Ukwakha igophe lekhalibhreshini ye-methylene blue: iisampulu ezingenadayi, uluhlu, nokulandela umgca othe ngqo

Abahlalutyi ababini basebenzisa ibhotile enye yesisombululo sesitokhwe se-methylene blue, aze ngamnye akhe igophe lekhalibhreshini. Owokuqala ufumana umgca othe ngqo odlula kwindawo engu-zero aze alinganise iisampulu ezinezixinano ezingaziwayo ngokuzithemba. Owesibini ufumana umgca ogobayo kwisiphelo esiphezulu, indawo yokunqumla i-axis engasondeliyo ku-zero, kunye neziphumo zeesampulu ezitshintshayo xa zixutywa. Idayi iyafana, isixhobo siyafana, nobude bamaza buyafana. Umahluko awukho kwisakhono. Ukuyilo: oko kufakwe kwisampulu engenadayi, izixinano ezikhethiweyo, nokuba uluhlu oluvavanyiweyo luhlale ngaphakathi kummandla apho imodeli enye elula isasebenza khona.

Igophe lekhalibhreshini sisivumelwano phakathi kwempendulo yesixhobo nesixinano, esisebenza kuphela phantsi kweemeko elakhiwe kuzo. Kwi-methylene blue, eso sivumelwano sinobuthathaka obaziwayo. Iimolekyuli zedayi ziyadibana emanzini, ngoko uhlobo olufunxa ukukhanya luyatshintsha ngokwesixinano. Igophe elakhiwe kuluhlu olunqumla olo tshintsho aliyi kuba ngumgca othe ngqo, kwaye akukho zibalo ezinokuyilungisa loo nto. Eli nqaku libonisa indlela yokuyila igophe ukuze umgca ube nentsingiselo: khetha isampulu engenadayi efanelekileyo, gcina uluhlu kummandla olawulwa ziimonomer, yenza iziphindaphindo ngokunyanisekileyo, uze utolike ukugoba njengolwazi kunokuba ukuthathe njengengxolo.

Qala ngesampulu engenadayi, hayi imigangatho

Isampulu engenadayi ichaza u-zero, kwaye kwi-methylene blue kufuneka iqulathe yonke into ngaphandle kwedayi. Umgangatho wamanzi ofanayo, isithinteli se-pH ne-pH efanayo, iityuwa ezifanayo, umlinganiselo ofanayo wesinyibilikisi esongezelelweyo, nobushushu obufanayo. Lungisa izisombululo zekhalibhreshini kuloo matrix ifanayo. Isizathu sisekhemistri, hayi kwimigaqo yolawulo. Ityuwa nesinyibilikisi zitshintsha ulungelelwano phakathi kweemonomer, iidimer, namaqela amakhulu eemolekyuli, nto leyo etshintsha ispektram kubude bamaza bokulinganisa. Isampulu engenadayi enamanzi kuphela ayinakulungisa utshintsho kwiintlobo zeekhemikhali olubangelwa yimatrix. Uhlalutyo oludibeneyo lweespektram ze-methylene blue emanzini, olufumaneka kwirekhodi yalo enokufikelelwa simahla, lwatshintsha i-sodium chloride ukusuka ku-0 ukuya ku-0.15 M kanye kuba amandla eeyoni ehambisa ulungelelwano, yaye ukudibana kweemolekyuli kwanda ngokwanda kwetyuwa.

Esinye isiphumo esisebenzayo: ungaze wenze ikhalibhreshini emanzini asuswe iiyoni uze ulinganise iisampulu kwisithinteli se-pH se-phosphate okanye kulwelo oluhluzwe kumanzi amdaka. Xa kungekho matrix engenanto ehlalutywayo, ukongeza umgangatho kwisampulu ngokwayo kungcono kunegophe langaphandle elingahambelaniyo nayo. Isikhokelo esinxulumene nokugcina iimvavanyo zezinyibilikisi zamanzi zithembekile sikwingxoxo yematrix nokuphazamiseka, kwaye imigaqo ngokubanzi yeemvavanyo zokulinganisa ubungakanani bedayi ichazwe kumbono obanzi woyilo lweemvavanyo.

Khetha uluhlu olulawulwa luhlobo olunye

Ukulinganisa ubungakanani kwibhendi yobude bamaza obude kufutshane no-664 nm kusekelwe ekubeni uninzi lwedayi lukwimo yemonomer. Emanzini angenazo ezinye izongezo, le ngcinga isebenza kuphela xa izisombululo zixutywe kakhulu, malunga nommandla osezantsi wezixinano zemicromolar. Amathambeka afunyenwe ngokulinganisa apapashiweyo abonisa umda: iikhalibhreshini ezigcinwe ngaphantsi malunga no-7 micromolar kwiiseli ze-1 cm zinika ithambeka elikufutshane no-82,000 M-1 cm-1, ngelixa ikhalibhreshini eyandiswe ukuya malunga no-37 micromolar iyehla iye malunga no-67,100 kuba imigangatho ephezulu sele iqulethe isixa esibonakalayo sedimer. Iimeko ze-coefficient nganye ekhankanywe apha zichazwe kwisikhokelo seespektram ze-methylene blue, eyindawo efanelekileyo yokuthelekisa amanani; eli nqaku liwasebenzisa kuphela ukumisela uluhlu.

Imigaqo yoyilo ilandela ngqo:

1. Gcina umgangatho ophezulu uxutywe ngokwaneleyo ukuze ukumila kwespektram kuzinze. Ngexesha lophuhliso, skena lonke uluhlu lokukhanya olubonakalayo (malunga no-550 ukuya ku-750 nm) kwinqanaba ngalinye endaweni yokufunda ubude bamaza obunye. Ukuba iispektram ezibekwe kwisikali esifanayo zitshintsha ukumila ngokwesixinano, ikhemistri iyatshintsha kwaye uluhlu lubanzi kakhulu.
2. Landela umlinganiselo wokufunxa ukukhanya kufutshane no-610 nm xa kuthelekiswa nokufunxa kufutshane no-665 nm kuwo onke amanqanaba. Umlinganiselo onyuka ngokwanda kwesixinano uthetha ukuba igxalaba ledimer liyakhula kwaye imodeli yemonomer ayisasebenzi. Olu phawu lokuxilonga luchazwe ngakumbi kwisikhokelo sokudibana kweemolekyuli nemiphumo yesixinano.
3. Sebenzisa iseli ye-1 cm xa usebenza ngezixinano ezisezantsi zemicromolar. Xa ithambeka likufutshane no-82,000, i-5 micromolar inika umlinganiselo wokufunxa ukukhanya okufutshane no-0.41 kwaye i-10 micromolar inika malunga no-0.82, omabini amanani alungele izixhobo ezininzi.
4. Kwizisombululo zesitokhwe ezixinene kakhulu, nciphisa ubude bendlela yokukhanya endaweni yokwamkela umlinganiselo wokufunxa ukukhanya ongaphezulu kakhulu ko-1, kodwa khumbula ukuba indlela emfutshane ilungisa umlinganiselo wokukhanya, hayi ikhemistri. Ukudibana kweemolekyuli kuxhomekeke kwisixinano, hayi kubude bendlela yokukhanya, ngoko isisombululo sesitokhwe esixinene kakhulu sisafuna indlela yaso ethathela ingqalelo iintlobo zeekhemikhali kunegophe lemonomer kwisinyibilikisi esixutywe kakhulu.

Ukufaniswa kwemodeli kuzo zonke iinkcukacha okuxhasa uninzi lwalo mfanekiso kwakuquka iispektram eziyi-224 ukusuka ku-1.1 micromolar ukuya ku-3.4 millimolar kwaye kwafumanisa ukuba uphawu lwetetramer lwalusele lunokubonwa ngaphantsi ko-34 micromolar. Esi siphumo sibonisa indlela yokujonga lo msebenzi: uluhlu olulandela umgca othe ngqo lumxinwa kunokuba kubonakala xa uqikelela, ngoko luqinisekise kunokuba uluthathe njengoluqinisekileyo.

Amanqanaba, iziphindaphindo, nolandelelwano

Igophe elinamanqaku amathathu alingqini nto. Isikhokelo sokuqinisekisa kwi-ICH Q2(R2) sicela ubuncinane izixinano ezintlanu ukuvavanya ukulandela umgca othe ngqo, kwaye amanqanaba amathandathu ukuya kwasibhozo angengu-zero anika igophe lokusebenza eliqinisekisayo ngakumbi, ingakumbi kufutshane nesiphelo esiphezulu apho ukudibana kweemolekyuli kuqala khona. Cwangcisa amanqanaba ukuze agubungele uluhlu olucetyiweyo lweesampulu, uwasondeze ngakumbi apho ukugoba kunokwenzeka khona, kunokusebenzisa uthotho lwejometri olukhethwe kuba lulula.

Linganisa inqanaba ngalinye kabini okanye kathathu, ulilungise ngokuzimeleyo endaweni yokufunda kathathu kwiflaski enye. Iziphindaphindo ezizimeleyo ziveza impazamo yokulungiselela; ukufunda okuphindaphindiweyo kwiflaski enye kuveza kuphela ingxolo yesixhobo. Uphononongo oludibeneyo lwango-2020 lwalinganisa isisombululo ngasinye kathathu, nto leyo iseyindlela efanelekileyo yokuqalisa. Lungisa ulandelelwano lokufunda ngokungakhethiyo ukuze ukutyibilika kwemilinganiselo, ukubambelela kumphezulu weglasi, okanye ukutshintsha kobushushu kungabonakali ngathi kukugoba. Uphononongo lwekhiyuvethi ye-quartz lwafumanisa ukuba malunga ne-3 pesenti yedayi yayibambelela kumphezulu isuka kwisisombululo se-34 micromolar kwisithuba semizuzu eyi-10, ngoko gcina ixesha lokudibana, ikhiyuvethi, ukucoca, nolandelelwano zingatshintshi kulo lonke uthotho lwemilinganiselo.

Umzekelo oselusetyenzweni wokubonisa indlela (ayizizo iinkcukacha ezilinganisiweyo)

Amanani angezantsi ayiliwe ukubonisa ukubala. Ayiyomilinganiselo yaselebhu kwaye akufuneki acatshulwe njengeenkcukacha ze-methylene blue. Okubalulekileyo yindlela yokuwahlola.

Masithi kukho imigangatho emithandathu kwisithinteli se-pH esihambelanayo, ubude bendlela yokukhanya buyi-1 cm, ifundwa ku-665 nm kuthelekiswa nesampulu engenadayi ehambelanayo:

Ukufanisa umgca othe ngqo nomndilili wokufunxa ukukhanya xa kuthelekiswa nesixinano kunika ithambeka elikufutshane neeyunithi zokufunxa ukukhanya eziyi-0.0805 nge-micromolar kunye nendawo yokunqumla i-axis ekwiiyunithi ezimbalwa zamawaka eyunithi yokufunxa ukukhanya ukusuka ku-zero. Ukwahlula ithambeka ngobude bendlela yokukhanya kuliguqula libe yi-coefficient yokufunxa ukukhanya ebonakalayo: i-0.0805 nge-micromolar ilingana malunga no-80,500 M-1 cm-1, ehambelana noluhlu olulawulwa ziimonomer emanzini. Oko kubalulekileyo ekuhlolweni asingu-R ophindwe ngaye, oya kubonakala emhle kakhulu nokuba kunjalo okanye akunjalo. Yindawo yokunqumla i-axis ekufutshane no-zero, umlinganiselo we-A610/A665 ongatshintshiyo ukusuka ku-S2 ukuya ku-S5, kunye neentsalela ezisasazekileyo ezingenalo utyekelo oluthile. Ukunyuka okuncinci komlinganiselo ku-S6 luphawu oluthembekileyo lokuba ususe elo nqanaba okanye uphawule i-7 micromolar njengomda ophezulu wethutyana wale matrix nesi sixhobo xa zisetyenziswa kunye.

Isampulu enesixinano esingaziwayo efundwe ku-0.245 phantsi kweemeko ezifanayo iguqulwa ngqo: i-0.245 yahlulwe ngo-0.0805 nge-micromolar inika malunga ne-3.0 micromolar, ngaphambi kokuba kusetyenziswe nayiphi na ifektha yokuxutywa kwesampulu. Xela isiphumo kunye neemeko zaso: ubude bamaza, indlela yokukhanya, imatrix yesampulu engenadayi, uluhlu lwekhalibhreshini, nomhla.

Iphepha lomsebenzi wekhalibhreshini elinokuzaliswa

Khuphela le thempleyithi kwirekhodi yaselebhu kwimatrix nganye entsha. Imatrix enye, iphepha elinye, igophe elinye.

Umqolo wemo yereyajenti unesizathu sokuba ubekho. I-methylene blue ethengiswayo inokuba yityuwa engenamanzi okanye ihydrate efana netrihydrate, kwaye ubunzima bemolar obungachanekanga butshintsha zonke izixinano ezichaziweyo kungabonakali. Ukuhlolwa kococeko nokuhambelana neenkcukacha zobugcisa zezinto zokuqala kuchazwe kwisikhokelo somgangatho nenqanaba.

Qinisekisa ngomgangatho wokuhlola ozimeleyo

Igophe lekhalibhreshini akufuneki lizihlolele ngokwalo. Emva kokufanisa, lungisa umgangatho omnye wokuhlola kwisisombululo sesitokhwe esilungiswe ngokulinganisa ubunzima ngokwahlukileyo, ngesixinano esikufutshane embindini woluhlu olwamkelweyo, uze uwufunde ngokungathi isixinano sawo asaziwa. Ukuvumelana ngaphakathi kweepesenti ezimbalwa kuqinisekisa ikhonkco lokulungiselela; ukungavumelani kukhomba kwisisombululo sesitokhwe okanye ekuxutyweni kunakwisixhobo. Phinda usebenzise umgangatho wokuhlola kwibhetshi nganye yeesampulu. Ukuba uyatyibilika ngelixa igophe lingatshintshanga, rhanelwa izisombululo zokusebenza okanye ikhiyuvethi ngaphambi kokurhanela iisampulu. Lo mkhwa mnye ubamba uninzi lweempazamo ezingaqaphelekiyo: ubunzima bemolar behydrate obusetyenziswe kwibhotile engenamanzi, imicropipette engakhalibhreshwanga kakuhle, isithinteli se-pH esilungiswe ngamandla eeyoni angachanekanga. Rekhoda ipesenti yokubuyiselwa komgangatho wokuhlola kwiphepha lomsebenzi ecaleni kwethambeka, ukuze umfundi wamva abone ukuba umgca uvavanyiwe, awuzotywanga nje kuphela.

Ukutolika ukungalandeli umgca othe ngqo njengendlela yokufumanisa unobangela

Xa igophe ligoba, ukumila kwengxaki kuchaza unobangela. Ukugoba ukuya ezantsi xa umlinganiselo wokufunxa ukukhanya uphezulu kodwa ukumila kwespektram kungatshintshanga kukhomba kwisixhobo, amaxesha amaninzi kukukhanya okungafunekiyo okubangela ukuba imilinganiselo ephezulu yokufunxa ukukhanya ifundwe iphantsi. Ukugoba okuhamba nokunyuka kwegxalaba ku-610 nm kukhomba kwikhemistri: iidimer namaqela amakhulu eemolekyuli afunxa ukukhanya ngendlela eyahlukileyo kwiimonomer. Isampulu engenadayi engafikeleli ku-zero, okanye imigangatho enemilinganiselo eyehlayo ngokuhamba kwexesha, ikhomba kwisikhongozeli: ukubambelela kumphezulu weglasi okanye weekhiyuvethi, ingakumbi xa amaxesha okudibana ebengafani phakathi kwemilinganiselo. Kwaye umgca ococekileyo odlula kumanqaku kodwa usanika iziphumo ezingachanekanga zeesampulu ukhomba kwimatrix: iisampulu zahlukile kwizisombululo zekhalibhreshini, ngoko yakha kwakhona igophe kwimatrix yesampulu okanye utshintshele ekongezeni umgangatho. Zoba iintsalela xa zithelekiswa nesixinano kuzo zonke iimeko. Ukusasazeka okungakhethiyo kuxhasa imodeli yomgca othe ngqo; ukumila okufana noncumo okanye umlomo ojongise ezantsi kuyayiphikisa ngokuthembekileyo kunawo nawuphi na umda ka-R ophindwe ngaye, ingongoma evunywa ngokucacileyo sisikhokelo sokuqinisekisa.

I-methylene blue iyaluvuza olu qeqesho lomsebenzi. Ibhendi yayo yemonomer yomelele, ikhemistri yayo iqondwa kakuhle, kwaye iindlela enokusilela ngazo zibonakala kwispektram ngaphambi kokuba zonakalise amanani. Yakha igophe kuluhlu olumxinwa, sebenzisa isampulu engenadayi ngokunyanisekileyo, yenza iziphindaphindo ngokuzimeleyo, uze uvumele ukumila kweespektram kube negalelo ekukhetheni uluhlu. Umgca ophumayo mfutshane, kodwa inqaku ngalinye kuwo liyinyani.

Ubalo lokubonisa indlela kweli nqaku lusebenzisa amanani ayiliweyo ukubonisa inkqubo. Ii-coefficient zesalathiso ezilinganisiweyo kunye neemeko zazo ezipheleleyo zifumaneka kwisikhokelo seespektram esiqhagamshelweyo, kwaye uphononongo lokudibana kweemolekyuli olusisiseko ludweliswe ku-PMID 33251415.