CN1867822A - Direct observation of molecular modifications in biological test systems by measuring fluorescence lifetime - Google Patents
Direct observation of molecular modifications in biological test systems by measuring fluorescence lifetime Download PDFInfo
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Abstract
The invention relates to a method for directly detecting the modification of a molecule containing a fluorescent dye by measuring the fluorescence lifetime.
Description
The present invention relates to by measuring the method that fluorescence lifetime directly detects the modification of the molecule that contains fluorescent dye.
The introduction of fluorescence spectrometry
During excited molecule transits to its energy ground state, be called luminous and be divided into fluorescence and phosphorescence usually with all processes of emitted radiation.In addition, this excitation energy can discharge by various non-radiative processes.
From excited singlet state S
1The lowest vibration energy level transition to monoenergetic ground state S
0Vibrational energy level during fluorescence can take place.Transition speed k
fBe 10
7To 10
12s
-1Compare with fluorescent emission, fluorescence excitation produces under lower wavelength because in the absorption of emittance and between discharging owing to non-radiative process has been lost energy.
Fluorescence lifetime (FLT) is measuring of molecule on average stopped in excited state before fluorescent emission took place time quantum.Radiation lifetime τ
fBe equivalent to fluorescent transition speed k
fInverse.Opposite with this radiation lifetime of excited molecule, for the reality of investigating excited molecule-measurable-FLT τ must consider described non-radiative process:
K wherein
IcTransition speed between=vibrational state, k
Isc=transit to speed, the k of triplet
Q=quencher speed.Especially apparent thus, fluorescence quencher reduces FLT.Similarly function shows by so-called " acceptor dye ", and they absorb the excitation energy of donor dye and discharge the absorption energy in non-radiative mode or as fluorescence in non-radiative mode via resonance effect.This similarly reduces the FLT of donor dye.
Measure the method for fluorescence lifetime (FLT)
Use two kinds fundamentally diverse ways measure FLT: measurement in time domain (TD) and the measurement in frequency field (FD).
In TD-FLT, by short pulse optical excitation sample and measure the fluorescence decay curve.On the one hand, can write down the complete decay curve of each flash of light (flash) in principle.Yet this need have high time resolution and the transient recorder of bandwidth in the kilo-mega cycles per second scope.Yet in most of the cases, use (TCSPC) method of what is called " single photon counter time correlation ".TCSPC is a digital technology of calculating temporarily relevant with excitation pulse photon.In the method, experiment begins with excited sample and the excitation pulse that starts the hypervelocity clock.In case first emitted fluorescence photon arrives detecting device, clock will stop and storing this time.Repeat this process repeatedly.Because the process of fluorescent emission is a stochastic process, so can obtain the different time.The frequency of drawing these Measuring Time obtains the fluorescence decay curve as the figure of the function of this Measuring Time, and its time constant is this FLT (referring to Fig. 1).
The replacement scheme that FLT measures in time domain is the measurement in frequency field, and they also are called phase modulation (PM).By the continuous laser excited sample, the light intensity of this continuous laser is used the sinusoidal curve modulation.Usually use the frequency in the order of magnitude of fluorescent transition speed.When fluorescence excitation dyestuff in this way, its emission has to follow described modulation.Depend on this FLT, with respect to exciting, emission can postpone.This delay measurements is phase shift, and can calculate FLT according to this phase shift.In addition, the maximum of modulated transmission signals and the maximum difference between the minimum value increase with FLT and reduce, so that also can calculate this FLT thus.
The detection fluorescent measurement of biological test system
Especially prove in following method aspect high throughput and the high stability and to be suitable for detecting the biochemical test system:
Measure fluorescence intensity and can for example be used for measuring the fluorescence increment that carries out mmp reaction with the fluorescence peptide substrates, fluorescence aminocoumarin (AMC) is removed by cracking in this reaction.But the common autofluorescence of measuring large-signal screening material (screening substances) can disturb.In addition, this fluorescence intensity signals is to " self-filtering effect " sensitivity, if this solution comprises absorbing material.Except that the bleaching or volume/meniscus (meniscus) effect of fluorescent dye, also can disturb owing to the Dynamic Fluorescence quencher of the light scattering in molecular collision and the turbid solution.This fluorescence signal also depends on the concentration and the temperature of fluorescent dye in addition.As with regard to the purposes of screening method, all these interference sources have problems with regard to the stability of these tests and they.On the other hand, the standard among the HTS can be carried out and therefore develop into to this class test easily under very short Measuring Time.
If little fluorescence molecule for example is bonded on the significantly bigger molecule (for example protein), then can record deceleration in the rotation diffusion of macromolecule complex by measuring static fluorescence polarization.This method also becomes the standard of the bonding reaction among the HTS simultaneously.Disturbing effect owing to " self-filtering effect ", light scattering, concentration and temperature is unobtrusive.Yet fluorescence polarization also is subjected to the volume and the meniscus influence of true property (genuine) collisional quenching, autofluorescence, solution.
The another kind of method of bonding incident is utilized the fluorescence resonance energy transmission (FRET) between donor and acceptor dye, and wherein the excitation spectrum of the emission spectrum of this donor dye and this receptor dyestuff is overlapping.Compatibility agent agent must be carried donor dye and another compatibility agent agent must be carried acceptor dye in the reaction of described bonding.Because the space is approaching, FRET only produces when bonding takes place." self-filtering effect ", quencher and autofluorescence material can disturb FRET to measure.On the contrary, light scattering, photobleaching, volume and meniscus effect and concentration and temperature can not disturbed.Therefore, compare with fluorescence intensity, fluorescence polarization is the relative practical method that is used to measure interaction of molecules with FRET.
Compare with mentioned fluorescence method, fluorescence lifetime (FLT) is practical more.Only under a little situation, can be subjected to interference from strong autofluorescence material with suitable FLT.But FLT neither is subjected to " self-filtering effect " also can not be collided influence of quencher, photobleaching, bulk effect or concentration.These performances be doomed this practical method be used for the screening.On the other hand, do not have shaker test to build on FLT, mainly be so far since instrumental analysis than small throughput and higher cost.Powerful and the stable laser instrument and the modernization development of detection architecture have made FLT measure recently and can be incorporated into microtiter plate and therefore screen material.Therefore, Tecan company sold the commercial apparatus that is used to read microtiter plate, UltraEvolution first late period in 2002.
Known FLT uses:
The FLT measurement is applied to large number of biological knowledge topic.Used in more such fluorescent probe molecules any at this, when described molecule is bonded to kation such as Ca
2+(Schoutteten L., Denjean, P., Joliff-Botrel G., Bernard C., Pansu D., Pansu R.B., Photochem.Photobiol.70,701-709 (1999)), Mg
2+(Szmacinski H., Lakowicz J.R, J.Fluoresc.6,83-95 (1996)), H
+(Lin H.J., Szamacinski, Anal.Biochem.269,162-167 (1999)), Na
+(Nowaczyk K., Lederer W.J., Kirby M.S., Johnson M.L., CellCalcium 15,7-27 (1994) for Lakowicz J.R, SzamacinskiH.), K
+(Szmacinski H., Lakowicz J.R.in, " Topics in Fluorescence Spectroscopy " Vol.IV, (Lakowicz, J.R., Ed), 295-334 (1994)) or negative ion such as Cl
-When (A.S.Verkman, Am.J.Physiol253, C375-C388 (1990)), their performance and especially fluorescence lifetime can change.The variation of fluorescence lifetime also can be reacted by the bonding of molecule and be realized, this or since resonance energy transmission (quencher or FRET) produce littler donor dye FLT or, once in a while, cause bigger FLT.The activity that receives body tyrosine activating enzymes is for example measured (CurrentBiology 9 for F.S.Wouters, P.I.H.Bastiaens, 1127-1130,1999) by means of the anti-phosphotyrosine antibody of bonding Cy3-mark.
The application of also not describing the change of using FLT to measure molecular modification before this and not comprising the biological test system of bonding reaction.On the other hand, wherein directly measure enzyme the test of the modification of for example substrate molecule is had significant advantage, do not need the enzyme cascade because the substrate conversion of substrate can directly measure or make the visible bonding reaction of elementary substrate conversion indirectly.The screening substances advantage is that the material of testing no longer can the Interference Detection reaction.This can prevent pseudo-sampling (vorget uschte Hits) or the material that can not estimate owing to described interference.
The shaker test pattern of kinases/phosphatase
(taking off) phosphorylation of protein is general regulation mechanism, and cell is optionally modified with it outside conditioning signal is imposed on nuclear protein.The protein that carries out these biochemical modifications belongs to kinases or phosphatase.PDE hydrolysis second messenger cAMP or cGMP and can influence the cell signalling passage equally like this.Therefore these enzymes are the target molecules with big interest of medicine and plant protection research.
Screen kinase whose various pattern and set up, they all have such fact, and promptly phosphorylation reaction is always measured (except that the radiation method) indirectly.Therefore, these methods are basically to disturbing downstream enzyme cascade or the interference sensitivity of the material of the reaction that bonds.Certain methods even only limit to tyrosine kinase.
The classic method of measuring the cell protein phosphorylation state is based upon the introducing radioactivity
32On the basis of P-orthophosphate.Separating on gel should
32The P-phosphorylating protein also uses phosphoric acid photographic developer (phosphoimager) developing subsequently.Perhaps, the anti-phosphotyrosine antibody of phosphorylated tyrosine residue by radioactivity bonding mark bonds and measures by immunity test, for example immunoprocipitation or soak into (blotting) and detect.These methods are consuming time, because radioactive isotope needs to detect, and also are unsuitable for high flux screening (uHTS, high flux sieve), and this is because the consideration of the secure context relevant with handling radiomaterial.
More recent method replaces the radioimmunoassay test with ELISAs (enzyme-linked immunosorbent adsorption test).These methods are used purifying substrate protein white matter or the synthetic peptide substrates that is fixed on the substrate surface.After handling with activating enzymes, phosphorylation degree quantizes by the anti-phosphotyrosine antibody that is coupled to reinforcing agent enzyme (for example peroxidase), and this enhancing enzyme is bonded to the fixing substrate of phosphorylation.
Epps. wait people (US 6203994) to describe protein kinase and phosphatase and use HTS test, wherein use the phosphorylation reporter molecule of fluorescence-mark and the antibody of the described phosphorylation reporter molecule of special bonding based on fluorescence.Bonding is measured by fluorescence polarization, fluorescent quenching or fluorescence correlation spectroscopy (FCS).This method latent defect be the general preferably antibody that only can utilize the phosphotyrosine substrate (clone PT66 for example, PY20, Sigma).The example of the anti-phosphoserine that is fit to of minority or anti-threonine antibody be in the news (for example people such as Bader B., Journal ofBiomolecular Screening, 6,255 (2001), Panvera-Kit No.P2886) only.Yet these antibody have not only to be discerned phosphoserine but also discerns the adjacent amino acid whose performance as epi-position.Yet, the activating enzymes function be very substrate-single-minded and substrate sequence to differ widely be known.Therefore anti-phosphoserine antibody can not be used as general reaction reagent.
Perkin Elmer (Wallac) provide the test that is used for tyrosine kinase, and it is based upon on the basis of time-resolved fluorescence and the NE BY ENERGY TRANSFER from the europium chelate to allophycocyanin (also referring to EP929810).Same at this owing to use antibody, so this method is restricted to tyrosine kinase substantially.
Recently, Molecular Devices provides has the cationic nano particle of live metal in its surface as general bonding reaction reagent, and it is suitable for the phosphorylation reaction on tyrosine and serine and threonine simultaneously.Yet this bonding is reflected to be approximately under 5 highly acid pH and the higher ion concentration carries out.Therefore the bonding of nano particle need this be reflected at dilution strongly in the target buffering, and this is debatable under overall test volume with 1536 patterns, 10 μ l in uHTS.Here bonding is still by fluorescence polarization measurement.
As measuring method, fluorescence polarization relative complex and do not allow any horizontal survey of microtiter plate (MTP) at present.The Measuring Time of 1536-MTP therefore can grow very much and the horizontal survey of enzyme kinetics is impossible.In addition, the fluorescence polarization method is limited to very little fluorogenic substrate.
Kinase activity can form by downstream enzyme cascade by firefly luciferase or via ADP via ATP consumption in addition and measure.These test models are unfavorable to be: because indirect measurement method, they not only produce bigger data dispersion but also can run into the problem that material suppresses described cascade enzyme.
Measure if phosphorylation/dephosphorylation can be directly detects by FLT, then the measurement meeting is more direct and therefore can contain still less system or stochastic error.In addition, some test models are confined to tyrosine kinase or phosphatase can be removed, because no longer need specific antibodies.
Current test problem:
In many cases, might use the fluorogenic substrate that contains C-end-blocking dyestuff, for example be used for the aminocoumarin of proteinase, wherein the amino acid of C-end-blocking is removed.Protein incision enzyme in the cutting of the middle part of peptide sequence can be measured in the FRET test usually preferably, and wherein donor (for example EDANS) and acceptor dye (for example Dabcyl) are positioned at the end of substrate.Substrate cracking meeting increases fluorescence intensity, because acceptor dye no longer can the quencher donor dye.Yet, still have the proteinase that can not construct fluorogenic substrate for it.In the case, this enzyme reaction must rely on complicated chemical analysis (for example HPLC/MS GC/MS) or indirectly measures by chemical reaction or enzyme cascade.Therefore, must accept with regard to test stability and under detection reaction the screening material uncertain reaction with regard to any shortcoming.The analysis of this complexity is unsuitable for high flux screening.The enzyme that can not directly measure of reaction is included in and for example carries out following those that modify on the substrate in required flux: phosphorylation/dephosphorylation, sulphation/desulfurization acidifying, methylate/demethylation, the coupling of the removal/amino acid/peptide of oxidation/reduction, acetylation/deacetylated, amidation/desamidization, cyclisation/decyclizationization, conformation change, amino acid/peptide, encircle the contraction of expansion/ring, rearrangement, replacement, cancellation, addition reaction etc.
The present invention describes:
Fluorescence lifetime (FLT) changes with the change of chemical environment in principle.Yet these changes of FLT can't be predicted so far usually, especially when molecular modification hour.Therefore any FLT test of delivering before this always comprise with sensor molecule or with the bonding reaction of the compatibility agent molecule of quencher.
Surprisingly, we find that in experiment difference only is that the peptide of phosphorylation has had visibly different FLT.More detailed experiment has shown that this conclusion (statement) can expand to other peptide.For obtaining this acceptable FLT difference between phosphorylation and non-phosphorylating peptide, must test diversified condition in advance.Yet experiment also clearly reveals the FLT difference and can optimize by changing parameter.Based on these experiments, possible spread F LT measures all kinases and phosphatase enzymes.In addition, also allow in other reaction immeasurable aspect the HTS adaptability or that only can measure very indirectly by previous method.Generally speaking, below should use:
For example, if the phosphorylation state of reactant changes with changing into its product, then compatibly be coupled to dyestuff on it and should change by FLT this molecular modification is described.This method has the possibility that generally is applicable to tyrosine and serine/threonine kinase and phosphatase.This principle should also be applicable to other modification reaction, as sulphation/desulfurization acidifying, methylate/demethylation, the coupling of the removal/amino acid/peptide of oxidation/reduction, acetylation/deacetylated, amidation/desamidization, cyclisation/decyclizationization, conformation change, amino acid/peptide, the contraction of ring expansion/ring, rearrangement, replacement, cancellation, addition reaction etc.In fact might very rapidly carry out FLT and measure (be sometimes 50ms or still less/hole (well)), make this method be suitable for high flux screening.Significant practicality is to be particularly conducive to conflict such as the self-filtering effect concerning HTS uses, the influence of the concentration of autofluorescence, light scattering, photobleaching, volume/meniscus effect, fluorogenic substrate.
Only drawing 2 components (substrate and enzyme) from this application must mix with starting and measure this reaction.Conventional test method requires to add other reaction reagent (such as the cascade enzyme) usually reacting by survey record.Each suction moves (pipetting) step and causes the additive error of inhaling shift error and therefore causing measurement result, and this is also referred to as error propagation.The error of these propagation causes the difference of measurement result to increase.
Suction at minimum volume moves down, and in screening substances, the error of each independent process no longer can be ignored.Therefore, for wherein needing to inhale any test system that moves small size, and screening substances especially, be necessary to reduce the number of error source and therefore also be to inhale the rapid number of moving one's steps.
Draw from this point: the present invention compares with the conventional test method and can obtain simply more practical and more accurate measurement result.It is noticeable that these advantages especially become in screening substances.
Be characterised in that according to even test method of the present invention or according to the method for the present invention of directly and quantitatively measuring molecular modification: the fluorescence lifetime that molecule carries fluorescent dye and described molecule is different from the fluorescence lifetime of the molecule of modified.The fluorescence lifetime of the molecule that the fluorescence lifetime of the molecule of modified is crossed greater than unmodified.Yet the present invention also comprises according to test method of the present invention, wherein the fluorescence lifetime of the molecule crossed less than unmodified of the fluorescence lifetime of the molecule of modified.
This molecule can be for example organic molecule, especially peptide or peptide mimics, or inorganic molecule.This fluorescent dye can be for example cumarin, fluorescein, rhodamine, piperazine or cyanine dye.Employed fluorescent dye can be coupled to molecule with covalent manner or non-covalent mode.Between this fluorescent dye and molecule, spacer molecule can be set.The invention still further relates to according to test method of the present invention or be used to quantize the purposes of the method according to this invention of biochemical test.Can be used for quantizing biochemical test according to test method of the present invention or the method according to this invention, wherein enzyme can carry out for example following modification reaction: phosphorylation/dephosphorylation, sulphation/desulfurization acidifying, methylate/demethylation, the coupling of the removal/amino acid/peptide of oxidation/reduction, acetylation/deacetylated, amidation/desamidization, cyclisation/decyclizationization, conformation change, amino acid/peptide, the contraction of ring expansion/ring, rearrangement, replacement, cancellation, addition reaction etc.In addition, can be used for high flux screening-be particularly useful for the differentiating high flux screening of medicine reactive compound in useful mode according to test method of the present invention or the method according to this invention.
The invention still further relates to reaction kit, it comprises fluorescent dye-molecular conjugates and other reaction reagent that need carry out according to test method of the present invention or the method according to this invention.
Accompanying drawing is described:
The fluorescence decay time curve (logarithmic scale) of fluorescein-peptide conjugates of Fig. 1: 15nM.Go up measurement by TCSPC at Ultra FLT prototype (TECAN).
Fig. 2: phosphorylation (1) and non-phosphorylating (2) peptide (1:Fl-P1, the difference of fluorescence lifetime 2:Fl-1).Measuring Time 1s.Average and the standard deviation of 10 measurements is shown.
Fig. 3: the time route map of having drawn fluorescence lifetime (FLT (ps)) according to the variation in reaction time (time (s)).Between the reaction period of PDElb phosphodiesterase and fluorescein-cAMP,
Fluorescence lifetime changed to about 3350ps from about 3500ps in 100 minutes.This variation shows that directly Fl-cAMP transforms in Fl-AMP.Concentration by increasing BAY 383045 is inhibitory enzyme reaction (green triangle: 20 μ M, red square: 10 μ M, purple cruciform: 5 μ M, brown circle: 2.5 μ M, pink square: 1.25 μ M, blue rhombus: 0.7 μ M, green plus sige: 0.35 μ M, mazarine minus sign: 0.17 μ M, light blue negative sign: 0.08 μ M) gradually.
Fig. 4:, drawn the phosphorylation of fluorescein-kemptide-peptide conjugates and the fluorescence lifetime difference plot between the non-phosphorylating form for different pH values and 200mM NaCl (1:pH13,2:pH9.5,3:pH8,4:pH7,5:pH200 mM NaCl, 7:pH6).
Fig. 5: under different condition (1: water, 2:pH6,3:7,4:pH8,5:pH9.5,6:00mM NaCl, 7:2M NaCl), measure the fluorescence lifetime of the conversion of potential reaction thing (FJ23, hash) and its product (FJ24, black) and TAFI enzyme.This fluorescence lifetime depends on test condition hardly.Yet FJ23 (552ps) is different significantly with the fluorescence lifetime of FJ23 (2194ps).
Embodiment
1. the difference material of the fluorescence lifetime of phosphorylation and non-phosphorylating peptide (FL-P1 is to FL1):
Fl-P1: fluorescein-C6-TEGQYpQPQP-COOH, Eurogentec, phosphorylation
Fl-1: fluorescein-C6-TEGQYQPQP-COOH, Eurogentec, non-phosphorylating
Program:
Be intended to investigate between the fluorescence lifetime (FLT) of whether fluorescein-peptide-conjugate Fl-P1 and Fl-1 difference is arranged.For this reason, 10nM Fl-P1 and Fl-1 are dissolved among the 50mM HEPES (pH7.5) in each case.Measure this fluorescence lifetime (FLT) by Ultra FLT prototype (Tecan).In each case, average 10 measurements of every 1s.
The result:
The fluorescence lifetime of Fl-P1 is that the FLT of 3880ps and Fl-1 is 3600ps.Because Measuring Time is the standard deviation of 1s be very little (＜25ps), so these two kinds of molecular energies are very well distinguished (referring to Fig. 2).Can calculate z ' factor (being used in the potential source biomolecule test performance of the FLT measurement window of Fl-P1 and Fl-1 demarcation) of about 0.5 from standard deviation and the average fluorescence lifetime of Fl-P1 and Fl-1, this is enough to satisfy screening stage (campaign).This z ' factor is introduced the performance that is used to calculate the HTS test (OldenburgKR, J.Biomol.Screen 4,67-73 (1999) for Zhang JH, Chung TDY) by people such as Zhang 1999.The kinases of phosphorylation Fl-1 is p60 for example
SrcActivity be fine measurement by the FLT measurement.
Employed many kinase assay are terminal point tests at present, and wherein this dynamics can not be monitored continuously.And the different reactions data that must stop in different periods and the be obtained kinetic curve of to collect to provide then.
The measurement of fluorescence lifetime makes the phosphorylation dynamics can be directly and monitoring and need not detect the enzyme cascade immediately.This especially also is convenient to be provided with the induction time of remote control (robot) screening stage.
2. the optimization of the FLT difference between the phosphorylation of fluorescein-mark and non-phosphorylating kemptide peptide.
Material:
Fl-P-kemptide: fluorescein-C6-LRRApSLGCONH
2, Eurogentec, phosphorylation
Fl-kemptide: fluorescein-C6-LRRASLGCONH
2, Eurogentec, non-phosphorylating
0.1M NaOH, 50mM borate buffer solution pH9.5,50mM HEPES pH of buffer 8.0,50mM HEPES pH of buffer 7.0,50mM MES pH of buffer 6.0,200mMNaCl (low)
Program:
The quality of FLT test is improved with the increase of the difference of the fluorescence lifetime of reactant and product.In all cases, best big FLT difference can not measured immediately.On the other hand, should be for example by selection with in conjunction with the spacer molecule between various parameters such as fluorescent dye, dyestuff and substrate molecule or polarity, pH, the ionic strength of solvent or the FLT difference that other adjuvant improves initial acquisition.This embodiment shows how to improve the acidifying of fluorescein-kemptide-peptide conjugates (Fl-P-kemptide, Fl-kemptide) and the FLT difference between the non-phosphorylating variant significantly by improving pH.In each case, 50nM Fl-P-kemptide and Fl-kemptide are dissolved in the solution that material is partly described, their FLTs utilizes the transmission signal to measure to the improved Nanoscan device (IOM GmbH, Berlin, Germany) of instantaneous record device.Get the mean value of 16 decay curves for each data point.The sloping portion of the curve of logarithmic scale is estimated by linear regression and this negative slope converts FLT to mathematical way.
The result:
Fig. 4 shows Fl-P-kemptide and Fl-kemptide FLT difference under various conditions.The result here shows when the phosphorylation of pH kemptide when 6.0 are increased to 9.5 and the non-phosphorylating form difference with the FLT form and is improved.The result who is obtained shows in conjunction with the discovery of first embodiment: for many, even be not nearly all phosphorylation and non-phosphorylating peptide substrates concerning, can be by selecting correct fluorescent dye, sept and solvent nature or adjuvant to find to cause to have between the fluorescence lifetime between reactant and product the phosphatase or the kinases condition of big difference, this difference is enough to satisfy screening.Therefore for this fermentoid of being mentioned, can construct the ordinary test that is very easy to exploitation.In case understood the correct response condition of this enzyme, then this reaction only requires mixed enzyme and substrate.Dynamics subsequently can be monitored immediately and directly.This can easily be provided with the induction time on the HTS remote control equipment.Because the practical parameter of fluorescence lifetime, the minor fluctuations of volume and concentration of substrate only slightly influence the result of this measurement.In addition, compare, have and seldom inhale this class test of moving one's steps rapid and be commonly referred to be practical more with having additional other code test of inhaling move one's steps rapid (as detecting those of enzyme cascade requirement often).
3.PDE reaction
Material:
Fl-cAMP:8-fluoro-cAMP, BIOLOG Life Science Institute
PDE lb: phosphodiesterase lb (Laboratory of Dr.A.Tersteegen, Bayer AG) BAY 383045:Bayer AG
Program:
With phosphatase discussed above and kinases seemingly, PDE is the very important target of a class, especially in the indication field of cardiovascular, metabolism disorder, central nervous system, cancer and respiratory disease.Therefore have energy measurement cAMP or cGMP and change into separately that the ordinary test pattern of monophosphate has vital interests.Usually use and detect the enzyme cascade.This embodiment shows might directly measure this di-phosphate ester enzyme reaction.In this experiment, at first in the presence of the inhibitor B AY 383045 of variable concentrations, mix the PDE1b of 1 μ M Fl-cAMP and dilution in 1: 360.The dynamics of enzyme reaction at room temperature utilizes Ultra FLT prototype (Tecan) to measure.
The result:
Obtain in 100 minutes, do not having the FLT of Fl-cAMP under the inhibitor to change to about 3350ps in the process of Fl-AMP in reaction from about 3500ps.The concentration that improves BAY 383045 little by little suppresses described enzyme reaction (referring to Fig. 3).The variation of the fluorescence lifetime of the announcement of the concentration dependent clearly Fl-cAMP of inhibition di-phosphate ester enzyme reaction is relevant with enzymatic activity significantly.This proof can be used to this method screen the material that suppresses PDE in principle.Yet this measuring principle can also expand to kinases and phosphatase test and other enzyme test, takes place during the enzyme modification of substrate if measurable FLT changes.With regard to phosphatase discussed above and kinase assay, because interference-blunt measuring-signal and suction are seldom moved one's steps suddenly, the di-phosphate ester enzyme test with direct FLT detection of substrate modification is very practical.Described test method can be used for eliminating material to detecting the interference of enzyme.Below be applied to be based upon the described test method on the fluorescence lifetime measurement basis usually: for the remote control high flux screening stage, because directly and measure enzyme kinetics immediately, can be easily in experiment and the induction time of phosphodiesterase, kinases and phosphatase test and other enzyme test accurately is set.
4.TAFI the reactant of enzyme reaction and the fluorescence lifetime difference between product
Material: FJ23:Evoblue30-Ttds (sept)-IFTR-COOH, Jerini PeptideTechnologies
FJ24:Evoblue30-Ttds (sept)-IFT-COOH, Jerini PeptideTechnologies
Program:
The enzyme fibrin ferment that activates with fibrinolysis inhibitor (TAFI) is the carboxypeptidase that plays an important role in blood coagulation.The arginine of TAFI cleavage of peptide sequence IFTR.This reaction can detect by mass spectroscopy or chromatography.Two kinds of methods all are unsuitable for the high throughput materials test.Perhaps, can use some complicated enzyme cascade or chemical reaction, they produce measurable absorption, fluorescence or luminous signal.So far also do not describe and to be used for directly measuring the TAFI reaction and to be suitable for more high-throughout method simultaneously.Therefore, measured the fluorescence lifetime of conjugate FJ23 and FJ24, their boths carry the fluorescent dye that can activate at 630nm (Evoblue30, Mobitec) and their difference only is that the FJ24 conjugate does not have C-end-blocking arginine.FJ23 is the potential reaction thing of TAFI reaction, and FJ24 will be corresponding reaction product.FJ23 and FJ24 conjugate with the concentration of 60nM in various damping fluids (having 6,7,8 and 9.5 pH value) and in the presence of 200mM and 2M NaCl, dissolve.
The result:
The fluorescence lifetime of FJ23 is (552 ± 45) ps and the fluorescence lifetime of FJ24 is (2194 ± 18) ps, does not rely on pH value and NaCl concentration (referring to Fig. 5).Thus, can calculate fabulous is z ' factor of 0.89, and this shows the test that expectability is very powerful.Embodiment as first prokinase, phosphatase and PDE is the same, and showing can the synthetic reaction thing and the fluorescence conjugated thing of product, and their (under situations of TAFI) have very large fluorescence lifetime difference.This big fluorescence lifetime difference facilitates structure to have large-signal stability and the different tests that have fabulous difference between inhibiting substances by force.In addition, this embodiment has also shown the solution of TAFI-selectivity problem, and this problem is that permission is directly measured enzyme reaction and do not needed the high-throughout method that is suitable for of secondary detection reaction also not describe so far for TAFI.
Claims (8)
1. measure the method for molecular modification equably, directly and quantitatively, be characterised in that: the fluorescence lifetime that this molecule carries fluorescent dye and described molecule is different from the fluorescence lifetime of the molecule of modified.
2. the method for claim 1, wherein this molecule is an organic molecule, especially peptide or peptide mimics, or inorganic molecule.
3. as claim 1 and 2 described methods, wherein this fluorescent dye can be for example cumarin, fluorescein, rhodamine, a kind of cicada mentioned in ancient books, cyanine dye.
4. as the described method of claim 1 to 3, wherein this fluorescent dye is coupled to molecule with covalent manner or non-covalent mode.Spacer molecule can be arranged between fluorescent dye and the molecule.
5. as the described method that is used to quantize biochemical test of claim 1 to 4.
6. method as claimed in claim 5, wherein enzyme can carry out following modification reaction: phosphorylation/dephosphorylation, sulphation/desulfurization acidifying, methylate/demethylation, coupling, the contraction of ring expansion/ring, rearrangement, replacement, cancellation, the addition reaction of the removal/amino acid/peptide of oxidation/reduction, acetylation/deacetylated, amidation/desamidization, cyclisation/decyclizationization, conformation change, amino acid/peptide.
7. as the described method that is used for high flux screening of claim 1 to 6.
8. reaction kit, it comprises fluorescent dye-molecular conjugates and need carry out other reaction reagent as the described test method of claim 1 to 6.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE10348949.5 | 2003-10-18 | ||
| DE2003148949 DE10348949A1 (en) | 2003-10-18 | 2003-10-18 | Quantitative determination of molecular modifications, e.g. in high throughput screening, comprises labeling molecules with fluorescent dye, change in lifetime of fluorescence with modification being measured |
| DE200410022107 DE102004022107A1 (en) | 2004-05-05 | 2004-05-05 | Quantitative determination of molecular modifications, e.g. in high throughput screening, comprises labeling molecules with fluorescent dye, change in lifetime of fluorescence with modification being measured |
| DE102004022107.3 | 2004-05-05 | ||
| PCT/EP2004/011100 WO2005043137A1 (en) | 2003-10-18 | 2004-10-05 | Direct observation of molecular modifications in biological test systems by measuring fluorescence lifetime |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1867822A true CN1867822A (en) | 2006-11-22 |
| CN1867822B CN1867822B (en) | 2011-03-09 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN2004800306399A Expired - Fee Related CN1867822B (en) | 2003-10-18 | 2004-10-05 | Direct observation of molecular modifications in biological test systems by measuring fluorescence lifetime |
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| CN (1) | CN1867822B (en) |
| DE (1) | DE10348949A1 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293929A (en) * | 2014-09-28 | 2015-01-21 | 南京诺唯赞生物科技有限公司 | Method for quantitatively determining activity of T4 polynucleotide kinase |
| CN107112333A (en) * | 2014-08-08 | 2017-08-29 | 宽腾矽公司 | The integrating device in time point storehouse is carried out for the photon to reception |
| US10845308B2 (en) | 2016-12-22 | 2020-11-24 | Quantum-Si Incorporated | Integrated photodetector with direct binning pixel |
| US11344200B2 (en) | 2016-02-17 | 2022-05-31 | Tesseract Health, Inc. | Sensor and device for lifetime imaging and detection applications |
| US11391626B2 (en) | 2018-06-22 | 2022-07-19 | Quantum-Si Incorporated | Integrated photodetector with charge storage bin of varied detection time |
-
2003
- 2003-10-18 DE DE2003148949 patent/DE10348949A1/en not_active Withdrawn
-
2004
- 2004-10-05 CN CN2004800306399A patent/CN1867822B/en not_active Expired - Fee Related
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|---|---|---|---|---|
| US11719636B2 (en) | 2014-08-08 | 2023-08-08 | Quantum-Si Incorporated | Integrated device for temporal binning of received photons |
| CN107112333A (en) * | 2014-08-08 | 2017-08-29 | 宽腾矽公司 | The integrating device in time point storehouse is carried out for the photon to reception |
| US10775305B2 (en) | 2014-08-08 | 2020-09-15 | Quantum-Si Incorporated | Integrated device for temporal binning of received photons |
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| US11209363B2 (en) | 2014-08-08 | 2021-12-28 | Quantum-Si Incorporated | Integrated device for temporal binning of received photons |
| CN104293929A (en) * | 2014-09-28 | 2015-01-21 | 南京诺唯赞生物科技有限公司 | Method for quantitatively determining activity of T4 polynucleotide kinase |
| US11344200B2 (en) | 2016-02-17 | 2022-05-31 | Tesseract Health, Inc. | Sensor and device for lifetime imaging and detection applications |
| US10845308B2 (en) | 2016-12-22 | 2020-11-24 | Quantum-Si Incorporated | Integrated photodetector with direct binning pixel |
| US11719635B2 (en) | 2016-12-22 | 2023-08-08 | Quantum-Si Incorporated | Integrated photodetector with direct binning pixel |
| US12111261B2 (en) | 2016-12-22 | 2024-10-08 | Quantum-Si Incorporated | Integrated photodetector with direct binning pixel |
| US11112361B2 (en) | 2016-12-22 | 2021-09-07 | Quantum-Si Incorporated | Integrated photodetector with direct binning pixel |
| US11391626B2 (en) | 2018-06-22 | 2022-07-19 | Quantum-Si Incorporated | Integrated photodetector with charge storage bin of varied detection time |
| US12123772B2 (en) | 2018-06-22 | 2024-10-22 | Quantum-Si Incorporated | Integrated photodetector with charge storage bin of varied detection time |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1867822B (en) | 2011-03-09 |
| DE10348949A1 (en) | 2005-05-19 |
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