{"id":9917,"date":"2022-02-28T12:10:52","date_gmt":"2022-02-28T12:10:52","guid":{"rendered":"https:\/\/cotslab.com\/fluorescence-cenny-doplnek-k-absorbanci\/"},"modified":"2022-02-28T12:11:00","modified_gmt":"2022-02-28T12:11:00","slug":"fluorescence-cenny-doplnek-k-absorbanci","status":"publish","type":"post","link":"https:\/\/cotslab.com\/cs\/fluorescence-cenny-doplnek-k-absorbanci\/","title":{"rendered":"Fluorescence: cenn\u00fd dopln\u011bk k absorbanci?"},"content":{"rendered":"\n

Kvantifikace nukleov\u00fdch kyselin a protein\u016f se b\u011b\u017en\u011b prov\u00e1d\u00ed pomoc\u00ed UV-Vis spektrofotometrie; n\u011bkdy se v\u0161ak m\u016f\u017ee uk\u00e1zat jako v\u00fdhodn\u011bj\u0161\u00ed detekce pomoc\u00ed fluorescence. V tomto p\u0159\u00edpad\u011b molekuly nejsou detekov\u00e1ny absorbanc\u00ed, ale jsou detekov\u00e1ny nep\u0159\u00edm\u00fdm zp\u016fsobem \u2013 prost\u0159ednictv\u00edm sign\u00e1lu fluorescen\u010dn\u00edho barviva. Nejen, \u017ee fluorescence nab\u00edz\u00ed vysoce citlivou metodu detekce, ale umo\u017e\u0148uje selektivn\u00ed m\u011b\u0159en\u00ed jednotliv\u00fdch analyt\u016f.<\/p>\n\n

P\u0159\u00edpad 1: Jak m\u00e1m postupovat s roztoky vzork\u016f s n\u00edzkou koncentrac\u00ed?<\/strong><\/p>\n\n

UV-Vis spektrofotometrie nen\u00ed p\u0159\u00edli\u0161 citliv\u00e1 \u2013 pro z\u00edsk\u00e1n\u00ed p\u0159esn\u00fdch a p\u0159esn\u00fdch \u00fadaj\u016f by proto koncentrace vzork\u016f nem\u011bla klesnout pod ur\u010ditou koncentra\u010dn\u00ed hranici. Pro dsDNA je tato spodn\u00ed hranice p\u0159ibli\u017en\u011b 1 \u00b5g\/ml. I kdy\u017e nebylo dosa\u017eeno doln\u00ed meze detekce fotometru, dopad nep\u0159esnosti m\u011b\u0159en\u00ed v tomto rozsahu je zna\u010dn\u00fd a v\u00fdsledn\u00e9 hodnoty mohou b\u00fdt n\u00e1chyln\u00e9 k podstatn\u00fdm zm\u011bn\u00e1m. Fluorometrick\u00e1 anal\u00fdza, kter\u00e1 je 1000kr\u00e1t citliv\u011bj\u0161\u00ed ne\u017e metoda absorbance, je schopna velmi p\u0159esn\u011b kvantifikovat roztoky vzork\u016f s extr\u00e9mn\u011b n\u00edzk\u00fdmi koncentracemi.<\/p>\n\n

P\u0159\u00edpad 2: Jak mohu prov\u00e9st p\u0159esnou kvantifikaci navzdory kontaminaci vzorku?<\/strong><\/p>\n\n

Krom\u011b kvantifikace nukleov\u00fdch kyselin p\u0159i 260 nm nab\u00edz\u00ed UV-Vis spektrofotometrie mo\u017enost stanoven\u00ed \u010distoty vzorku vyu\u017eit\u00edm dal\u0161\u00edch vlnov\u00fdch d\u00e9lek (230 nm, 280 nm, 320 nm), resp. Anal\u00fdza dat umo\u017en\u00ed u\u010dinit z\u00e1v\u011bry t\u00fdkaj\u00edc\u00ed se p\u0159\u00edtomnosti l\u00e1tek, jako je s\u016fl, proteiny nebo pevn\u00e9 \u010d\u00e1stice. Pokud jsou naopak absorban\u010dn\u00ed spektra p\u0159\u00edli\u0161 podobn\u00e1, jako je tomu u r\u016fzn\u00fdch nukleov\u00fdch kyselin, samotn\u00e1 absorbance nebude schopna rozli\u0161it nap\u0159\u00edklad RNA a DNA. V tomto p\u0159\u00edpad\u011b fluorescen\u010dn\u00ed m\u011b\u0159en\u00ed umo\u017en\u00ed specifickou kvantifikaci analyt\u016f bez interference s jin\u00fdmi molekulami.<\/p>\n\n

Na jak\u00e9m principu je tato metoda zalo\u017eena?<\/strong><\/p>\n\n

M\u011b\u0159en\u00ed fluorescence vyu\u017e\u00edv\u00e1 jevu, \u017ee ur\u010dit\u00e9 molekuly jsou schopny vyza\u0159ovat energii ve form\u011b sv\u011btla. Pro detekci biomolekul, jako jsou nukleov\u00e9 kyseliny a proteiny, se vybere fluorescen\u010dn\u00ed barvivo, kter\u00e9 se nav\u00e1\u017ee na analyt s nejvy\u0161\u0161\u00ed mo\u017enou specificitou. Klasick\u00fdm p\u0159\u00edkladem je vazba \u010dinidla PicoGreen \u00ae<\/sup> na dsDNA. Po excitaci komplexu sv\u011btlem o specifick\u00e9 vlnov\u00e9 d\u00e9lce (p\u0159ibli\u017en\u011b 480 nm pro PicoGreen) bude fluorofor emitovat sv\u011btlo s ni\u017e\u0161\u00ed energi\u00ed, tj. s del\u0161\u00ed vlnovou d\u00e9lkou (p\u0159ibli\u017en\u011b 520 nm pro PicoGreen) (obr\u00e1zek 1). Sv\u011btlo se m\u011b\u0159\u00ed jako relativn\u00ed fluorescence (RFU), kde intenzita fluorescence je \u00fam\u011brn\u00e1 koncentraci vzorku.<\/p>\n\n

\"\"
Obr\u00e1zek 1:A) Princip fluorescen\u010dn\u00edch m\u011b\u0159en\u00ed nukleov\u00fdch kyselin (vlevo)B) Sv\u011btlo, kter\u00e9 se pou\u017e\u00edv\u00e1 k excitaci fluoroforu (EX), m\u00e1 krat\u0161\u00ed vlnovou d\u00e9lku ne\u017e emitovan\u00e9 sv\u011btlo (EM) (vpravo)<\/figcaption><\/figure><\/div>\n\n

Jak\u00e9 materi\u00e1ly jsou pro tuto metodu zapot\u0159eb\u00ed?<\/strong><\/p>\n\n

Kvantifikace nukleov\u00fdch kyselin a protein\u016f t\u011b\u017e\u00ed z v\u00fdb\u011bru fluorescen\u010dn\u00edch barviv, kter\u00e1 se v\u00e1\u017eou na p\u0159\u00edslu\u0161nou c\u00edlovou molekulu s vysokou specificitou. Krom\u011b toho mus\u00ed b\u00fdt k dispozici alespo\u0148 jeden standard o zn\u00e1m\u00e9 koncentraci. Excitace fluoroforu a m\u011b\u0159en\u00ed intenzity fluorescence emitovan\u00e9ho sv\u011btla vy\u017eaduje bu\u010f samostatn\u00fd fluorometr, nebo fluorescen\u010dn\u00ed modul, kter\u00fd je integrov\u00e1n v jin\u00e9m p\u0159\u00edstroji. Vybaven\u00ed a fluorescen\u010dn\u00ed barvivo mus\u00ed b\u00fdt kompatibiln\u00ed, aby zdroj sv\u011btla uvnit\u0159 p\u0159\u00edstroje poskytoval po\u017eadovanou vlnovou d\u00e9lku (y) excitace a detektor m\u011b\u0159il emitovan\u00e9 sv\u011btlo. V z\u00e1vislosti na typu p\u0159\u00edstroje jsou roztoky \u010dinidel um\u00edst\u011bny bu\u010f v tenkost\u011bnn\u00fdch reak\u010dn\u00edch n\u00e1dobk\u00e1ch nebo v kyvet\u00e1ch. Je t\u0159eba vz\u00edt v \u00favahu kompatibilitu s konkr\u00e9tn\u00ed aplikac\u00ed s ohledem na p\u0159izp\u016fsoben\u00ed, pr\u016fhlednost, autofluorescenci a objem.<\/p>\n\n

Jak se metoda prov\u00e1d\u00ed?<\/strong><\/p>\n\n

Obr\u00e1zek 2 ukazuje sekvenci fluorometrick\u00e9ho m\u011b\u0159en\u00ed. P\u0159\u00edprava slep\u00e9ho vzorku, standard\u016f a vzorku je zah\u00e1jena p\u0159id\u00e1n\u00edm fluorescen\u010dn\u00edho barviva, po kter\u00e9m n\u00e1sleduje kr\u00e1tk\u00e1 inkuba\u010dn\u00ed doba. Nejprve se zm\u011b\u0159\u00ed jeden nebo v\u00edce standard\u016f o zn\u00e1m\u00fdch koncentrac\u00edch, aby se vytvo\u0159ila standardn\u00ed k\u0159ivka. Stanoven\u00ed koncentrace vzorku n\u00e1sledn\u011b prob\u00edh\u00e1 ve druh\u00e9m kroku, kde jsou nam\u011b\u0159en\u00e9 intenzity fluorescence vzork\u016f hodnoceny ve vztahu ke standardn\u00ed k\u0159ivce.<\/p>\n\n

\"\"
Fluorescence pracovn\u00edho postupu<\/figcaption><\/figure><\/div>\n\n

Fluorescence \u2013 metoda volby?<\/strong><\/p>\n\n

I kdy\u017e fluorometrie nab\u00edz\u00ed v ur\u010dit\u00fdch p\u0159\u00edpadech v\u00fdhody, klasick\u00e1 UV-Vis spektrofotometrie nad\u00e1le z\u016fst\u00e1v\u00e1 standardn\u00ed metodou, pokud jde o kvantifikaci nukleov\u00fdch kyselin a protein\u016f. Spektrofotometrie umo\u017e\u0148uje ov\u011b\u0159en\u00ed \u010distoty vzorku a vysoce koncentrovan\u00e9 roztoky lze analyzovat p\u0159\u00edmo. V z\u00e1vislosti na kvalit\u011b vzorku a tak\u00e9 na po\u017eadavc\u00edch n\u00e1sledn\u00fdch aplikac\u00ed m\u016f\u017ee b\u00fdt vhodn\u00e9 ob\u011b techniky kombinovat. <\/p>\n\n

PicoGreen \u00ae<\/sup> je registrovan\u00e1 ochrann\u00e1 zn\u00e1mka spole\u010dnosti Molecular Probes, Inc. Corporation, Eugene, OR, USA. Eppendorf \u00ae<\/sup> , Eppendorf Brand Design, Eppendorf BioSpectrometer \u00ae<\/sup> , Uvette \u00ae<\/sup> a Eppendorf \u00b5Cuvette \u00ae<\/sup> jsou registrovan\u00e9 ochrann\u00e9 zn\u00e1mky spole\u010dnosti Eppendorf AG, N\u011bmecko. V\u0161echna pr\u00e1va v\u010detn\u011b grafiky a obr\u00e1zk\u016f vyhrazena. Copyright \u00a9 2019 Eppendorf AG.<\/em><\/p>\n\n

Zdroj<\/a><\/p>\n","protected":false},"excerpt":{"rendered":"

Kvantifikace nukleov\u00fdch kyselin a protein\u016f se b\u011b\u017en\u011b prov\u00e1d\u00ed pomoc\u00ed UV-Vis spektrofotometrie; n\u011bkdy se v\u0161ak m\u016f\u017ee uk\u00e1zat jako v\u00fdhodn\u011bj\u0161\u00ed detekce pomoc\u00ed fluorescence. V tomto p\u0159\u00edpad\u011b molekuly nejsou detekov\u00e1ny absorbanc\u00ed, ale jsou detekov\u00e1ny nep\u0159\u00edm\u00fdm zp\u016fsobem \u2013 prost\u0159ednictv\u00edm sign\u00e1lu fluorescen\u010dn\u00edho barviva. Nejen, \u017ee fluorescence nab\u00edz\u00ed vysoce citlivou metodu detekce, ale umo\u017e\u0148uje selektivn\u00ed m\u011b\u0159en\u00ed jednotliv\u00fdch analyt\u016f. P\u0159\u00edpad 1: Jak […]\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":[],"categories":[483],"tags":[],"_links":{"self":[{"href":"https:\/\/cotslab.com\/cs\/wp-json\/wp\/v2\/posts\/9917"}],"collection":[{"href":"https:\/\/cotslab.com\/cs\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/cotslab.com\/cs\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/cotslab.com\/cs\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/cotslab.com\/cs\/wp-json\/wp\/v2\/comments?post=9917"}],"version-history":[{"count":1,"href":"https:\/\/cotslab.com\/cs\/wp-json\/wp\/v2\/posts\/9917\/revisions"}],"predecessor-version":[{"id":9925,"href":"https:\/\/cotslab.com\/cs\/wp-json\/wp\/v2\/posts\/9917\/revisions\/9925"}],"wp:attachment":[{"href":"https:\/\/cotslab.com\/cs\/wp-json\/wp\/v2\/media?parent=9917"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/cotslab.com\/cs\/wp-json\/wp\/v2\/categories?post=9917"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/cotslab.com\/cs\/wp-json\/wp\/v2\/tags?post=9917"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}