Auna nau'ikan oxygen masu saurin amsawa a gwaje-gwajen methylene blue

Dakunan gwaje-gwaje biyu suna gudanar da abin da kowannensu yake kira gwajin methylene blue na hana oxidation. Ɗaya yana bayar da rahoton cewa rinin yana rage nau'ikan oxygen masu saurin amsawa. Ɗayan yana bayar da rahoton cewa yana samar da su. Dukkansu suna nuna zane-zanen alaƙar adadin sinadari da amsarsa masu tsafta. Dukkansu suna faɗin abin da suka samu da gaskiya. Bambancin ba yaudara ba ne ko rashin ƙwarewa. Abin da ya faru shi ne ƙungiyoyin biyu sun auna abubuwa dabam a ƙarƙashin haske dabam, da sinadaran gano abu waɗanda suke amsa tambayoyi dabam, sannan suka bayyana amsoshin biyu da kalma ɗaya: ROS.

Methylene blue ƙwayar sinadari ce mai wahalar aunawa daidai saboda tana shiga cikin amsoshin sinadarai da ake tsammanin ta lura da su. A duhu tana jigilar electrons tsakanin masu bayarwa da masu karɓa, sannan ta koma siffarta ta farko, a shirye ta sake zagayawa. A ƙarƙashin haske tana zama sinadarin da ke tayar da amsoshi ta hanyar haske, tana kaiwa yanayin triplet mai ƙarin kuzari, inda za ta iya miƙa kuzari ga oxygen don samar da singlet oxygen ko ta miƙa electrons don samar da radicals da hydrogen peroxide. Duk wani sinadarin gano abu da aka saka cikin wannan tsarin yana iya fuskantar oxidation, reduction, gushewar launi, ko kuma rinin kansa ya hana ganin siginarsa ta haske. Don haka tambayar farko game da kowane karatun ROS ba ita ce menene lambar ba. Ita ce ainihin abin da gwajin yake ganowa, da kuma abin da rinin yake yi wa gwajin.

Amplex Red: gwajin auna hydrogen peroxide mai daidaitaccen ma'auni

Haɗin methylene blue da Amplex Red shi ne haɗin da ya fi samun ingantacciyar hujja a waɗannan wallafe-wallafen bincike, idan an shirya shi daidai. Amsar sinadarin takamaimai ce: horseradish peroxidase yana amfani da hydrogen peroxide don yin oxidation ga substrate na Amplex ya zama resorufin, wani samfurin da ke fitar da kyallin fluorescence, da kusan rabon ɗaya ga ɗaya. Peroxide daga samfurin gwaji yana zama kyallin fluorescence a na'urar ganowa. Babu wani abu dabam a rukunin ROS na yau da kullum da ke haifar da wannan amsa kai tsaye.

Babban abin da ya ba shi fifiko shi ne daidaita ma'auni. Mai aiki da kulawa yana gwada sanannun ma'aunan peroxide a cikin ruwan buffer ɗaya, da tarin peroxidase da substrate iri ɗaya, lokacin barin amsar ta gudana iri ɗaya, da saitunan na'urar haske iri ɗaya, sannan ya kwatanta karatun samfuran da wannan lanƙwasar ma'auni. A binciken mitochondria da aka keɓe daga ƙwaƙwalwa wanda yake ginshiƙin wannan hanyar, marubutan sun yi haka daidai, suna ƙara sanannun adadin peroxide a ƙarshen kowane zagayen gwaji. An bayar da lambobin a matsayin saurin samarwa, a picomoles a minti ɗaya ga kowane milligram na furotin, maimakon raka'o'in kyallin fluorescence marasa daidaitaccen ma'auni. Wannan ne yake sauya kyalli zuwa aunawa.

Abubuwa uku daga wannan binciken sun cancanci a yi koyi da su. Na farko, marubutan sun tabbatar da siginar da catalase, wanda yake lalata peroxide. Ƙaruwar kyallin fluorescence da methylene blue ya haifar ta tsaya lokacin da aka ƙara catalase, ta tabbatar da cewa siginar peroxide ce maimakon sakamakon ƙarya na na'urar haske. Na biyu, sun gwada tasirin haske kai tsaye ta hanyar rufe katakon hasken tayarwa, ta yadda samfuran suka karɓi kashi ɗaya cikin takwas na adadin hasken da aka saba amfani da shi, kuma siginar ta ragu da kashi tara cikin ɗari kawai. Fiye da kashi casa'in cikin ɗari na peroxide da suka bayar da rahotonsa bai dogara da hasken aunawa ba. Na uku, sun auna kawar da peroxide dabam da electrode, suka gano cewa rinin yana ƙara samarwa kuma yana rage saurin kawarwa. Sinadarin gano abu da yake bayar da jimillar kyallin fluorescence bayan cire raguwa kawai zai haɗa waɗannan tasirai biyu ya ɗauke su a matsayin abu ɗaya.

Duk da haka, gwajin yana da hanyoyin da zai iya kasa, kuma methylene blue yana haifar da da dama daga cikinsu. Rinin yana tsotse haske a faɗin yankin ja ɗaya da resorufin yake fitar da haske a ciki, don haka idan tarin rinin ya yi yawa, samfurin zai iya ɓoye siginarsa ta hanyar tasirin tace haske a cikin samfurin. Saboda haka ma'aunan daidaitawa ya kamata su ƙunshi tarin rini iri ɗaya da samfuran. Amplex Red kuma yana iya fuskantar oxidation ta haske da kansa, kuma samfurinsa resorufin zai iya shiga zagayowar redox da haske ke haifarwa wadda take samar da superoxide da peroxide. Wannan yana da matuƙar muhimmanci idan gwajin kuma yana haskaka sinadarin da ke tayar da amsoshi ta haske. Mafita a aikace ita ce a fara haskaka samfuran methylene blue, sannan a ɗauki ƙananan kaso daga cikinsu a auna da sinadaran Amplex a duhu, maimakon haskaka rinin da sinadarin gano abu tare. A ƙarshe, superoxide dismutase ba kawai yana rage siginar Amplex ba: mayar da superoxide zuwa peroxide zai iya ƙara ta. Gwajin tabbatarwa na SOD da yake ƙara kyallin fluorescence bai gaza ba. Ya gano cewa superoxide yana cikin matakan farko na hanyar amsar.

DCFDA: alamar oxidation marar takamaiman nau'i

Haɗin methylene blue da DCFDA shi ne tushen yawancin ruɗanin. DCFH-DA wani sinadarin farko ne da ake shigarwa cikin tantanin halitta. Esterases a cikin tantanin suna cire ƙungiyoyin acetate ɗinsa su bar DCFH marar kyallin fluorescence, sannan oxidation na DCFH ya samar da DCF mai kyallin fluorescence. Abubuwan da suke yin oxidation gare shi suna da yawa: hydroxyl radicals, matsakaitan samfuran peroxidase da heme, nitrogen dioxide, hypochlorous acid, da radicals da suka samo asali daga peroxynitrite. Hydrogen peroxide shi kaɗai ba ya yin oxidation gare shi kai tsaye da wani sauri mai amfani; kyallin fluorescence da ya dogara da peroxide yawanci yana buƙatar iron, furotin masu heme, ko amsoshin peroxidase a cikin tsarin.

Binciken suka da ya kamata kowane sashe na hanyoyin bincike ya ambata ya fito daga nazarin DCFH a matsayin sinadarin gano abu da Kalyanaraman da abokan aikinsa suka yi, kamar yadda aka taƙaita a binciken mitochondria na ƙwaƙwalwa wanda ya ƙi amfani da DCFDA saboda wannan dalili. Sun nuna cewa rinin yana amsawa a hankali, da rabon sinadarai da ba a tantance ba, ba tare da ingantaccen daidaita ma'auni ba, kuma yana amsawa da sinadarai waɗanda ba ROS ba ne. Sinadarin gano abu kuma yana ƙara abin da yake aunawa: oxidation na DCFH ta hanyar rasa electron ɗaya yana samar da DCF radical wanda yake amsawa da oxygen don samar da superoxide, sannan superoxide ya sauya ta dismutation zuwa ƙarin peroxide. Karatun yana samar da wani ɓangare na amsoshin sinadarai da yake bayar da rahotonsu.

Babu ɗaya daga cikin waɗannan da yake sa DCFDA ya zama marar amfani. Suna sa ya zama gwajin farko na gano alama, ba na tantance abin da ke akwai ba. Ƙaruwar kyallin fluorescence na DCF tana nufin cewa oxidation na sinadarin gano abu da ya dogara da masu haifar da oxidation ya ƙaru. Ba tana nufin peroxide ya ƙaru ba, kuma tabbas ba tana nufin jimillar ROS ta ƙaru ba; babu wani sinadarin gano abu guda ɗaya da yake auna wannan jimillar. Binciken methylene blue na baya wanda ya dogara da oxidation na DCF a sama da micromolar ɗaya ya bayar da rahoton tasirin ƙara oxidation, yayin da wasu tsarin gwaji a wannan lokacin suka bayar da rahoton kariya daga mutuwar tantanin halitta da peroxide ya haifar. Duk waɗannan abubuwan biyu na iya zama gaskiya game da rini ɗaya a yanayin redox dabam, kuma sakamakon DCF shi kaɗai ba zai iya bayyana wane sinadarin haifar da oxidation ne ya samar da siginar ba. Muhimmancin binciken mitochondria na ƙwaƙwalwa na baya shi ne cewa ya maye gurbin wannan gwajin farko da aunawar peroxide mai daidaitaccen ma'auni, ya kuma gano nauyin samarwar kai tsaye: ko da rini a nanomolar ɗari ɗaya ya ƙara sakin peroxide sau da yawa a duk substrates da yanayin numfashin tantanin da aka gwada.

DCFDA yana da wata matsala ta musamman da ta shafi methylene blue. Rinin yana da launi mai ƙarfi kuma yana shiga amsoshin redox, don haka yana iya gusar da kyallin fluorescence, kashe fitar haske, ko haifar da oxidation ga sinadarin gano abu ba tare da dogaro da ROS na tantanin halitta ba. Gwajin tabbatarwa mai rini da sinadarin gano abu amma babu ƙwayoyin halitta ba wani ƙarin ado na zaɓi ba ne. Shi ne gwajin da yake gaya maka ko rinin yana mu'amala da sinadarin gano abu.

Tarkon spin da shan oxygen: tantance nau'i da saurin kwarara

Yayin da sinadaran gano abu masu kyallin fluorescence suke bayar da rahoton cewa wani abu mai iya fuskantar oxidation ya bayyana, resonance na spin na electron tare da tarkunan spin yana bayar da rahoton da ya fi kusantar tantance abin da yake. Shahararren binciken amsoshin methylene blue da haske ke haifarwa ya yi amfani da ESR don gano ascorbate radical daga rini, ascorbate da haske, sannan ya nuna samar da peroxide a gwaje-gwajen shan oxygen da kuma samar da hydroxyl radical da iron ya hanzarta a gwaje-gwajen tarkon spin. Kowace hanya ta amsa tambayar da sauran ba su iya amsawa ba: ESR ya tantance nau'in radical, shan oxygen ya nuna saurin kwararar amsar ba tare da dogaro da wani sinadarin gano abu mai kyallin fluorescence ba, kuma haɗin hanyoyin ya nuna cewa hanyoyin singlet oxygen da radicals duk suna aiki a tsarin da aka haskaka ɗaya.

Wannan sakamakon na 1984 har yanzu yana kama da gargaɗi. Lokacin da haske da sinadarin reduction suke akwai, rinin ya samar da peroxide, kuma lokacin da iron yake akwai, tsarin ya samar da hydroxyl radical, nau'in da ya fi saurin amsawa a cikin rukunin. Marubutan sun danganta wannan da ascorbate a ido kuma suka yi shakku kan amfani da shi a ido ba tare da kulawa ba. Masu karatu na yanzu ya kamata su lura da sharuɗɗan: haske tare da sinadarin reduction tare da iron. Cire kowanne daga cikinsu, kuma hanyar amsar za ta raunana ko ta ɓace.

Electrodes na oxygen suna samun muhimmancinsu ta wata hanya dabam. Electrode na nau'in Clark yana auna jimillar oxygen da aka cinye bayan lissafin duk wani oxygen da aka samar. Wannan yana tabbatar da cewa amsoshin redox da rinin yake haifarwa suna cinye oxygen na ƙwayoyin sinadari, kuma yana bayar da bayanan saurin amsa waɗanda babu wani sakamakon ƙarya na kyallin fluorescence da zai iya kwaikwaya. Ba ya tantance samfurin da aka samu. Peroxide, superoxide, ruwa, da lalacewar singlet oxygen duk suna cinye oxygen. Sanin saurin kwarara ba tare da tantance nau'i ba bai cika ba, kamar yadda tantance nau'i ba tare da sanin saurin kwarara ba bai cika ba. Shi ya sa bincike masu ƙarfi suke haɗa electrode da gwajin auna peroxide mai daidaitaccen ma'auni ko da tarko.

Dalilin da ya sa sakamakon hana oxidation yake saɓa wa juna

Idan aka jera waɗannan karatun uku a gefe da gefe, saɓanin da ake gani a wallafe-wallafen methylene blue yana bayyana a matsayin bambance-bambancen hanyoyi tare da gaskiyar ilimin halitta guda uku.

Na farko, rinin yana aiki a matsayin mai jigilar electrons da kuma tushen peroxide. Ta hanyar karkatar da electrons daga wuraren numfashin tantanin inda oxygen zai zama superoxide, zai iya rage wani ROS, yayin da siffarsa da ta fuskanci reduction take yin reduction ga oxygen kai tsaye zuwa peroxide, tana ƙara wani ROS dabam. Saboda haka dakin gwaje-gwajen da yake nazarin amsoshin redox na mitochondria da rawar methylene blue wajen samar da hanyar kaucewa ya kamata ya sa ran amsoshi da suka dogara da sinadarin gano abu: karatun da yake jin superoxide yana raguwa yayin da karatun peroxide yake ƙaruwa a cikin cuvette ɗaya.

Na biyu, peroxide a ƙananan adadi sigina ne, ba lalacewa kawai ba. Binciken mitochondria na ƙwaƙwalwar bera wanda ya biyo bayan aikin farko ya gano samar da peroxide daga rini mai ƙarancin nanomolar hamsin ba tare da raunukan DNA na mitochondria a matakan magani ba, tare da kariya daga raunukan da rotenone ya haifar bayan an yi amfani da rinin tun farko. Sai a micromolar ɗari ɗaya, wanda yake nesa sama da zangon magani, rinin ya lalata wasu sassan DNA da aka zaɓa. Kuma jan haske tare da rini na micromolar ashirin ya samar da singlet oxygen wanda ya lalata duk sassan da aka gwada. Tsarin da aka gabatar na hormesis ne: samar da peroxide a ɗan ƙaramin mataki yana tayar da hanyoyin kariya daga oxidation da suke da alaƙa da Nrf2 da kuma samar da sababbin mitochondria. Don haka karatun ƙara oxidation na ɗan gajeren lokaci yana zuwa kafin sakamakon hana oxidation na dogon lokaci. Gwajin da aka karanta bayan awa ɗaya da gwajin da aka karanta bayan yini ɗaya na iya saɓa wa juna saboda yanayin halitta ya canza a tsakaninsu.

Na uku, haske yana tantance ko wace ƙwayar sinadari ce ma take nan. Haskakawa tana cire ƙungiyoyin methyl daga rinin, tana sauya shi zuwa Azure B da samfura masu alaƙa, kuma fitilar microscope da take kan kwanon gwaji ta isa ta tayar da amsoshinsa ta haske. Gwaje-gwajen duhu da na haske gwaje-gwaje ne dabam, kuma sashen gwaje-gwajen tabbatarwa na haskakawa da sarrafa rini a kowace ƙa'idar gwaji ya kamata ya ɗauke su haka.

Mafi ƙarancin gwaje-gwajen tabbatarwa, da yadda za a karanta kowace iƙirari

Kwatancin da ke ƙasa shi ne asalin kayan dubawa na wannan maƙala. Kafin karɓar wani sakamakon ROS na methylene blue, duba wane layi marubutan suka cika da kuma wanne suka tsallake.

Daga nan akwai ƙa'idar karatu mai amfani. Amplex tare da peroxidase, bayan daidaita ma'auni da binciken catalase, hujja ne game da peroxide. DCFDA hujja ne game da oxidation marar takamaiman nau'i na sinadarin gano abu, ba wani abu fiye da haka ba. Tarkon spin tare da gwaje-gwajen tabbatarwa masu sinadaran kama radicals hujja ne game da nau'in radical. Shan oxygen hujja ne game da saurin kwarara. Jumlar da take iƙirarin fiye da abin da gwajinta zai iya tabbatarwa, ko da an rubuta ta da kwarin gwiwa, jumla ce game da sinadarin gano abu, ba game da rinin ba. Tattaunawa masu alaƙa game da dogaro da adadi da zangon hormesis, kwararar electrons a mitochondria, tsangwamar gwajin auna a samfuran da aka yi wa rini, da sakamakon ƙarya na haskakawa a microscope suna faɗaɗa kowanne daga cikin waɗannan batutuwa, kuma kowanne yana ci gaba da kasancewa ingantacce bayan an maye gurbin manazartarsa ta wucin gadi.