肽-铽配合物中磷光寿命检测方案(分子荧光光谱)

FRET with a HORIBA Jobin Yvon Phosphorimeter Introduction A phosphorimeter integral to theFluoroMax -P spectrofluorometer or asthee FiL-1042 accessory on theFluorolog~ system can uncoverimportant information on varioussystems of chemical and biochemicalinterest by revealing luminescence datanormally masked by intense but rapidtluorescence. This Technical Note describes anexample of Forster resonance energy-transfer (FRET) from a peptide-terbium-complexdonorto aa fluoresceinacceptor， using the HORIBA Jobin Yvonphosphorimeter. The peptide in thecomplex absorbs light at 280 nm， andthe terbium phosphoresces at 485 nm，where fluorescein dye absorbs.Thefluorescence of fluorescein can beobserved when the sample is excited at280)nm. usingour phosphorimeteraccessory. Experimental procedure Peptide-terbium complex wasdissolved in aqueous solution， alongwith fluorescein in some samples. Themeasurements were taken con aFluorolog@-3-22 spectrofluorometer，including double-grating excitation andemission monochromators.Fluorescencee measurements； used a450-W CW xenon lamp as the photon-Source. and phosphorescencemeasurements used a xenon flash lampas thephoton-source. An R928photomultiplier tube， operated at 950 Vin the photon-counting mode， was thedetector. The FL-1042 phosphorimeteraccessory includes a dual-lamp housing(containing both CW and pulsed Xe lamps)， and all control electronics. Alight-pulse excites the sample，. andvariabledelayscontroll when thedetection window opens， and for howlong. Sample excitation was at 280 nm，with 100 flashesmeasured.Forluminescence spectra， the integrationtime was 0.2 s， except as noted. Thescans were taken under ambient roomconditions. Results and discussion Fig. 1 comparesluminescencefrom the peptide-Tb complex with nodelay and a 50-us delay following theexcitation light pulse. With the delay， thefluorescence at 363 nm from the peptidedisappears， isolating the Tbphosphorescence at 486 and 540 nm.Fig. 2 (next page) shows fluorescencefrom a solution of peptide + fluoresceindye， also with and without delay. With a50-ps delay， all fluorescence from thepeptide + fluorescein vanishes. Takentogether， Figs. 1 and 2 show that thephosphorescent species is Tb. Fig. 1. Luminescence spectra of peptide +terbium with (green) and without (red) a 50-usdelay. Nexc=285 nm， and bandpass =2 nm forboth excitation and emission. Fig. 2. Luminescence spectra of peptide +fluorescein with (green) and without (red) a 50-ps delay. All bandpasses =5 nm. Fig. 3 combines all three species，in a plot of peptide-terbium complex withand without 0.67-uM fluorescein， withoutandwitha50-usSdelaybetweenexcitationwith a )xenon1flashlamp.Again， the spurious fluorescence at 363nm is removed by delaying the samplewindow for 50 ps. The blue and greencurves directly compare fluorescein-containing and fluorescein-lacking solu-tions， showing fluorescein phosphores-cence at 511 nm from energy-transferfrom complex to fluorescein. Fig. 4 shows phosphorescence-decay curves of peptide with terbium ionalone (T=3.8 ms)， and in the presenceof fluorescein (T = 3.0 ms). The fasterphosphorescence decay in the presenceof fluorescein (blue curve in Fig. 4)confirmsenergy-transfer trom thepeptide-T)b donor1to the ffluoresceinacceptor. Similar eexperiments can beperformed with the： FluoroMax@-pspectrofluorometer， which contains anintegrated phosphorimeter along withCW lamp. Fig. 3. Luminescence spectra of complex， with(red) 0.67-pM fluorescein and no delay afterexcitation， (green) 0.67-pM fluorescein and a 50-ps delay’ after excitation， and (blue)nofluorescein and a 50-us delay after excitation.Bandpass= 5 nm for excitation and emission. Fig. 4. Phosphorescence decay curve from anaqueous solution of： (red) complex alone， and(blue) complex +fluorescein. Integration time =0.4 s； bandpass = 5 nm on excitation (280 nm)and emission (540 nm). Conclusions Use of a HORIBA Jobin Yvonphosphorimeter， including gated delayof signal-acquisition， can reveal extrainformation， such as FRET， about thephysical and chemical properties of ma-terials. Copyright C HORIBA Jobin Yvon； version .HORIBAExplore the future HORIBAJOBIN YVONChina： +() ORIBAExplore the future Use of a HORIBA Jobin Yvon phosphorimeter, including gated delay of signal-acquisition, can reveal extra information, such as FRET, about the physical and chemical properties of materials.