I-spectrum Yokumuncwa Kwe-Methylene Blue ne-Coefficient Yokuncipha Kokukhanya Ngokwemol

I-spectrum ye-methylene blue ibonakala ilula uze uzame ukuphinde usebenzise inani elithathwe kuyo. Enye ilabhorethri ibika i-coefficient yokuncipha kokukhanya ngokwemol eseduze kuka-82,000. Enye ibika u-67,000. Eyesithathu ibika inani elingaphezu kuka-100,000. Womathathu angaba izincazelo ezinembile zezilinganiso zawo, kodwa alikho eliyisilinganiso esingaguquki salo dayi kuzo zonke izimo.

Ukudideka kubangelwa ukuhlanganisa imiqondo emibili ehlukene. Isilinganiso sokumuncwa kokukhanya silinganiswa esixazululweni esithile esisesitsheni sokulinganisa esithile. Ikhono lokumunca ngokwemol, elivame ukubhalwa ngokuthi i-epsilon, liyipharamitha kumodeli yaleso sixazululo. Lapho i-methylene blue ishintsha indlela ama-molecule ayo ahlangana ngayo, imodeli iyashintsha, ngakho nepharamitha iyashintsha. Umkhuba owusizo ukuphatha i-coefficient ngayinye njengenani elinezimo ezichaziwe: ubude begagasi, isincibilikisi, i-pH noma i-buffer, izinga lokushisa, uhla lokugxila nobude bendlela yokukhanya kufanele kuhambisane nenani.

Ibhendi ye-monomer emanzini lapho ingagxilile kakhulu

Esixazululweni samanzi esingagxilile kakhulu, ibhendi evelele ye-cation ye-methylene blue itholakala eduze kuka-664 kuya ku-665 nm, futhi kuvame ukubikwa ukuthi iphakathi kuka-664 kuya ku-668 nm kuye ngezinsimbi nezindlela zokulinganisa. Leyo bhendi iyona efanele ukukala inani uma isixazululo singagxilile ngokwanele ukuba ingxenye enkulu yodayi ibe sesimweni se-monomer.

Amanani amathathu asezincwadini zesayensi abonisa ukuthi kungani izimo zibaluleke ngaphezu kwenani eliqokonyiswayo. Abacwaningi bobumba abagcina amasampula amanzi engaphansi cishe kuka-7 micromolar ezitsheni zokulinganisa ezingu-1 cm babika i-epsilon ecishe ibe ngu-82,000 M-1 cm-1 ku-665 nm. Leli nani lichazwe kahle ngokungavamile njengomthambeka wokulinganisa otholwe ngokuhlolwa kwamasampula amanzi angagxilile kakhulu. Kamuva, ukulinganisa okuyisisekelo emanzini akhishwe ama-ion, kusetshenziswa u-2.34 kuya ku-37.52 micromolar nobude bendlela yokukhanya obungu-1 cm, kwaveza cishe u-67,100 M-1 cm-1 ku-665 nm wodayi ophelele. Umphumela ophansi ulindelekile, awuphikisani nowokuqala, ngoba ingxenye ephezulu yalolo hlu lokulinganisa isivele iqukethe inani elibonakalayo le-dimer. Inani lesithathu, cishe u-73,460 M-1 cm-1 ku-664 nm, livela emsebenzini owenziwa ku-pH 6.86 naku-293 K, owahlukanisa i-spectrum ebonwayo yaba izingxenye ze-monomer ne-dimer, njengoba kufingqiwe ekubuyekezweni kwakamuva okuhlaziya ngokucophelela i-photophysics ye-methylene blue. Lelo nani liyingxenye ye-monomer ehlukaniswe ngokuhlaziywa, hhayi umthambeka oqondile wesilinganiso sokumuncwa kokukhanya uma siqhathaniswa nenani likadayi ophelele.

Iphuzu elisebenzisekayo: khetha i-coefficient enezimo ezifana nezamasampula akho, noma uzenzele ukulinganisa. Ungathathi u-82,000 uwusebenzise ekuhlolweni kwe-phosphate buffer engu-30 micromolar bese ucabanga ukuthi ubudlelwano bomugqa oqondile busasebenza.

Iqhubu elisebudeni begagasi obufushane liyingxube

Njengoba ukugxila kukhuphuka, iqhubu eliseduze kuka-600 kuya ku-615 nm liyakhula uma liqhathaniswa nesiqongo esiku-664 nm. Ucwaningo lwamanje luvame ukubeka umnikelo we-dimer yohlobo lwe-H eduze kuka-605 kuya ku-610 nm. Ukuhlaziywa okuhlanganisiwe kwe-spectrum ye-methylene blue emanzini kwehlukanisa isiqongo se-dimer esiku-607 nm nesiqongo seqoqo elikhulu esiku-600 nm, kube nezingxenye ze-resonance ye-monomer ku-664 naku-650 nm. Umbhalo wayo ophelele uyatholakala erekhodini elifinyeleleka mahhala le-PMID 33251415.

Lelo qhubu akufanele lichazwe njengokumuncwa kokukhanya kwe-dimer kuphela. Isakhiwo se-monomer esihlanganisa ukudlidliza nokushintsha kwezimo zama-electron naso sinomnikelo kuleyo ndawo, ngakho ukuphakama okulinganisiwe kuwumphumela wezingxenye ezigqagqene. Okunye ukufaniswa kwemodeli nayo yonke idatha kwathola ukuthi imodeli ye-monomer ne-dimer ayisebenzanga kulo lonke uhla olusuka ku-1.1 micromolar kuya ku-3.4 millimolar, kanti ukwengeza i-tetramer kwanikeza ukufaniswa okungcono kakhulu, nesignali ye-tetramer isivele ibonakala ngaphansi kuka-34 micromolar. Amandla ezingxenye atholwe ngokufanisa ayemakhulu: cishe u-107,800 M-1 cm-1 ku-664 nm wengxenye eyinhloko ye-monomer, cishe u-101,400 we-dimer ku-607 nm, kanye no-138,500 we-tetramer ku-600 nm, emanzini akhishwe ama-ion angenayo i-buffer, ane-sodium chloride esuka ku-0 kuya ku-0.15 M namazinga okushisa asuka ku-282 kuya ku-333 K. Lezi yizingxenye ezihlukaniswe ngemodeli, ngakho akufanele zisetshenziswe esikhundleni somthambeka we-Beer Lambert otholwe ngokuhlolwa.

Amandla okwakheka kwama-dimer alinganisiwe ngokuqondile. Ku-pH 6.86 naku-293 K, olunye ucwaningo lwathola isilinganiso sokulingana sokwakheka kwama-dimer esiseduze kuka-3,900 M-1. Lobo bukhulu buchaza ukuthi kungani isixazululo sokusebenza esingu-5 micromolar silandela cishe ubudlelwano bomugqa oqondile, kanti isixazululo esiyisisekelo esingu-50 micromolar asenzi kanjalo. Ukuhlola okuwusizo lapho kuthuthukiswa indlela yokulinganisa yisilinganiso phakathi kokumuncwa kokukhanya eduze kuka-610 nm nokumuncwa kokukhanya eduze kuka-665 nm. Uma leso silinganiso sikhuphuka njengoba ukugxila kukhuphuka, i-epsilon ku-664 nm ayisachazi uhlobo olulodwa olungaguquki.

Ohlwini lokugxila olubuswa yizinhlobo ezimbili ezishintshana zibe olunye uhlobo, ama-spectrum angaba nephuzu elifanayo le-isosbestic, elitholakala eduze kuka-625 nm ekuhlolweni kochungechunge lwezixazululo zamanzi. Iphuzu elicacile elinganyakazi lihambisana nokuba khona kwezinhlobo ezimbili ezivelele. Uma lelo phuzu lisuka noma linyamalala njengoba ukugxila kukhuphuka, kuphathe njengesixwayiso sokuthi sekuvele amaqoqo amakhulu noma ukulingana sekuyashintsha, hhayi njengephutha lensimbi yokulinganisa.

Isincibilikisi, i-pH nezinga lokushisa kushintsha i-spectrum

Amanzi akhuthaza ukuhlangana kwama-molecule. Ukwengeza i-dimethyl sulfoxide kunciphisa ukwakheka kwama-dimer, kususa iziqongo zokumuncwa kokukhanya nezokukhanya kwe-fluorescence ziye ebudeni begagasi obude, futhi kwandisa isivuno se-quantum se-fluorescence cishe ngokuphindwe kane, ngokusho kocwaningo lwesincibilikisi se-methylene blue emanzini nasezingxubeni ze-DMSO, olufakwe ohlwini njengo-PMID 30933781. Lowo msebenzi uphatha ama-spectrum asemanzini njengokulingana phakathi kwe-monomer ne-dimer, lapho ukumuncwa kokukhanya kwe-monomer kuseduze kuka-650 kuya ku-670 nm nokwe-dimer kuseduze kuka-580 kuya ku-615 nm, futhi uthola izilinganiso zokulingana zokwakheka kwama-dimer kuma-spectrum aku-293.15 K.

Ama-alcohol ashintsha kokubili indawo yesiqongo namandla abonakalayo, kodwa awaqedi ukwakheka kwamaqoqo. Kokunye ukuqhathanisa okuyisisekelo okwenziwa ku-31.3 micromolar nasezingeni lokushisa lasekamelweni, okwabikwa ocwaningweni lwesincibilikisi se-methylene blue, isiqongo esisebudeni begagasi obude sasiku-665 nm emanzini kodwa siku-655 nm ku-methanol, kube namanani abonakalayo esixazululo sonke aseduze kuka-37,100 emanzini no-60,800 ku-methanol. Okunye ukuqinisekiswa okwenziwa ku-ethanol engenamanzi ku-298 K kwaveza isiqongo esiku-655 nm, ne-epsilon eseduze kuka-68,000.

Izinguquko ezivamile ze-pH zishintsha i-spectrum kancane kunalokho okucatshangwa yizinqubo eziningi. Ukubuyekezwa okuhlaziya ngokucophelela okucashunwe ngenhla kubika ukuthi kusukela cishe ku-pH 2 kuya ku-pH 12, isiqongo sahlala eduze kuka-665 nm, amandla eshintsha kancane kuphela kuze kube ku-pH 11. Uhlobo lwe-buffer namandla ama-ion kungashintsha imiphumela ngendlela efanayo ne-pH ebekiwe, ngakho bhala i-buffer nosawoti abasetshenzisiwe, hhayi kuphela lokho okufundwe yimitha ye-pH. I-asidi enamandla noma i-alkali enamandla kuyisimo esihlukile ngoba udayi ungashintsha ngokwekhemikhali kunokuba ama-molecule awo ahlangane ngendlela engahlehliswa. Izinga lokushisa nosawoti nakho kushintsha ukulingana, yingakho ucwaningo lwamanzi lwango-2020 lwakushintsha kokubili ngamabomu.

Ukulinganisa ukuze inani lihlale lisebenza

Umsebenzi wokulinganisa amanani usekelwe ebudlelwaneni be-Beer Lambert: isilinganiso sokumuncwa kokukhanya silingana ne-epsilon ephindwe ngokugxila nangobude bendlela yokukhanya, kanye nobufakazi bokuthi uhla lobudlelwano bomugqa oqondile luyasebenza ngempela ngaphansi kwezimo zakho.

Sebenzisa isitsha sokulinganisa esingu-1 cm emsebenzini wokugxila okuphansi kwe-micromolar. Nge-epsilon eseduze kuka-82,000, u-5 micromolar unikeza isilinganiso sokumuncwa kokukhanya esiseduze kuka-0.41, kanti u-10 micromolar unikeza cishe u-0.82. Ukuze ukulinganisa kugxile ku-monomer emanzini angenazo izithasiselo, ukuhlala eduze noma ngaphansi cishe kuka-5 kuya ku-7 micromolar kuphephe kakhulu kunokulinganisa kuze kufike emashumini amaningi e-micromolar. Ezixazululweni eziyisisekelo ezigxilile, finyeza indlela yokukhanya esikhundleni sokwamukela isilinganiso sokumuncwa kokukhanya esingaphezulu kakhulu kuka-1.

Setha isilinganiso esingenadayi usebenzisa ingxube eyisisekelo efana ncamashi: izinga elifanayo lamanzi, ingxenye efanayo yesincibilikisi esengeziwe, i-buffer efanayo nosawoti abafanayo, kodwa ngaphandle kukadayi. Lungiselela izixazululo zokulinganisa kuleyo ngxube efanayo nasezingeni lokushisa elifanayo. Hlola lonke uhla lokukhanya okubonakalayo ngesikhathi sokuthuthukisa indlela, okungcono cishe u-550 kuya ku-750 nm, kunokufunda ubude begagasi obubodwa kuphela. Landelela isimo se-spectrum, hhayi ukuphakama kuphela. Uma ukuhlanjululwa okulandelanayo kushintsha isimo se-spectrum esilinganiselwe ngendlela efanayo, uhlelo luncike ekugxileni ngokwekhemikhali. Isiqongo esizinzile nesilinganiso esizinzile phakathi kuka-610 no-665 nm kuwubufakazi obuqinile bohla olwamukelekayo kunenani eliphezulu lika-R ophindwe ngesikwele kuphela.

Bhala phansi isimo sekhemikhali serejenti. Impahla ethengiswayo ingaba yi-methylthioninium chloride engenamanzi noma i-hydrate efana ne-trihydrate, ngakho isisindo ngokwemol esisetshenziselwa ukubala izixazululo eziyisisekelo kufanele sihambisane nokuqukethwe yibhodlela langempela. Lesi sinyathelo sisodwa sichaza ingxenye yokungavumelani kokugxila okubekiwe ngokwemol phakathi kwamalabhorethri. Ukuqaphela okufanayo kuyasebenza lapho imiphumela isetshenziswa ekubaleni ukuhlolwa kwamanani, lapho ama-spectrum esekela ukuhlolwa kokuzinza kwengxube, nalapho amaphrofayili okungcola edinga ukubuyekezwa kobumsulwa nemininingwane ebekiwe.

Isifinyezo esinezimo ezichaziwe

Sebenzisa leli thebula njengesifanekiso sokuqala serekhodi laselabhorethri, hhayi njengomthombo wamanani angashintshaniswa.

Uhlobo nobude begagasi	Izimo	I-epsilon noma ukuziphatha okubikiwe
I-monomer, 665 nm	Amanzi, ngaphansi cishe kuka-7 micromolar, 1 cm, i-pH ayibikwanga	Cishe u-82,000 M-1 cm-1, umthambeka wodayi ophelele otholwe ngokuhlolwa
Ingxenye ye-monomer, 664 nm	Amanzi, pH 6.86, 293 K, ihlukaniswe ngokuhlaziywa ku-dimer	Cishe u-73,460 M-1 cm-1, ingxenye ye-monomer kuphela
Udayi ophelele, 665 nm	Amanzi akhishwe ama-ion, 2.34 kuya ku-37.52 micromolar, 1 cm	Cishe u-67,100 M-1 cm-1, umthambeka wokulinganisa ohlwini olunokwakheka kwamaqoqo engxenyeni yalo
I-resonance ye-monomer, 664 nm	Amanzi akhishwe ama-ion angenayo i-buffer, uchungechunge olusuka ku-1.1 micromolar kuya ku-3.4 millimolar, NaCl 0 kuya ku-0.15 M, 282 kuya ku-333 K	Cishe u-107,800 M-1 cm-1, ingxenye ehlukaniswe ngemodeli
I-dimer, 607 nm	Uchungechunge olufanayo nolungenhla	Cishe u-101,400 M-1 cm-1, ingxenye ehlukaniswe ngemodeli
I-tetramer, 600 nm	Uchungechunge olufanayo nolungenhla	Cishe u-138,500 M-1 cm-1, ingxenye ehlukaniswe ngemodeli
Udayi ophelele, 665 nm emanzini uma kuqhathaniswa no-655 nm ku-methanol	31.3 micromolar, izinga lokushisa lasekamelweni	Cishe u-37,100 emanzini, cishe u-60,800 ku-methanol, amanani abonakalayo kulokho kugxila
Ukulingana phakathi kwe-monomer ne-dimer	Izingxube zamanzi ne-DMSO, 293.15 K	I-DMSO inciphisa i-dimer, iziqongo ziya ebudeni begagasi obude, isivuno sokukhishwa kokukhanya siyakhuphuka

Ngakho-ke ubude begagasi lokumuncwa kwe-methylene blue buyimpendulo emfushane edinga incazelo ende ehambisana nayo. Bika isiqongo osilinganisile, esixazululweni osilinganisile, nasekugxileni osilinganisile. Lowo mkhuba uvimbela inani elilodwa lesixazululo samanzi esingagxilile kakhulu eliseduze kuka-664 nm ukuba liphenduke isilinganiso samanga esingaguquki kuzo zonke izimo.