TW201606285A - Method for measuring real-time kinetics of chemical reactions - Google Patents

Abstract

The present invention discloses a method for measuring real-time kinetics of chemical reactions. The biomolecules of interest should be labeled by the donor and acceptor fluorophores of a Forster resonance energy transfer (FRET) pair. When the biomolecules interact with each other, the donor and acceptor would give FRET signals. The fluorescence intensity signals of FRET and non-FRET are measured to calculate the concentrations of bounded FRET pair, dissociated donor alone molecule, and dissociated acceptor alone molecule. Then, chemical equilibrium constant can be achieved from calculated results of the concentrations. The present invention can be integrated into any instrument setup that can detect fluorescence intensity, and applied for both in vitro and in vivo samples to achieve fast, real-time and continuous measurements.

Description

Instant measurement method for biochemical reaction

The present invention relates to a technique for measuring intermolecular interactions, and more particularly to an instant measurement method for biochemical reactions for instantaneous quantitative measurement of intermolecular interactions in in vivo samples or chemical solution samples.

Generally, in the way of measuring the binding efficiency between molecules and molecules, a common surface plasmon resonance system is used, in which the measured molecular concentration must be known in advance and changed. The concentration of one of the molecules and the reaction are measured multiple times to quantitatively measure the binding efficiency between the molecules. However, in order to repeat multiple measurements of different concentrations, a sufficient number of biological samples must be used, which is difficult to quantify accurately for rare samples. Furthermore, the structure of the surface plasmonic resonance system is incapable of measuring in vivo, because the molecular concentration in the living body is difficult to control or change; and the system architecture cannot observe the continuous action of biomolecules in living organisms. Next, the combination of efficiency changes with time.

In addition to using the surface co-plasma resonance system described above to measure the intermolecular binding efficiency, Förster resonance energy transfer (FRET) related techniques can also be used.

For example, US Patent US 7,456,954 B2 and related papers (Biophysical) Journal, Vol. 88, April 2005, 2939-2953), which uses Foster resonance energy transfer fluorescent molecules to calibrate the molecules to be measured. This measurement method respectively excites Foster resonance. The fluorescence intensity of the energy-shifted fluorescent molecules, and the signals measured at different excitation wavelengths in different fluorescent bands; this way, the number of molecules in different states such as separation or combination can be judged. However, this measurement method is limited to single-molecule measurement, that is, only a single-molecule fixed on the surface of the slide can be measured using a total reflection fluorescent microscopy system. In other words, for a solution or a sample in a living body, the interaction between the molecules cannot be measured, and no immediate or continuous measurement is possible.

Another example is the US patent US 2013/0052656A1 and the European patent EP. 2196794A1, which measures the Foster resonance energy transfer under the framework of a flow cytometer. This patent is based on measuring the fluorescence half-life of fluorescent molecules and distinguishing the change of Foster resonance energy transfer by the value of the fluorescence half-life. Therefore, the fluorescence signal of Foster resonance energy transfer is also used to judge the Calibrate the interaction between molecules. However, since only the relative change of the fluorescence half-life is observed, the absolute value cannot be quantified, so it is necessary to carry out multiple measurements, and the concentration of the molecules is changed during the measurement, and the chemical reaction is calculated by using different concentrations of data. Binding constants and separation constants. This technology is similar to the above-mentioned surface plasmonic resonance system, which means that there must be multiple measurements of different concentrations, so it is not applicable to the measurement in the living body, and the instantaneous dynamic measurement cannot be performed.

In view of this, the present invention proposes an instant measurement method for biochemical reactions, Solve these shortcomings that existed in the prior art.

The main object of the present invention is to provide an instant measurement method for biochemical reactions. The method utilizes the quantification of the fluorescence intensity of the Foster resonance energy transfer to obtain the respective molecular concentrations of the two biomolecules in the separated and bound state, thereby obtaining the reaction rate of the biological reaction, so the present invention can indeed be in vivo In the sample, the reaction rate of the biochemical reaction is measured quantitatively in order to study the interaction between the molecules in the living body and the binding efficiency, which is a very convenient and practical biochemical reaction quantitative detection technology.

Another object of the present invention is to provide an instant measurement method for biochemical reactions. The method is not limited to the type of sample, the living sample or the solution sample, and the measurement method can be applied for immediate and continuous measurement, and the dynamic or excited chemical reaction process can be observed.

For the above purposes, the present invention proposes an instant measurement method for biochemical reactions, The steps include: firstly providing a sample to be tested, wherein there is a biomolecule in which a binding reaction occurs; and respectively, the bioluminescent molecule is calibrated by the donor fluorescent molecule and the acceptor fluorescent molecule having different excitation wavelengths, and the donor body is applied. And the receptor fluorescent molecule has known fluorescent molecular characteristics; after the calibration is completed, the donor and acceptor fluorescent molecules can be excited at the donor or acceptor absorption wavelengths at different time points, and The fluorescence signal is measured instantaneously at the emission wavelength of the body or the receptor. The fluorescent signal is derived from the self-fluorescent signal that is calibrated to the biomolecule donor or the receptor does not produce Foster resonance energy transfer, and when the biomolecule When combined, the Foster resonance energy transfer signal generated by the donor and acceptor fluorescent molecules forms a complex Foster resonance energy transfer pair; after obtaining these fluorescent signals, the combined Foster resonance is calculated accordingly Three concentrations of energy transfer pair, donor fluorescent molecules that do not produce Foster resonance energy transfer, and acceptor fluorescent molecules that do not generate Foster resonance energy transfer, and then according to these three concentrations To obtain a reaction rate between the biological molecules.

Among them, these fluorescent signals are measured under three measurement conditions: The hair is absorbed at the donor absorption wavelength and measured at the donor emission wavelength; the other is excited at the donor absorption wavelength and measured at the acceptor emission wavelength; and excited at the acceptor absorption wavelength and emitted at the receptor The wavelength is measured to thereby measure the fluorescence signal to obtain the aforementioned three concentrations.

The purpose, technical contents, features, and effects achieved by the present invention will become more apparent from the detailed description of the appended claims.

10‧‧‧ sample

LED1, LED2‧‧‧Lighting diode

PMT1, PMT2‧‧‧photomultiplier

DM1~DM4‧‧‧ dichroic filter

D‧‧‧body fluorescent molecule

A‧‧‧Receptor Fluorescent Molecules

X, Y‧‧ biomolecules

Figure 1 is a schematic diagram of a measurement architecture for an embodiment of the invention.

Figure 2 is a schematic flow chart of the instant measurement biochemical reaction of the present invention.

Figure 3 is a schematic diagram of the irradiation wavelength and biomolecule of the measurement conditions used in the present invention, wherein (A) is a time-series diagram of the irradiation method used in the present invention, and (B)-(D) is at the emission wavelength The fluorescent signal is detected, and the (E)~(G) map is a schematic diagram of the biomolecule reaction.

Figure 4 is a schematic illustration of one embodiment of a fluorescence excitation and emission spectrum for selecting a Foster resonance energy transfer pair of the present invention.

Figure 5 is a schematic illustration of the biomolecules X and Y of the calibrated fluorescent molecules used in the present invention in a separated state.

Figure 6 is a schematic diagram showing the binding state of the biomolecules X and Y of the calibrated fluorescent molecules used in the present invention.

Figure 7 is a schematic diagram showing the reaction efficiency of different reactions in the energy transfer between Foster resonance energy transfer and donor D and receptor A in the present invention.

In biomolecular reactions, Foster can be utilized between biomolecules that attract each other. The Förster resonance energy transfer (FRET) pairs of the donor fluorescent molecule and the acceptor fluorescent molecule are separately calibrated. When the molecules are mutually reacted, the labeled donor fluorescent molecule The FRET signal is generated by the fluorescent molecule of the receptor, and the fluorescence intensity of the FRET signal can indicate the concentration (number) of the biological molecules that have been combined with each other, and the fluorescence intensity of the signal of the single donor and the individual receptor is It is related to the concentration of biomolecules in the separated state. In the method of the present invention, the measured FRET signal and the fluorescence intensity of the non-FRET signal are used to calculate the concentration of reactants and products in a chemical reaction. The rate of chemical reaction can then be obtained from the calculations of these concentrations, and several basic fluorescence characteristics of the FRET pair, including the extinction coefficient of both the donor and the acceptor, should be obtained prior to use of the method of the invention ( Extinction coefficient) and fluorescence quantum yield and FRET efficiency between donor and acceptor.

The invention can be applied to any instrumentation apparatus for measuring FRET fluorescence signals, such as a fluorescence microscope, a flow cytometer or a microanalytical tray. 1 is a schematic diagram of a measurement architecture for use in an embodiment of the present invention for obtaining FRET. As shown, the device includes donor fluorescent molecules and acceptor fluorescence having at least two different excitation wavelengths. Molecules, according to the present embodiment, two light-emitting diodes LED1, LED2 are used to illuminate the sample 10, the LED1 excites the donor fluorescent molecule at the donor absorption wavelength D _EX , and the LED 2 excites the receptor fluorescence at the acceptor absorption wavelength A _EX molecule. In sample 10, the biomolecule in which the binding reaction occurs is calibrated using the donor fluorescent molecule and the acceptor fluorescent molecule, and the sample can be a living cell or a chemical solution. Then, photomultiplier tubes (PMTs) PMT1, PMT2 are used to detect the fluorescence signals of the donor and acceptor fluorescent molecules, PMT1 detects the fluorescent signal at the donor emission wavelength D _EM , and PMT2 is detected at the receptor. A fluorescent signal at the wavelength A _EM is emitted. The dichroic filters DM1 to DM4 as shown in Fig. 1 are used to reflect and transmit the selected wavelength of light. Among them, the LEDs LED1 and LED2 are alternately activated at different time points, so that the donor fluorescent molecules and the acceptor fluorescent molecules are excited at different time points. Therefore, the photomultiplier tubes PMT1 and PMT2 are recorded at different times. The FRET pair of signals in different states, the illumination and detection method is designed to distinguish the fluorescent intensity signals that have been combined with the fluorescent molecules and the separated fluorescent molecules.

After explaining the measurement architecture used in the present invention, the method of the present invention will be described The method uses FRET to obtain the chemical reaction concentration and the reaction rate. For the mutually interactive molecular interaction reaction, in order to calculate the reaction rate according to the molecular concentration measured according to the present invention, the reaction rate of the reaction must be obtained first. form. Please refer to the schematic diagram of FIG. 1 and the flowchart shown in FIG. 2. First, as shown in step S10, a sample to be tested is first provided, and at least two biomolecules in which a binding reaction can occur are present therein. For example, proteins, DNA, RNA, peptides, protein fragments, and the like. Then, as shown in step S12, a FRET pair having different excitation wavelengths is included. The FRET pair includes a donor fluorescent molecule and an acceptor fluorescent molecule, and the number of biomolecules is 1:1 for the number of fluorescent molecules. The ratios are separately calibrated for the two biomolecules, so the intensity of the fluorescent signal of the donor fluorescent molecule and the acceptor fluorescent molecule can be used to represent the concentration of the biomolecule to which it is calibrated. Moreover, the donor fluorescent molecule and the acceptor fluorescent molecule have known fluorescent molecular characteristics, including the extinction coefficient of the donor fluorescent molecule and the acceptor fluorescent molecule, the donor fluorescent molecule and the acceptor fluorescent device. Fluorescence quantum yield of light molecules and FRET efficiency between donor and acceptor. Then, as shown in step S14, the sample 10 is illuminated by the LEDs LED1 and LED2 to sequentially excite the donor fluorescent light and the acceptor fluorescent molecule at a donor or acceptor absorption wavelength at different time points, and Different fluorescent signals are measured instantaneously at the donor or acceptor emission wavelength. The measured fluorescent signals include the Foster resonance energy transfer signals generated by the donor and the receptors to which the bound biomolecules are bound and form a complex Foster resonance energy transfer pair, and are calibrated on the biomolecule but not A fluorescent signal produced by a donor fluorescent molecule or a receptor fluorescent molecule that produces a Foster resonance energy transfer. Wherein, the measured fluorescent signals are carried out under three measurement conditions, one is excitation at the donor absorption wavelength, and is measured at the donor emission wavelength; the other is excitation at the donor absorption wavelength, It is measured at the emission wavelength of the acceptor; the last one is excited at the absorption wavelength of the acceptor and measured at the emission wavelength of the acceptor. With these three sets of measurement conditions, three sets of fluorescent signals can be obtained correspondingly. Finally, as shown in step S16, based on the fluorescent signals, the concentration of the bound FRET pair, the concentration of the unbound (isolated) donor fluorescent molecule, and the concentration of the unbound (isolated) acceptor fluorescent molecule can be calculated. The three concentrations, and the reaction form of the binding reaction which has been obtained in advance, further obtain the reaction rate of mutual reaction between the biomolecules.

Figure 3 is a schematic diagram of the irradiation wavelength and biomolecule of the measurement conditions used in the present invention. As shown in the figure, the above three sets of fluorescent signals are measured to solve three unknown concentrations: the FRET pair has been combined, The concentration of the donor fluorescent molecule bound (separated), and the unbound (isolated) acceptor fluorescent molecule. Figure 3(A) shows a time-series schematic of the illumination method used in the present invention, set at different time points to excite the D _EX and A _EX wavelengths. Figures 3(B) and 3(E) show that when the sample is excited at the donor absorption wavelength D _EX , the signal detected at the donor emission wavelength D _EM includes two cases: calibration in the binding molecule XY The donor D does not have the fluorescence energy left by the Foster resonance energy transfer to the receptor A, and the fluorescent signal emitted after the donor D is excited on the molecule X in the separated state. . Figures 3(C) and 3(F) show that the excitation is at the absorption wavelength D _EX and the fluorescence signal is detected at the receptor emission wavelength A _EM . The signal source includes three types: receptors from the binding molecule XY. A is excited by the FRET to emit the fluorescent light, and is calibrated to the molecule Y in the bound state and the receptor A in the unbound state (separated) molecule Y, because the donor absorption wavelength D _EX is directly excited and generates fluorescence. Signal. In the 3(D) and 3(G) diagrams, the sample is excited at the acceptor absorption wavelength A _EX and detected at the acceptor emission wavelength A _EM , so that it can be in either the bound state or the separated state. Receptor A is activated and a fluorescent signal is emitted. In the present invention, only the three sets of fluorescent signals are required to calculate the reaction rate of the chemical concentration and the chemical reaction. In Figure 3, the continuous rotation excitation architecture is designed to measure the dynamic or concentration changes of the donor and acceptor, so using Figure 3 with the measurement architecture shown in Figure 1 can be achieved quickly. Instantly observe the reaction.

Having described the main technical features of the present invention in detail, the present invention will be described hereinafter. The technical principles used and the calculation methods.

As shown in FIG. 4, it is a schematic diagram of one embodiment of fluorescence excitation and fluorescence spectra of a FRET pair selected in the present invention, which comprises a donor excitation spectrum D1, a donor emission spectrum D2, and an acceptor excitation spectrum. A1 and the acceptor emit spectrum A2, and the spectral overlap region between the donor emission spectrum D2 and the acceptor excitation spectrum A1 is quite necessary for the occurrence of FRET. When the donor fluorescent molecule is excited at the wavelength D _EX , the donor fluorescent molecule emission can be detected between the bands D _EM , and the excitation wavelength and emission spectrum of the acceptor fluorescent molecule are labeled as A _EX and A _EM . Fluorescence detection of D _EM and A _EM can be resolved using a spectrometer, or all fluorescent signals in the D _EM and A _EM bands can be measured. Both detection methods can be used. Select the appropriate values for D _EX and A _EX so that they can only be used to excite the donor or acceptor and avoid exciting other fluorescent molecules. As for D _EM and A _EM , the spectral bands should also be avoided from other fluorescent molecular weight measurements. Fluorescent signal.

The biomolecules X and Y are calibrated with the donor fluorescent molecule D and the acceptor fluorescent molecule A, respectively, and the binding reaction rate and chemical kinetics of the interaction between them can be utilized. The FRET method of the invention was measured. As shown in Fig. 5, when the biomolecules X and Y are separated, the donor D and the receptor A are far apart from each other, so there is no Foster resonance energy transfer between the donor D and the acceptor A, therefore, The measured fluorescent signal is derived from a separate donor fluorescent molecule and acceptor fluorescent molecule. As shown in Fig. 6, when the biomolecules X and Y are combined, the donor D and the receptor A will be close to each other, and the FRET signal can be detected at this time. Since the biomolecule and the fluorescent molecule are calibrated in a 1:1 ratio, the concentration of the bound biomolecules X and Y is equal to the concentration of the donor D and the acceptor A having FRET, which is represented by [DA]; The concentration of the individual biomolecule X is equal to the concentration of the donor D [D], and the concentration of the individual biomolecule Y is equal to the concentration of the receptor A [A].

The reaction between biomolecules is as shown in formula (1), and the fluorescence signal measured by the FRET is used, and the chemical reaction rate in the formula (1) can be expressed as the formula (2), and the reaction rate can be in the form of Set before measurement and calculation. As described above, the concentrations of biomolecule X and biomolecule Y are equal to the concentration of the corresponding donor and acceptor fluorescent molecules, respectively, and can be calculated by quantifying the concentration of donor D and acceptor A in the separated state. The chemical reaction rate K _{a is obtained} .

Figure 7 is a schematic diagram showing the fluorescence emission process of FRET versus donor D and acceptor A in the present invention. The excited state and the ground state of the donor are labeled D* and D, respectively, and the excited and ground states of the acceptor are shown. Marked as A* and A, respectively, the reaction dynamic constant of FRET is k _et , and the reaction dynamic constants of donor and acceptor fluorescence emission are respectively with And the dynamic constant of the donor reaction in the non-radiative decay process And receptor reaction dynamic constant . When FRET ( k _et =0) does not occur between D and A, the fluorescence reaction functions I _D (t) and I _A (t) of this receptor and donor can be expressed as the following formula (3) and formula ( 4):

When FRET( k _et ≠0) occurs between D and A, the fluorescence reaction functions I _D ' (t) and I _A ' (t) of the FRET receptor and the donor can be expressed as the following formula (5), respectively. And equation (6):

The fluorescence intensity of the donor and acceptor fluorescent molecules is determined by the molecular concentration of the FRET pair separated and bound, and the fluorescence intensity measured from the donor emission spectrum is measured by the above formulas (3) to (6). Is D _EM , and the fluorescence intensity measured from the acceptor emission spectrum is A _EM , which are respectively expressed as shown in formula (7) and formula (8):

Here, the quantum yield of energy transferred from the donor to the receptor, also referred to as FRET efficiency, is defined as . The fluorescence quantum yields of donor and acceptor are with They are all functions of the wavelength of the fluorescent emission. The extinction coefficients of the donor and the receptor are with It is also a function of the absorption wavelength. In the method of the present invention, it is first necessary to obtain the fluorescence quantum yield, extinction coefficient and FRET efficiency of the FRET pair in order to calculate the chemical reaction rate obtained from the fluorescence measurement. To calculate the chemical reaction rate, three kinds of fluorescence should be obtained. Signal: 1. Excitation at the donor absorption wavelength At the emission wavelength of the acceptor Measurement, its system can be expressed as 2, excitation at the donor absorption wavelength Excipient emission wavelength Detection, which can be expressed as ; 3, excitation at the receptor absorption wavelength At the emission wavelength of the acceptor Detection, which can be expressed as This is .

The calculation of the foregoing steps is used to distinguish signals 1 to 3, and the ratio of the receptor signals (R _A ) at different excitation wavelengths is as shown in equation (9): From equation (9), the measured fluorescence signals (R _A ) and extinction coefficients are measured. with The molecular concentration ratios d ₊ and α can be calculated as shown in the following formulas (10) and (11):

Then, the concentration [DA] of the bound FRET pair and the unbound receptor concentration [A] can be calculated. According to the formula (11), the formula (12), the formula (13) and the formula (14) can be obtained:

Similarly, the signal 2 is divided by the signal 3 as the donor signal ratio (R _D ), as shown in equation (15):

The concentration ratio β of the bound FRET pair on the unbound donor pair is as shown in equation (16): According to formula (13) and formula (16), the unbound donor fluorescent molecule concentration [D] can be calculated, as shown in formula (17):

According to the results of the above formula (13), formula (14) and formula (17), [DA], [A] and [D] can be obtained, thereby obtaining a reaction rate ( ).

Therefore, the present invention can distinguish the fluorescent signal of the biomolecule in the separated or bound state, and can calculate the molecular concentration corresponding to the intensity of the fluorescent signal. Furthermore, the present invention can be applied to any device structure capable of measuring fluorescence, and is not limited to the type of sample, such as a living sample or a solution sample, and the measurement method can be applied.

The invention only needs to measure the three kinds of fluorescent signals mentioned above, and through the calculation of the formulas (9) to (17), the instantaneous biomolecule concentration and the biochemical reaction rate at the measuring time point can be obtained, and the weight is not required. The measurement of the concentration of different samples is repeated several times. Therefore, the present invention can indeed perform rapid and immediate measurement, and by the continuity measurement method shown in FIG. 3, it is possible to observe a chemical reaction process which is dynamic or changed by excitation, and is widely used.

The embodiments described above are merely illustrative of the technical spirit and the features of the present invention, and those skilled in the art can understand the contents of the present invention and implement them. That is, the equivalent variations or modifications made by the spirit of the present invention should still be included in the scope of the present invention.

Claims (6)

An instant measurement method for a biochemical reaction, comprising the steps of: providing a sample to be tested, wherein at least two biomolecules are mutually reactive; and the donor fluorescent molecule and the acceptor fluorescent molecule having different excitation wavelengths respectively The two biomolecules are calibrated, and the donor fluorescent molecule and the acceptor fluorescent molecule have known fluorescent molecular characteristics; the donor fluorescent is excited at the donor or acceptor absorption wavelength at different time points. The photomolecule and the acceptor fluorescent molecule produce the following three fluorescent signals: a Foster resonance energy transfer pair generated by the donor and the receptor fluorescent molecule calibrated in the bound state, and labeled in the biomolecule but a fluorescent signal generated by the donor fluorescent molecule or the acceptor fluorescent molecule that does not generate Foster resonance energy transfer; and based on the fluorescent signals, the combined Foster resonance energy transfer pair is calculated and not generated The Foster resonance energy transfer of the donor fluorescent molecule and the acceptor fluorescent molecule and other three concentrations, thereby obtaining the reaction rate of the two biomolecules. The method for immediate measurement of a biochemical reaction according to claim 1, wherein the sample to be tested is a living cell or a chemical solution. An immediate measurement method for a biochemical reaction according to claim 1, wherein the fluorescent molecular characteristic comprises an extinction coefficient of the donor fluorescent molecule and the acceptor fluorescent molecule, a fluorescence quantum yield, and the Foster resonance energy transfer efficiency. . An immediate measurement method for a biochemical reaction according to claim 1, wherein the fluorescent signals are performed under the following three measurement conditions: excitation at the absorption wavelength of the donor, and measurement at a donor emission wavelength; excitation At the donor absorption wavelength, and at the receptor emission wavelength; The absorption wavelength at the receptor is measured at the emission wavelength of the acceptor. The method for immediate measurement of a biochemical reaction according to claim 1, wherein the donor fluorescent molecule and the acceptor fluorescent molecule respectively calibrate the biomolecule in a ratio of 1:1. An immediate measurement method for a biochemical reaction according to claim 1, wherein the biomolecule can be a protein, a DNA, an RNA, a peptide or a protein fragment.