JP2007120996A - Nano light-emitting probe - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new nano-size light-emitting probe capable of facilitating detection in a visible light zone with high sensitivity, without using a metal material such as a noble metal or a semiconductor, which is expensive, not perfect in handling or safety, and difficult to be prepared as a probe. <P>SOLUTION: This nano light-emitting probe includes at least either of fullerol and its derivative. Fullerol and its derivative are expressed by a following formula: Cn(OR)m(OH)p. (Cn shows fullerene, n shows the number of carbon atoms constituting the fullerene, R shows a hydrocarbon group or an acyl group, m and p show the number of bonds respectively, and neither of m and p becomes zero). <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a fullerene derivative nanoluminescent probe that is useful for molecular identification in various fields such as biochemistry, medicine, and environment, and that can be used as a luminescent probe in a wide range of visible light.

  Conventionally, in fields such as cell imaging, organic (fluorescent) dye-containing and surface-hydrophilic polystyrene fine particles having a diameter of several tens of nanometers have been frequently used as luminescent probes. However, it has been pointed out that probes of several tens of nanometers are very large in terms of molecules such as proteins to be labeled, and that the influence of attaching the probe is exerted. On the other hand, there is a method of using a fluorescent molecule as a luminescent probe in units of one molecule, but it is a measurement of weak luminescence, and an expensive and sensitive detection system is required, and the fluorescent molecule can be quickly formed by strong excitation light. Disassembly and fading are pointed out as problems.

  Under such circumstances, studies on nano-sized luminescent probes have been promoted in recent years. For example, a luminescent probe in which noble metal nanoclusters are encapsulated with a dendrimer or peptide (Patent Document 1) has been proposed. A polychromatic (light emission wavelength varies depending on the diameter) luminescent probe using semiconductor fine particles is manufactured and marketed by Quantum Dot. However, these luminescent probes are expensive and use a group 4 atom such as CdS, CdSe, CdTe or the like as a noble metal or a semiconductor which is not perfect in terms of handling and safety. In addition, these conventional probes have a problem that their preparation is not always easy.

Although there are quantum dots using silicon, the disadvantage is that the emission becomes near-infrared and the detection system becomes expensive.
Special table 2005-530517

  The present invention eliminates the problems of the prior art from the background as described above, such as noble metals and semiconductors, is expensive, is not perfect in terms of handling and safety, and is not easy to prepare as a probe. It is an object of the present invention to provide a new nano-sized light-emitting probe that can be easily detected in the visible light region without using a light source.

  The present invention provides the following means for solving the above-described problems.

  1st: The nanoluminescent probe characterized by including at least any one of a fullerol and its derivative (s).

Second: fullerol and its derivatives have the following formula C _n (OR) _m (OH) _p
(C _n represents fullerene, n represents the number of carbon atoms constituting the fullerene, R represents a hydrocarbon group or an acyl group, m and p represent the number of bonds, and m and p are both It will never be 0.)
The above-described nano-light-emitting probe, which is represented by

  Third: A nano-light-emitting probe comprising plural kinds of fullerol and derivatives thereof and capable of polychromatic light emission.

  Fourth: A nanoluminescent probe composition characterized by being an aqueous solution or an organic solvent solution of any of the above nanoluminescent probes.

Fifth: A molecular detection method using any one of the above-described nanoluminescent probes or compositions, comprising at least the following processes.
(A) Fullerol or a derivative thereof, or both are bound to a molecule to be detected.
(B) The molecule to be detected is recognized by detecting the light emission of the bound fullerol or its derivative upon irradiation with excitation light.

  Sixth: A method for preparing any one of the above-described nanoluminescent probes, wherein a nanoluminescent probe having a required emission wavelength is separated from fullerol or a derivative thereof, or a mixture of both of them. .

  Seventh: A method for preparing a nano-light-emitting probe, comprising mixing a plurality of kinds of separately prepared light-emitting probes to enable required polychromatic light emission.

  The nano luminescent probe of the present invention can be used not only in an aqueous solution system but also in a wide range of solvents, and can be used in a wide range of visible light as a luminescent probe that can be bonded to a wide variety of molecules. Is possible. Furthermore, it is superior to the prior art in that it contains carbon, hydrogen and oxygen as elements and does not contain toxic elements such as cadmium and arsenic.

  Due to its hydrophilicity, fullerol is encapsulated in heavy metal atoms and used as a contrast agent, or used as a metal ion ligand to measure metal ion concentration by luminescence, and some researches on physiological activity have also been conducted. However, it has not been used actively because of its low reactivity. In recent years, carbon nanotubes have been widely used, but research has not progressed on their harmfulness, and fullerenes are expected to be less toxic than nanotubes. desirable. Further, since fullerol and a fullerol ester as a derivative thereof contain many hydroxyl groups, they are easily stabilized by ionization in an aqueous solution and hardly aggregate even under a high ion (particularly metal ion) concentration. Further, fullerol esters are easily neutralized and stabilized in an organic solvent. With respect to the emission wavelength (emission color), derivatives such as fullerol and fullerol ester show a wide emission wavelength with respect to the preparation (synthesis) method, but by separating this by electrophoresis distance, for example, by electrophoresis, A wide range of colors can be selected from red or orange red to yellow to blue. The excitation wavelength (irradiation light) can be selected from short wavelengths (UV) to visible light by appropriately selecting and mixing fullerol and fullerol ester (or the degree of esterification).

  Embodiments of the present invention having the features as described above will be described below.

  The nanoluminescent probe according to the present invention is characterized in that it contains at least one of fullerol and a derivative thereof. In this case, the fullerol is hydroxylated to fullerene to convert a hydroxyl group (OH) to a carbon atom of the fullerene skeleton. Are combined. Various reaction methods for hydroxylation of fullerene are already known, and the fullerol of the present invention may be synthesized by any of these methods.

  Normally, fullerenes synthesized by hydroxylation of fullerenes are produced in a plurality of types having different numbers of hydroxyl groups (OH) bonded to fullerene carbon atoms and different bonding positions of hydroxyl groups (OH). Since each of these multiple types often has different emission wavelengths and emission spectra, a mixture of these multiple types may be used as a polychromatic emission probe, or those with specific emission wavelengths may be separated. Further, if necessary, these can be mixed to form a nano-emitting probe that emits light at a specific wavelength.

On the other hand, the derivative of fullerol in the present invention
1) The hydroxyl group (OH) of fullerol is esterified or etherified,
2) As the fullerol, its ester, or ether, those having other organic groups are considered.

  Depending on the type and composition of fullerol as the hydroxylated fullerene, these derivatives may be obtained as a mixture of a plurality of types. This can also be used as a nano-emitting probe in the same manner as described above.

  Fullerol is a water-soluble fullerene derivative. For example, fullerol esters are also soluble in organic solvents. Fullerol, fullerol ester, and the like can be separated from the mixture for each emission wavelength by gel electrophoresis, particularly PAGE. Specific results of gel electrophoresis of fullerol and the result of emission spectrum of the separated fullerol are shown in the examples described later.

The fullerol and derivatives thereof of the present invention can be represented by the formula: C _n (OR) _m (OH) _p as described above. Here, regarding C _n indicating fullerene, not only typical C ₆₀ but also higher-order fullerene having a larger number of carbon atoms and organic groups such as hydrocarbon groups are bonded to these unless the function as a luminescent probe is inhibited. It may be what you did.

  The symbol R is a hydrocarbon group or an acyl group, but these may similarly have an organic group.

Among them, the present in invention, Furaroru and hydroxides of _{C 60 C} 60 _{(OH) p} or _C 60 esterified some of the hydroxyl groups _(OR) m _{(OH) p} suitably as a derivative thereof Can be used. Here, as the number of hydroxyl groups (OH): p, for example, about 50 is preferably considered. As the number: m, for example, about 20 is preferably considered.

  The nanoluminescent probe of the present invention as described above can be used as a luminescent probe in a wide range of visible light by binding to various molecules. In this case, for example, contact with a test substance in the form of an aqueous solution or an organic solvent solution, a luminescent probe is chemically or physically bonded to the test molecule, and fluorescence emission or the like is detected by irradiation with excitation light. Thus, it is possible to identify molecules in the test substance.

  Then, an Example is shown below and the light emission capability of the nano light-emitting probe of this invention is demonstrated.

<1> C ₆₀ fullerene is reacted with fuming sulfuric acid and phosphorus pentoxide at a temperature of 60 ° C. for about 1 hour, and then hydrolyzed at a temperature of 80 ° C. for about 10 hours to obtain an average number of hydroxyl (OH) bonds, that is, Fullerol-A having an average number of bonds per molecule of C60 of 12 was synthesized.
<2> On the other hand, C ₆₀ fullerene was reacted with tetrabutylammonium hydroxide and an aqueous sodium hydroxide solution at room temperature for about 3 hours to synthesize a fullerol-B having a hydroxyl group (OH) average bond number of 20 to 25. .
<3> The results of gel electrophoresis of the fullerol synthesized by the above method <1><2> are shown in FIG. The electrophoresis conditions were 12% -polyacrylamide gel, 100 V, 30 minutes, and use of tris-borate buffer.

  Lanes A, B, C, D, and E in FIG. 1 indicate the following.

A: BRC
B: fullerol-B
C: fullerol-A
D: ARC
E: ARA
In either case, 25 μg is used.

  FIG. 2 shows emission spectra of the fullerol compounds separated by electrophoresis for each of fullerol-A and fullerol-B. The symbol in the figure indicates the separation distance of electrophoresis.

A: 0.5cm
B: 2cm
C: 3cm
D: 4cm
From the above, it can be seen that those having different fluorescence emission wavelengths (spectrums) can be used as individual probes or as polychromatic luminescent probes by a desired combination.

It is the figure which illustrated the result of gel electrophoresis. It is the figure which illustrated the emission spectrum of the separated fullerol compound.

Claims (7)

  A nanoluminescent probe comprising at least one of fullerol and its derivatives. Fullerol and its derivatives have the formula C _n (OR) _m (OH) _p
(C _n represents fullerene, n represents the number of carbon atoms constituting the fullerene, R represents a hydrocarbon group or an acyl group, m and p represent the number of bonds, and m and p are both It will never be 0.)
The nanoluminescent probe according to claim 1, wherein   The nanoluminescent probe according to claim 1 or 2, wherein the nanoluminescent probe includes plural kinds of fullerol and derivatives thereof and can emit polychromatic light.   A nanoluminescent probe composition comprising an aqueous solution or an organic solvent solution of the nanoluminescent probe according to any one of claims 1 to 3. 5. A molecular detection method using the nanoluminescent probe according to claim 1 or a composition thereof, comprising at least the following process.
(A) Fullerol or a derivative thereof, or both are bound to a molecule to be detected.
(B) The molecule to be detected is recognized by detecting the light emission of the bound fullerol or its derivative upon irradiation with excitation light.   A method for preparing a nanoluminescent probe according to any one of claims 1 to 3, wherein a nanoluminescent probe having a required emission wavelength is separated from fullerol or a derivative thereof, or a mixture of both of them. Preparation method.   A method for preparing a nano-luminescent probe, comprising mixing a plurality of types of luminescent probes separated and prepared by the method of claim 6 to enable required polychromatic light emission.