Ukwakha ijika lokulinganisa i-methylene blue: amasampula angenadayi, ibanga, nokuhambisana nomugqa oqondile

Abahlaziyi ababili babelana ngebhodlela elilodwa lesixazululo esiyisisekelo se-methylene blue, futhi ngamunye wakha ijika lokulinganisa. Owokuqala uthola umugqa oqondile odlula lapho zombili izilinganiso zinguziro, bese ekala amasampula angaziwa ngokuzethemba. Owesibili uthola umugqa ogoba phezulu, indawo yokunqamula i-eksisi engasondeli kuziro, nemiphumela yamasampula eshintshashintsha lapho ehlanjululwa. Udayi uyafana, ithuluzi liyafana, nobude begagasi buyafana. Umehluko awukho ekhonweni. Usekuhleleni: lokho okufakwe kusampula engenadayi, amazinga okugxila akhethiwe, nokuthi ibanga elihloliwe lihlale yini endaweni lapho imodeli eyodwa elula isasebenza khona.

Ijika lokulinganisa liyisivumelwano phakathi kwempendulo yethuluzi nokugxila, esisebenza kuphela ngaphansi kwezimo elakhiwe kuzo. Ku-methylene blue, leso sivumelwano sinendawo ebuthakathaka eyaziwayo. Ama-molecule kadayi ahlangana wodwa emanzini, ngakho uhlobo olumunca ukukhanya lushintsha ngokugxila. Ijika elakhiwe linqamule kulolo shintsho ngeke liqonde, futhi akukho zibalo zokuhlaziya ezingakulungisa lokho. Lesi sihloko sibonisa indlela yokuhlela ijika ukuze umugqa ube nencazelo: khetha isampula engenadayi efanele, gcina ibanga endaweni okubusa kuyo ama-monomer, phinda izilinganiso ngendlela efanele, futhi ubheke ukugoba njengolwazi kunokuba kube umsindo wokulinganisa.

Qala ngesampula engenadayi, hhayi ngamasampula ayizilinganiso

Isampula engenadayi ichaza uziro, futhi ku-methylene blue kufanele iqukathe konke ngaphandle kukadayi. Izinga lamanzi elifanayo, isixazululo esigcina i-pH kanye ne-pH efanayo, usawoti ofanayo, ingxenye efanayo yesincibilikisi esengeziwe, nezinga lokushisa elifanayo. Lungisa amasampula okulinganisa kuleyo matrix efanayo. Isizathu sisekhemistrini, hhayi emigomeni yokuphatha. Usawoti nesincibilikisi kushintsha ibhalansi phakathi kwe-monomer, i-dimer, namaqoqo amakhulu, okushintsha i-spectrum ebudeni begagasi obusetshenziswa ekulinganiseni. Isampula engenadayi enamanzi kuphela ayikwazi ukulungisa ushintsho ezinhlotsheni zamakhemikhali olubangelwa yi-matrix. Ukuhlaziywa okuhlanganisa ama-spectrum e-methylene blue emanzini, okutholakala erekhodini lakho elivulekele ukufundwa, kwashintsha i-sodium chloride kusukela ku-0 kuya ku-0.15 M ngoba amandla ama-ion ashintsha ibhalansi, futhi ukuhlangana kwama-molecule kwanda ngokwanda kukasawoti.

Umphumela owodwa osebenzayo: ungalokothi wakhe ijika lokulinganisa emanzini akhishwe ama-ion bese ukala amasampula esixazululweni se-phosphate esigcina i-pH noma oketshezini oluhlungiwe lwamanzi angcolile. Uma ingekho i-matrix engenayo into ehlaziywayo, ukwengeza inani elaziwayo laleyo nto kusampula uqobo kungcono kunejika langaphandle elingahambisani nayo. Iziqondiso ezihlobene nokugcina ukuhlolwa kwezixazululo zamanzi kuthembekile zitholakala engxoxweni ye-matrix nokuphazamiseka, kanti imigomo evamile yokuhlolwa kokulinganisa inani likadayi ihlanganiswe embonweni jikelele wokuhlela ukuhlolwa.

Khetha ibanga lapho kubusa uhlobo olulodwa

Ukulinganisa inani ngebhendi yobude begagasi obude eseduze no-664 nm kuthatha ngokuthi iningi likadayi lingama-monomer. Emanzini angenazo izengezo, lokhu kusebenza kuphela uma izixazululo zihlala zihlanjululwe kakhulu, cishe endaweni yamazinga aphansi e-micromolar. Imithambeka eshicilelwe etholwe ngokulinganisa ibonisa umngcele: amajika okulinganisa agcinwe ngaphansi cishe kuka-7 micromolar kumaseli angu-1 cm anikeza umthambeka oseduze no-82,000 M-1 cm-1, kanti ijika elinwetshwe lafika cishe ku-37 micromolar lehlela cishe ku-67,100 ngoba amasampula ayizilinganiso aphezulu asevele equkethe inani elibonakalayo lama-dimer. Ikhoyefishiyenti ngayinye ecashunwe lapha ihambisana nezimo zayo enkombeni ye-spectrum ye-methylene blue, okuyindawo efanele yokuqhathanisa izinombolo; lesi sihloko sizisebenzisa kuphela ukunquma ibanga.

Imithetho yokuhlela ilandela ngokuqondile:

1. Gcina isampula eyisilinganiso ephezulu ihlanjululwe ngokwanele ukuze ukuma kwe-spectrum kungashintshi. Ngesikhathi uthuthukisa indlela, hlola lonke ibanga lokukhanya okubonakalayo (cishe u-550 kuya ku-750 nm) ezingeni ngalinye kunokuba ufunde ubude begagasi obubodwa. Uma ama-spectrum abekwe esikalini esifanayo eshintsha ukuma ngokugxila, ikhemistri iyashintsha futhi ibanga libanzi kakhulu.
2. Landelela isilinganiso se-absorbance eduze no-610 nm uma iqhathaniswa ne-absorbance eduze no-665 nm kuwo wonke amazinga. Isilinganiso esikhuphuka ngokugxila sisho ukuthi iqhubu le-dimer liyakhula futhi imodeli ye-monomer ayisasebenzi. Le ndlela yokuthola imbangela ichazwe kabanzi esiqondisweni semiphumela yokuhlangana kwama-molecule nokugxila.
3. Sebenzisa iseli elingu-1 cm emsebenzini wamazinga aphansi e-micromolar. Ngomthambeka oseduze no-82,000, u-5 micromolar unikeza i-absorbance eseduze no-0.41, kanti u-10 micromolar unikeza cishe u-0.82; kokubili kulula ukukala ngamathuluzi amaningi.
4. Ezixazululweni eziyisisekelo ezigxilile, finyeza indlela yokukhanya kunokuba wamukele i-absorbance engaphezu kakhulu kuka-1, kodwa khumbula ukuthi indlela emfushane ilungisa ukulinganiswa kokukhanya, hhayi ikhemistri. Ukuhlangana kwama-molecule kuncike ekugxileni, hhayi ebudeni bendlela yokukhanya, ngakho isixazululo esiyisisekelo esigxilile sisadinga indlela yaso yokuhlaziywa ebheka izinhlobo zamakhemikhali kunokuba kusetshenziswe ijika lama-monomer ahlanjululwe kakhulu.

Ukufaniswa kwemodeli nayo yonke idatha okusekela okuningi kwalokhu kwahlanganisa ama-spectrum angu-224 kusukela ku-1.1 micromolar kuya ku-3.4 millimolar, futhi kwathola ukuthi uphawu lwe-tetramer lwalusengabonakala ngaphansi kuka-34 micromolar. Lokho okutholakele kunikeza isixwayiso: ibanga elihambisana nomugqa oqondile lincane kunalokho umuntu angakulindela, ngakho liqinisekise kunokuba ulithathe njengeliqinisekile.

Amazinga, izilinganiso eziphindaphindiwe, nokulandelana

Ijika elinamaphuzu amathathu alifakazeli lutho. Iziqondiso zokuqinisekisa indlela ku-ICH Q2(R2) zidinga okungenani amazinga amahlanu okugxila ukuze kuhlolwe ukuhambisana nomugqa oqondile, kanti amazinga ayisithupha kuya kwayisishiyagalombili angeyena uziro anikeza ijika lokusebenza elisekelwe kangcono, ikakhulukazi ngasezingeni eliphezulu lapho ukuhlangana kwama-molecule kuqala khona. Hlela izikhala phakathi kwamazinga ukuze zimboze ibanga lamasampula elihlosiwe, zibe mincane lapho ukugoba kulindeleke khona kakhulu, kunokuba usebenzise uchungechunge olukhula ngesilinganiso esingaguquki olukhethwe ngoba lulula.

Kala izinga ngalinye kabili noma kathathu, ngamasampula alungiswe ngokuzimela kunokuba ufunde kathathu kusuka eflaskini elilodwa. Izilinganiso eziphindaphindiwe ezilungiswe ngokuzimela ziveza amaphutha okulungisa; ukufunda ngokuphindaphindiwe eflaskini elilodwa kuveza kuphela umsindo wethuluzi. Ucwaningo oluhlanganisa yonke idatha lwango-2020 lwakala isixazululo ngasinye kathathu, okuyindlela efanele yokuqala nanamuhla. Hlela ukufundwa kwamasampula ngokungahleliwe ukuze ukushintsha kancane kwempendulo, ukunamathela engilazini, noma ukushintsha kwezinga lokushisa kungabonakali njengokugoba kwejika. Ucwaningo lwama-cuvette e-quartz lwathola ukuthi cishe amaphesenti angu-3 kadayi anamathela ebusweni be-cuvette esuka esixazululweni esingu-34 micromolar phakathi nemizuzu engu-10, ngakho gcina isikhathi sokuthintana, i-cuvette, ukuhlanza, nokulandelana kufana kuwo wonke umjikelezo wokulinganisa.

Isibonelo esibaliwe sokuchaza indlela (akuyona idatha elinganisiwe)

Izinombolo ezingezansi ziqanjwe ukuze kuboniswe izibalo. Azizona izilinganiso zaselebhu futhi akufanele zicashunwe njengedatha ye-methylene blue. Okubalulekile yindlela yokuhlola ehambisana nazo.

Ake sithi kunamasampula ayizilinganiso ayisithupha esixazululweni esifanayo esigcina i-pH, indlela yokukhanya ingu-1 cm, futhi afundwa ku-665 nm kusetshenziswa isampula engenadayi ene-matrix efanayo njengereferensi:

Ukufanisa umugqa oqondile ne-absorbance emaphakathi uma iqhathaniswa nokugxila kunikeza umthambeka oseduze no-0.0805 wamayunithi e-absorbance nge-micromolar, nendawo yokunqamula i-eksisi eqhele kuziro ngamayunithi ambalwa ezinkulungwane ze-absorbance. Ukuhlukanisa umthambeka ngobude bendlela yokukhanya kuwuguqula ube yikhoyefishiyenti yokumunca ebonakalayo: u-0.0805 nge-micromolar ulingana cishe no-80,500 M-1 cm-1, okuhambisana nebanga elibuswa ama-monomer emanzini. Ukuhlola okubalulekile akuwona u-R oyisikwele, ozobukeka umuhle kakhulu noma kunjalo. Okubalulekile yindawo yokunqamula i-eksisi eseduze noziro, isilinganiso se-A610/A665 esingashintshi kusukela ku-S2 kuya ku-S5, nomehluko phakathi kwamanani alinganisiwe nabikezelwe osakazeke ngaphandle kwephethini. Ukukhuphuka okuncane kwesilinganiso ku-S6 kuwuphawu olufanele lokususa lelo zinga noma ukumaka u-7 micromolar njengomkhawulo ophezulu wesikhashana wale nhlanganisela ye-matrix nethuluzi.

Isampula engaziwa efundwa ku-0.245 ngaphansi kwezimo ezifanayo iguqulwa ngokuqondile: u-0.245 uhlukaniswe ngo-0.0805 nge-micromolar unikeza cishe u-3.0 micromolar, ngaphambi kokusebenzisa noma iyiphi ifektha yokuhlanjululwa kwesampula. Bika umphumela kanye nezimo zawo: ubude begagasi, indlela yokukhanya, i-matrix yesampula engenadayi, ibanga lokulinganisa, nosuku.

Ishidi lokusebenza lokulinganisa eligcwalisekayo

Kopisha leli thempulethi kurekhodi laselebhu nge-matrix ngayinye entsha. I-matrix eyodwa, ishidi elilodwa, ijika elilodwa.

Umugqa wohlobo lwe-reagent ubalulekile. I-methylene blue ethengiswayo ingaba usawoti ongenamanzi noma i-hydrate njenge-trihydrate, futhi isisindo ngemola esingalungile senza wonke amazinga okugxila ahlosiwe abe nephutha elingabonakali. Ukuhlolwa kobumsulwa nemininingwane ebekiwe yezinto zokuqala kuchazwe esiqondisweni sekhwalithi nezinga.

Qinisekisa ngesampula eyisilinganiso yokuhlola elungiswe ngokuzimela

Ijika lokulinganisa akufanele lizihlulele umsebenzi walo. Ngemva kokufanisa umugqa nedatha, lungisa isampula eyisilinganiso eyodwa yokuhlola ngesixazululo esiyisisekelo esinesisindo esikalwe ngokwehlukana, ekugxileni okuseduze nendawo ephakathi yebanga elamukelwe, bese uyifunda njengesampula engaziwa. Ukuvumelana okungaphakathi kwamaphesenti ambalwa kuqinisekisa uchungechunge lokulungisa; ukungavumelani kukhomba isixazululo esiyisisekelo noma ukuhlanjululwa kunokuba kukhombe ithuluzi. Phinda isampula eyisilinganiso yokuhlola ngeqoqo ngalinye lamasampula. Uma ishintsha kancane kodwa ijika lingashintshile, sola izixazululo ezisetshenziswayo noma i-cuvette ngaphambi kokusola amasampula. Lo mkhuba owodwa uthola amaphutha amaningi angabonakali: isisindo ngemola se-hydrate esisetshenziswe ebhodleleni lento engenamanzi, i-micropipette engasalinganisi kahle, noma isixazululo esigcina i-pH esilungiswe ngamandla ama-ion angalungile. Bhala izinga lokubuyiselwa kwenani elaziwayo lesampula yokuhlola eshidini lokusebenza eduze komthambeka, ukuze ofunda kamuva abone ukuthi umugqa uhloliwe, awudwetshiwe nje.

Ukusebenzisa ukungahambisani nomugqa oqondile ukuthola imbangela

Uma ijika ligoba, ukuma kokuphambuka kukhomba imbangela. Ukugoba okubheke phansi ku-absorbance ephezulu, kube ukuma kwe-spectrum kungashintshile, kukhomba ithuluzi; ngokuvamile ukukhanya okunhlanhlathayo kwenza amanani e-absorbance aphezulu abonakale ephansi. Ukugoba okuhambisana nokukhuphuka kweqhubu ku-610 nm kukhomba ikhemistri: ama-dimer namaqoqo amakhulu amunca ukukhanya ngendlela ehlukile kuma-monomer. Isampula engenadayi engakwazi ukufundeka njengoziro, noma amasampula ayizilinganiso afundeka ngaphansi ngokuhamba kwesikhathi, kukhomba isitsha: ukunamathela engilazini noma kuma-cuvette, ikakhulukazi uma izikhathi zokuthintana zihluke phakathi nomjikelezo wokulinganisa. Futhi umugqa ohlanzekile odlula emaphuzwini kodwa usalokhu unikeza imiphumela yamasampula engalungile ukhomba i-matrix: amasampula ahlukile kumasampula okulinganisa, ngakho yakha kabusha ijika ku-matrix yesampula noma ushintshele ekwengezeni inani elaziwayo lento ehlaziywayo kusampula. Kuzo zonke izimo, dweba umehluko phakathi kwamanani alinganisiwe nabikezelwe uma uqhathaniswa nokugxila. Ukusakazeka okungahleliwe kusekela imodeli yomugqa oqondile; ukuma okufana nokumamatheka noma ukuhwaqabala kuyayiphikisa ngendlela ethembeke kakhulu kunanoma yimuphi umkhawulo ka-R oyisikwele, okuyiphuzu elisekelwa ngokusobala yiziqondiso zokuqinisekisa indlela.

I-methylene blue inikeza imiphumela ethembekile uma kulandelwa lokhu kuhleleka. Ibhendi yayo ye-monomer iqinile, ikhemistri yayo iqondwa kahle, futhi izindlela eziphambuka ngazo zibonakala ku-spectrum ngaphambi kokuba zonakalise izinombolo. Yakha ijika ebangeni elincane, sebenzisa isampula engenadayi ngendlela efanele, phinda izilinganiso ngamasampula alungiswe ngokuzimela, futhi uvumele ukuma kwama-spectrum kunqume ibanga. Umugqa owumphumela mfushane, kodwa wonke amaphuzu awo ayiqiniso.

Izibalo zokuchaza indlela kulesi sihloko zisebenzisa izinombolo eziqanjiwe ukuze kuboniswe indlela. Amakhoyefishiyenti ereferensi alinganisiwe kanye nazo zonke izimo zawo atholakala enkombeni ye-spectrum exhunywe lapha, kanti ucwaningo lokuhlangana kwama-molecule oluyisisekelo salokhu lubhaliswe ku-PMID 33251415.