JP2015048385A - Fractionation method for composite fluorescent material and fraction of composite fluorescent material - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of fractionating a composite fluorescent material usable as a sensor for quantitative environmental measurements and a fraction of a composite fluorescent material.SOLUTION: A method of fractionating a composite fluorescent material includes: a composite fluorescent material formation step of synthesizing a composite fluorescent material composed of a semiconductor quantum dot fluorescent material and an organic molecule capable of causing fluorescence resonance energy transfer between the semiconductor quantum dot fluorescent material and the molecule, bonded together from a first view point; and a fractionation step of fractionating the composite fluorescent material by chromatography. In the fractionation step, there is applied such an amount of the composite fluorescent material as to secure the condition that the ratio of the organic molecule to the semiconductor quantum dot fluorescent material in a fraction fractionated by chromatography changes as the elution area increases.

Description

  The present invention relates to a composite phosphor fractionation method and a composite phosphor fractionation solution, and more specifically, a composite phosphor fractionation method in which a semiconductor quantum dot phosphor and an organic molecule are combined, The present invention also relates to a fraction of the composite phosphor obtained using the fractionation method.

  In recent years, organic dyes that emit light by fluorescence resonance energy transfer between semiconductor quantum dot phosphors or fluorescent molecules that emit light by fluorescence resonance energy transfer between semiconductor quantum dot phosphors. A fluorescent complex in which organic molecules labeled with (hereinafter referred to as “organic dye bodies”) are bound has attracted attention particularly in applications for bioimaging (see Non-Patent Document 1). This is because the luminous efficiency of organic pigments often changes depending on the surrounding environment (pH, temperature, solvent, interaction and bonding with surrounding molecules, etc.), whereas semiconductor quantum dot phosphors In such a case, there are few changes depending on the environment (less environmental dependency), and the characteristics are strong and low in photobleaching, while the molecular size is in the nanometer unit and excellent compatibility with biomolecules. .

  Utilizing these properties of semiconductor quantum dot phosphors and organic pigments, the excitation light excites semiconductor quantum dot phosphors in the complex phosphor in the living body, and from semiconductor quantum dot phosphors to organic pigments. The organic pigment body is caused to emit light by generating a fluorescence resonance energy transfer (hereinafter referred to as “Fluorescence Resonance Energy Transfer”, sometimes referred to as “FRET”). And, it is expected to enable quantitative and real-time monitoring of the in vivo environment by measuring the fluorescence intensity ratio between the fluorescence intensity of the semiconductor quantum dot phosphor and the fluorescence intensity of the organic dye body. .

  In such real-time monitoring of the in vivo environment, the creation of composite phosphors that can be used as sensors has been extensively studied (see Non-Patent Document 2: hereinafter referred to as “conventional example”). The technique described in this conventional example basically uses a chemical reaction between a functional group on the surface of a semiconductor quantum dot phosphor and an organic dye, and a simple ratio of semiconductor quantum dot phosphor and Some of them mix with organic pigments to advance chemical reactions.

Phys. Chem. Phys., 2009, 11 pp. 17-45 Advanced Drug Delivery Reviews 64 (2012), pp.138-166

  By the way, in the conventional technology, the reaction between the semiconductor quantum dot phosphor and the organic dye does not necessarily occur with the same probability, so the number of organic dyes bound to the surface of the semiconductor quantum dot phosphor is inevitably. A large variation occurs. In addition, the number of functional groups on the surface of the semiconductor quantum dot phosphor is not uniform and is prepared in a state having a certain width and is commercially available.

For this reason, when the composite phosphor prepared by the conventional technique is used, even if the composite phosphor is prepared at the same time, the measured fluorescence intensity ratio varies greatly. This large variation is quantitative using the ratio between the intensity of the fluorescence generated by the semiconductor quantum dot phosphor and the intensity of the fluorescence generated by the organic dye that has undergone fluorescence resonance energy transfer from the semiconductor quantum dot phosphor. This is a fatal defect as a sensor for environmental measurement.
In addition, with a commercially available simple column recommended by the manufacturer and distributor of quantum dots, the separation of molecular weight is quite rough, and usually only the degree of separation from unreacted excess organic molecules is expected. In other words, it is difficult to think that the quantum dots to which the organic molecules are not bonded and the quantum dots to which the organic molecules are bonded are sufficiently distinguished.

  The present invention has been made in view of the above circumstances, and provides a fractionation method of a composite phosphor that can be used as a sensor for quantitative environmental measurement, and a fraction of the composite phosphor. Objective.

  From the first aspect, the present invention synthesizes a composite phosphor in which a semiconductor quantum dot phosphor and an organic molecule capable of generating fluorescence resonance energy transfer between the semiconductor quantum dot phosphor are combined. A composite phosphor forming step; and a fractionation step of fractionating the composite phosphor by a chromatography method. In the fractionation step, the semiconductor quantum dot fluorescence in the fraction fractionated by the chromatography method A method for fractionating a composite phosphor, comprising applying the amount of the composite phosphor, wherein the ratio of the organic molecules to the body changes with an increase in elution volume.

  In this composite phosphor fractionation method, in the composite phosphor forming step, a composite in which a semiconductor quantum dot phosphor and an organic molecule capable of generating fluorescence resonance energy transfer between the semiconductor quantum dot phosphor are combined. A phosphor is synthesized. In this composite phosphor forming step, as in the case of the above-described conventional example, a certain proportion of semiconductor quantum dot phosphors and organic dyes are simply mixed to advance a chemical reaction.

Subsequently, in the fractionation step, the ratio of the organic molecule to the semiconductor quantum dot phosphor in the fraction fractionated by the chromatographic method is applied by applying the amount of the composite phosphor that changes as the elution volume increases. To fractionate the composite phosphor. As a result, a fraction with improved uniformity of fluorescence resonance energy transfer can be obtained.
Therefore, according to the composite phosphor fractionation method of the present invention, a composite phosphor that can be used as a sensor for quantitative environmental measurement can be obtained.

  In the method for fractionating a composite phosphor of the present invention, the chromatography method can be a gel filtration column chromatography method. In this case, since it can be fractionated using an aqueous solution, aqueous dispersion, organic solvent solution, organic solvent dispersion, etc. as a buffer, it can be used as a sensor for quantitative environmental measurement for bioimaging. A composite phosphor suitable for use can be fractionated.

  In addition, in the fractionation method of the composite phosphor of the present invention, when the gel filtration column chromatography method is adopted, in the fractionation step, the semiconductor quantum dot phosphor contained in the fraction to be fractionated is described above. The amount of the composite phosphor having the range of the elution volume in which the proportion of organic molecules increases with the increase of the elution volume can be applied and fractionated within the range.

  According to the knowledge obtained by the present inventors as a result of research, usually, when a certain amount of composite phosphor is applied to a gel filtration column and subjected to chromatography, the molecular weight of the molecules contained in the obtained fraction is: It becomes smaller as the elution volume increases. On the other hand, even when the same column is used, there is an elution volume range in which the ratio of organic molecules to the semiconductor quantum dot phosphor increases with an increase in the elution volume when the amount of application of the composite phosphor is increased. When fractionation is performed within the range, a composite phosphor with improved uniformity of occurrence of fluorescence resonance energy transfer can be efficiently obtained. Furthermore, there is a correlation between the amount of the complex phosphor molecules in the fraction and the fluorescence peak intensity.

When the gel filtration column chromatography method is employed as the method for fractionating the composite phosphor of the present invention, the organic molecule used is a fluorescent organic material capable of generating fluorescence resonance energy transfer with the semiconductor quantum dot phosphor. Either a dye or a non-fluorescent organic molecule labeled with a fluorescent molecule capable of generating fluorescence resonance energy transfer with the semiconductor quantum dot phosphor. In this case, the composite phosphor in the measurement target environment was irradiated with predetermined excitation light, and the fluorescence peak intensity generated by the semiconductor quantum dot phosphor and the fluorescence resonance energy transfer from the semiconductor quantum dot phosphor were received. By measuring the fluorescence peak intensity generated by the organic dye and utilizing the ratio thereof, it is possible to quantitatively measure the surrounding environment of the composite phosphor.
Here, the “organic molecule” is a generic term for molecules composed of elements such as carbon, hydrogen, oxygen, nitrogen, and sulfur. In the present specification, the “organic molecule” includes various biomolecules. Included, including biopolymers.

Further, in the fractionation method of the composite phosphor of the present invention, when gel filtration column chromatography is used, in the fractionation step, fluorescence resonance energy transfer between the semiconductor quantum dot phosphor and the organic molecular body is performed. Any of an aqueous solution, an aqueous dispersion, an organic solvent solution, and an organic solvent dispersion having a predetermined characteristic can be used. In this case, it is possible to obtain a fraction having the generation characteristics of fluorescence resonance energy transfer corresponding to the combination of the characteristics of the aqueous solution, the organic solvent, or the dispersion and the elution volume.
Here, a solution means a liquid in which a solute and a solvent are mixed and homogeneous, and a dispersion is a particle in which particles of about 1 nm to 1 μm are suspended or suspended in the liquid. It means what is being done.

  The organic molecule used in the method for fractionating a composite phosphor of the present invention is any one selected from the group consisting of a fluorescein derivative, a rhodamine derivative, a cyanine derivative, an alexafluoro derivative, a delight fluoro derivative, and a body pie derivative. It is preferable that the molecule is

  In the method for fractionating a composite phosphor of the present invention, a fluorescence resonance energy transfer efficiency specifying step can be further provided. In this step, the fluorescence spectrum of each of the fractionated composite phosphors is measured, and the average value of the fluorescence resonance energy transfer efficiency in each of the fractionated composite phosphors is specified based on the measurement result. To do.

  From the second aspect, the present invention is a fraction of a composite phosphor obtained using the composite phosphor fractionation method of the present invention. This fraction of the composite phosphor is obtained using the method for fractionating a composite phosphor of the present invention described above. Therefore, quantitative environmental measurement can be performed by using the fraction of the composite phosphor of the present invention as a sensor.

  As described above, according to the method for fractionating a composite phosphor of the present invention, a composite phosphor that can be used as a sensor for quantitative environmental measurement can be obtained. Moreover, according to the fraction of the composite phosphor of the present invention, quantitative environmental measurement can be performed using this fraction as a sensor.

It is process drawing for demonstrating the fractionation method of the composite fluorescent substance which concerns on Example 1 of this invention. The difference between (A) and (E) is the volume and contents of the separation resin packed in the column. Therefore, when performing fractionation, the set of (C) and (D), (F) and (G) is repeated. It is a figure which shows the pH dependence of the fluorescence spectrum of the solution collect | recovered 3rd in Example 1. FIG. In a customary commercial column, the separation is said to end with the third solution. It is a figure which shows the pH dependence of FRET efficiency estimated from the fluorescence spectrum of the solution collect | recovered 3rd in Example 1. FIG.

It is a figure which shows the pH dependence of the fluorescence spectrum of the solution collect | recovered 4th in Example 1. FIG. It is a figure which shows the pH dependence of FRET efficiency estimated from the fluorescence spectrum of the solution collect | recovered 4th in Example 1. FIG. It is a figure which shows the pH dependence of the fluorescence spectrum of the solution collect | recovered 5th in Example 1. FIG. It is a figure which shows the pH dependence of FRET estimated from the fluorescence spectrum of the solution collect | recovered 5th in Example 1. FIG.

6 is a diagram for explaining a difference in fluorescence spectrum depending on presence / absence of column separation according to Example 1. FIG. It is process drawing for demonstrating the fractionation method of the composite fluorescent substance which concerns on Example 2 of this invention. The difference between (A) and (E) is the volume and content of the separation resin packed in the column. It is a figure which shows the pH dependence of the fluorescence spectrum of the solution collect | recovered 3rd in Example 2. FIG. In a customary commercial column, the separation is said to end with the third solution. It is a figure which shows the pH dependence of FRET efficiency estimated from the fluorescence spectrum of the solution collect | recovered 3rd in Example 2. FIG. It is a figure which shows the pH dependence of the fluorescence spectrum of the solution collect | recovered 4th in Example 2. FIG.

It is a figure which shows the pH dependence of FRET estimated from the fluorescence spectrum of the solution collect | recovered 4th in Example 2. FIG. It is a figure which shows the pH dependence of the fluorescence spectrum of the solution collect | recovered 5th in Example 2. FIG. It is a figure which shows the pH dependence of FRET estimated from the fluorescence spectrum of the solution collect | recovered 5th in Example 2. FIG.

6 is a diagram for explaining a difference in fluorescence spectrum depending on presence / absence of column separation according to Example 2. FIG. It is process drawing for demonstrating the fractionation method of the composite fluorescent substance which concerns on Example 3 of this invention. The difference between (A), (E) and (J) is the volume and contents of the separation resin packed in the column. It is a figure which shows the pH dependence of the fluorescence spectrum of the solution collect | recovered 2nd in Example 3. FIG. In a customary commercial column, the separation is said to end with the second number of solutions.

It is a figure which shows the pH dependence of FRET estimated from the fluorescence spectrum of the solution collect | recovered 2nd in Example 3. FIG. It is a figure which shows the pH dependence of the fluorescence spectrum of the solution collect | recovered 3rd in Example 3. FIG. It is a figure which shows the pH dependence of FRET efficiency estimated from the fluorescence spectrum of the solution collect | recovered 3rd in Example 3. FIG.

It is a figure which shows the pH dependence of the fluorescence spectrum of the solution collect | recovered 4th in Example 3. FIG. It is a figure which shows the pH dependence of FRET efficiency estimated from the fluorescence spectrum of the solution collect | recovered 4th in Example 3. FIG. In Example 4, it is a figure which shows the relationship between buffer addition amount and FRET efficiency when dye addition amount is 5 microliters.

In Example 4, it is a figure which shows the relationship between buffer addition amount and FRET efficiency when dye addition amount is 10 microliters. In Example 4, it is a figure which shows the relationship between buffer addition amount and FRET efficiency when dye addition amount is 20 microliters. It is a figure which shows the result of Example 5 which observed the time-dependent change on the same conditions as Example 1 (however, pH of the buffer at the time of column separation was fixed to "8.5").

Hereinafter, an embodiment of the present invention will be described with reference to FIGS. In the following description, “column” refers to a column used for gel filtration column chromatography.
FIG. 1 is a process diagram of a method for fractionating a composite phosphor according to an embodiment. First, a predetermined amount of the quantum dot solution is applied to the fractionation column. Next, fractionation is performed using a buffer. Here, the pH of the buffer used does not significantly affect the fractionation. The pigment | dye couple | bonded with the quantum dot shows the fluorescence characteristic according to pH as an intrinsic characteristic.

  Here, the quantum dots used are CdSe / ZnS quantum dots, InP / ZnS quantum dots, ZnSe / ZnS quantum dots, CdS / ZnS quantum dots, CdSeTe / ZnS quantum dots, PbS / ZnS quantum dots, PbSe / ZnS quantum dots A dot etc. can be mentioned, However, CdSe / ZnS quantum dot fluorescent substance of amino coordination, CdSe / ZnS quantum dot fluorescent substance of carboxyl group coordination, etc. can be used conveniently. This is because these quantum dots have a high fluorescence quantum yield, excellent photobleachability and durability, and high biocompatibility.

The organic molecule used in the method for fractionating a composite phosphor of the present invention includes a fluorescein derivative, a rhodamine derivative, a cyanine derivative, an alexafluoro derivative, a recently developed near-infrared fluorescent dye group, a delight fluoro derivative, And any molecule selected from the group consisting of body pie derivatives.
Here, examples of the fluorescein derivative include fluorescein isothiocyanate (FITC), N-hydroxysuccinimide (NHS), carboxyfluorescein, carboxy fluorescein diacetate (CFDA), and the like. .

Examples of rhodamine derivatives include tetraethylrhodamine-5- (and 6) -isothiocyanate (Tetramethylrhodamine-5- (and 6) -isothiocyanate, TRITC), NHS-rhodamine (NHS-Rhodamin), and the like. Examples of the cyanine derivative include carbocyanine dyes having long hydrocarbon chains and indocarbocyanine dyes. As Alexafluoro derivatives, various Alexa Flour (registered trademark) products (manufactured by Life Technology Co., Ltd.), and as body pie (BODIPY, abbreviation for boron-dipyrromethene) derivatives, BODIPY FL, BODIPY RGG, BODIPY TMR, BODIPY 581 / 591 (manufactured by Invitrogen) and the like.
Among these, it is preferable to use body peas because of high fluorescence peak intensity and high stability after binding.

In addition to the open column using silica gel, the column used here is a commercially available gel filtration column such as NICK column, NAP-5 column, PD-10 column (all manufactured by GE Healthcare Bioscience). Can be mentioned.
These are equilibrated in a column fractionation buffer, and a predetermined amount of the quantum dots are applied in a predetermined amount to perform fractionation. First, equilibration is performed by adding 3 to 25 times as much buffer as the applied amount. This amount is the amount described in the instructions attached by the manufacturer.
Subsequently, the same buffer is added to apply, elute and fractionate the quantum dot complex in a predetermined amount. Examples of the buffer used here include a buffer including a Good's buffers in addition to a boric acid buffer and a phosphate buffer. Here, “good buffer” refers to the following buffering agent disclosed by Norman Good et al. In 1966.

  Specifically, ACES (N- (2-acetamido) -2-aminoethanesulfonic acid, N- (2-Acetamido) -2-aminoethanesulfonic acid); ADA (N- (2-acetamido) iminodiacetic acid, N- (2-Acetamido) iminodiacetic acid); BICINE (N, N-di (2-hydroxyethyl) glycine); BES (N, N-bis (2-hydroxyethyl)) -2-aminoethanesulfonic acid, N, N-Bis (2-hydroxyethyl) -2-aminoethanesulfonic acid); Bis-Tris (bis (2-hydroxyethyl) aminotris (hydroxymethyl) methane; Bis (2-hydroxyethyl) aminotris (hydroxymethyl) methane); DIPSO (3- [N, N-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid, 3- [N, N-Bis (2-hydroxyethyl) amino] -2 -hydroxypropanesulfonic acid); CHES (2-Cyclohexylaminoethanesulfonic acid); CAPS (3-Cyclohexylaminopropanesulfonic acid, Acid 3-Cyclohexylaminopropanesul fonic Acid);

2- [4- (2-Hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (2- [4- (2-Hydroxyethyl) -1-piperazinyl] ethanesulfonic acid); HEPES (4- (2-hydroxyethyl)- 1-piperazinepropanesulfonic acid, 4- (2-Hydroxyethyl) -1-piperazinepropanesulfonic acid); MES (2-morpholinoethanesulfonic acid); MOPSO (2-hydroxy-3-morpholinopropanesulfonic acid, 2-Hydroxy-3-morpholinopropanesulfonic acid); POPSO Dihydrate (piperazine-1,4-bis (2-hydroxypropanesulfonic acid) dihydrate); PIPES ( Piperazine-1,4-bis (2-ethanesulfonic acid); Piperazine-1,4-bis (2-ethanesulfonic acid)); Tricine (N- [Tris (hydroxymethyl) methyl] glycine, N- [Tris (hydxymethyl) ) methyl] glycine); TES (N-Tris (hydroxymethyl), N-Tris (hydroxymethyl) TAPS (N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid); TAPSO (3- [N-Tris (hydroxymethyl)] ) Methylamino] -2-hydroxypropanesulfonic acid, 3- [N-Tris (hydroxymethyl) methylamino] -2-hydroxypropanesulfonic acid)

  In the method of the present invention, the buffer is not limited, but it is preferable to use a buffer corresponding to the quantum dot to be used because there is a buffer that does not easily aggregate depending on the type of quantum dot to be used. When CdSe / ZnS quantum dots are used, it is preferable to use a boric acid buffer because the quantum dots are less likely to aggregate.

  Here, fractions can be obtained for each molecular weight within a certain range by a simple operation. Therefore, NICK column, NAP-5 column, PD-10 column (all are GE Healthcare Biosciences) Use a gel filtration column such as It is preferable to apply 2 to 10 times the buffer applied amount described in the instructions attached by the manufacturer of these columns.

Subsequently, 1 to 10 μL of 5 to 15 N NaOH for pH adjustment, 1 to 10 μL of a dye having a concentration of about 10 mg / ml to accelerate the reaction, and, if necessary, a quantum dot are added to the fractionated fraction. Depending on the functional group to be coordinated, reagents such as 50-150 mM hexadiamine and 1-alkyl-3-dialkylaminoalkyl-carbodiimide hydrochloride must be added. After the reaction is complete, load the entire amount of this reaction mixture (hereinafter referred to as “QD1-dye1”) onto a NAP-5 column (manufactured by GE Healthcare Biosciences). It was removed (see FIG. 1 (E)).
Here, the difference between FIG. 1A and FIG. 1E is the volume and contents of the separation resin packed in the column. Accordingly, when fractionation is performed, the operation is repeated with the set of FIG. 1 (C) and FIG. 1 (D), FIG. 1 (F) and FIG. 1 (G).

In addition, the initial amount of application required for the columns shown in FIGS. 1 (A) and 1 (D) is 100 μL and 500 μL, respectively. Adjust by adding a buffer.
Here, in this way, by applying the amount of buffer required for the elution described in the above instructions to the NAP-5 column, first, the complex has a relatively low molecular weight or hydrophilicity. Sex molecules are eluted, and large molecular weight or hydrophobic molecules are eluted later. In order to promote the reaction between the quantum dots and the fluorescent dye efficiently, the fluorescent dye is preferably added in an excess amount, that is, 100 times or more, and more preferably about 1,000 times.

Examples of the dye used here include a fluorescein derivative, a rhodamine derivative, a body pie derivative, a cyanine derivative, an alexafluoro derivative, and a delight fluoro derivative as described above. Any of the above phosphors preferably uses a phosphor capable of causing FRET because it has an absorption spectrum that matches the emission spectrum from the quantum dots.
The reaction is preferably performed at room temperature for about 30 minutes to 1 day from the viewpoint of reaction efficiency, and 0.5 to 1 day is preferable from the viewpoint of efficiency of the experimental operation. Here, the hexanediamine used for the reaction with the quantum dot that coordinates the carboxyl group is not limited thereto, and when a molecule having two amino groups is used, the alkyl group depends on the dye. It is preferable to select appropriately, and there is no need for a straight chain.

However, it is preferable to use 1-ethyl-3-dimethylaminopropyl-carbodiimide hydrochloride as a catalyst in order to efficiently bind the quantum dot phosphor and the amine compound. This reaction is not necessary for amino group coordinated quantum dots.
Preferably, 1 μL of 10N NaOH, 5 to 10 μL of 10 mg / ml of the above dye, 1-ethyl-3-dimethylaminopropyl-carbodiimide hydrochloride (EDC), and an amine compound are added and reacted at room temperature for about 1 day. Let By reacting under such conditions, it is possible to obtain a composite phosphor in which the dye is efficiently bonded to the functional group on the surface of the quantum semiconductor dot.

After completion of the reaction, the entire amount of the reaction liquid is applied to the column. Thereafter, a predetermined amount of a buffer having a predetermined pH is added for fractionation, and a desired fraction is collected in a test tube (see FIGS. 1 and 17). The buffer used here is as described above.
The above operation is repeated to perform fractionation, and the obtained fraction is collected. The size of the fraction is not particularly limited, but is preferably a column elution volume as instructed by the manufacturer. The elution volume of this column depends on the column size. For example, when the column of FIG. 1 is about 400 to about 500 μL, it is excellent in separation according to the method of the present invention and is used in subsequent experiments. In both cases, it is easy to handle and easy to use.

Next, the fluorescence spectrum of the obtained fraction is measured, and the FRET efficiency in the composite phosphor is estimated. For example, if the excitation wavelength is 450 nm, the above dye cannot be directly excited by 450 nm excitation light, so in the fractionated composite phosphor, check whether energy transfer from the complex to the dye occurs. (See FIGS. 2 to 16 and FIGS. 18 to 23).
Here, the FRET efficiency can be roughly expressed by the fluorescence peak intensity of the dye / the fluorescence peak intensity of the quantum dot phosphor, and this value is used when used as a sensor. However, it can be accurately obtained only by measuring the fluorescence lifetime of the quantum dots.

By performing the above fractionation operation, the molecular weight of the molecular body contained in the obtained fraction increases as the elution volume increases. This is an elution profile completely opposite to the fractionation by a normal gel filtration column.
In the case of normal gel filtration, due to the interaction with porous gel electrophoresis, molecules with a large molecular weight are eluted quickly, and molecules with a small molecular weight are eluted with a delay. However, in the case of the above-described fractionation, a molecule having a small molecular weight is eluted quickly and a molecule having a large molecular weight is eluted with a delay. In addition, the FRET efficiency of the composite phosphor of the obtained fraction depends on the molecular weight of the composite phosphor contained therein, and there is a quantitative correlation between the FRET efficiency and the molecular weight of the composite phosphor. It has the characteristic in having (refer FIGS. 24-26).

Each of the obtained fractions is excited at a predetermined excitation wavelength, a fluorescence spectrum is measured, and FRET efficiency (dye1 fluorescence intensity) / (QD2 fluorescence intensity), which is the intensity ratio of the fluorescence maximum value, is estimated.
Here, it is preferable to set the wavelength of the excitation light to be 400 to 500 nm in order to confirm energy transfer between the quantum dot and the fluorescent substance. When 450 nm is used, the fluorescent substance used has this wavelength. Since a fluorescent dye that is not directly excited by the excitation light and can cause FRET is selected, it is more preferable in that the energy transfer can be reliably observed.

  In addition, by confirming the change over time in the FRET efficiency of the composite phosphor formation reaction when fractionation is not performed, the composite has stable FRET efficiency in the reaction time during the fractionation from the above 30 minutes to 1 day. A phosphor can be used (see FIG. 27). Thereby, the experimental accuracy can be further improved. The composite phosphor having such FRET efficiency can be suitably used as a sensor for quantitative environmental measurement.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.
Example 1 CdSe / ZnS quantum dot phosphor with amino group coordination (hereinafter referred to as “QD1”)

  First, 8 μg / ml of 25 μL of QD1 solution was added to a NICK column (manufactured by GE Healthcare Bioscience) equilibrated with borate buffer, and then 75 μL of borate buffer (pH 8.5) was added. The first eluate of the NICK column was removed from the process of separating the sample in the column according to the instructions (see FIG. 1 (A)). In Example 2 and later, the first eluate was discarded for the same reason as above. Subsequently, 400 μL of borate buffer (pH 8.5) was added, and the eluate was similarly removed because it was a waste solution in the process of separating the sample in the column (see FIG. 1 (B)).

Subsequently, 1 μL of 10N NaOH and 5 μL of 5- (and-6) -carboxynaphthylfluorescein (succimidyl ester derivative) (manufactured by Life Science Technology) (hereinafter referred to as “dye1”) were added to the solution collected in the test tube. The aqueous solution (pH 8.5) was added and reacted at room temperature for 1 day (see FIG. 1 (D)). After the reaction is complete, apply the entire amount of this reaction mixture (hereinafter referred to as “QD1-dye1”) to a NAP-5 column (manufactured by GE Healthcare Biosciences). It was removed (see FIG. 1 (E)).
Here, the difference between FIG. 1A and FIG. 1E is the volume and contents of the separation resin packed in the column. Therefore, when fractionation was performed, the operation was repeated with the set of FIG. 1 (C) and FIG. 1 (D), FIG. 1 (F) and FIG. 1 (G).

In this case, since the reaction solutions were 25 μL and 400 μL, after applying the entire amount, the buffer was adjusted so that the required application amounts were 100 μL and 500 μL, respectively. In order to advance the reaction between the quantum dots and the fluorescent dye efficiently, an excessive amount (about 1,000 times) of the fluorescent dye was added. By applying an amount of buffer necessary for elution to the NAP-5 column in this manner, first, the molecular weight is relatively small or the hydrophilic molecule is eluted in the complex, and then the molecular weight is large. Alternatively, hydrophobic molecules were eluted with a delay.
That is, 500 μL of borate buffer (pH 8.5) is added to the NAP-5 column, and the eluate is collected in a test tube (see FIG. 1 (F)), and an aqueous solution containing a composite phosphor in which QD1 and dye1 are bound is prepared. Obtained (see FIG. 1 (G)).

After collecting the buffer, the batch operation of supplying 500 μL of buffer to the NAP-5 column and collecting the eluate was repeated. The eluate was collected for each batch of supplied buffer, and a fraction containing a composite phosphor in which QD1 and dye1 were bound was fractionated.
In Example 1, the pH of the borate buffer was set in the range of 7.5 to 9.5 as shown in FIG. Then, in each case of the pH value, the fluorescence spectrum of the eluate collected 3rd to 5th was measured with a NAP-5 column (excitation wavelength = 450 nm), and the FRET efficiency in the composite phosphor was estimated.

FIGS. 2 and 3 show the results of measuring the fluorescence spectrum of the third solution, FIGS. 6 and 7 and the results of estimating the FRET efficiency, respectively. Show.
In FIGS. 3, 5, and 7, the FRET efficiency is expressed as (dye1 fluorescence peak intensity) / (QD1 fluorescence peak intensity).
As shown in FIG. 2, FIG. 4, and FIG. 6, the light emission from dye 1 increased as the pH became alkaline. This indicates that energy transfer from QD1 to dye1 occurs in the fractionated composite phosphor because dye1 cannot be excited directly with 450 nm excitation light.

In addition, as shown in FIGS. 3, 5, and 7, the FRET efficiency increased as the pH shifted to the alkali side regardless of the number of times of buffer recovery during fractionation. Thus, it was shown that the sensor ability can be shown as a change in FRET efficiency by adjusting the pH of the buffer.
Moreover, as comprehensively shown in FIG. 3, FIG. 5, and FIG. 7, the FRET efficiency changed depending on the fraction of the composite phosphor recovered. From this, it was shown that the FRET efficiency can be controlled by changing the elution volume at the time of fractionation (that is, the number of separations by the column).

Even in the case where the fractionation (separation) according to Example 1 was not performed, the fluorescence spectrum was measured with an excitation wavelength of 450 nm. The measurement result of the fluorescence spectrum is shown in FIG. 8 together with the measurement result of the fluorescence spectrum of the third to fifth recovered solutions when fractionated as described above using a borate buffer (pH 8.5).
From FIG. 8, as the recovery number progresses to the latter half, the emission intensity derived from dye1 becomes stronger with reference to the emission intensity derived from QD1 (that is, the FRET efficiency is increased), so that it binds to QD1 by the above-described fractionation. It is shown that the average value of the number of dye1s is considerably larger than the case where the fractionation is not performed, and the reaction product is proportional to the molecular weight (number of dye1 bound to QD1). It was shown that it can be quantitatively fractionated. In the original method of using the column, the second recovered sample is used as a purified sample, but here, it is a consistent separation in which the low FRET efficiency is outstanding.

Example 2 Carboxyl coordinated CdSe / ZnS quantum dot phosphor (hereinafter referred to as “QD2”)
As a solution of QD2, Life Technologies (trade name: Qdot (registered trademark) 605 ITK (trademark) Carboxyl Quantum Dots) was used.
First, 8 mg / ml 20 μL of QD2 solution and 6 μL of 1-ethyl-3-dimethylaminopropyl-carbodiimide hydrochloride (EDC, 10 mg / mL) and 0.5 μl of 100 mM hexanediamine were added and reacted at room temperature for 1 day. After applying the solution to a NICK column (GE Healthcare Bioscience), 80 μL of borate buffer (pH 8.5) was added to remove the first eluate. Subsequently, 400 μL of borate buffer (pH 8.5) was added to remove the eluate (see FIG. 9B).

Thereafter, as shown in FIG. 9 (C), 400 μL of borate buffer (pH 8.5) was added, and the eluate was collected in a test tube. The quantum dot fractionation is performed as in the case of Example 1 because it is a waste liquid in the process of separating the sample in the column.
Subsequently, 10 mg / ml and 5 μL of dye1 were added to the solution collected in the test tube together with 1 μL of 10N NaOH and reacted at room temperature for 1 day (see FIG. 9D). After completion of the reaction, the entire amount of the reaction solution (hereinafter referred to as “QD2-dye1”) was applied to the NAP-5 column, 100 μL of borate buffer (pH 8.5) was added, and the first eluate was removed (FIG. 9 ( See F)).
Thereafter, 500 μL of borate buffer (pH 8.5) was added to the NAP-5 column, and the eluate was collected in a test tube (see FIG. 9 (G)). As a result, a fraction containing a composite phosphor in which QD2 and dye1 were bound was obtained (see FIG. 9 (H)).

Thereafter, the steps of FIGS. 9 (G) and 9 (H) were repeated with the test tube replaced. Thus, a composite phosphor in which QD2 and dye1 were bound was fractionated.
As in Example 1, the pH of the borate buffer was in the range of 7.5 to 9.5, and fractionation was performed in the same manner as described above with each pH buffer. The collected eluate was measured for fluorescence spectrum (excitation wavelength = 450 nm), and the FRET efficiency in the composite phosphor was estimated.

FIGS. 10 and 11 show the measurement results of the fluorescence spectrum and the FRET efficiency estimation results of the third solution, the fourth in FIGS. 12 and 13 and the fifth in FIGS. 14 and 15, respectively. Show. In FIGS. 11, 13, and 15, the FRET efficiency is expressed as (dye1 fluorescence peak intensity) / (QD2 fluorescence peak intensity).
As shown in FIGS. 10, 12, and 14, as in the case of Example 1, the light emission from dye 1 was strong as the pH became alkaline. This indicates that energy transfer from QD2 to dye1 occurs in the fractionated composite phosphor since dye1 cannot be directly excited by 450 nm excitation light.

In addition, as shown in FIGS. 11, 13, and 15, the FRET efficiency increased as the pH shifted to the alkali side in any fraction collection times, as in Example 1. . Therefore, it was considered that the sensoring ability can be shown as a change in FRET efficiency by adjusting the pH of the buffer.
From FIG. 11, FIG. 13, and FIG. 15, the FRET efficiency varied depending on the fraction of the composite phosphor collected by batch operation, as in the case of Example 1. For this reason, it was shown that FRET efficiency can be controlled by the elution volume at the time of fractionation (that is, the number of column separations).

Even in the case where the fractionation (separation) according to Example 2 described above was not performed, fluorescence spectrum measurement was performed with an excitation wavelength of 450 nm. FIG. 16 shows the measurement results of the third to fifth recovered solutions when a borate buffer (pH 8.5) was used.
As shown in FIG. 16, when the emission intensity derived from QD2 is used as a reference, the emission intensity derived from dye1 becomes strong (that is, the FRET efficiency is high). It was shown that the average value of the number of dye1s is larger compared to the case where the fractionation is not performed. The second fraction, a normal fraction, contained complexes with particularly low FRET efficiency.

[Example 3] InP / ZnS quantum dot phosphor with amino coordination (hereinafter referred to as "QD3")
First, as shown in FIG. 17 (A), 6 μL of 10 mg / ml EDC and 0.5 μL of 100 mM hexadiamine (hereinafter sometimes referred to as “HDA”) are added to 20 μL of a QD3 solution. The reaction was allowed to proceed for half a day at room temperature. After completion of the reaction, after applying the reaction solution (hereinafter referred to as “QD3-H”) to the NICK column (GE Healthcare Bioscience), 74 μL borate buffer (pH 8.5) was added, and the reaction solution from the NICK column was added. The eluate was removed. Subsequently, 400 μL of borate buffer (pH 8.5) was added to remove the eluate (see FIG. 17 (B)).
Thereafter, 400 μL of borate buffer (pH 8.5) was added, and the eluate was collected in a test tube. Such quantum dot fractionation is performed for the same reason as in the second embodiment.

Subsequently, 1 μL of 10N NaOH was added to these solutions, 10 mg / mL, 5 μL of dye 2 was added, and the mixture was reacted at room temperature for one day. After completion of the reaction, the entire amount of the reaction solution (hereinafter referred to as “QD3-dye2”) was added to the NAP-5 column, and the eluate was removed. Further, 100 μL of borate buffer was added.
Thereafter, 500 μL of borate buffer was added to the NAP-5 column, and the eluate was collected in a test tube. As a result, a fraction containing a composite phosphor in which QD3 and dye2 were bound was obtained (see FIG. 17 (L)).

Thereafter, the steps shown in FIGS. 17K and 17L were repeated while exchanging the test tubes. In this way, a fraction containing a composite phosphor in which QD3 and dye2 were bound was fractionated.
In Example 3, as in Example 1, fractionation was performed with the borate buffer pH set to 7.5 to 9.5, and the fluorescence spectrum of the second to fourth solution collected on the NAP-5 column was measured ( Excitation wavelength = 450 nm), and FRET efficiency of the composite phosphor was estimated.
18 and 19 show the second, FIG. 20 and FIG. 21 the third, and FIG. 22 and FIG. 23 show the fluorescence spectrum measurement results and FRET efficiency estimation results of the fourth recovered solution. Shown respectively. In these figures, the FRET efficiency is expressed as (dye2 fluorescence peak intensity) / (QD3 fluorescence peak intensity).

As shown in FIGS. 18, 20, and 22, the light emission intensity from dye2 changed according to the pH value. Since dye2 cannot be directly excited by 450 nm excitation light, this indicates that energy transfer from QD3 to dye2 occurs in the fractionated composite phosphor.
Further, as shown in FIG. 19, FIG. 21, and FIG. 23, the FRET efficiency changed depending on the pH of the buffer. This indicates that the sensing ability can be expressed as a change in FRET efficiency by changing the pH of the buffer.
Further, as comprehensively shown in FIG. 19, FIG. 21, and FIG. 23, the FRET efficiency varied depending on the fraction of the recovered composite phosphor. This indicates that the FRET efficiency can be controlled by the elution volume at the time of fractionation (that is, the number of column separations).

[Example 4] Relationship between applied amount of complex phosphor, elution volume during fractionation, and FRET efficiency In Example 4, as in Example 1, the amount of QD1-dye1 solution was 5 μL, 10 μL, and 20 μL. The fluorescence spectrum of the recovered solution was measured in the same manner as in Example 1 while finely changing the amount of borate buffer (pH 8.5) added for each batch, and the FRET efficiency was estimated.
FIG. 24 shows the results when the amount of QD1-dye1 solution is 5 μL, FIG. 25 shows the results when 10 μL, and FIG. 26 shows the results when 10 μL. The results shown in FIGS. 24 to 26 show that the FRET efficiency changes according to the change in the amount of buffer added.

  Further, from the results shown in FIGS. 24 to 26, it was confirmed that there is a range in which the FRET efficiency increases as the buffer addition amount increases. Furthermore, although there is a difference in the relative positions of the fractions depending on the amount of QD-dye1 applied, the tendency is very constant, indicating the generality of the present invention.

[Example 5] Time change of FRET efficiency of fraction of composite phosphor In this example, it was observed by fluorescence spectrum measurement how the reaction without separation at all changes with time.
In FIG. 27, the example of the investigation result of the time change of FRET efficiency is shown. From this result, it was shown that the reaction was completed and the FRET efficiency was stabilized in about 50 minutes.

  As described above, the method for fractionating a composite phosphor of the present invention can be applied to obtaining a composite phosphor that can be used as a sensor for quantitative environmental measurement. The fraction of the composite phosphor of the present invention can be applied to a sensor for quantitative environmental measurement.

11 ... NICK column 12, 13 ... Test tube 14, 15 ... NAP-5 column 16 ... Test tube

Claims (8)

A composite phosphor forming step of synthesizing a composite phosphor in which a semiconductor quantum dot phosphor and an organic molecule capable of generating fluorescence resonance energy transfer between the semiconductor quantum dot phosphor are combined;
A fractionation step of fractionating the composite phosphor by a chromatographic method;
In the fractionation step, the amount of the composite phosphor that ensures that the ratio of the organic molecules to the semiconductor quantum dot phosphor in the fraction fractionated by the chromatography method changes with an increase in elution volume is determined. Apply,
And a method for fractionating a composite phosphor.   The method for fractionating a composite phosphor according to claim 1, wherein the chromatography method is a gel filtration column chromatography method.   In the fractionation step, the ratio of the organic molecules to the semiconductor quantum dot phosphor in the fraction fractionated by the gel filtration column chromatography method has a range of the elution volume that increases as the elution volume increases. 3. The method for fractionating a composite phosphor according to claim 2, wherein the amount of the composite phosphor is applied and fractionated within the range.   The organic molecule is a fluorescent organic dye capable of generating fluorescence resonance energy transfer with the semiconductor quantum dot phosphor, or a fluorescence capable of generating fluorescence resonance energy transfer with the semiconductor quantum dot phosphor. 4. The method for fractionating a composite phosphor according to claim 2, wherein the method is a non-fluorescent organic molecule labeled with a molecule.   In the fractionation step, any one of an aqueous solution, an aqueous dispersion, an organic solvent, and an organic solvent dispersion having characteristics predetermined for fluorescence resonance energy transfer between the semiconductor quantum dot phosphor and the organic molecular body is used. The method for fractionating a composite phosphor according to any one of claims 2 to 4, wherein the composite phosphor is fractionated by gel filtration column chromatography.   The organic molecule is any one selected from the group consisting of a fluorescein derivative, a rhodamine derivative, a cyanine derivative, an alexafluoro derivative, a delight fluoro derivative, and a body pie derivative. 6. The method for fractionating a composite phosphor according to any one of 5 above.   Fluorescence resonance energy for measuring the fluorescence spectrum of each of the fractionated composite phosphors and identifying an average value of the fluorescence resonance energy transfer efficiency in each of the fractionated composite phosphors based on the measurement result The method for fractionating a composite phosphor according to any one of claims 1 to 6, further comprising a transfer rate specifying step.   The fraction of the composite fluorescent substance obtained using the fractionation method of the composite fluorescent substance in any one of Claims 1-7.

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