike ijikwa mmiri mmiri dị iche iche wettability na viscosity n'otu ngwaọrụ ka bụ ihe ịma aka maka ngwa POCT.Mmiri mmiri na-adịghị mma nwere ike iduga nkwụsị ma ọ bụ omume na-asọ oyi ndị ọzọ a na-atụghị anya ya na ọwa, na ngwá ọrụ enyemaka dị ka vortex mixers, centrifuges na nzacha na-achọkarị iji kwadebe nnukwu mmiri mmiri viscous 52 .Anyị nwalere mmekọrịta dị n'etiti nrụgide dị oke egwu na akụrụngwa mmiri (nke nwere oke mmiri na viscosity).E gosipụtara nsonaazụ ya na tebụl 1 na vidiyo S5.Enwere ike ịhụ na mmiri mmiri nke dị iche iche wettability na viscosity nwere ike mechie n'ime ụlọ ahụ, na mgbe a na-etinye nrụgide, ọbụna mmiri mmiri nwere viscosity ruo 5500 cP nwere ike ibufe n'ọnụ ụlọ dị n'akụkụ, na-eme ka o kwe omume ịchọpụta ihe nlele ndị nwere elu. viscosity (ya bu sputum, ihe nlere viscous nke ukwuu eji nchoputa nke oria iku ume).
Site n'ijikọta ngwaọrụ ndị na-ekesa multifunctional dị n'elu, enwere ike ịmepụta ọtụtụ ngwaọrụ POCT dabere na FAST.E gosipụtara ihe atụ na eserese 1. Osisi ahụ nwere ebe a na-edebe ebe nchekwa, ụlọ ngwakọta, ụlọ mmeghachi omume na ụlọ mkpofu.Enwere ike ịchekwa reagents n'ime ụlọ a na-ebu ụzọ echekwa ruo ogologo oge wee tụba ya n'ime ụlọ agwakọta.Site na nrụgide ziri ezi, enwere ike ịnyefe ndị na-eme ihe na-agwakọta ọnụ na ụlọ mkpofu ma ọ bụ ụlọ mmeghachi omume.
N'ihi na nchọpụta PCR bụ ọkọlọtọ ọla edo maka ịchọpụta ọrịa nje dị ka H1N1 na COVID-19 yana gụnyere ọtụtụ usoro mmeghachi omume, anyị jiri usoro FAST-POCT maka nchọpụta PCR dị ka ngwa.Na fig.4 na-egosi usoro nyocha PCR site na iji ngwa ngwa FAST-POCT.Nke mbụ, a na-atụnye reagent eluting, magnetik microbead reagent, ngwọta ịsacha A na ihe ịsa ahụ W n'ime ụlọ nchekwa E, M, W1 na W2 n'otu n'otu.E gosipụtara usoro mgbasa ozi RNA na fig.4a ma bụrụ ndị a: (1) mgbe a na-etinye nrụgide P1 (= 0.26 mmanya), ihe nlele ahụ na-abanye n'ime ụlọ M ma wepụ ya n'ime ụlọ ngwakọta.(2) A na-enye nrụgide ikuku P2 (= 0.12 mmanya) site n'ọdụ ụgbọ mmiri A jikọtara na ala nke ụlọ ngwakọta.Ọ bụ ezie na a ọnụ ọgụgụ nke Ịgwakọta ụzọ gosiri na ha nwere ike ịgwakọta mmiri mmiri na POCT nyiwe (eg serpentine Ịgwakọta 53, random Ịgwakọta 54 na ogbe Ịgwakọta 55), ha Ịgwakọta arụmọrụ na irè ka na-adịghị afọ ojuju.Ọ na-anabata usoro ngwakọta afụ, nke a na-ebute ikuku n'ime ala nke ọnụ ụlọ iji mepụta egosipụta na mmiri mmiri, mgbe nke ahụ gasịrị, vortex dị ike nwere ike nweta ngwakọta zuru ezu n'ime sekọnd.Emere nnwale ịgwakọta afụ wee gosipụta nsonaazụ ya na foto mgbakwunye S6.Enwere ike ịhụ na mgbe a na-etinye nrụgide nke 0.10 mmanya, ngwakọta zuru ezu na-ewe ihe dịka 8 sekọnd.Site n'ịbawanye nrụgide na 0.20 mmanya, a na-enweta ngwakọta zuru ezu n'ime ihe dịka 2 sekọnd.E gosipụtara usoro maka ịgbakọ arụmọrụ ngwakọta na ngalaba Usoro.(3) Jiri magnet rubidium wepụ beads, wee pịa P3 (= 0.17 mmanya) site n'ọdụ ụgbọ mmiri P iji bugharịa reagents n'ime ụlọ mkpofu.Na fig.4b, c na-egosi usoro ịsa ahụ iji wepụ ihe ndị na-adịghị ọcha site na ihe nlele ahụ dị ka ndị a: (1) A na-ewepụ ihe ngwọta ịsacha A site na ụlọ W1 n'ime ụlọ ngwakọta nrụgide P1.(2) Mgbe ahụ mee usoro ịgwakọta afụ.(3) A na-ebufe ihe ngwọta ịsacha A na ụlọ mmiri na-ekpofu ihe mkpofu, a na-ewepụkwa microbeads n'ime ụlọ ngwakọta site na magnet.Ịsacha W (Fig 4c) yiri ịsacha A (Fig 4b).Ekwesiri iburu n'uche na a na-eme usoro ịsa ahụ A na W ugboro abụọ.Onyonyo 4d na-egosi usoro elution iji wepụ RNA site na beads;Usoro mmalite na ngwakọta ngwakọta bụ otu ihe mgbasa ozi RNA na usoro ịsa ahụ akọwara n'elu.Dị ka a na-ebufe elution reagents n'ime ụlọ mmeghachi omume PCR n'okpuru nrụgide P3 na P4 (= 0.23 mmanya), a na-enweta nrụgide dị egwu iji mechie ogwe aka nke ụlọ mmeghachi omume PCR.N'otu aka ahụ, nrụgide P4 na-enyekwa aka mechie ụzọ ahụ na ụlọ mkpofu.Ya mere, a na-ekesa reagents niile n'otu n'otu n'etiti ụlọ mmeghachi omume PCR anọ iji malite mmeghachi omume PCR multiplex.E gosipụtara usoro a dị n'elu na ihe nkiri mgbakwunye S6.
N'ime usoro mgbasa ozi RNA, a na-ewebata ihe nlele ahụ n'ime mbanye M wee tinye ya n'ime ụlọ ngwakọta yana ngwọta bead echekwara na mbụ.Mgbe agwakọta ma wepụ granules, a na-ekesa reagents n'ime ụlọ mkpofu.b na c na-asacha usoro, webata ụdị reagents dị iche iche echekwara n'ime ụlọ a na-agwakọta, na mgbe agwakọta ma wepụ beads, nyefee reagents n'ime ụlọ mmiri na-ekpofu ihe.d Elution nzọụkwụ: Mgbe ewebata elution reagents, ịgwakọta na bead mmịpụta, ndị reagents na-ebufe na PCR mmeghachi omume ụlọ.Ngwongwo ndị ahụ na-egosi usoro ọrụ na ihe ndị yiri ya nke ọkwa dị iche iche.Nrụgide bụ nrụgide a na-eme site na ọnụ ụlọ nke ọ bụla.Olu bụ olu mmiri mmiri n'ime ụlọ ngwakọta.Ogwe ọnụ ọgụgụ niile bụ 1 cm.A na-enye data raw dị ka faịlụ data raw.
Emere usoro ule PCR yana S7 mgbakwunye na-egosi profaịlụ okpomọkụ gụnyere nkeji 20 nke oge ntụgharị ntụgharị na nkeji 60 nke oge ịgba ígwè (95 na 60 Celsius C), yana otu okirikiri okpomọkụ bụ 90 s (Ihe Nkwukọrịta Ihe nkiri S7)..FAST-POCT na-achọ obere oge iji wuchaa otu okirikiri okpomọkụ (sekọnd 90) karịa RT-PCR a na-emekarị (sekọnd 180 maka otu okirikiri okpomọkụ).Enwere ike ịkọwa nke a site na mpaghara elu dị elu na oke olu yana obere ọkụ ọkụ nke ụlọ mmeghachi omume micro-PCR.Elu ụlọ ahụ bụ 96.6 mm2 na ọnụ ụlọ ahụ bụ 25 mm3, na-eme ka elu na nha nha dịka 3.86.Dị ka a na-ahụ na S10 nke mgbakwunye, mpaghara ule PCR nke ikpo okwu anyị nwere oghere na azụ azụ, na-eme ka ala nke ụlọ PCR dị 200 µm.A na-ejikọta pad na-agbanwe ihe na-ekpo ọkụ n'elu ikpo ọkụ nke onye na-ahụ maka okpomọkụ, na-eme ka njikọ dị n'azụ igbe ule ahụ.Nke a na-ebelata ikpo ọkụ ọkụ nke ikpo okwu ma na-eme ka ọkụ ọkụ / dị jụụ rụọ ọrụ.N'oge ịnya igwe na-ekpo ọkụ, paraffin nke etinyere n'elu ikpo okwu na-agbaze wee banye n'ime ụlọ mmeghachi omume PCR, na-eme dị ka ihe mkpuchi iji gbochie evaporation reagent na mmetọ gburugburu ebe obibi (lee ihe nkiri S8 mgbakwunye).
Usoro nchọpụta PCR niile akọwara n'elu bụ akpaaka zuru oke site na iji ngwa emebere ngwa ngwa FAST-POCT, nke nwere ngalaba njikwa nrụgide akwadoro, ngalaba mmịpụta magnetik, ngalaba njikwa okpomọkụ, yana njide na nhazi mgbama fluorescent.N'ịma ama, anyị jiri usoro FAST-POCT maka ikewapụ RNA wee jiri ihe nlele RNA ewepụtara maka mmeghachi omume PCR site na iji sistemụ FAST-POCT na PCR desktọọpụ maka ntụnyere.Nsonaazụ ọ fọrọ nke nta ka ọ bụrụ otu egosiri na foto mgbakwunye S8.Onye na-arụ ọrụ na-arụ ọrụ dị mfe: na-ewebata ihe nlele n'ime ime ụlọ M ma tinye ikpo okwu n'ime ngwa.Nsonaazụ ọnụọgụ ọnụọgụ dị na ihe dịka nkeji iri asatọ na abụọ.Enwere ike ịchọta ozi gbasara ngwa FAST-POCT na ọnụ ọgụgụ mgbakwunye ahụ.C9, C10 na C11.
Influenza nke influenza A (IAV), B (IBV), C (ICV) na D (IDV) kpatara bụ ihe a na-ahụkarị n'ụwa niile.N'ime ndị a, IAV na IBV na-ahụ maka ikpe kachasị njọ na ọrịa na-efe efe nke oge, na-ebute 5-15% nke ndị bi n'ụwa, na-akpata 3-5 nde ikpe siri ike ma na-akpata ọnwụ 290,000-650,000 kwa afọ.Ọrịa iku ume56,57.Nchọpụta mmalite nke IAV na IB dị mkpa iji belata ọrịa na ibu akụ na ụba jikọtara ya.N'ime usoro nchọpụta dịnụ, a na-ewere reverse transcriptase polymerase chain reaction (RT-PCR) dị ka nke kachasị emetụ n'ahụ, kpọmkwem na nke ziri ezi (> 99%) 58,59.N'ime usoro nchọpụta dịnụ, a na-ewere reverse transcriptase polymerase chain reaction (RT-PCR) dị ka nke kachasị emetụ n'ahụ, kpọmkwem na nke ziri ezi (> 99%) 58,59.Среди доступных диагностических методов полимеразная цепная реакция с тельной, специфичной и точной (> 99%)58,59.N'ime ụzọ nchọpụta dịnụ, a na-ewere reverse transcriptase polymerase chain reaction (RT-PCR) dị ka nke kacha emetụ n'ahụ, kpọmkwem na nke ziri ezi (> 99%)58,59. Из доступных диагностических методов полимеразная цепная реакция с обратной транскриптазой льной, специфичной и точной (>99%)58,59.N'ime ụzọ nchọpụta dịnụ, a na-ewere reverse transcriptase polymerase chain reaction (RT-PCR) dị ka nke kacha nwee mmetụta, kpọmkwem na nke ziri ezi (> 99%) 58,59.Otú ọ dị, ụzọ RT-PCR omenala chọrọ pipetting ugboro ugboro, ịgwakọta, ịnyefe na ịnyefe mmiri mmiri, na-amachi ndị ọkachamara na-eji ha eme ihe na ntọala ndị nwere oke akụ.N'ebe a, ejiri usoro FAST-POCT mee ihe maka nchọpụta PCR nke IAV na IBV, n'otu n'otu, iji nweta njedebe dị ala nke nchọpụta (LOD).Tụkwasị na nke ahụ, IAV na IBV ejirila ọtụtụ ihe ịkpa ókè n'etiti ụdị pathotypes dị iche iche n'ofe ụdị, na-enye ohere maka nyocha mkpụrụ ndụ ihe nketa na ikike ịgwọ ọrịa ahụ nke ọma.
Na fig.5a na-egosi nsonaazụ HAV PCR site na iji 150 µl nke nje RNA dị ọcha dị ka ihe nlele.Na fig.5a (i) na-egosi na na ntinye HAV nke 106 mbipụta / ml, ike ọkụ ọkụ (ΔRn) nwere ike iru 0.830, na mgbe a na-ebelata ntinye uche na 102 mbipụta / ml, ΔRn ka nwere ike iru 0.365, nke kwekọrọ na elu karịa nke ahụ. nke otu nchịkwa ọjọọ efu (0.002), ihe dị ka 100 ugboro elu.Maka ntule dabere na nnwale isii nọọrọ onwe ha, a na-emepụta usoro nhazi usoro n'etiti ntinye ntinye na okirikiri okirikiri (Ct) nke IAV (Fig. 5a(ii)), R2 = 0.993, sitere na 102-106 mbipụta/mL.Nsonaazụ dị na nkwekọrịta dị mma na usoro RT-PCR a na-emekarị.Na fig.5a (iii) na-egosi foto fluorescent nke nsonaazụ ule mgbe okirikiri 40 nke ikpo okwu FAST-POCT.Anyị chọpụtara na ikpo okwu FAST-POCT nwere ike ịchọpụta HAV dị ala dị ka 102 mbipụta / mL.Otú ọ dị, usoro ọdịnala enweghị uru Ct na 102 mbipụta / ml, na-eme ka ọ bụrụ LOD nke ihe dịka 103 mbipụta / ml.Anyị chere na nke a nwere ike ịbụ n'ihi oke arụmọrụ nke ngwakọta afụ.Emere nnwale PCR na IAV RNA dị ọcha iji nyochaa ụzọ ngwakọta dị iche iche, gụnyere ịgwakọta shake (otu usoro ngwakọta dị ka ọ na-arụ ọrụ RT-PCR a na-emekarị), ngwakọta vial (usoro a, 3 s na 0.12 mmanya) na enweghị ngwakọta dị ka otu njikwa. ..Enwere ike ịhụ nsonaazụ ya na foto mgbakwunye S12.Enwere ike ịhụ na na ntinye RNA dị elu (106 mbipụta / mL), ụkpụrụ Ct nke ụzọ agwakọta dị iche iche fọrọ nke nta ka ọ bụrụ otu maka ịgwakọta afụ.Mgbe mkpokọta RNA gbadara na mbipụta 102 / mL, ngwakọta shake na njikwa enweghị ụkpụrụ Ct, ebe usoro ngwakọta afụ ka na-enye uru Ct nke 36.9, nke dị n'okpuru ọnụ ụzọ Ct nke 38. Nsonaazụ na-egosi njirimara agwakọta kachasị. vesicles, nke egosipụtakwara na akwụkwọ ndị ọzọ, nke nwekwara ike ịkọwa ihe kpatara nghọta nke FAST-POCT ikpo okwu dị ntakịrị elu karịa RT-PCR omenala.Na fig.5b na-egosi nsonaazụ PCR nke ihe nlele IBV RNA dị ọcha sitere na 101 ruo 106 mbipụta/ml.Nsonaazụ ndị ahụ yiri ule IAV, na-enweta R2 = 0.994 yana LOD nke 102 mbipụta/mL.
Nyocha PCR nke nje influenza A (IAV) na ntinye IAV sitere na 106 ruo 101 mbipụta / mL site na iji TE buffer dị ka njikwa na-adịghị mma (NC).(i) Ogologo oge fluorescence.(ii) Usoro nhazi usoro n'etiti logarithmic IAV RNA itinye uche na oke okirikiri (Ct) maka ngwa ngwa na ụzọ nnwale.(iii) Foto fluorescent IAV FAST-POCT mgbe okirikiri 40 gachara.b, Nchọpụta PCR nke nje influenza B (IBV) nwere (i) ụdị fluorescence dị adị.(ii) Usoro nhazi usoro na (iii) Foto fluorescence FAST-POCT IBV mgbe okirikiri 40 gachara.Obere njedebe nke nchọpụta (LOD) maka IAV na IBV site na iji ngwa ngwa FAST-POCT bụ 102 mbipụta / mL, nke dị ala karịa usoro ndị a na-emekarị (103 mbipụta / mL).c nsonaazụ ule Multiplex maka IAV na IBV.A na-eji GAPDH mee ihe dị ka njikwa dị mma na TE buffer ejiri mee ihe nchịkwa na-adịghị mma iji gbochie mmetọ nwere ike ime na nkwalite ndabere.Enwere ike ịmata ụdị ihe atụ anọ dị iche iche: (1) GAPDH-naanị ihe na-adịghị mma ("IAV-/IBV-");(2) ọrịa IAV ("IAV+/IBV-") nwere IAV na GAPDH;(3) ọrịa IBV ("IAV-/IBV+") nwere IBV na GAPDH;(4) Ọrịa IAV/IBV ("IAV+/IBV+") nwere IAV, IBV na GAPDH.Ahịrị nwere ntụpọ na-anọchi anya ahịrị ọnụ ụzọ.n = A na-eme nnwale nnwere onwe nke ndụ 6, egosiri data dị ka ± ọkọlọtọ ọkọlọtọ.A na-ewepụta data raw dị ka faịlụ data raw.
Na fig.5c na-egosi nsonaazụ nke ule multiplexing maka IAV/IBV.N'ebe a, a na-eji lysate nje mee ihe dị ka ihe nlele anya n'ọnọdụ RNA dị ọcha, na primers anọ maka IAV, IBV, GAPDH (nchịkwa dị mma) na TE buffer (nchịkwa na-adịghị mma) ka agbakwunyere na ụlọ mmeghachi omume anọ dị iche iche nke ngwa ngwa FAST-POCT.A na-eji njikwa dị mma na nke na-adịghị mma ebe a gbochie mmetọ nwere ike ime na nkwalite ndabere.E kewara ule ndị ahụ ụzọ anọ: (1) GAPDH-adịghị mma sample ("IAV-/IBV-");(2) oria IAV (“IAV+/IBV-”) megide IAV na GAPDH;(3) IBV-.oria (“IAV-”) -/IBV+”) IBV na GAPDH;(4) IAV/IBV ("IAV+/IBV+") na-ebute ọrịa IAV, IBV na GAPDH.Na fig.5c na-egosi na mgbe etinyere ihe nlele na-adịghị mma, ọkụ ọkụ ọkụ ΔRn nke ụlọ nchịkwa dị mma bụ 0.860, na ΔRn nke IAV na IBV yiri njikwa na-adịghị mma (0.002).Maka otu IAV +/IBV-, IAV-/IBV + na IAV +/ IBV +, igwefoto IAV/GAPDH, IBV/GAPDH na IAV/IBV/GAPDH gosipụtara ike ọkụ dị ukwuu, n'otu n'otu, ebe igwefoto ndị ọzọ na-egosipụtakwa ike ọkụ na ndabere. ọkwa nke 40 mgbe ịnya igwe thermal.Site na ule ndị dị n'elu, ikpo okwu FAST-POCT gosipụtara nkọwapụta pụtara ìhè ma nye anyị ohere ka anyị na-akpakọrịta nje influenza dị iche iche n'otu oge.
Iji kwado ngwa ngwa ụlọ ọgwụ nke FAST-POCT, anyị nwalere ụdị ọgwụgwọ 36 (ụdị imi swab) sitere na ndị ọrịa IB (n=18) na njikwa ndị na-abụghị IB (n=18) (Nyocha 6a).E gosipụtara ozi onye ọrịa na tebụl mgbakwunye 3. A kwadoro ọnọdụ ọrịa IB n'onwe ya na ụlọ ọgwụ mbụ nke Mahadum Zhejiang kwadoro (Hangzhou, Zhejiang).E kewara ihe nlele ọ bụla nke ndị ọrịa ụzọ abụọ.Ejiri FAST-POCT were hazie nke ọzọ site na iji sistemụ PCR desktọpụ (SLAN-96P, China).Nnwale abụọ a na-eji otu ngwa nchacha na nchọpụta nchọpụta.Na fig.6b na-egosi nsonaazụ FAST-POCT na PCR reverse transcription PCR (RT-PCR).Anyị tụlere ike nke fluorescence (FAST-POCT) na -log2 (Ct), ebe Ct bụ ọnụ ụzọ okirikiri maka RT-PCR.Enwere ezigbo nkwekọrịta n'etiti ụzọ abụọ a.FAST-POCT na RT-PCR gosipụtara njikọ dị mma siri ike yana uru nha Pearson (r) nke 0.90 (Njirimara 6b).Anyị wee nyochaa izi ezi nke nchoputa nke FAST-POCT.Enyere nkesa ọkụ ọkụ (FL) maka ihe ndị dị mma na nke na-adịghị mma dị ka ihe nyocha nke onwe ya (Fig 6c).Ụkpụrụ FL dị elu nke ukwuu na ndị ọrịa IB karịa njikwa (**** P = 3.31 × 10-19; t-test ọdụdụ abụọ) (Fig 6d).Na-esote, IBV na-arụ ọrụ nke ọma (ROC) kpara nkata.Anyị chọpụtara na izi ezi nke diagnostic dị ezigbo mma, na mpaghara n'okpuru usoro nke 1 (Fig 6e).Biko mara na n'ihi iwu nkpuchi amanyere iwu na China n'ihi COVID-19 dị ka nke 2020, anyị achọpụtabeghị ndị ọrịa nwere IBD, yabụ ụdị ụdị ọgwụgwọ niile dị mma (ya bụ, ụdị swab imi) bụ naanị maka IBV.
Nhazi ọmụmụ ụlọ ọgwụ.Ngụkọta ihe nlele 36, gụnyere ihe nlele ndị ọrịa 18 na njikwa 18 na-abụghị influenza, nyochara site na iji ngwa ngwa FAST-POCT na RT-PCR.b Nyochaa ndakọrịta nyocha n'etiti FAST-POCT PCR na RT-PCR omenala.Ejikọtara nsonaazụ ya nke ọma (Pearson r = 0.90).c Fluorescence ike ọkwa na ndị ọrịa 18 IB na njikwa 18.d N'ime ndị ọrịa IB (+), ụkpụrụ FL dị elu karịa na otu njikwa (-) (**** P = 3.31 × 10-19; t-test ọdụdụ abụọ; n = 36).Maka mpempe square ọ bụla, akara ojii dị n'etiti na-anọchite anya etiti, na ala na ahịrị elu nke igbe ahụ na-anọchi anya 25th na 75th percentiles, n'otu n'otu.Nkịta ndị ahụ na-agbatị ruo kacha nta na isi data kachasị, nke a na-ewereghị dị ka ndị ọzọ.e ROC curve.Ahịrị nwere ntụpọ d na-anọchi anya uru ọnụ ụzọ echere na nyocha ROC.The AUC maka IBV bụ 1. Raw data na-nyere dị ka raw data faịlụ.
N'isiokwu a, anyị na-eweta FAST, nke nwere njirimara achọrọ maka POCT dị mma.Uru nke nkà na ụzụ anyị gụnyere: (1) usoro ọgwụgwọ dị iche iche (cascade, n'otu oge, usoro na nhọrọ), ntọhapụ na ọchịchọ (ntọhapụ ngwa ngwa na nke nha nke nrụgide etinyere) na ọrụ a pụrụ ịdabere na ya (vibration na 150 degrees) (2) nchekwa ogologo oge. (afọ 2 nke nyocha ngwa ngwa, ọnwụ dị arọ banyere 0.3%);(3) ike na-arụ ọrụ na mmiri mmiri nwere ọtụtụ mmiri mmiri na viscosity (viscosity ruo 5500 cP);(4) Ego (Egodoro ọnụ ahịa ihe onwunwe nke ngwa PCR FAST-POCT bụ ihe dịka US$1).Site na ijikọ ndị na-ekesa multifunctional, agbakwunyere ngwa ngwa FAST-POCT maka nchọpụta PCR nke nje influenza A na B ka egosiri wee tinye ya.FAST-POCT na-ewe naanị nkeji 82.Nnwale ụlọ ọgwụ nwere ụdị swab 36 na-egosi ezigbo nkwenye na ike fluorescence na ọkọlọtọ RT-PCR (Pearson coefficients> 0.9).Nnwale ụlọ ọgwụ nwere ụdị swab 36 na-egosi ezigbo nkwenye na ike fluorescence na ọkọlọtọ RT-PCR (Pearson coefficients> 0.9).Клинические тесты 36 образцами мазков из носа? ЦР (nkịtị Пирсона> 0,9).Nnwale ụlọ ọgwụ na ihe nlele 36 nke imi imi gosipụtara nkwekọrịta dị mma na ike ọkụ nke ọkọlọtọ RT-PCR (Pearson's coefficients> 0.9) .RT-PCR Клинические испытания 36 образцов мазков ПЦР ( kọmpụta Пирсона > 0,9).Nnwale ụlọ ọgwụ nke ụdị swab imi 36 gosipụtara ezigbo nkwekọrịta nke ike fluorescence na ọkọlọtọ RT-PCR (Pearson's coefficient> 0.9).N'otu aka ahụ na ọrụ a, ụzọ dị iche iche nke biochemical na-apụta (dịka ọmụmaatụ, ịgba ígwè ikuku plasma, immunoassays na-enweghị nkwalite, na nyocha ọrụ nanobody) egosila ikike ha nwere na POCT.Agbanyeghị, n'ihi enweghị ikpo okwu POCT zuru oke na nke siri ike, ụzọ ndị a enweghị atụ chọrọ usoro nhazi ụzọ dị iche iche (dịka ọmụmaatụ, RNA isolation44, incubation45 na wash46), nke na-emeju ọrụ ugbu a na ụzọ ndị a iji mejuputa ọrụ POCT dị elu. paramita achọrọ.rụpụta ọrụ nzaghachi-na-aza.N'ime ọrụ a, ọ bụ ezie na mgbapụta ikuku na-eme ka valvụ FAST rụọ ọrụ dị ntakịrị iji tinye ya na ngwá ọrụ benchtop (Fig. S9, S10), ọ ka na-eri ike dị ukwuu ma na-emepụta mkpọtụ.N'ụkpụrụ, obere ụdị nfuli pneumatic nwere ike iji ụzọ ndị ọzọ dochie ya, dị ka iji ike electromagnetic ma ọ bụ mmegharị mkpịsị aka.Nkwalite ndị ọzọ nwere ike ịgụnye, dịka ọmụmaatụ, ngwa ngbanwe maka nyocha biochemical dị iche iche na nke akọwapụtara, na-eji ụzọ nchọpụta ọhụrụ nke na-achọghị usoro ikpo ọkụ / oyi, si otú a na-enye ikpo okwu POCT na-enweghị ngwá ọrụ maka ngwa PCR.Anyị kwenyere na enyere na ikpo okwu FAST na-enye ụzọ iji megharịa mmiri mmiri, anyị kwenyere na teknụzụ FAST a na-atụ aro na-enye ike ịmepụta ikpo okwu nkịtị ọ bụghị naanị maka nyocha biomedical, kamakwa maka nlekota gburugburu ebe obibi, nyocha mma nri, ihe onwunwe na njikọ ọgwụ. ..
Kọmiti ụkpụrụ omume nke ụlọ ọgwụ mbụ jikọrọ mahadum Zhejiang (IIT20220330B) akwadola mkpokọta na ojiji nke ụdị imi imi mmadụ.A nakọtara ihe nlele imi 36, gụnyere ndị okenye iri na isii <30 afọ, ndị okenye 7> 40 afọ, na ụmụ nwoke 19, ụmụ nwanyị 17.A nakọtara ihe nlele imi 36, gụnyere ndị okenye iri na isii <30 afọ, ndị okenye 7> 40 afọ, na ụmụ nwoke 19, ụmụ nwanyị 17.Было собрано 36 образцов мазков из носа. na 17 женщин.A nakọtara ụdị swab imi iri atọ na isii site n'aka ndị okenye iri na isii <30 afọ, ndị okenye 7 karịrị afọ 40, ndị nwoke 19 na ụmụ nwanyị 17..E gosipụtara data ọnụọgụgụ na tebụl mgbakwunye 3. E nwetara nkwenye ọmụma sitere n'aka ndị niile sonyere.A na-enyo ndị niile sonyere na ha nwere influenza ma nwalee ha n'afọ ofufo na-enweghị ụgwọ ọrụ.
Ihe ndabere FAST na mkpuchi bụ nke polylactic acid (PLA) wee bipụta ya site na ngwa nbipute Ender 3 Pro 3D (Shenzhen Transcend 3D Technology Co., Ltd.).A zụtara teepu n'akụkụ abụọ n'aka Adhesives Research, Inc. Model 90880. PET ihe nkiri 100 µm gbara ọkpụrụkpụ ka zụtara n'aka McMaster-Carr.A na-egbutu ma nrapado na ihe nkiri PET site na iji Silhouette Cameo 2 cutter si Silhouette America, Inc. Ihe nkiri na-agbanwe agbanwe bụ ihe PDMS site na ịkpụzi injections.Nke mbu, a na-eji sistemu laser bee 200µm gbara ọkpụrụkpụ PET frame wee tinye ya na mpempe PMMA gbara ọkpụrụkpụ 3 mm site na iji teepu nrapado nwere akụkụ abụọ 100 µm.PDMS precursor (Sylgard 184; Nkebi A: Nkebi B = 10:1, Dow Corning) wee wụsa n'ime ihe ebu ma were mkpanaka iko wepụ PDMS ngafe.Mgbe gwọchara na 70 ° C. maka awa 3, ihe nkiri 300 μm gbara ọkpụrụkpụ PDMS nwere ike ịpụpụ na ebu.
A na-eji igwefoto na-agba ọsọ (Sony AX700 1000 fps) were were foto maka nkesa dịgasị iche iche, mbipụta achọrọ na arụmọrụ a pụrụ ịdabere na ya.A zụtara ihe na-eme mkpọtụ nke ejiri na ule ntụkwasị obi sitere na SCILOGEX (SCI-O180).A na-emepụta nrụgide ikuku site na ikuku compressor, a na-ejikwa ọtụtụ ndị na-ahụ maka nrụgide nkenke dijitalụ mee ihe iji dozie uru nrụgide.Usoro nnwale omume ịgba ọsọ bụ nke a.Agbara mmiri mmiri a kara aka ekpebi n'ime ngwaọrụ nnwale ahụ wee jiri igwefoto ọsịsọ dị elu wee dekọọ omume mgbaba ahụ.E wepụrụ ihe onyonyo a ka na-eme site na vidiyo nke omume mgbaba n'oge a kapịrị ọnụ, wee gbakọọ mpaghara fọdụrụ site na iji sọftụwia Image-Pro Plus, nke ejiri omimi igwefoto mụbaa iji gbakọọ olu.Enwere ike ịhụ nkọwapụta nke sistemu nnwale omume na-aga n'ihu na onyonyo S4.
Tinye 50 µl nke microbeads na 100 µl nke mmiri deionized n'ime ngwaọrụ ngwakọta vial.A na-eji igwefoto dị elu were foto arụmọrụ agwakọtara agwakọta na 0.1 sekọnd na nrụgide nke 0.1 mmanya, mmanya 0.15 na 0.2 mmanya.Enwere ike nweta ozi Pixel n'oge usoro ngwakọta site na onyonyo ndị a site na iji ngwa nhazi foto (Photoshop CS6).Enwere ike ijikwa usoro 53 na-esonụ rụzuo arụmọrụ nke ọma.
Ebe M bụ ngwakọta nke ọma, N bụ ngụkọta nke pikselụ sample, na ci na \(\bar{c}\) bụ mkpokọta ahaziziri ahazi na atụ anya.Ịgwakọta arụmọrụ sitere na 0 (0%, enweghị agwakọta) ruo 1 (100%, agwakọta nke ọma).E gosipụtara nsonaazụ ya na foto mgbakwunye S6.
Ngwa RT-PCR ezigbo oge maka IAV na IBV, gụnyere IAV na IBV RNA sample (cat. no. RR-0051-02/RR-0052-02, Liferiver, China), Tris-EDTA buffer (TE buffer no. B541019). , Sangon Biotech, China), Ngwa njikwa RNA dị ọcha (Nkebi nke Z-ME-0010, Liferiver, China) na GAPDH Solution (Nkebi nke M591101, Sangon Biotech, China) na-ere ahịa.Ngwa RNA dị ọcha gụnyere ihe nkpuchi, ịsacha A, wash W, eluent, microbeads magnetik na ihe acrylic.Ngwa IAV na IBV ezigbo oge RT-PCR gụnyere ngwakọta nchọpụta PCR nucleic acid IFVA na enzyme RT-PCR.Tinye 6 µl nke AcrylCarrier na 20 µl nke magnetik beads na 500 µl nke ngwọta ihe nkpuchi, maa jijiji nke ọma wee dozie ngwọta bead.Tinye 21 ml nke ethanol ka ị na-asa A na W, maa jijiji nke ọma iji nweta ngwọta nke ịsacha A na W, n'otu n'otu.Mgbe ahụ, 18 µl nke fluorescent PCR ngwakọta na IFVA nucleic acid na 1 µl nke RT-PCR enzyme agbakwunyere na 1 µl nke ngwọta TE, maa jijiji na centrifuged ruo ọtụtụ sekọnd, na-enweta 20 µl nke IAV na IBV primers.
Soro usoro nchacha RNA ndị a: (1) mgbasa ozi RNA.Pipette 526 µl nke pellet ngwọta n'ime tube 1.5 ml centrifuge ma gbakwunye 150 µl nke sample, wee jiri aka kpalie tube ahụ elu na ala ugboro 10.Nyefee 676 µl nke ngwakọta na kọlụm mmekọ na centrifuge na 1.88 x 104 g maka 60 sekọnd.A na-atụfukwa drains ndị na-esote.(2) Oge mbụ nke ịsa ahụ.Tinye 500 µl nke ngwọta ịsacha A na kọlụm mmekọ, centrifuge na 1.88 x 104 g maka 40 s, ma tụfuo ihe ngwọta emefuru.A na-emegharị usoro ịsa ahụ ugboro abụọ.(3) ogbo nke abụọ nke ịsa ahụ.Tinye 500 µl nke ngwọta ịsacha W na kọlụm mmekọ, centrifuge na 1.88 × 104 g maka 15 s wee tụfuo ngwọta emefu.A na-emegharị usoro ịsa ahụ ugboro abụọ.(4) Elu.Tinye 200 µl nke eluate na kọlụm mmekọ na centrifuge na 1.88 x 104 g maka 2 min.(5) RT-PCR: A na-agbanye eluate n'ime 20 μl nke ngwọta primer na PCR tube, mgbe ahụ, a na-etinye tube ahụ n'ime ngwa nyocha PCR nke oge (SLAN-96P) iji mee usoro RT-PCR.Usoro nchọpụta niile na-ewe ihe dịka nkeji 140 (nkeji 20 maka ịdị ọcha RNA yana nkeji 120 maka nchọpụta PCR).
526 µl nke ngwọta bead, 1000 µl nke ngwọta ịsacha A, 1000 µl nke ngwọta W, 200 µl nke eluate na 20 µl nke ngwọta primer bụ nke agbakwunyere na mbụ ma chekwaa na ụlọ M, W1, W2, E na PCR nchọpụta ụlọ.Mgbakọ Platform.Mgbe ahụ, 150 µl nke ihe nlele ahụ bụ ọkpọkọ n'ime ụlọ M wee tinye usoro FAST-POCT n'ime ngwa nnwale egosiri na S9 mgbakwunye.Mgbe ihe dị ka nkeji iri asatọ na abụọ gachara, rịzọlt a dị.
Ọ gwụla ma akọwapụtaghị ya, a na-egosipụta nsonaazụ ule niile ka ọ pụtara ± SD ka opekempe nke isii gachara site na iji naanị FAST-POCT ikpo okwu yana ihe nlele nnwere onwe nke ndu.Enweghị data ewepụrụ na nyocha ahụ.Nnwale ndị a abụghị enweghị usoro.Ndị nchọpụta ahụ kpuru ìsì na-arụ ọrụ otu n'oge nnwale ahụ.
Maka ozi ndị ọzọ gbasara imepụta ọmụmụ ihe, lee akụkọ nyocha nke okike ejikọtara na edemede a.
Data na-akwado nsonaazụ ọmụmụ a dị na Ozi Mgbakwụnye.Edemede a na-enye data izizi.
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