基于FRET生物传感器的灵敏度评价

智能文字提取功能测试中

荧光共振能量转移（FRET）是一种控制相邻两个分子之间能量转移的机制。供体最初处于激发态，可以通过非辐射电子共振将能量转移到受体分子上。FRET使用荧光分光计监测，测量受体或激发供体的荧光/猝灭。FRET效率取决于以下因素：供体和受体之间的距离，供体和受体之间的光谱重叠，以及它们的偶极矩的对齐。本文中的结果表明，抗tnt单抗是一种高敏感性的硝基化合物生物传感器，不需要使用荧光标记。Application NoteFP-0022 Sensitivity Evaluation of aBiosensor using FRET2/4Application Note Sensitivity Evaluation of a Biosensor using FRET In troduc t ion F l uorescence resonance energy transfer (FRET) is a mechanism gove r ning the ene r gy transfer between two neighboring mo l ecules. A donor ， in i t i ally in its excited state， may t ransfe r energy to an accepto r molecule through non-radiative e l ectron resonance. FRET i s monitored using a spectrofluorometer ， wh i c h measu r es the fluorescence/quenching of an accepto r or excited donor. FRET efficiency depends o n the following factors： the dista n ce between the donor and acceptor， the spectral overlap between t he donor and acceptor， and the alignme n t of their dipole moments. FP-8500 3D Fl uo r escence Hig h -Speed Measuremen t System Figure 1. J a blo n ski diag r a m il l ust r at in g dif f erent t ransi ti on s b etwee n a molecule's energy states . Th e eff ici e ncy i s i n v e rs e l y propor ti onal to th e s i xth power of t h e d i stanc e betwe e n th e donor and acceptor ， making the t ec h nique e x tr eme l y sensi ti ve to smal l c h ang e s i n distance. Wh e n t h e ove r lap a r ea of the donor f l uorescence spec t rum and t h e acceptor absorption spec t rum i s l arger， FRE T ef fi ciency i s high e r . The FRET efficiency is also at a maximum when the two dipole mome n t s are paralle l o r anti-paral l el to e ach oth er ， and no e nergy transfer occurs when the dipole moments are perpen d icular to one another. Typically when t h e d i stance between the donor and acceptor is between 1and 10 nm ， FRET occurs. An excellent example of a FRET applicat i on i s the monitor i ng the complexing of murine anti-TNT monoclonal antibody (ant i -TNT mAb) with aromatic nitrogen based explosives such as tr i nitrotoluene (TNT). Anti-TNT mAb absorbs excitation light of 280 nm， and produces f luorescence l ight of 340 nm. When a n ti-NT mAb forms a complex with TNT ， FRET occurs between t he anti-TNT mAb (donor) and TNT (acceptor). The energy is transferred f rom the anti-TNT mAb excited at 280nm to the TNT ， and t h e fluo r escence at 340 nm fro m the anti-TNT mAb is quenched (Figure 2). By utilizing this i nteraction as biosensor， this application note shows t h e evaluation of the sens i tivity between the aromatic nitro compounds and anti-TNT mAb [2]. Figure 2. Schematic il l ust r ation of f l uo r escence qu en c h ing caused by T N T detection of a n ti-T N T. K eyword s F P -8500， F l u oresce n ce ，Fluorescence Reso n a n ce Energ y Tran s fe r， FRET， TNT， I mmunosenso r s ， Biochem i stry， Mate r ia l s E xper i men t al 10 pL of 4.4x 10* M TNT s ol u ti o n w as added t o 800 p L of 1.6x 10°M a n ti-TNT m A b ， an d the f lu o rescence m ea su remen t s w ere perfo rm ed u sing th e p a r ame t e r s belo w . After each meas u remen t， th e f in al concentration o f TNT was in c reased s t e p -b y-s tep b y add i ng eac h 10 p L of TNT solu t io n (4.4x 10M， 4.4x10-6M， 4.4x10-5.5 M an d 4.4x10-5M )， an d f lu o r esce n ce m ea s u r e m en t of eac h d ifferen t TN T c on c e n tra ti o n s ol u ti o n w a s per f o r me d . Excitation Wavelength 280nm Emission Range 300-500nm Excitation Bandwidth 1nm Emission Bandwidth 20nm Data Interval 0.5nm Response 1 sec Sensitivity 500V Spectral Correction ON Results The f luorescence s p ec t ra o f a m i x ture sol u tion o f an ti -TNT mAb an d TNT a re shown in F i g ur e 3. As t h e TNT conce n tra ti on i ncrease s ， the f luorescence in t e n s ity of an ti -TN T mA b b eco m es w eaker. Simil ar m e asur e m e n t s w ere per fo rmed us in g o t h e r a r oma ti c n itro com p o u nds a s samples . The degree o f progress o f complex f orma t i on r eaction wa s determined by th e d e c r e a se i n the f luo r escence in tensit y a t 340 n m . 28600 Mary's Cour t ， E a s to n ， M D 21601US A Figure 3. F l u or escence s p ect ra of s o l ut i o ns co n t a i ni n g a n t i -TNT mAb a n d var i o us concentra ti o n of TNT. F i g u re 4 sh o w s the rel a ti o n s hip b e tween t h e do s e of a romatic n it r o c omp o u n d s a n d t h e r ea c ti o n rat i o. F r o m th is cu r ve， th e h alf m a xi mal i n h i bito r y co n cen tr ation (I C50) was estimate d (Ta bl e 1). Th i s r esu lt shows th at a n ti-T NT mAb h a s s e nsi ti vi ty to T N T， TNB， N T an d 2，6-DN T ， an d i s h ig h l y sen s i tiv i t y to TN T a n d TNB howeve r i t cannot detect RDX (c y cl ot r i me th y l e n etrin i t r a min e). T h e se r es ult s demon s tr a t e th a t an ti-TNT m A b i s a h ig h ly se n si tiv e bi o se n s o r f o r n itr o c o mp ou n ds witho u t t he re q u i rem e n t to use a f lu o r esce n t la b el . Fina l concentration [mol/L] Figure 4. Dose -re spo n se curve fo r the immunocomp l ex for m ation of a n ti b odies wi t h TNT an d other ex p losive compo u nd fo r mat i o n s . Table 1. An a ly sis o f b i n d ing of dif f ere n t explo si ve c o m p ound s to ant i -TNT. Name Structure ICso(M) TNT CHs ON NO NO2 4.0x108 TNB NO2 ON NO2 3.8x10-8 2，6-DNT CHON NO2 7.9x106 NT CH3NO2 3.6x104 RDX ON、 NO22 not detected 28600 Mary's Cour t ， E a s to n ， M D 21601US A Refer en c es Speci a l th a n k s t o H ir osh i Naka y ama of Pan a s onic Co rp o r ation w h o ass i sted w ith the exper i men t . 1. S a t o k o Su z u k i ， T osh i f u mi U c hi y a m a， Ke n -i chi Ak a o ， Kou s hi Naga m o r i ， H ir o s h i Nak a y am a a n d Y u ji I t o， TNT sensi n g u s ing no n -l a b el ing FR ET， Jap an -Ta iw a n M edical Sp ectros c o py I n t e rn a ti o n a l S ympo s i u m /14th An n ual M eet i n g o f t h e J ap a n Associa t io n of Medical Spectroscopy，AWAJ I ， JA P AN 2016. 28600 Mary's Cour t ， E a s to n ， M D 21601US A