KR20110121070A - Method for the detection of fluorescence resonance energy transfer by measuring fluorescence lifetime and intensity - Google Patents

Abstract

PURPOSE: A fluorescence resonance energy transfer detecting method using fluorescent lifetime and fluorescence intensity is provided to measure fluorescent lifetime and fluorescence intensity and to detect fluorescence resonance energy transfer. CONSTITUTION: A fluorescence resonance energy transfer detecting method using fluorescent lifetime and fluorescence intensity is as follows. The fluorescent lifetime and magnetic covariance are used. A fluorescence signal extraction for measuring the fluorescent lifetime uses a time correlation single photon count method. The fluorescent lifetime is extracted by consisting histogram. Histogram is constituted in order to measure the fluorescence intensity.

Description

Method for the detection of fluorescence resonance energy transfer by measuring fluorescence lifetime and intensity

The present invention relates to the development of a method for detecting fluorescence resonance energy transfer (FRET) more accurately by measuring fluorescence lifetime and fluorescence intensity.

As a post-genome-related technology in the medical, drug development, and food industries, the interpretation of protein function is becoming important. In particular, in order to analyze the action of cells, studies on the interaction (binding and separation) between proteins, which are biological substances in living cells, and other proteins and small molecule compounds are necessary.

Regarding the interaction between such proteins, a method of analyzing using a fluorescence resonance energy transfer (FResonance Energy, hereinafter referred to as "FRET") phenomenon has been recently performed. In other words, the interaction between molecules in the several nanometer region is detected using fluorescence, and the detection using such FRET phenomenon is mainly performed using a microscope system.

In FRET, when the distance between the donor and acceptor lies below a certain level, an energy transfer occurs between the donor and the acceptor, resulting in a wavelength band different from that of the excitation light at the acceptor. The technique of measuring the distance between a donor and a recipient as it emits light.

When the energy state of the molecule is in an excited state and changes to the ground state, it gives energy corresponding to the energy difference between the two states. Fluorescence occurs when energy is released in the form of photons. On the other hand, if proper conditions are provided, the dipoles of the fluorescent molecules in the excited state can transmit energy by vibrating the dipoles of other fluorescent molecules in the surroundings. That is, energy is transferred by resonance phenomenon. The fluorescent molecules around the energy are excited and then change back to the ground to emit light corresponding to their emission spectrum. In this case, a fluorescent molecule that delivers energy is a donor, and a fluorescent molecule that is delivered is called an acceptor. The donor has a shorter wavelength of absorption or emission than the recipient.

On the other hand, there is a conventional method for detecting the FRET phenomenon, a method of simply measuring the light intensity, FRET detection method using the light intensity has a disadvantage that the accuracy is lower depending on the background noise and the concentration of the fluorescent material.

Recently, a method of measuring and detecting the lifetime of fluorescent materials has also been introduced. However, in calculating FRET efficiency, we need to know the lifetime of the fluorescent material that participates in FRET and the lifetime of the material that does not participate in FRET. Actually, the lifetime of the fluorescent material rarely has only one component, and most of them are two or more. Because of its composition, it is often used as the representative value using the average lifetime, which is the average of several fluorescence lifetimes. However, some components of the lifetime of the fluorescent material participate in the FRET, and some components do not participate, so it is not easy to determine whether the FRET has a simple life expectancy and the average life has not changed. In addition, there is a problem that the fluorescence lifetime may be changed even if it is not exchanged with the recipient because the fluorescence lifetime is also changed by other environmental factors.

In order to solve the problems of the prior art, the present inventors came to complete the present invention by using the light intensity as another parameter in addition to the fluorescence lifetime to detect the FRET more efficiently.

Accordingly, an object of the present invention is to provide a fluorescence resonance energy transfer detection method using fluorescence lifetime and fluorescence intensity.

In order to achieve the object of the present invention as described above, the present invention provides a fluorescence resonance energy transfer detection method using fluorescence lifetime and intensity measurement, characterized in that using the fluorescence lifetime and auto covariance.

In one embodiment of the present invention, the fluorescence signal extraction for measuring the fluorescence lifetime may use time correlated single photon counting (TCSPC).

In one embodiment of the present invention, the fluorescence lifetime can be extracted by constructing a histogram.

In an embodiment of the present invention, after constructing the histogram, the fluorescence lifetime may be extracted using any one of an exponential model, a maximum likelihood method, and a maximum entropy method.

In one embodiment of the present invention, the fluorescence intensity may be measured by constructing the histogram and integrating.

In one embodiment of the present invention, the magnetic covariance can be calculated directly from the data measured by time-correlated single photon counting or by obtaining partial fluorescence intensity.

In one embodiment of the present invention, the fluorescence lifetime can be calculated by the following equation (1).

(Equation 1)

In Equation 1, α _i represents a fractional amplitude and k represents the number of components of the fluorescence lifetime.

In one embodiment of the present invention, the magnetic covariance can be calculated by the following equation (2).

(Equation 2)

In Equation 2, N represents the number of data, x (n) represents TCSPC data.

In one embodiment of the present invention, the fluorescence resonance energy transition can be detected by obtaining a function F as shown in Equation 3 using the fluorescence lifetime and magnetic covariance.

(Equation 3)

In Equation 3, the fluorescence lifetime is represented by Equation 1, and auto covariance is represented by Equation 2.

In one embodiment of the present invention, the fluorescence resonance energy transfer detection method using the fluorescence intensity measurement is detected with a ratio before and after FRET occurs, the efficiency of the FRET can be expressed by the following equation (4). .

(Equation 4)

In one embodiment of the present invention, the fluorescence resonance energy transfer detection method using the fluorescence lifetime measurement is detected with a ratio of the fluorescence lifetime before and after FRET occurs, the efficiency of the FRET is expressed by the equation Can be.

(5)

The fluorescence resonance energy transfer detection method according to the present invention can increase the signal-to-noise ratio by reducing the influence on the background noise or the common noise by simultaneously using the fluorescence intensity and the fluorescence lifetime, and can detect FRET more accurately by reducing the false detection. It has an effect.

The present invention relates to a method for detecting fluorescence resonance energy transfer using fluorescence lifetime and fluorescence intensity, and more particularly, fluorescence resonance energy transfer using fluorescence lifetime and intensity measurement, characterized by using fluorescence lifetime and auto covariance. It is characterized by providing a detection method.

As used herein, the term "fluorescence resonance energy transfer (FRET)" means that when an energy donor, which is a shorter wavelength dye, absorbs energy from the outside, the excitation energy of the donor is released as light energy. It refers to a phenomenon in which only the long-wavelength fluorescence of the receptor is emitted by radiationless delivery to an energy receptor, a fluorescent protein (longer wavelength excitation dye). For the generation of acceptor fluorescence by FRET or photobleaching of donor emission light, the energy donor and acceptor must be located within a very short distance (<10 nm). When the donor, which is a fluorescent protein that emits light of a certain wavelength, and the receptor, which is a fluorescent protein that can absorb energy by resonating with the luminescence energy, is brought within a predetermined distance, the light energy irradiated from the donor to excite the donor from the outside comes from the donor. Radiationless transmission to the receptor reduces emission of the intrinsic wavelength of the donor, and FRET phenomenon occurs in which the receptor receiving energy from the donor emits light in its own wavelength range.

What is important in the FRET process is how closely the emission wavelength of the donor matches the absorption wavelength of the recipient. The more similar the wavelengths, the better the energy transfer. However, in practice, the donor can determine the pair of phosphors that FRET occurs well, depending on how much the donor's emission spectrum overlaps with the recipient's absorption spectrum because the light emitted or absorbed by the donor appears in the form of a spectrum rather than a single wavelength. . The equation for the degree of overlap between the donor and the recipient is as follows.

(Equation 1)

In the formula 1, when the wavelength is in the range of λ and λ + Δλ F D _(λ) is the fluorescence intensity of the donor, f D _(λ) is the fluorescence intensity of the normalized donor, ∈ _{A (λ)} represents the absorption rate of the grantor .

In the FRET phenomenon, the arrangement of the donor and donor transition dipoles is very important. The relative arrangement of donor and donor transition dipole directions is denoted by κ ^{2. In} general, if random averaging of donor and donor directions is possible, the approximation is 2/3.

Considering the overlapping degree of the spectrum and the direction of the transition dipole arrangement, κ _T , the ratio of energy transfer from donor to recipient, can be obtained as follows.

(Equation 2)

In Equation 2, _{Ｄ D} is the quantum efficiency of the donor in the absence of the donor, τ _D is the life time of the donor in the absence of the donor, N is the number of avogadro, n is the refractive index. In general, the refractive index of the biomolecule in the aqueous solution is set to 1.4.

Here, the energy transfer efficiency κ _T is related to the distance r.

(Equation 3)

In Equation 3, R _O represents the distance between the donor and the donor when the energy transfer efficiency is 50%.

The efficiency E of FRET can be expressed as the ratio of photons transmitted to the recipient among photons absorbed by the donor as follows.

(Equation 4)

Since FRET has a strong dependence on distance, two kinds of fluorescent materials can be attached to an object to be observed to observe the change in the intensity of the spectrum as the wavelength changes. Using this property, one can observe various phenomena occurring inside and outside the cell.

On the other hand, when the reaction ratio k ₁ , AB is dissociated and divided into A and B by k ₂ is the reaction rate for combining the two substances A and B to form a binder AB can be expressed by the following equation 5.

(Eq. 5)

If P _n (t) is defined as the probability that the number of ABs is n at time t, the rate of change of P _n (t) with time is given by Equation 6 below.

(Equation 6)

The probability mean M (t) and the deviation Ｖ (t) are defined as in Equation 7 below.

(Eq. 7)

After deriving M (t) and Ｖ (t) with respect to time and putting together the above equation for dp _n / dt, two equations can be obtained:

(Eq. 8)

Through these two equations, when there is a change in the number of particles due to stochastic chemical reactions, the reaction rate constant value can be obtained by considering only the probability average and the deviation without considering the variation.

On the other hand, when irradiating a light source corresponding to the absorption wavelength of the donor to the sample mixed with the donor and the receiver, it is necessary to observe the emission spectrum according to the wavelength to determine whether FRET occurred. If the distance between the donor and the donor is so large that it rarely occurs in the FRET, the donor goes to an excited state because the incident light is its absorption wavelength, and emits fluorescence. Only a few will fluoresce. Therefore, only the fluorescence spectrum of the donor appears strongly in the emission spectrum.

On the other hand, if FRET occurs well because the distance between the donor and the donor is close, the donor's fluorescence energy is transferred to the recipient, so the donor receives more energy from the donor, More molecules are going to be excited by the donor's energy, which in turn causes more fluorescence. Therefore, the fluorescence spectrum of the donor is lowered and the fluorescence spectrum of the donor is much larger than when the distance between the donor and the recipient is far.

In this way, the efficiency of FRET can be calculated using the change in intensity of fluorescence. The fluorescence intensity of when the only donor F _D, donor and grantor is the fluorescence intensity of F _DA la When FRET is the FRET efficiency E because that as donor fluorescence intensity is reduced in takes place is the following formula 9 or formula 10, when such Can be written as:

(Eq. 9)

(Eq. 10)

In Equation 10, τ ₀ represents the fluorescence life of a donor not participating in FRET, τ _fret represents the fluorescence life of a donor participating in FRET.

That is, in the present invention, the fluorescence resonance energy transfer detection method using fluorescence intensity measurement is detected with a ratio before and after FRET occurs, and the efficiency of FRET is expressed by Equation 9, and fluorescence resonance using fluorescence lifetime measurement The energy transfer detection method detects with a ratio of fluorescence lifetime before and after FRET occurs, and the efficiency of FRET is expressed by the following equation.

In the present invention, the fluorescence lifetime of the fluorescent material refers to the time taken for the fluorescent material to fall from the excited state to the ground state when it is excited by the light source and then goes up to the excitation state and falls back to the ground state. This fluorescence lifetime becomes a unique characteristic of each fluorescent material. The fluorescence lifetime is also changed by the environment of the material and also when the FRET phenomenon occurs by energy exchange between the two fluorescent materials. Therefore, in the present invention, the FRET can be detected using the fluorescence lifetime.

There are two methods of measuring fluorescence lifetime in the time domain and the other in the frequency domain. Recently, fluorescence lifetime is measured in the time domain using time-correlated single photon counting (TCSPC). Many methods are used.

In the present invention, it is preferable to extract the fluorescent signal using time correlation single photon counting (TCSPC) to measure the fluorescence lifetime. The histogram is constructed by measuring the photons of the donor fluorescent substance for a certain time by the TCSPC method, and the fluorescence lifetime is extracted from the histogram by using the constant method, and the average thereof is obtained.

(Eq. 11)

In Equation 11, α _i represents a fractional amplitude and k represents the number of components of the fluorescence lifetime.

 In the present invention, it is preferable to use any one of an exponential model, a maximum likelihood method, and a maximum entropy method to extract the fluorescence lifetime.

The histogram data obtained above are integrated to calculate fluorescence intensities and average over time of these intensities. Then, the normalized auto covariance is obtained using the TCSPC data and the mean of the shape and intensity. TCSPC data has the advantage of allowing simultaneous measurement of fluorescence lifetime and intensity.

(Eq. 12)

In Equation 12, N represents the number of data, x (n) represents TCSPC data.

In the present invention, it is preferable to calculate the magnetic covariance directly from the data measured by the time-correlated single photon counting method or to obtain a partial intensity.

Next, a new function F is derived by using Equations 11 and 12 as shown in Equation 13.

(Eq. 13)

The function F according to the present invention increases in value when the FRET is generated, and less than the conventional method due to the concentration of the fluorescent material, the background noise and the surrounding environment, it is possible to detect the FRET more efficiently.

Meanwhile, different fluorescent substances may be attached to antigens and antibodies, and antigen-antibody reactions may be observed, or fluorescent substances may be attached to enzymes and proteins to determine whether proteins react with enzymes. In addition, the FRET reaction can be traced through the FRET reaction by attaching a fluorescent material to the expected path of the material to be observed. In addition, by precisely estimating the FRET occurrence by attaching a fluorescent material at a specific position of a protein, the distance between two fluorescent materials can be determined. In this way, the distance between various positions of the protein can be determined. . When a specific part of a gene is expressed, the technique of manipulating the sequence of DNA so that the fluorescent protein is expressed together, such as green fluorescent protein (GFP) or red fluorescent protein (RFP), can be observed in any environment. It is easy to see if the gene is expressed. In addition, the change in ion concentration can be observed by using the property that FRET is expressed according to its concentration.

Therefore, the method of the present invention can increase the signal-to-noise ratio by simultaneously measuring the fluorescence lifetime and the fluorescence intensity, thereby reducing the influence on the background noise or the common noise, and more accurately the fluorescence resonance energy transfer by reducing the false detection. (FRET) has the advantage of being able to detect effectively. In addition, the FRET detection method can be useful for discovering the interaction between molecules in proteins or compounds in biotechnology, medical field, food industry, etc., as well as for detecting or screening various substances for the purpose of heavy metal detection. Can be used.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (11)

A fluorescence resonance energy transfer detection method using fluorescence lifetime and intensity measurement, characterized by using fluorescence lifetime and auto covariance. The method of claim 1,
The fluorescence resonance energy transfer detection method using fluorescence lifetime and intensity measurement, characterized in that the fluorescence signal extraction for measuring the fluorescence lifetime using time-correlated single photon counting (TCSPC). The method according to claim 1 or 2,
The fluorescence lifetime is a fluorescence resonance energy transfer detection method using fluorescence lifetime and intensity measurement, characterized in that by extracting the histogram. The method of claim 3,
After constituting the histogram, a fluorescence resonance energy transfer detection method using fluorescence lifetime and intensity measurement, characterized in that to extract the fluorescence lifetime using any one of an exponential model, the maximum likelihood method and the maximum entropy method. The method of claim 3,
Fluorescence resonance energy transfer detection method using fluorescence lifetime and intensity measurement, characterized in that for measuring the fluorescence intensity by constructing the histogram and taking the integral. The method of claim 1,
The magnetic covariance is calculated directly from the data measured by time-correlated single photon counting method or by calculating the fluorescence intensity (intensity), characterized in that fluorescence resonance energy transfer detection method using fluorescence lifetime and intensity measurement. The method of claim 1,
The fluorescence lifetime detection method using the fluorescence lifetime and intensity measurement, characterized in that the fluorescence lifetime is calculated by the following equation:
[Equation 1]

In the above, α _i represents the fractional amplitude, k represents the number of components of the fluorescence lifetime. The method of claim 1,
The magnetic covariance is calculated by the following Equation 2 fluorescence resonance energy transfer detection method using the fluorescence lifetime and intensity measurement:
[Equation 2]

In the above, N represents the number of data, x (n) represents TCSPC data. The method of claim 1,
A fluorescence resonance energy transfer detection method using fluorescence lifetime and intensity measurement, characterized by detecting the fluorescence resonance energy transition by obtaining a function F as shown in Equation 3 using the fluorescence lifetime and magnetic covariance:
[Equation 3]

In the above, fluorescence lifetime is represented by Equation 1, and auto covariance is represented by Equation 2. The method of claim 1,
The fluorescence resonance energy transfer detection method using the fluorescence intensity measurement is detected using a ratio before and after the occurrence of FRET, the efficiency of the FRET is expressed by the following equation using the fluorescence lifetime and intensity measurement Fluorescence Resonance Energy Transfer Detection Method:
[Equation 4]

. The method of claim 1,
The fluorescence resonance energy transfer detection method using the fluorescence lifetime measurement detects with a ratio of fluorescence lifetime before and after FRET occurs, and the efficiency of FRET is expressed by the following equation (5). Method for detecting fluorescence resonance energy transfer using measurement:
&Quot; (5) &quot;

.