U.S. patent application number 13/518966 was filed with the patent office on 2012-11-29 for fluorescent particle, with semiconductor nanoparticles dispersed therein, fabricated by the sol-gel process.
Invention is credited to Masanori Ando, Norio Murase, Ping Yang.
Application Number | 20120301971 13/518966 |
Document ID | / |
Family ID | 44226448 |
Filed Date | 2012-11-29 |
United States Patent
Application |
20120301971 |
Kind Code |
A1 |
Murase; Norio ; et
al. |
November 29, 2012 |
FLUORESCENT PARTICLE, WITH SEMICONDUCTOR NANOPARTICLES DISPERSED
THEREIN, FABRICATED BY THE SOL-GEL PROCESS
Abstract
An object of the present invention is to prepare a fine particle
with high durability and high brightness, in which semiconductor
nanoparticles are assembled. The present invention provides
fluorescent fine particles comprising Cd- and Se-containing
semiconductor nanoparticles dispersed in silicon-containing fine
particles, wherein the average particle size of the
silicon-containing fine particles is 20 to 100 nm, and the number
of semiconductor nanoparticles dispersed in the silicon-containing
fine particles is 10 or more.
Inventors: |
Murase; Norio; (Ikeda-shi,
JP) ; Yang; Ping; (Ikeda-shi, JP) ; Ando;
Masanori; (Ikeda-shi, JP) |
Family ID: |
44226448 |
Appl. No.: |
13/518966 |
Filed: |
December 17, 2010 |
PCT Filed: |
December 17, 2010 |
PCT NO: |
PCT/JP2010/072777 |
371 Date: |
August 14, 2012 |
Current U.S.
Class: |
436/172 ;
977/773; 977/810 |
Current CPC
Class: |
C09K 11/565 20130101;
C09K 11/025 20130101; C09K 11/883 20130101; H01L 33/0083 20130101;
Y10S 977/824 20130101; Y10S 977/779 20130101; Y10S 977/774
20130101 |
Class at
Publication: |
436/172 ;
977/773; 977/810 |
International
Class: |
G01N 21/64 20060101
G01N021/64 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 28, 2009 |
JP |
2009-296596 |
May 12, 2010 |
JP |
2010-109885 |
Claims
1. Fluorescent fine particles comprising Cd- and Se-containing
semiconductor nanoparticles dispersed in silicon-containing fine
particles, wherein the average particle size of the
silicon-containing fine particles is 20 to 100 nm, and the number
of semiconductor nanoparticles dispersed in each silicon-containing
fine particle is 10 or more.
2. The fluorescent fine particles according to claim 1, wherein the
average particle size of the silicon-containing fine particles is
40 to 100 nm, and the number of semiconductor nanoparticles
dispersed in each silicon-containing fine particle is 20 or
more.
3. The fluorescent fine particles according to claim 1, wherein the
semiconductor nanoparticles dispersed in each silicon-containing
fine particle are regularly arranged.
4. The fluorescent fine particles according to claim 1, wherein the
PL efficiency is 20% or higher.
5. The fluorescent fine particles according to claim 1, wherein the
PL efficiency is 20% or higher when the semiconductor nanoparticles
are dispersed at a concentration of 10 nmol/L in a pH 7.4 solution
of HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid)
serving as a Good's buffer.
6. The fluorescent fine particles according to claim 1, wherein
after the semiconductor nanoparticles are dispersed in a pH 7.4
solution (HEPES concentration of 10 mmol/L) of HEPES
(2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) serving
as a Good's buffer, at a concentration of X nmol/L, and maintained
at room temperature for 15 hours, Y nanograms that indicate the
dissolved amount of Cd in the HEPES solution (the weight of
dissolved Cd in a solution of 1 mL) satisfies
10.times.Y/X<1.
7. The fluorescent fine particles according to claim 1, wherein the
semiconductor nanoparticles are CdSe and/or CdSe/ZnS in which CdSe
is coated with ZnS.
8. The fluorescent fine particles according to claim 1, having a
silicon-containing layer which has a thickness of 2 nm or more.
9. The fluorescent fine particles according to claim 1, comprising
on the surface at least one selected from the group consisting of
COOH, NH.sub.2, SH, salts thereof, and groups originated from
polyethyleneglycol.
10. The fluorescent fine particles according to claim 1 for
fluorescent probes.
11. The fluorescent fine particles according to claim 1 for
electronic materials.
12. The fluorescent fine particles according to claim 1, wherein
the fluorescent fine particles exhibit electroluminescence and/or
cathodeluminescence.
13. A method of preparing the fluorescent fine particles according
to claim 1, comprising the steps of: (A) adding a metal alkoxide
(1) to an organic solvent in which Cd- and Se-containing lipophilic
semiconductor nanoparticles are dispersed, and stirring the
mixture, thereby obtaining organic solution A; (B) mixing aqueous
solution B containing a metal alkoxide (2) whose hydrolysis rate is
lower than that of the metal alkoxide (1) with the organic solution
A and stirring the mixture, thereby obtaining an semiconductor
nanoparticle assembly; and (C) adding a solution containing a metal
alkoxide (3) to an alkaline aqueous solution containing the
semiconductor nanoparticle assembly, thereby forming a coating
layer on the surface of the semiconductor nanoparticle
assembly.
14. The method of preparing fluorescent fine particles according to
claim 13, wherein the metal alkoxide (2) is a compound represented
by Formula (II): X.sub.n--Si(OR.sup.2).sub.4-n (II) wherein X
represents a group represented by CH.sub.2.dbd.CH--, a group
containing oxirane, a group represented by
H.sub.2NC.sub.mH.sub.2m--, a group represented by
CH.sub.2.dbd.C(CH.sub.3)COOC.sub.pH.sub.2p--, a group represented
by HSC.sub.qH.sub.2q--, or a phenyl group; R.sup.2 represents a
lower alkyl group; n is an integer of 1, 2, or 3; m is an integer
of 1 to 6; p is an integer of 1 to 5; and q is an integer of 1 to
10.
15. The method of preparing fluorescent fine particles according to
claim 13, wherein heating is performed during Step (C).
16. A method of preparing the fluorescent fine particles according
to claim 1, comprising the steps of: (A1) adding a metal alkoxide
(1) and a metal alkoxide (2) whose hydrolysis rate is lower than
that of the metal alkoxide (1) to an organic solvent in which Cd-
and Se-containing lipophilic semiconductor nanoparticles are
dispersed, and stirring the mixture, thereby obtaining organic
solution A; and (B1) adding a metal alkoxide (3) and an alkaline
aqueous solution to the organic solution A, thereby forming a
semiconductor nanoparticle assembly.
17. A method of preparing the fluorescent fine particles according
to claim 1, comprising the steps of: (A2) adding two types of metal
alkoxides to a non-polar solvent in which Cd- and Se-containing
lipophilic semiconductor nanoparticles are dispersed, and stirring
the mixture, thereby obtaining organic solution X; and (B2)
bringing the organic solution X into contact with solution Y
containing a metal alkoxide and water so as to transfer the
semiconductor nanoparticles in the organic solution X to the
solution Y.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for preparing
highly durable silica glass fine particles in which numerous
semiconductor nanoparticles with high photoluminescence (PL)
efficiency are dispersed therein, and an application thereof.
BACKGROUND ART
[0002] Because fluorescent materials (phosphors) obtained by
dispersing rare-earth ions, transition metal ions, and the like in
inorganic materials have better durability than organic dyes, these
fluorescent materials have been conventionally used for lights,
displays, and the like. However, because the brightness and
color-rendering properties thereof are not always sufficient, there
has been a demand for a fluorescent material with higher
brightness. In recent years, semiconductor nanoparticles (particle
size of several nanometers; without doping of rare-earth ions or
transition metal ions; hereinafter also simply referred to as
"nanoparticles" or "quantum dots") are gaining attention as a
high-performance fluorescent material that embodies the above
demand because of the following reasons: semiconductor
nanoparticles are excellent in color-rendering properties because
these particles emit bright fluorescence of various wavelengths
according to the particle size even when irradiated with
ultraviolet light of the same wavelength, and the brightness of
these nanoparticles can be increased because their emission decay
time is short. If semiconductor nanoparticles are carefully
prepared, the brightness becomes high to the degree that the
emission of each particle can be separately detected and
spectroscopically analyzed. Consequently, in addition to the use
for displays and light, there is the beginning of a great
development in the field of application where semiconductor
nanoparticles are conjugated to biomolecules and used as
fluorescent probes for the elucidation of the mechanism of the
life, the diagnosis of diseases, and the like.
[0003] Primary examples of semiconductors that serve as the
above-described fluorescent materials include II-VI semiconductors
(cadmium sulfide (CdS), zinc selenide (ZnSe), cadmium selenide
(CdSe), zinc telluride (ZnTe), cadmium telluride (CdTe), mixed
crystals thereof, etc.) and III-V semiconductors (indium phosphide
(InP), etc.). These are direct transition semiconductors, and their
emission lifetime is about 10 nanoseconds, which is about five
orders of magnitude smaller than conventional forbidden transition
fluorescent materials that use a rare-earth ions or transition
metal ions. Consequently, fluorescence with much higher brightness
can be achieved.
[0004] There are two synthesis methods for semiconductor
nanoparticles that emit such high-intensity light (fluorescence):
one method for synthesizing in an aqueous solution (hydrophilic
nanoparticles are synthesized), and another method for synthesizing
in an organic solution (non-polar solvent) in which water is
removed at a high level (hydrophobic nanoparticles are
synthesized). Because nanoparticles have a large specific surface
area, these nanoparticles are gradually agglomerated in the
solution in order to reduce the surface energy, and the PL
efficiency is thus decreased. Therefore, there was a problem with
the nanoparticles synthesized by both methods in that it was
difficult to put them to practical use when these nanoparticles
were in the form of a solution. In order to solve this problem,
semiconductor nanoparticles must be incorporated in a transparent
matrix in such a manner that the nanoparticles are dispersed and
fixed therein so as to obtain a solid material that maintains the
initial properties for a long period of time under various
environments. As a solid matrix therefor, there are two materials:
glass, and organic polymer materials. Between these, glass,
particularly silica glass, has a higher transparency and a higher
tolerance to ultraviolet irradiation than organic polymers.
Additionally, moisture and oxygen cannot easily permeate through
silica glass when silica glass is formed in a network structure,
making it possible to prevent degradation of dispersed
nanoparticles for a long period of time. A sol-gel method is
favorable for the preparation of such glass because in the sol-gel
method, vitrification progresses under mild conditions at or close
to normal temperature and pressure; thus, if a preparation method
is improved, semiconductor nanoparticles can be dispersed and fixed
in a transparent glass while maintaining high PL efficiency that
was achieved immediately after synthesis by the solution method.
Because the sol-gel method uses water, it is preferable to use
hydrophilic nanoparticles from the viewpoint of preventing
agglomeration and quenching.
[0005] The term "silica glass" is explained here. Although glass
prepared by the sol-gel method contains an organic materials and
water, such a product is called glass, silica, silica glass,
silica-based glass, amorphous silica, SiO.sub.2, etc., in related
scientific societies. This is because other metal ions that modify
the network structure formed by silicon are not contained in the
prepared solid matrix. Therefore, a matrix containing silicon
prepared by the sol-gel method is also called glass, silica, or
silica glass in the present description.
[0006] The present inventors developed a bulk-like glass (Patent
Literature 1), glass fine particles (Patent Literature 2, 3, and
4), and a glass thin film (Patent Literature 5) as the fluorescent
glass described above. Of these, glass fine particles (particle
size of 10 nm to 2 .mu.m; when the particle is not a complete
sphere, for example, a rugby ball shape (spheroid elongated in the
direction of the symmetrical axis), pancake shape (flattened
spheroid), and the like, the average length of three principal axes
of inertia is defined as the particle size in the present
description; when the particle is a complete sphere, the diameter
is the particle size) can be used as powdered fluorescent material
for light-emitting devices such as displays, lights, and the like.
In addition, such glass fine particles have an important
application as fluorescent probes by being bound to biomolecules.
The description is given below, by limiting to the fluorescent
silica glass fine particles.
[0007] In Patent Literature 2, 3, and 4 of the present inventors,
the sol-gel method in which alkoxide is hydrolyzed and
dehydration-condensed is used. In particular, a reverse micelle
method (a method in which the sol-gel method is performed in minute
water droplets dispersed in the oil phase, wherein
water-dispersible nanoparticles are dispersed in the water droplets
in advance) or Stober method (a method in which hydrolyzed alkoxide
is deposited on the nanoparticle surface) was used to develop a
technology to disperse multiple semiconductor nanoparticles in
silica glass fine particles with high PL efficiency (25% or
higher).
[0008] However, when the prepared silica glass fine particles are
applied as fluorescent probes in the field of biotechnology,
conditions for the evaluation of fluorescence properties are
usually significantly different from when the particles are
prepared as usual phosphor such as for light-emitting
materials.
[0009] Because silica glass fine particles have less scattering
when the particle size is about 100 nm or less, the particles are
introduced into a quartz cell having a light path of 1 cm while
being dispersed in a solution, and the quartz cell is measured
using an absorption spectrophotometer and a fluorescence
spectrophotometer for general purposes. Thereby, the absorbance and
the fluorescence intensity of each wavelength are determined. An
integrating sphere is used when the influence of scattering is a
concern; however, in this case, there is an increase in errors in
both absorbance and fluorescence intensity, compared to the case
where there is no scattering. Also in this case, general-purpose
measurement devices have recently been commercially available (for
example, C9920-02 by Hamamatsu Photonics K.K.).
[0010] The concentration of semiconductor nanoparticles just after
synthesis is usually about 1 to about 10 .mu.M (.mu.mol/L; this
indicates the number of semiconductor nanoparticles, rather than
the number of atoms constituting the nanoparticles). These
particles are stored as-is in a cool, dark space. When measuring
the PL efficiency, the concentration is diluted to about 200 to
about 300 nM because the above concentration is too high.
Consequently, the signal level that can be most easily measured
using a general-purpose absorption spectrophotometer or
fluorescence spectrophotometer is obtained. Pure water is often
used as the solvent. On the other hand, when semiconductor
nanoparticles are applied as fluorescent probes, the fluorescence
is often separately detected from one or several nanoparticles; and
in that case, the nanoparticle concentration is about 10 nM at
most, and the nanoparticles are dispersed in a highly concentrated
salt solution such as saline. Further, the irradiated light
intensity is also usually 10 W/cm.sup.2 or greater, which is more
intense compared to irradiated light intensity when measuring using
a spectrometer by a different order of magnitude. In this way, it
became clear that, in terms of material synthesis as described
above, when the concentration of dispersed nanoparticles is
extremely low and a large amount of salts are contained in the
solution, there is a case where nanoparticles are degraded even if
glass is used for coating of the nanoparticles. In order to prevent
such degradation, developing a glass network structure is one
effective means. Further, it is more preferable, from the viewpoint
of increasing the brightness, to incorporate multiple nanoparticles
in glass to form one glass fluorescent fine particle than to coat
glass with one nanoparticle.
[0011] As described later, the present inventors found that, among
the nanoparticles described in Patent Literature 2 to 4 above, it
is effective to use nanoparticles containing Cd and Se, for
example, CdSe nanoparticles, in order to prevent degradation.
However, CdSe nanoparticles having high PL efficiency are prepared
by an organic solution method in which water is removed at a high
level, and the CdSe nanoparticles are quenched when they are
dispersed as-is in an aqueous solution. Therefore, it is desirable
to disperse CdSe nanoparticles in glass fine particles while
maintaining the PL efficiency thereof. In particular, it is
desirable to disperse numerous nanoparticles in order to increase
the brightness as much as possible.
[0012] Further, in the search of a possible application in the
field of biotechnology, it became clear that fluorescent fine
particles having a particle size of 100 nm or less are effective.
Cells are typically 10 to 30 .mu.m in size, and when the particle
size exceeds 100 nm, the possibility of the particles being
internalized by cells through phagocytosis decreases. Further, when
the cell interior is stained in various colors, the particle size
of over 100 nm and close to 200 nm is not suitable for clear
staining, because the shape thereof can be seen under the optical
microscope when the particle size is in that range. On the other
hand, the particle size of semiconductor nanoparticles is a few to
several nanometers, and fine particles in which 10 or more
nanoparticles are dispersed are necessary in order to increase the
brightness by a different order of magnitude. In order to do so,
the particle size must be about 20 nm or more.
[0013] Next, the present inventors examined particles known as
silica glass fine particles in which nanoparticles containing Cd
and Se are dispersed, and the emission properties thereof.
[0014] Bawendi et al. reported fluorescent silica glass fine
particles in which CdSe/ZnS nanoparticles are dispersed and fixed
in glass by the sol-gel method, and a method for preparing the
particles (Non-Patent Literature 1). This preparation method is a
method in which the surface of nanoparticles that have been
synthesized in an organic solvent in advance is coated with
alkoxide having an amino group (3-aminopropyltrimethoxysilane) and
alcohol having an amino group (5-amino-1-pentanol), and the
resulting product is bound as a layer having a thickness of about
50 nm to the surface of separately prepared silica glass fine
particles having a diameter of about a few hundred nanometers. This
method provides fluorescent glass fine particles having a structure
in which the surface of silica glass fine particles not containing
nanoparticles is coated with a sol-gel glass layer containing
nanoparticles. However, because the nanoparticles are present only
around and on the surface of the glass fine particles, and no
nanoparticles are contained in the core of the glass fine
particles, it was not possible to increase the concentration of
dispersed nanoparticles in the glass fine particles. Additionally,
the PL efficiency was about 13%.
[0015] As another preparation method, a method in which alkoxide
having a thiol group or the like is formed on the surface of
CdSe/ZnS nanoparticles, and silica glass fine particles containing
one nanoparticle per one silica glass fine particle are prepared
(Non-Patent Literature 2) has been reported. The PL efficiency in
this case is reported to be 5 to 18%. There is a report on a silica
glass fine particle prepared by a similar method, wherein the
particle has a particle size of 30 nm to 1 .mu.m and contains one
CdSe/ZnS nanoparticle; however, this report is silent about the PL
efficiency (Non-Patent Literature 3).
[0016] Meijerink et al. introduced
CdSe/CdS/Cd.sub.0.5Zn.sub.0.5S/ZnS (CdSe as the core is
sequentially coated with CdS, Cd.sub.0.5Zn.sub.0.5S, and ZnS)
nanoparticles into silica glass fine particles by a reverse micelle
method in order to introduce one nanoparticle into one silica glass
fine particle. However, based on the examination of the mechanism,
it was found that because hydrolyzed alkoxide has a high affinity
for nanoparticles, the ligands arranged on the nanoparticle surface
at the time of preparation are replaced by the hydrolyzed alkoxide,
thus quenching the emission. Accordingly, in regard to the silica
glass fine particle containing only one nanoparticle, the PL
efficiency of the nanoparticle was rapidly decreased immediately
after preparation, and was further gradually decreased. One week
after preparation, the PL efficiency was about 2% of what it was
before being introduced into the silica glass (a drop from the
initial value of 60% to 1.2% in the absolute value). In order to
suppress such quenching effect of silica glass, a nanoparticle with
a specially made thick shell was used. As a result, the PL
efficiency was increased to a maximum of 35% (Non-Patent Literature
4). However, such a nanoparticle with a specially made thick shell
has a large particle size, and is not suitable for application in
the field of biotechnology; additionally, it is difficult to
prepare such nanoparticles.
[0017] There is known research in which a water-dispersible CdSe
nanoparticle (citric acid coating) was prepared, and several of
these nanoparticles were introduced into silica glass fine
particles. However, the PL efficiency of water-dispersible CdSe
nanoparticles is 0.1 to 0.15%, which is extremely low (Non-Patent
Literature 5). The PL efficiency of the nanoparticle when it is
introduced into a silica matrix is nowhere described; however, the
PL efficiency is usually further decreased in that case. Therefore,
such a product cannot be called a "fluorescent material
(phosphor)." Further, in the case of relatively recent literature
(Non-Patent Literature 6) in which a water-dispersible CdSe
nanoparticle was similarly introduced into a silica particle by a
reverse micelle method, the PL efficiency was 1.48% at most, and
this nanoparticle cannot be called a "fluorescent material
(phosphor)." As illustrated in Patent Literature 6, as a rough
standard, the PL efficiency should be 20% or higher for a
nanoparticle to be called a fluorescent material (phosphor).
[0018] As described above, a method for preparing fluorescent
silica fine particles having a particle size of 20 to 100 nm, in
which 10 or more nanoparticles containing Cd and Se are dispersed,
has not been developed.
[0019] Meanwhile, recently, there is a report on a method for
preparing an assembly of multiple nanoparticles using a linear
polymer (Non-Patent Literature 7). Polymer particles prepared by
this method are reported to have an average particle size of 112
nm, as measured by dynamic light scattering. At present, it is
difficult to prepare nanoparticles having a particle size of 100 nm
or less. It is possible to glass-coat the surface of the assembly;
however, this further increases the particle size. Further, a glass
material not containing a polymer generally has better durability,
and produces a smaller amount of dissolved Cd released from
particles. Therefore, there is a demand to prepare a glass material
in which numerous semiconductor nanoparticles are dispersed,
without using a polymer.
CITATION LIST
Patent Literature
[0020] PTL 1: Japanese Patent No. 4366502 [0021] PTL 2: Japanese
Patent No. 3677538 [0022] PTL 3: Japanese Patent No. 3755033 [0023]
PTL 4: Japanese Domestic Re-Publication of PCT International
Application No. 2007/034877 [0024] PTL 5: Japanese Unexamined
Patent Publication No. 2006-282977 [0025] PTL 6: Japanese Patent
No. 4555966
Non-Patent Literature
[0025] [0026] NPL 1: Chan, Zimmer, Stroh, Steckel, Jain, and
Bawendi, Advanced Materials, Vol. 16, p. 2092 (2004) [0027] NPL 2:
Gerion, Pinaud, Williams, Parak, Zanchet, Weiss, and Alivisatos,
Journal of Physical Chemistry B, Vol. 105, p. 8861 (2001) [0028]
NPL 3: Nann and Mulvaney, Angewandte Chemie International Edition,
Vol. 43, p. 5393 (2004) [0029] NPL 4: Koole, Schooneveld, Hilhorst,
Donega, Hart, Blaaderen, Vanmaekelbergh, and Meijerink, Chemistry
of Materials, Vol. 20, p. 2503 (2008) [0030] NPL 5: Rogach,
Nagesha, Ostrander, Giersig, and Kotov, Chemistry of Materials,
Vol. 12, p. 2676 (2000) [0031] NPL 6: Chu, Sun, and Xu, Journal of
Nanoparticle Research, Vol. 10, p. 613 (2008) [0032] NPL 7: Yang,
Dave, and Gao, Journal of American Chemical Society, Vol. 130, p.
5286 (2008)
SUMMARY OF INVENTION
Technical Problem
[0033] An object of the present invention is to provide silica
glass fluorescent fine particles having high durability and an
average particle size of 20 to 100 nm, in which numerous
nanoparticles having high PL efficiency are dispersed. Further,
another object of the present invention is to show means to apply
the silica glass fluorescent fine particles in the field of
biotechnology.
Solution to Problem
[0034] In order to solve the above-described problems, first, the
present inventors examined the stability of nanoparticles dispersed
at a low concentration in a buffer solution. As a result, it was
found that nanoparticles containing Cd and Se are most excellent,
and the present inventors thus decided to use such nanoparticles.
It is even more excellent when the nanoparticle has a shell of ZnS,
ZnSe, CdS, and the like on the surface. The shell may have a
gradient composition in which the compositions of Cd, Zn, Se, S,
and the like vary in the direction of the shell thickness. Further,
in regard to silica glass fine particles for dispersing
nanoparticles, it was found that the Stober method, rather than a
reverse micelle method, is suitable to obtain a uniform particle
size in the desired particle size range (20 to 100 nm); and a
synthesis method comprising the following three steps was
developed. Steps (A), (B), and (C) described below respectively
correspond to steps (A), (B), and (C) recited in the claim. Metal
that contains silicon is preferably used as metal to be contained
in a metal alkoxide (1) and a metal alkoxide (2) (described later),
in terms of controlling the hydrolysis rate.
[0035] Step 1 (Step (A) in the Claim)
[0036] An adequate amount of the metal alkoxide (1) is added to
lipophilic semiconductor nanoparticles containing Cd and Se and
appropriately hydrolyzed, thereby replacing the ligands on the
nanoparticle surface by a hydrolysate of the metal alkoxide (1).
The thus-obtained solution is regarded as organic solution A. In
this step, the nanoparticle surface is coated with the metal
alkoxide (1) like a surfactant, thereby suppressing a decrease in
the PL efficiency.
[0037] Step 2 (Step (B) in the Claim)
[0038] The metal alkoxide (2) is dispersed in an aqueous solution
to cause partial hydrolysis, thereby obtaining aqueous solution B.
As the metal alkoxide (2), a metal alkoxide whose hydrolysis rate
is lower than that of the metal alkoxide (1) is selected. The
aqueous solution B is mixed with organic solution A, thereby
further forming a layer of the metal alkoxide (2) on the surface of
semiconductor nanoparticles coated with the metal alkoxide (1).
When the semiconductor nanoparticles are in contact with water, the
metal alkoxide on the surface is further hydrolyzed, becomes
hydrophobic, and moves to the aqueous phase. At this time, the
nanoparticles form an assembly. Because the metal alkoxide (2)
present around and on the surface has a lower hydrolysis rate
compared to the metal alkoxide (1) used in step 1, the metal
alkoxide (2) plays a role in preventing formation of large lumps of
alkoxide caused by sudden dehydration-condensation and aggregation
when alkoxide moves to the aqueous phase. Accordingly, there is a
tendency that small assemblies are formed when the amount of metal
alkoxide (2) is large and large assemblies are formed when the
amount is small. Adding alcohol to the aqueous solution B used
herein increases the contact area with the organic solution A,
thereby reducing the reaction time. Examples of alcohols include
methanol, ethanol, isopropanol, and the like.
[0039] Step 3 (Step (C) in the Claim)
[0040] A silica glass layer is further deposited on the assemblies
in the aqueous phase, thereby preparing silica fine particles in
which semiconductor nanoparticles are dispersed. This is performed
by a regular Stober method in which a slight amount of metal
alkoxide (3) is hydrolyzed by a large amount of water and alcohol
in the alkaline region, and deposited on each nanoparticle assembly
that serves as the core. The thus-obtained fine particles are
separated and washed, if necessary. The metal alkoxide (3) may be
the same as the metal alkoxide (1) or (2).
[0041] It is also possible to modify the above-described
preparation method so as to simultaneously add the metal alkoxide
(1) and the metal alkoxide (2) whose hydrolysis rate is lower than
that of the metal alkoxide (1) in step 1, thereby obtaining the
organic solution A (step (A1) in the claim). At this time, in step
2, the metal alkoxide (3) and an alkaline aqueous solution are
added to render the nanoparticle surface in the organic solution A
hydrophilic so as to transfer the nanoparticles to the aqueous
phase, and assemblies are formed at the same time (step (B1) in the
claim). In this case, the nanoparticle surface is coated with two
types of metal alkoxides (1) and (2). Because of its effect, there
was a case where assemblies in which the nanoparticles are
regularly arranged were obtained. It is possible to further protect
the surface of the nanoparticle assemblies with a silica glass
layer by applying the above-described step 3 after step 2.
[0042] This preparation method is generally described as
follows.
[0043] First, the metal alkoxides A and B are added to a non-polar
solvent in which lipophilic semiconductor nanoparticles are
dispersed; and the mixture is stirred, thereby obtaining organic
solution X. Herein, it is possible to control the mutual distance
between each assembly during formation of assemblies in the next
stage and the PL efficiency by simultaneously adding the metal
alkoxide A and the metal alkoxide B whose hydrolysis rate is lower
than that of the metal alkoxide A. Examples of non-polar solvents
include toluene, hexane, benzene, diethyl ether, chloroform, ethyl
acetate, methylene chloride, and the like (step (A2) in the claim).
In this step, the surface of lipophilic semiconductor nanoparticles
is replaced by partially hydrolyzed metal alkoxides A and B. Next,
this organic solution X is brought into contact with solution Y
containing the metal alkoxide A or B and water, thereby
transferring the semiconductor nanoparticles in the organic
solution X to the solution Y (step (B2) in the claim). In this
step, the phase transfer is performed using characteristics that
the alkoxides A and B that are bound to the surface of the
semiconductor nanoparticles in the organic solution X are further
hydrolyzed and become hydrophilic by contact with the water. Once
in the water, hydrolysis or dehydration-condensation of the
alkoxide further progresses, resulting in the formation of
assemblies of the semiconductor nanoparticles. Subsequently, the
surface of the assembly may be coated with silica glass or modified
with a functional group, if necessary.
[0044] The present inventors confirmed that fluorescent silica
glass fine particles in which semiconductor nanoparticles
containing Cd and Se are dispersed, which are prepared by the
sol-gel method modified as described above, achieve an average
particle size of 20 to 100 nm, and form assemblies in which
nanoparticles are densely packed while substantially maintaining
the PL efficiency obtained in the organic solution; and that even
when the concentration is diluted to about 10 nM in a buffer
solution for biotechnology, a decrease in the PL efficiency and
degradation such as dissolution of cadmium ions are less likely to
occur because of the silica network structure. The present
invention was completed based on the above findings.
[0045] Specifically, the present invention provides highly durable
fine particles in which semiconductor nanoparticles with high
brightness are dispersed, and a method for preparing the fine
particles by a sol-gel method, as described below. In the present
invention, the PL efficiency of prepared fluorescent fine particles
is close to 30% by the use of nanoparticles whose PL efficiency in
an organic solution is 30%. It is possible to obtain fluorescent
fine particles whose PL efficiency is close to 70% by the use of
nanoparticles whose PL efficiency in an organic solution is
70%.
Item 1. Fluorescent fine particles comprising Cd- and Se-containing
semiconductor nanoparticles dispersed in silicon-containing fine
particles, wherein the average particle size of the
silicon-containing fine particles is 20 to 100 nm, and the number
of semiconductor nanoparticles dispersed in each silicon-containing
fine particles is 10 or more. Item 2. The fluorescent fine
particles according to Item 1, wherein the average particle size of
the silicon-containing fine particles is 40 to 100 nm, and the
number of semiconductor nanoparticles dispersed in each
silicon-containing fine particles is 20 or more. Item 3. The
fluorescent fine particles according to Item 1 or 2, wherein the
semiconductor nanoparticles dispersed in each silicon-containing
fine particles are regularly arranged. Item 4. The fluorescent fine
particles according to any one of Items 1 to 3, wherein the PL
efficiency is 20% or higher. Item 5. The fluorescent fine particles
according to any one of Items 1 to 4, wherein the PL efficiency is
20% or higher when the semiconductor nanoparticles are dispersed at
a concentration of 10 nmol/L in a pH 7.4 solution of HEPES
(2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) serving
as a Good's buffer. Item 6. The fluorescent fine particles
according to any one of Items 1 to 5, wherein after the
semiconductor nanoparticles are dispersed in a pH 7.4 solution
(HEPES concentration of 10 mmol/L) of HEPES
(2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) serving
as a Good's buffer, at a concentration of X nmol/L, and left to
stand at room temperature for 15 hours, Y nanograms that indicate
the dissolved amount of Cd in the HEPES solution (the weight of
dissolved Cd in a solution of 1 mL) satisfies 10.times.Y/X<1.
Item 7. The fluorescent fine particles according to any one of
Items 1 to 6, wherein the semiconductor nanoparticles are CdSe
and/or CdSe/ZnS in which CdSe is coated with ZnS. Item 8. The
fluorescent fine particles according to any one of Items 1 to 7,
having a silicon-containing layer which has a thickness of 2 nm or
more. Item 9. The fluorescent fine particles according to any one
of Items 1 to 8, comprising on the surface at least one selected
from the group consisting of COOH, NH.sub.2, SH, salts thereof, and
groups originated from polyethyleneglycol. Item 10. The fluorescent
fine particles according to any one of Items 1 to 9 for fluorescent
probes. Item 11. The fluorescent fine particles according to any
one of Items 1 to 9 for electronic materials. Item 12. The
fluorescent fine particles according to any one of Items 1 to 11,
wherein the fluorescent fine particles exhibit electroluminescence
and/or cathodeluminescence. Item 13. A method of preparing the
fluorescent fine particles according to any one of Items 1 to 12,
comprising the steps of: (A) adding a metal alkoxide (1) to an
organic solvent in which Cd- and Se-containing lipophilic
semiconductor nanoparticles are dispersed, and stirring the
mixture, thereby obtaining organic solution A; (B) mixing aqueous
solution B containing a metal alkoxide (2) whose hydrolysis rate is
lower than that of the metal alkoxide (1) with the organic solution
A and stirring the mixture, thereby obtaining an semiconductor
nanoparticle assembly; and (C) adding a solution containing a metal
alkoxide (3) to an alkaline aqueous solution containing the
semiconductor nanoparticle assembly, thereby foisting a coating
layer on the surface of the semiconductor nanoparticle assembly.
Item 14. The method of preparing fluorescent fine particles
according to Item 13, wherein the metal alkoxide (2) is a compound
represented by Formula (II):
X.sub.n--Si(OR.sup.2).sub.4-n (II)
wherein X represents a group represented by CH.sub.2.dbd.CH--, a
group containing oxirane, a group represented by
H.sub.2NC.sub.mH.sub.2m--, a group represented by
CH.sub.2.dbd.C(CH.sub.3)COOC.sub.pH.sub.2p--, a group represented
by HSC.sub.qH.sub.2q--, or a phenyl group; R.sup.2 represents a
lower alkyl group; n is an integer of 1, 2, or 3; m is an integer
of 1 to 6; p is an integer of 1 to 5; and q is an integer of 1 to
10. Item 15. The method of preparing fluorescent fine particles
according to Item 13 or 14, wherein heating is performed during
Step (C). Item 16. A method of preparing the fluorescent fine
particles according to any one of Items 1 to 12, comprising the
steps of: (A1) adding a metal alkoxide (1) and a metal alkoxide (2)
whose hydrolysis rate is lower than that of the metal alkoxide (1)
to an organic solvent in which Cd- and Se-containing lipophilic
semiconductor nanoparticles are dispersed, and stirring the
mixture, thereby obtaining organic solution A; and (B1) adding a
metal alkoxide (3) and an alkaline aqueous solution to the organic
solution A, thereby forming a semiconductor nanoparticle assembly.
Item 17. A method of preparing the fluorescent fine particles
according to any one of Items 1 to 12, comprising the steps of:
(A2) adding two types of metal alkoxides to a non-polar solvent in
which Cd- and Se-containing lipophilic semiconductor nanoparticles
are dispersed, and stirring the mixture, thereby obtaining organic
solution X; and (B2) bringing the organic solution X into contact
with solution Y containing a metal alkoxide and water so as to
transfer the semiconductor nanoparticles in the organic solution X
to the solution Y.
ADVANTAGEOUS EFFECTS OF INVENTION
[0046] The fluorescent fine particles of the present invention
contain a dispersion of a considerable amount of semiconductor
nanoparticles having durability and high PL efficiency, and the
surface of thereof is coated with appropriately hydrolyzed
alkoxide. These semiconductor nanoparticles form an assembly of an
appropriate size, and the circumference of the assembly is coated
with silica glass. Therefore, the present invention achieves the
effect of providing two characteristics, i.e., high durability and
high brightness. Further, because the average particle size is 20
to 100 nm, the fluorescent fine particles are applicable as
fluorescent probes in the field of biotechnology. The semiconductor
nanoparticle assembly being coated with silica glass can be
confirmed by analysis of the coated portion using an analytical
electron microscope because the results show that silicon and
oxygen are contained. It can also be confirmed from the fact that
powder X-ray diffraction (irradiation with copper K.alpha. ray, at
1.5406 angstrom) of vacuum-dried powder sample shows a broad
diffraction peak (a full width at half-maximum of 5 degrees or
more) near an angle (20) of 23 degrees.
BRIEF DESCRIPTION OF DRAWINGS
[0047] FIG. 1A A schematic diagram showing step 1 in the
preparation process of the fluorescent fine particles prepared in
Example 1.
[0048] FIG. 1B A schematic diagram showing step 2 in the
preparation process of the fluorescent fine particles prepared in
Example 1.
[0049] FIG. 1C A schematic diagram showing step 3 in the
preparation process of the fluorescent fine particles prepared in
Example 1.
[0050] FIG. 2 A graph showing the concentration of water and the PL
efficiency of the semiconductor nanoparticles in step 1 in Example
1.
[0051] FIG. 3 A projection view (two-dimensional image) obtained by
projecting, in one direction, a three-dimensional tomography image
of the fluorescent fine particles prepared in Example 1. Silica
glass components are pale white. The view shows dark white
semiconductor nanoparticles in the silica glass components. In
regard to the scale of the view, the average particle size
(diameter) of one semiconductor nanoparticle is 5.5 nm.
[0052] FIG. 4 Transmission electron microscope images of silica
glass fine particles (1) to (4) prepared in Example 7. The images
(1L) to (4L) on the right side are enlarged images of (1) to (4),
respectively.
[0053] FIG. 5 A transmission electron microscope image of an
assembly in which nanoparticles prepared in Example 8 are regularly
arranged. The surface of the assembly can be coated with a layer of
silica glass.
DESCRIPTION OF EMBODIMENTS
[0054] Hereinafter, the present invention is described in the order
of preparation of fluorescent fine particles, application, and
evaluation.
[0055] I. Preparation of Fluorescent Fine Particles
[0056] The present invention prepares fluorescent fine particles in
which 10 or more Cd- and Se-containing fluorescent semiconductor
nanoparticles are dispersed in a matrix containing silicon having
an average particle size of 20 to 100 nm. The preparation process
comprises four steps: 1. preparation of semiconductor
nanoparticles, 2. coating of the nanoparticles, 3. formation of a
nanoparticle assembly, and 4. preparation of a fluorescent fine
particle by coating the assembly with glass. Further, if necessary,
another step, 5. modification of the surface of the fluorescent
fine particle, may be performed. When preparing fluorescent fine
particles as electronic materials, the preparation process may be
ended at the second, third, or fourth step. For the application
where electrons are passed through the nanoparticles in electronic
materials, a glass layer on the surface or a functional group on
the surface on the glass layer may be unnecessary. These steps are
described below in order.
[0057] First Step: Preparation of Semiconductor Nanoparticles
[0058] Examples of semiconductor nanoparticles used in the present
invention include those containing Cd and Se, specifically, CdSe,
CdSe/ZnS (CdSe nanoparticle coated with ZnS),
CdSe/CdS/Cd.sub.0.5Zn.sub.0.5S/ZnS (nanoparticle obtained by
sequentially coating CdSe as the core with CdS,
Cd.sub.0.5Zn.sub.0.5S, and ZnS), and CdSe.sub.xTe.sub.1-x
(0<x<1) having an alloy composition. These semiconductor
nanoparticles are prepared by the following ten known, typical
methods. [0059] Dmitri V. Talapin, Andrey L. Rogach, Ivo Mekis,
Stephan Haubold, Andreas Kornowski, Markus Haase, Horst Weller,
Colloids and Surfaces A, 202, 145 (2002). [0060] Dmitri V. Talapin,
Andrey L. Rogach, Andreas Kornowski, Markus Haase, and Horst
Weller, NANO LETTERS, 1, 207 (2001) [0061] Z. Adam Peng and
Xiaogang Peng, J. Am. Chem. Soc., 123, 1389 (2001). [0062] C. B.
Murray, D. J. Norris, M. G. Bawendi, J. Am. Chem. Soc., 115, 8706
(1993). [0063] B. O. Dabbousi, J. Rodriguez-Viejo, F. V. Mikulec,
J. R. Heine, H. Mattoussi, R. Ober, K. F. Jensen, and M. G.
Bawendi, J. Phys. Chem. B, 101 (46), 9463 (1997). [0064] Lianhua Qu
and Xiaogang Peng, J. Am. Chem. Soc., 124, 2049 (2002). [0065]
Xinhua Zhong, Yaoyu Feng, and Yuliang Zhang, J. Phys. Chem. C, 111,
526 (2007). [0066] Renguo Xie, Ute Kolb, Jixue Li, Thomas Basche,
and Alf Mews, J. Am. Chem. Soc., 127, 7480 (2005). [0067] Robert E.
Bailey and Shuming Nie, J. Am. Chem. Soc., 125, 7100 (2003). [0068]
S. Jun., E. Jang, J. E. Lim, Nanotechnology, 17, 3892 (2006).
[0069] The average particle size of semiconductor nanoparticles
prepared by these methods is about 2 to 9 nm.
[0070] All of the methods use a high-temperature reaction in an
organic solution from which water is removed. The ligands on the
nanoparticle surface prepared in the first step are replaced by the
metal alkoxide (1) hydrolyzed in the second step (step 1) described
below. Therefore, ligands with low binding energy are preferable in
this step. Examples of specific ligands include phosphate compounds
having an alkyl group (trioctylphosphine, trioctylphosphine oxide,
and the like), alkylamine, oleic acid, and the like.
[0071] Second Step: Coating of Nanoparticles (Step 1 (Step (A) in
the Claim))
[0072] In the second step, by a sol-gel method using the hydrolyzed
metal alkoxide (1), the surface of the semiconductor nanoparticles
prepared in the first step is coated with the hydrolyzed metal
alkoxide (1).
[0073] To coat the semiconductor nanoparticles with silica glass
while maintaining the PL efficiency of semiconductor nanoparticles
has been an interest of the academic community in recent years.
Therefore, there was a continuing debate regarding whether the
original ligands on the surface are removed when hydrophobic
nanoparticles are coated with silica glass. Later, known Non-Patent
Literature (Chemistry of Materials, Vol. 20, p. 2503 (2008))
demonstrated that hydrolyzed alkoxide has a high affinity for
nanoparticles, and coats the nanoparticles by replacing ligands
such as alkylamine bound to the surface at the time of preparation.
However, it has been reported that while the alkylamine ligands
improve the PL efficiency by eliminating surface defects, a ligand
((Et-O).sub.3--Si--O.sup.-) comprising a partially hydrolyzed
alkoxide has a quenching effect, which causes a rapid decrease in
the PL efficiency. At the same time, it has been unknown why
hydrolyzed alkoxide has a quenching effect.
[0074] On the other hand, it is considered necessary that the
ligands closely cover the nanoparticle surface in order to maintain
the PL efficiency. For this, unbranched primary alkylamines are
effective, as described in Non-Patent Literature (Colloids and
Surfaces A, Vol. 202, p. 145 (2002)).
[0075] As a result of a search for coating conditions based on
these literature, the present inventors found that there is hardly
any decrease in the PL efficiency of nanoparticles even after the
surface is replaced by alkoxide hydrolysates, as long as the
hydrolysis rate of alkoxide is reduced, the concentration of
semiconductor nanoparticles is diluted low, and the reaction time
is extended. Specifically, it became clear that hydrolyzed alkoxide
itself does not have a quenching effect, but quenching results from
random aggregation of alkoxides on the nanoparticle surface, an
insufficient number of alkoxide bound to the surface, and the like.
The present inventors revealed such a mechanism, and found a method
for coating the surface of nanoparticles with hydrolysis products
of alkoxide without quenching the nanoparticles.
[0076] Specifically, first, the hydrophobic nanoparticles prepared
in the above-described first step, covered with an alkyl
group-containing phosphate compound, alkylamine, oleic acid, and
the like are provided. At least three types of the following
alkoxides are provided: the metal alkoxide (1) used in the second
step, the metal alkoxide (2) used in the subsequent third step, and
the metal alkoxide (3) further used in the fourth step. In regard
to these metal alkoxides, it is advantageous that silicon is used
as at least one type of metal to facilitate control of hydrolysis.
Further, the metal alkoxides (1) and (2) are selected in such a
manner that the hydrolysis rate of the metal alkoxide (1) in the
alkaline solution is higher than that of the metal alkoxide (2). If
necessary, another metal alkoxide may further be added to improve
controllability of hydrolysis rate, fine particle size and form,
and the like.
[0077] The metal alkoxide (1) used in this step is not particularly
limited; however, a metal alkoxide with a high hydrolysis rate is
preferable because the metal alkoxide (1) must have a higher
hydrolysis rate than the metal alkoxide (2) as described above.
[0078] Specific examples include a metal alkoxide represented by
Formula (I):
Si(OR.sup.1).sub.4 (I)
wherein R.sup.1 represents a lower alkyl group.
[0079] In this step, for example, the synthesis can be performed by
the following procedure.
[0080] 2 to 400 .mu.L (more preferably 5 to 80 .mu.L, and most
preferably 10 to 30 .mu.L) of the metal alkoxide (1) is added to a
non-polar solvent (about 2.5 mL) such as toluene in which the
nanoparticles are dispersed, thereby obtaining organic solution A.
When tetraethoxysilane (tetraethyl orthosilicate) (TEOS) is
selected as the metal alkoxide (1), this solution is stirred for 1
to 8 hours (more preferably 2 to 5 hours, and most preferably 2.5
to 3.5 hours), and organic solution A can thereby be obtained.
[0081] When the stirring time is shorter than the above range,
there is a tendency for transfer to the aqueous phase to not be
easily carried out in the subsequent third step because the amount
of hydrolyzed metal alkoxides (1) bound to the nanoparticle surface
is small. Further, when the stirring time is longer than the above
range, there is a tendency for the PL efficiency to decrease and
agglomeration to occur because too many hydrolyzed metal alkoxides
(1) are bound to the nanoparticle surface.
[0082] The hydrolysis rate increases when, for example,
tetramethoxysilane (tetramethyl orthosilicate) (TMOS) is selected
instead of TEOS. Therefore, the stirring time can be shortened if
other hydrolysis conditions are the same. Specifically, the
stirring time (hydrolysis time) is preferably set to, for example,
about 30 minutes to about 4 hours. When silicon alkoxide is used, a
silica glass layer containing transparent silicon is formed. In the
case of TEOS, because it is better to decrease the hydrolysis rate,
it is effective to perform hydrolysis only using moisture drawn
from the air. A glovebox with adjusted atmosphere can also be used
to perform hydrolysis in a more controlled manner. As described
above, the stirring time and the like may be adjusted according to
the hydrolysis rate of the metal alkoxide (1) used.
[0083] Third Step: Formation of Nanoparticle Assembly (Step 2 (Step
(B) in the Claim))
[0084] In this step, first, an appropriate amount of an appropriate
type of metal alkoxide (2) is added to the aqueous phase to cause
hydrolysis, thereby obtaining aqueous solution B. The reaction at
this time preferably occurs in the alkaline region. For this
reason, ammonia, sodium hydroxide, or the like is added, with
ammonia being particularly preferable. Further, the aqueous
solution B and the organic solution A are mixed together, and
assemblies of the nanoparticles are formed while the nanoparticles
are transferred to the aqueous phase.
[0085] As described above, the metal alkoxide (2) in this step
preferably has a lower hydrolysis rate than the metal alkoxide (1)
in the second step. Specific examples include those represented by
Formula (II):
X.sub.n--Si(OR.sup.2).sub.4-n (II)
wherein X represents a group represented by CH.sub.2.dbd.CH--, a
group containing oxirane, a group represented by
H.sub.2NC.sub.mH.sub.2m--, a group represented by
CH.sub.2.dbd.C(CH.sub.3)COOC.sub.pH.sub.2p--, a group represented
by HSC.sub.qH.sub.2q--, or a phenyl group; R.sup.2 represents a
lower alkyl group; n is an integer of 1, 2, or 3; m is an integer
of 1 to 6; p is an integer of 1 to 5; and q is an integer of 1 to
10.
[0086] In this step, for example, the synthesis can be performed by
the following procedure.
[0087] The metal alkoxide (2) (0.2 to 5 .mu.L, more preferably 0.5
to 2 .mu.L, and most preferably 0.7 to 1.5 .mu.L) is mixed with an
organic solvent such as ethanol or the like (10 to 50 mL, more
preferably 15 to 30 mL, most preferably 20 to 27 mL) and alkaline
aqueous solution such as aqueous ammonia or the like (1 to 8 mL,
more preferably 2 to 6 mL, and most preferably 3 to 5 mL; the
concentration is about 3 wt %). The aqueous solution B can be
obtained using 3-mercaptopropyl trimethoxysilane (MPS) or the like
as the metal alkoxide (2). The hydrolysis rate of the metal
alkoxide (2) such as MPS as a typical example is lower than TEOS or
the like used as the metal alkoxide (1). The molar concentration of
the metal alkoxide (2) is preferably 1.9.times.10.sup.-5 to
4.7.times.10.sup.-4 mol/L, more preferably 4.7.times.10.sup.-5 to
1.9.times.10.sup.-1 mol/L, and most preferably 6.6.times.10.sup.-5
to 1.4.times.10.sup.-4 mol/L.
[0088] When the organic solution A and the aqueous solution B are
mixed together and stirred for 1 to 8 hours (more preferably 2 to 6
hours, and most preferably 2.5 to 3.5 hours), the hydrolysis of
alkoxides on the surface proceeds. Consequently, the semiconductor
nanoparticles are transferred to the aqueous phase; and further,
assemblies of the nanoparticles are formed in the aqueous
phase.
[0089] In these second and third steps, when the amount of the
organic solvent (toluene) described at the beginning of the second
step is changed from 1 mL, the amount of each solution may be
changed in a proportional manner.
[0090] Herein, if TEOS or the like used as the metal alkoxide (1)
is used as the metal alkoxide (2), the size of the assemblies will
rapidly increase in the aqueous phase, causing white turbidity.
This shows that the metal alkoxide (2) has an effect of adjusting
the size of the assemblies.
[0091] In this step, the alkoxides on the surface of the
hydrophobic nanoparticles prepared in the first and second steps
are further hydrolyzed, and the nanoparticles are thereby gradually
rendered hydrophilic and converted to the aqueous phase. When the
nanoparticles are converted to the aqueous phase, the hydrolysis
reaction proceeds rapidly and randomly, resulting in a large
assembly and white turbidity of the solution, if no countermeasure
is taken. Such a phenomenon is observed when a metal alkoxide with
a high hydrolysis rate, such as TEOS, is used as the metal alkoxide
(2). However, when an alkoxide with a low hydrolysis rate is added
to the aqueous phase, the alkoxide is bound to the surface of the
nanoparticles that are transferred to the aqueous phase, allowing
gradual aggregation of the nanoparticles. This causes a decrease in
the particle size distribution of the assemblies and the
distribution of the number of incorporated nanoparticles.
Accordingly, a decrease in the amount of the metal alkoxide (2)
added to the aqueous phase causes an increase in the size of the
assemblies and in the number of incorporated nanoparticles. The
present inventors revealed such a mechanism, and found a method for
controlling the number of nanoparticles to be dispersed and the
whole particle size, and forming an assembly in which silica glass
acts as the glue in the aqueous phase.
[0092] Fourth Step: Preparation of Fluorescent Fine Particles by
Coating the Assemblies with Glass (Step 3 (Step (C) in the
Claim))
[0093] In this step, the metal alkoxide (3) is hydrolyzed in an
alkaline aqueous solution containing alcohol by the so-called
Stober method, and the nanoparticle assemblies prepared in the
third step are used as seeds to deposit silica glass components on
the surface of the cores, thereby forming silica glass coating.
Ammonia, sodium hydroxide, or the like is used for alkalification,
with ammonia being particularly preferable. Examples of alcohols
include lower (the carbon number is 5 or less) primary alcohols,
with ethanol, methanol, and the like being preferable. Preferable
examples of the metal alkoxide (3) are tetrafunctional metal
alkoxydes, with TEOS being particularly preferable.
[0094] In this step, the solution obtained in the third step is
centrifuged, if necessary, to remove large particles. Further,
water is removed to concentrate the solution to render the
concentration of the semiconductor nanoparticles about 0.1 to about
5 .mu.M (more preferably 0.3 to 3 .mu.M, and most preferably 0.5 to
2 .mu.M). The thus-obtained aqueous solution (about 0.5 mL) is
extracted, and a lower alcohol (2 to 20 mL, more preferably 4 to 10
mL, and most preferably 7 to 9 mL) and an alkaline aqueous solution
(0.01 to 0.2 mL, more preferably 0.05 to 0.15 mL, and most
preferably 0.07 to 0.12 mL; the concentration is about 25 wt %) are
added to the solution. Further, the metal alkoxide (3) (5 to 40
.mu.L, more preferably 7 to 20 .mu.L, and most preferably 10 to 16
.mu.L) is added dropwise thereto. In this way, a silica glass layer
is formed on the surface of the semiconductor nanoparticle
assemblies, and silica glass fine particles are obtained. Further,
after stirring for 0.5 to 5 hours, more preferably 1 to 4 hours,
and most preferably 1.5 to 3 hours, the mixture is centrifuged to
extract the fluorescent fine particles. Subsequently, the
fluorescent fine particles may be dispersed in pure water at a
concentration of about 1 .mu.M. In this step, when the amount of
initially used aqueous solution is changed from 0.5 mL, the amount
of each solution may be changed in a proportional manner.
[0095] In order to prevent dissolution of constituents of the
nanoparticles, in particular, dissolution of Cd, from the
fluorescent fine particles, the thickness of the silica glass layer
(shell) prepared in this step is preferably 2 nm or more, and most
preferably 4 nm or more. When the average particle size of the
fluorescent fine particles becomes greater than 20 nm and reaches
about 50 nm, the thickness of the silica glass layer can be 10 nm
or more. Further, when the average particle size of the fluorescent
fine particles is close to 100 nm, the thickness of the silica
glass layer can be 20 nm or more. The thickness of the layer of
silica glass can be easily determined by measuring an outer portion
in which semiconductor nanoparticles are not contained by
transmission electron microscope observation. When the thickness
varies depending on the position along the circumference, the
average thickness determined from the entire circumference is
defined as the thickness of the silica glass layer.
[0096] Further, by performing heating in this step, it is possible
to develop the silica glass network structure so as to more
effectively prevent the dissolution of Cd. The heating temperature
is about 30 to about 85.degree. C., more preferably about 35 to
about 60.degree. C., and most preferably about 37 to about
50.degree. C.
[0097] Fifth Step: Surface Modification of Fluorescent Fine
Particles
[0098] Hydroxyl groups are present on the surface of the
water-dispersible fluorescent fine particles prepared in the
above-described fourth step, if left as-is. The surface can be
modified with a carboxyl group by further adding an alkoxide
containing a carboxyl group (for example, carboxyethylsilanetriol
sodium salt or the like) to the fluorescent fine particles. The
surface can also be modified with a thiol group by further adding
an alkoxide containing a thiol group (for example,
3-mercaptopropyltrimethoxysilane (MPS) or the like) to the
fluorescent fine particles. It is also possible to simultaneously
coat the surface with silica and modify the surface by mixing the
alkoxide used in this step with the metal alkoxide (3). Further,
the surface can also be modified using an amino group (for example,
3-aminopropyltrimethoxysilane (APS) or the like) or a group
originated from polyethyleneglycol (for example,
2-[methoxy(polyethylenoxy)propyl]-trimethoxysilane or the
like).
[0099] II. Application of Fluorescent Fine Particles
[0100] The fluorescent fine particles of the present invention
specifically bind to specific molecules in living organisms, using
the surface modification, and can be used as fluorescent probes for
observing the distribution, amount, behavior, and the like of the
specific molecules. Further, because the fluorescent fine particles
are high in brightness, PL efficiency, and durability, these
particles can also be used as fluorescent materials with good
energy efficiency for electronic materials. In addition to the
above-described application in which a short-wavelength light is
irradiated to cause excitation and emission, the fluorescent fine
particles can also be used as fluorescent materials for
electroluminescence (emission is caused by applying an alternating
voltage or direct voltage), cathodeluminescence (applying a
high-speed electron beam), and the like.
[0101] III. Evaluation of Fluorescent Fine Particles
[0102] 1. Concentration of Dispersed Nanoparticle in the
Solution
[0103] The concentration of the dispersed semiconductor
nanoparticles containing Cd and Se of the present description can
be determined by comparing the absorption spectrum of the
nanoparticles in light of literature (for example, Chemistry of
Materials, Vol. 15, p. 2854, 2003). When the composition is
changed, the concentration can be determined by utilizing the
additivity. Additionally, when the CdSe core is coated with ZnS as
the shell, the concentration can be determined using literature
(Journal of Physical Chemistry B, Vol. 101, p. 9463 (1997)).
[0104] 2. PL Efficiency of the Nanoparticles
[0105] The PL efficiency described in the present description
refers to the internal quantum efficiency, and is defined as the
possibility of emission of fluorescence photons after the
nanoparticles are excited by the light. In the case of a solution,
this value is determined by comparing the absorbance and the
emission intensity of the solution with those of a standard
substance (0.1N sulfuric acid solution of quinine) whose PL
efficiency is known. In order to determine the PL efficiency of a
dilute solution having a nanoparticle concentration of about 10 nM,
it is preferable to calibrate the sensitivity of absorption and
fluorescence spectrophotometers at each wavelength, and confirm the
stability of the baseline. Further, the temperature fluctuations in
the laboratory where measurement devices are placed are preferably
controlled to about .+-.2.degree. C. Specifically, it is preferable
to use the method described in literature (Journal of Luminescence,
Vol. 128, p. 1896 (2008)) by the present inventors. Although the
fluorescence of quinine is in the blue region, it is possible to
determine the PL efficiency of fluorescence in the red region in
the same manner as long as the sensitivity of the fluorescence
spectrophotometer is calibrated at each wavelength. For higher
accuracy, the value of the PL efficiency may be confirmed using a
standard substance (for example, Rhodamine 6G) emitting in the red
region.
[0106] The brightness of the silica glass fine particles is
proportionate to (PL efficiency.times.the number of nanoparticles
dispersed in one silica glass fine particle).
[0107] 3. The Number of Semiconductor Nanoparticles Dispersed in
One Silica Glass Fine Particle
[0108] The number of semiconductor nanoparticles dispersed in each
silica glass fine particle can be substantially accurately counted
by transmission electron microscope observation at an accelerating
voltage of 200 keV or more. However, when the size of the silica
glass fine particle is near 100 nm, the accelerating voltage is
preferably about 300 keV. Because the shape and size of each
nanoparticle are substantially constant, even when some particles
are overlapped in the moving direction of electrons of a
transmission electron microscope, it is possible to discern the
overlapping by a discontinuous change in the contour of the
particles, as long as it is not a complete overlap. A higher
accuracy can be achieved by taking images of the sample by
sequentially changing the angle using an electron beam tomography
method, and showing the images in 3D using dedicated software.
[0109] In any case, in order to determine the number of dispersed
nanoparticles, at least about 30 silica glass fine particles in the
field of vision are selected from among randomly selected
observation samples. Then, the number of dispersed nanoparticles in
the silica glass fine particles is counted, and a histogram is
prepared. After confirming that the distribution is not
disproportionate, the histogram is averaged.
[0110] As another method, it is possible to derive the number of
dispersed nanoparticles by dissolving the silica glass fine
particles with an acid, and determining the molar ratio of each
component by chemical analysis. At this time, if the composition of
the nanoparticles is also determined by chemical analysis, it is
possible to obtain an accurate value. Even if it is not possible to
obtain an accurate value, the rough composition of the
nanoparticles can be determined from the particle size of
nanoparticles, it is possible to estimate the number of dispersed
nanoparticles.
[0111] 4. Quantitative Determination of Dissolved Cadmium
[0112] As described in Background Art, because semiconductor
nanoparticles have a large specific surface area, a defect on the
surface significantly influences the emission properties. It is
preferable when the amount of dissolved cadmium is small under
conditions similar to those for the application of biotechnology
(the nanoparticle concentration in a buffer solution is low,
specifically, about 10 nM or less) in order to prevent cell death.
In addition, because dissolution inevitably occurs from the surface
of the semiconductor nanoparticles, when the dissolution amount is
small, it leads to less occurrence of surface defects, and the
prevention of a reduction in the PL efficiency. Therefore,
quantitative determination of the dissolution amount is important
to determine the degree of usefulness.
[0113] The most commonly used medium as a buffer solution or
culture solution for the above assessment is DMEM (Dulbecco's
Modified Eagle Medium). For the measurement, a solution obtained by
dispersing the prepared silica glass fine particles or commercially
available polymer-coated nanoparticles and allowing the dispersion
to stand for a certain period of time is filtered through a filter,
and the Cd concentration in the filtrate is measured by ICP mass
spectrometry. However, when the dissolved Cd is quantified using
the filtrate, the dissolution amount decreases with time. This is
because the dissolved Cd forms assemblies with the components in
DMEM, and the assemblies thereby grow to the same size of the
semiconductor nanoparticles and are stopped by the filter,
resulting in a decrease in the amount of Cd. This is also suggested
from the fact that there is no correlation between the amount of Cd
and the amount of Se that is simultaneously dissolved.
[0114] On the other hand, a HEPES solution is also a commonly used
buffer solution, and is ideal because the degree of binding to
metal has been reported to be undetectable. Therefore, a HEPES
buffer solution is used for the assessment of dissolved cadmium.
Further, the dissolution amount is converted to a dissolution
amount at a dispersed semiconductor nanoparticle concentration of
10 nM for comparison.
EXAMPLES
[0115] The present invention is described in more detail below with
reference to examples; however, the present invention is not
limited to these examples.
Example 1
Synthesis of Silica Glass Fine Particles
[0116] CdSe/ZnS nanoparticles (CdSe core, ZnS shell) whose surface
is coated with dodecylamine were prepared by a known method (Nano
Letters, Vol. 1, p. 207 (2001) and Colloids and Surfaces A, Vol.
202, p. 145 (2002)). The nanoparticles were dispersed at a
concentration of 20 .mu.M in a toluene solution. The PL efficiency
was measured to be 35%. The emission wavelength was about 620 nm,
and the half-bandwidth (full width at half-maximum) of the emission
spectrum was about 33 nm.
[0117] Next, silica glass fine particles in which fluorescent
nanoparticles are dispersed were prepared via 3 stages from step 1
to 3 shown in FIGS. 1A to 1C. Unless otherwise stated, the
synthesis was carried out at room temperature in the
atmosphere.
[0118] In step 1, tetraethoxysilane (TEOS, 10 .mu.L) was added to a
toluene solution in which nanoparticles are dispersed (0.4 mL, 1.5
.mu.mol/L), and the mixture was stirred for 3 hours, thereby
obtaining organic solution A. In regard to the nanoparticles in
this solution, only one ethoxy group of the tetrafunctional TEOS is
considered to be hydrolyzed and converted to
(Et-O).sub.3--Si--O.sup.- (wherein Et is ethyl group), which then
becomes a ligand and acts as a surfactant, orderly covering the
surface of the nanoparticles. In this state, a decrease in the PL
efficiency was hardly observed.
[0119] In order to search for the conditions under which a decrease
in the PL efficiency is not observed as described above, the
conditions for the above experiment were slightly changed. FIG. 2
shows plots of relative values of the PL efficiency obtained by
dispersing a slight amount of water in TEOS, and stirring for 3
hours. In the above-described experiment, the water concentration
was 0 M (M is mol/L), and it is clear that a decrease in the PL
efficiency begins around when the concentration exceeds 0.005 M.
When the amount of water is large, the hydrolysis rate is
increased. Because TEOS is a tetrafunctional alkoxide, TEOS
hydrolysate that coats the nanoparticle surface is also subjected
to hydrolysis at two or more positions, and hydrolysates tend to
bind to each other. Further, hydrolysis products are continuously
attached to the surface, causing the ligands on the surface to be
randomly arranged; this is considered to have led to a decrease in
the PL efficiency. TEOS is known to be gradually hydrolyzed by
absorbing moisture in the air, even when water is not added.
[0120] In step 2,3-mercaptopropyltrimethoxysilane (MPS, 1 .mu.L)
was mixed with ethanol (25 mL) and aqueous ammonia (4 mL, ammonia
concentration 10 wt %), thereby obtaining aqueous solution B. The
aqueous solution B was mixed with the organic solution A, and
stirred for 3 hours. As a result, the semiconductor nanoparticles
were transferred to the aqueous phase; further, nanoparticle
assemblies were formed in the aqueous phase. These assemblies were
extracted by centrifugation. Herein, when TEOS was used instead of
MPS, the assembly's size rapidly increased in the aqueous phase,
causing white turbidity. This shows that MPS has an effect of
adjusting the assembly size.
[0121] In step 3, 0.5 mL of aqueous solution in which the
above-described assemblies are dispersed was extracted, and ethanol
(8 mL), aqueous ammonia (0.1 mL, 25 wt %), and TEOS (14 .mu.L) were
added thereto. Thereby, a silica glass layer was formed on the
surface of the semiconductor nanoparticle assemblies, and in this
way, fluorescent silica glass fine particles were obtained.
Further, nanoparticles were dispersed at a concentration of about 1
.mu.M therein. The PL efficiency measured at this time was 31%. The
PL efficiency remained the same even when the above solution was
redispersed at a nanoparticle concentration of 10 nM in a pH 7.4
HEPES solution (HEPES concentration: 10 mM). Further, an equivalent
level of PL efficiency was obtained even when the solvent was
evaporated to give a dry powder, and the PL efficiency of the dry
powder was measured using an integrating sphere.
[0122] Further, for the purpose of developing a network structure
of the silica glass layer, the process in step 3 described above
was performed at 40.degree. C. to prepare silica glass fine
particles. The amount (weight) of dissolved cadmium was measured in
the same manner described below. Table 1 shows the results.
[0123] <Quantitative Determination of Dissolved Cadmium>
[0124] Preparation of HEPES Buffer Solution
[0125] 4.766 g of powder of commercially available HEPES (Dojindo
348-01372 produced by Dojindo Laboratories) was dissolved in
ultrapure water (160 mL). Next, 1N aqueous sodium hydroxide was
gradually added thereto to adjust the pH to 7.4. Further, ultrapure
water was added thereto to obtain an amount of 200 mL, and the
resulting product is stored as a stock solution in a refrigerator
at about 4.degree. C. An amount of 10 mL was extracted from this
stock solution, and diluted with ultrapure water to obtain an
amount of 100 mL. Thereby, 10 mM of HEPES buffer solution was
prepared.
[0126] Measurement of Dissolved Cadmium
[0127] The above-described HEPES solution (3 mL) was placed in a
container made of polypropylene. The prepared silica glass fine
particles were added thereto, and the concentration X of the
semiconductor nanoparticles was adjusted to 50 nM. The PL
efficiency was about 31%, which was the same before and after
dispersion. The thus-obtained product was allowed to stand at room
temperature for 15 hours. At this stage, the PL efficiency was
measured again, and the measurement gave substantially the same
value. 3 mL thereof was extracted and centrifuged at 10,000 rpm for
5 minutes using a centrifugal concentration filter (Vivaspin 6,
3000-MWCO produced by Sartorius), and only the solution components
that do not contain the silica glass fine particles were extracted
by filtration, and placed in a minitube made of polypropylene. The
minitube was tightly sealed, and the filtrate was used as a sample.
Subsequently, the filtrate was stored in a refrigerator at
4.degree. C. to prevent decomposition.
[0128] At this time, because the amount of dissolved cadmium was as
small as some parts per billion (some nanograms in 1 mL solution),
contamination caused by different factors must be removed to the
greatest possible extent. This time, a container made of
polypropylene was used as a container for a dissolution test, and a
control experiment was separately preformed without placing
semiconductor nanoparticles in the container to confirm that the
amount of dissolved cadmium was equal to or below the detection
limit (0.2 ppb). After confirmation, the sample was used in the
experiment. Caution is necessary because analytical accuracy will
decrease when molybdenum coexists in the solution.
[0129] Quantitative determination of the cadmium ion concentration
in the filtrate was performed by the following procedure.
[0130] First, the sample solution (0.1 mL) was pretreated by adding
ultrapure water (0.8 mL) and high purity nitric acid (0.1 mL;
Tamapure AA-100 (Tama Chemicals Co., Ltd.)), and quantified. Nitric
acid is effective in stabilizing elements in the sample and
preventing attachment of the sample to the wall of the analyzer. An
ICP mass spectrometer (Finnigan ELEMENT2, produced by Thermo Fisher
Scientific) was used for the measurement. A standard curve was
drawn in advance using a standard sample. Because there was a
concern that the measurement error would increase as the
concentration of dissolved cadmium decreased, a sample obtained
from a solution having a high concentration of dispersed
semiconductor nanoparticles was used, and a method in which
quantification is performed after the sample is concentrated as
needed was effective. In the confirmation of accuracy of the
measurement that used a standard sample having a known
concentration, the measurement error was confirmed to be within 10%
even near the lower limit of quantification (the weight in 1 mL
sample is 0.5 ng and 0.5 ppb). Further, although a solution
obtained from the semiconductor nanoparticles at a concentration of
50 nM was used as a sample this time, it was confirmed that the
concentration of the semiconductor nanoparticles and the
concentration of dissolved Cd ions in the sample are in a
proportional relationship at least in the range where the
semiconductor nanoparticle concentration is up to 100 nM.
[0131] Next, the same test was performed on commercially available
polymer-coated CdSe/ZnS nanoparticles. The following 3 types of
nanoparticles by Invitrogen, i.e., Q21321MP (the surface is COOH),
Q10021MP (the surface is streptavidin), and Q25021MP (commonly
called "Qtracker"; the surface is peptide) were provided, and each
type was dispersed in a HEPES buffer solution prepared by the
above-described procedure under conditions in which the
concentration X was 20 nM. Then, the amount Y (a value when the
unit is expressed in nanograms, which corresponds to a value
expressed in parts per billion) of cadmium dissolved in 1 mL
solution extracted from the above-obtained solution 15 hours after
dispersion was measured.
[0132] A formula, Z=10.times.Y/X, may be used to convert the
dissolution amount Y of each type to a dissolution amount Z at a
dispersed semiconductor nanoparticle concentration of 10 nM. The Z
was as shown based on the Cd weight concentration in Table 1.
Specifically, in the table, the Cd weight concentration expressed
in ppb is Z. It is clear from this table that the formula
10.times.Y/X<1 is satisfied only by the fluorescence silica
glass fine particles.
TABLE-US-00001 TABLE 1 Amount of cadmium dissolution from silica
glass fine particles in which CdSe/ZnS nanoparticles are dispersed
and from commercially available polymer-coated CdSe/ZnS
nanoparticles (when the concentration of dispersed nanoparticles is
10 nM) Amount of Cd Dissolution from Nanoparticles (CdSe/ZnS, 10
nM) in HEPES Cd weight concentration Relative Type (ppB) ratio
Remarks Q21321MP 1.9 1 COOH-coated Q10021MP 8.2 4.82
Streptavidin-conjugated Q25021MP 8.7 5.12 Qtracker, peptide-coated
Silica fine 0.41 0.24 OH-coated particles Silica fine 0.14 0.08
OH-coated particles prepared at 4.degree. C.
Example 2
[0133] Hydroxyl groups are present on the surface of the silica
glass fine particles prepared in Example 1. In order to apply the
silica glass fine particles as fluorescent probes in the field of
biotechnology, the surface of the silica glass fine particles were
modified with various functional groups; and further, antibodies
were conjugated thereto.
[0134] When MPS was used after the formation of a silica glass
layer in Example 1, fine particles coated with thiol groups were
prepared.
[0135] The fine particles after the formation of the silica glass
layer were dispersed in pure water, and 0.5 mL of fine particles
(nanoparticle concentration of 1 .mu.M) was extracted and added to
a mixture of MPS (2 .mu.L) and ethanol (30 .mu.L). After stirring,
precipitate was obtained by centrifugation, washed with pure water,
and dispersed in a PBS (concentration: 10 .mu.M) solution to obtain
an amount of 0.5 mL. 0.1 mL was extracted therefrom, and a PBS
buffer solution (10 .mu.L) in which the concentration of
streptavidin-conjugated maleimide was adjusted to 200 mM at
4.degree. C. was mixed with the above-described solution in which
the silica glass fine particles are dispersed. Subsequently,
precipitate was obtained by centrifugation, washed with a PBS
buffer solution, and redispersed again in the PBS buffer solution.
Biotinylated secondary antibodies were added to the resulting
product to allow binding to various primary antibodies. The fact
that the surface modification and attachment to the antibodies were
actually carried out was confirmed by the difference in the
electrophoretic velocity. As an example of biological applications,
embryonic rat hippocampal neurons (E18) were able to be stained
using anti-microtubule-associated proteins as primary
antibodies.
[0136] As another example, after bovine serum albumin was bound to
the surface of streptavidinylated silica glass fine particles to
remove non-specific adsorption, the silica glass fine particles
were bound to biotinylated antibodies against anti-influenza A
antigens. When the thus-obtained product was used as a fluorescent
probe (fluorescence reagent) in immunochromatography, it was
possible to determine whether influenza type A antigen is present
in the sample from the fluorescence intensity of a test line.
[0137] <Determination of the Particle Size of Silica Glass Fine
Particles and the Number N of Dispersed Nanoparticles by Electron
Microscope Observation>
[0138] After discharge treatment was applied to high resolution
carbon-support film grids produced by Okenshoji Co., Ltd. (made of
Cu, grid pitch of 100 .mu.m), one drop of the aqueous solution
having silica glass fine particles dispersed therein prepared in
Example 1 was dropped to allow a slight amount of fine particles to
be adsorbed on the surface, thereby preparing an observation
grid.
[0139] Observation by a transmission electron microscope (produced
by Topcon Corporation, EM-002B, accelerating voltage 200 kV) found
that the average particle size of silica glass fine particles was
about 47 nm. When the particle size is around 47 nm, electrons pass
through the fine particles in transmission electron microscope
observation. Therefore, it was relatively easy to count the number
of semiconductor nanoparticles incorporated. However, when the
particles are overlapped in the forward direction of the electron
beam, there is a case where count loss occurs. In order to avoid
such a situation, it is effective to determine that there is an
overlap when a discontinuous circumference is observed from the
shape of the particles. 30 silica glass fine particles were
randomly selected, and the images thereof were taken. Then, the
number of semiconductor nanoparticles distributed therein was
counted. In this way, a substantially accurate average distribution
number N.sub.2 was determined to be 22.
[0140] For higher accuracy, it is effective to take 3D images.
Using a Tecnai G2F20 (accelerating voltage: 200 kV) produced by FEI
Company, a series of dark-field images was taken at 79.000-fold
magnification while rotating the sample one degree at a time, in
the range of .+-.64 degrees. Reconstruction of a 3D image was
performed from these images using software (Inspect 3D) produced by
FEI Company, and an imaging process was performed using software
(Avizo5) produced by MCS Inc. While rotating the thus-created 3D
image, the number of semiconductor nanoparticles distributed in 60
randomly selected silica glass fine particles was counted, and the
accurate average distribution number N.sub.3 was determined to be
23. The reason why N.sub.2 observed in 2D is slightly lower than
N.sub.3 observed in 3D is assumed to be because minor count loss
occurs due to overlapping of nanoparticles.
[0141] <Determination of the Number of Dispersed Nanoparticles
by Chemical Analysis>
[0142] The chemical compositions of both silica glass fine
particles and semiconductor nanoparticles (CdSe/ZnS) prepared in
Example 1 were analyzed, thereby calculating the number of
semiconductor nanoparticles dispersed in each silica glass fine
particles.
[0143] After weighing a dry powder sample (about 4 mg in the case
of silica glass fine particles, and about 2 mg in the case of only
CdSe/ZnS nanoparticles) enveloped in paper for wrapping powdered
medicine, the sample was placed in a Teflon (registered trademark)
container; hydrofluoric acid and nitric acid were simultaneously
added thereto, and the container was sealed. Subsequently, the
sample was dissolved by heating at about 200.degree. C. using a
microwave sample pretreatment device (Milestone General). After
sufficient cooling, the resulting product was taken out and diluted
with ultrapure water. Subsequently, using a high-frequency
induction-coupled plasma quantometer (ICP-AES, Nippon Jarrell Ash
Co., Ltd., the current company name is Thermo Fisher Scientific
K.K., IRIS Advantage), Cd, Se, Zn, S, and Si in the solution were
quantified. A standard curve was drawn for each element, using a
sample whose concentration is already known. In this way, it was
ensured that the analytical error would be within 10%.
[0144] As a result, the analysis results shown in Table 2 were
obtained.
TABLE-US-00002 TABLE 2 Composition Ratio of Constituent Elements of
Silica Glass Fine Particles and CdSe/ZnS Nanoparticles Dry Molar
Ratio Sample weight/mg Cd Se Zn S Si Sample 1 4 1 0.23 2.39 3.31
28.99 Silica fine particles (CdSe/ZnS nanoparticles are dispersed)
Sample 2 2 1 0.25 1.46 1.97 0 CdSe/ZnS nanoparticles
[0145] The following procedure is performed to calculate the number
N of semiconductor nanoparticles dispersed in one silica glass fine
particle from the above results.
[0146] First, based on the observation of transmission electron
microscope images, the average particle size of one silica glass
fine particle was 47 nm, and the average particle size of
semiconductor nanoparticles was 5.8 nm. Additionally, based on the
speculations in known literature (Advanced Materials, Vol. 21, p.
4016, 2009; and New Journal of Chemistry, Vol. 33, p. 561, 2009),
the density of silica glass fine particles was 1.5 g/cm.sup.3. With
the size of the above-obtained semiconductor nanoparticles, 30% or
more of atoms would be located on the surface, and there would be a
deviation from the stoichiometric composition ratio. In order to
accurately estimate the number of semiconductor nanoparticles
dispersed in each silica glass fine particle regardless of such
deviation, a measurement must be made by the calculation as
described below.
[0147] From the analysis results of sample 2 in Table 2, the atomic
weights of cadmium selenide and zinc sulfide, which constitute the
nanoparticles, are determined to be 131.8 and 158.9, respectively,
assuming that their compositions are Cd.sub.1Se.sub.0.25 and
Zn.sub.1.46S.sub.1.97, respectively. Further, the atomic weight of
Si is determined to be 1737, assuming that every Si is present in
the form of SiO.sub.2. Here, by using the specific gravity of each
substance, the volume ratio can be determined as follows: CdSe:
ZnS:SiO.sub.2=32.1:27.4:1157.8 in the silica glass fine particle.
Accordingly, the volume fraction of the semiconductor nanoparticles
in the silica glass fine particle is 0.049
(=(32.1+27.4)/(32.1+27.4+1157.8)). Based on the volume of one
silica glass fine particle and the above-described volume fraction,
the volume of CdSe/ZnS nanoparticles in the silica glass fine
particle is determined to be 2660 nm.sup.3, and the volume of one
nanoparticle is 102 nm.sup.3. Accordingly, the number N of the
semiconductor nanoparticles in one silica glass fine particle was
determined to be 26.
Example 3
[0148] When the amount of MPS used in "Synthesis of Silica Glass
Fine Particles" in Example 1 was decreased to 0.5 .mu.L, the size
of nanoparticle assemblies formed was increased. The surface of
these assemblies was coated with silica glass in the same manner as
in Example 1, thereby obtaining fluorescent fine particles. At this
time, the PL efficiency when the semiconductor nanoparticles were
dispersed at a concentration of 10 nmol/L in a HEPES solution was
about 25%. It was found by electron microscope observation that the
fluorescent fine particles have an average particle size of 95 nm.
Further, it was confirmed that at least 160 nanoparticles are
dispersed.
Example 4
[0149] When the amount of MPS used in "Synthesis of Silica Glass
Fine Particles" in Example 1 was increased to 2 .mu.L, the size of
nanoparticle assemblies formed was decreased. The surface of the
assemblies was coated with silica glass in the same manner as in
Example 1, thereby obtaining fluorescent fine particles. At this
time, when the semiconductor nanoparticles were dispersed at a
concentration of 10 nmol/L in a HEPES solution, the PL efficiency
was about 25%. It was found by electron microscope observation that
the fluorescent fine particles have an average particle size of 21
nm. Further, it was confirmed that at least 11 nanoparticles are
dispersed.
Example 5
[0150] It was also possible to modify the surface with a carboxyl
group or a salt thereof by using carboxyethylsilanetriol sodium
salt (abbreviated as CES).
[0151] Specifically, TEOS was mixed with CES at a molar ratio of 5%
of TEOS, followed by stirring for 48 hours. The resulting mixture
was added in step 3 in Example 1, instead of pure TEOS, thereby
obtaining fluorescent fine particles whose surface is modified with
a sodium salt of a carboxyl group.
[0152] In a similar manner, aminopropyltrimethoxysilane was used,
and thereby the surface was able to be modified with an amino
group. Water-soluble carbodiimide was acted thereon to allow
binding to the carboxyl groups on the cell surface.
Example 6
[0153] After the fluorescent fine particles prepared in Example 1
were vacuum-dried, the fluorescent fine particles were bound to a
glass substrate. When irradiated with a commercially available
light-emitting diode (emission wavelength: 385 nm), the fluorescent
fine particles showed a red-light emission. Likewise, fluorescent
fine particles that showed a blue-light emission and a green-light
emission were also obtained by decreasing the average particle size
of nanoparticles embedded. Specifically, fluorescent fine particles
with a blue-light emission are obtained when the average particle
size of CdSe/ZnS nanoparticles is about 3.3 nm (the thickness of
the ZnS shell is about 0.5 nm, and the size of the CdSe core is
about 2.3 nm), and fluorescent fine particles with a green-light
emission are obtained when the average particle size of CdSe/ZnS
nanoparticles is about 5.1 nm (the thickness of the ZnS shell is
about 0.5 nm, and the size of the CdSe core is about 4.1 nm).
Further, the nanoparticles with a red-light emission (peak
wavelength: about 620 nm) used in Example 1 had an average particle
size of about 5.5 nm. Because the emission color is determined
based on both core size and shell thickness, there is a case where
the emission color is different even when the average particle size
is same. The average particle size of the nanoparticles can be
adjusted by the reaction time during synthesis by a known solution
method. It was found that these fluorescent fine particles are also
usable as fluorescent materials for electronic materials such as
lights and displays.
Example 7
[0154] The difference among prepared products by synthesis
conditions was systematically investigated. The experiment was
performed in the same manner as in Example 1. Table 3 shows
synthesis conditions for steps 2 and 3, the PL efficiency of the
synthesis products, and the particle size of the silica glass fine
particles. The reaction time in the table shows the reaction time
in step 3.
TABLE-US-00003 TABLE 3 Synthesis Conditions for Steps 2 and 3, and
the PL Efficiency and the Particle Size of Prepared Fluorescent
Silica Glass Fine Particles Results Silica Synthesis Conditions
Particle size glass TEOS/semi- Molar of silica fine conductor
concen- PL glass fine particle nanoparticles tration Reaction
efficiency particles number (molar ratio) of MPS time (h) (%) (nm)
(1) 2.81 .times. 10.sup.5 4.7 .times. 10.sup.-5 4.0 22 95 .+-. 9
(2) 2.39 .times. 10.sup.5 9.4 .times. 10.sup.-5 3.5 34 46 .+-. 6
(3) 1.32 .times. 10.sup.5 9.4 .times. 10.sup.-5 3.0 33 40 .+-. 5
(4) 2.68 .times. 10.sup.5 9.4 .times. 10.sup.-5 4.0 30 47 .+-.
7
[0155] FIG. 4 shows transmission electron microscope images of
synthesized silica glass fine particles (1) to (4). The figure
numbers (1L), (2L), (3L), and (4L) show enlarged images of the
silica glass fine particles (1), (2), (3), and (4),
respectively.
[0156] It is clear from these figures that these particles
individually have a silica glass layer thickness of 20 nm, 10 nm, 7
nm, and 13 nm. Among the synthesis conditions for silica glass fine
particles (1) in Table 3, when the molar concentration of MPS was
increased to 5.0.times.10.sup.-5 mol/L, the silica glass fine
particles had a PL efficiency of 25% and a particle size of 90.+-.9
nm.
Example 8
The Modified Preparation Method Described in "Solution to Problem"
was Used
[0157] MPS (0.5 .mu.L), ethanol (25 .mu.L), and TEOS (2 .mu.L) were
added to a toluene solution of CdSe/ZnS nanoparticles (0.5 mL;
concentration of 1.5 .mu.M), and the mixture was stirred for 2
days. In this step 1, nanoparticles coated with TEOS and MPS were
prepared.
[0158] In next step 2, ethanol (4 mL) and water (0.1 mL) were added
to the above solution, and further, TEOS (10 .mu.L) and aqueous
ammonia (6.3 wt %, 0.3 mL) were gradually added thereto. Thereby,
alkoxide attached to the surface of nanoparticles prepared in step
1 was hydrolyzed, became hydrophilic, and formed an assembly by
coming into contact with TEOS while moving in the aqueous solution.
After stirring for 3 hours and 30 minutes, particles were taken out
by centrifugation and observed under a transmission electron
microscope. FIG. 5 shows the results. FIG. 5 shows that the
nanoparticles in the assembly are regularly arranged, compared to,
for example, FIG. 4. In this case, about 30 nanoparticles are
observed in the assembly. The PL efficiency was about 25%. Because
of such regular arrangement, it was possible to increase the
concentration of nanoparticles dispersed in each glass bead, and
glass beads with high brightness were thereby obtained. The
emission wavelength was 652 nm. The particle size of the glass
beads was about 70 nm.
[0159] Further, in step 3, the surface of the glass beads was able
to be coated with a silica glass layer. The final particle size of
the glass beads was about 90 nm.
Example 9
[0160] A toluene dispersion of CdSe/ZnS nanoparticles
(concentration: 1.5 .mu.mol/L; volume: 0.4 mL) having an emission
wavelength of 610 nm was taken out; and further, TEOS (10 .mu.L)
and MPS (0.05 .mu.L) were added thereto. The mixture was stirred
for 3 hours, thereby obtaining organic solution A. Separately, MPS
(0.15 .mu.L), ethanol (25 mL), aqueous ammonia (6.25 wt %, 1.5 mL),
and water (2 mL) were mixed, thereby obtaining aqueous solution B.
When the organic solution A was mixed with the aqueous solution B,
the nanoparticles moved from the organic solution to the aqueous
solution. By further stirring the mixture for 3 hours, a
nanoparticle assembly was obtained. At this stage, the size of the
nanoparticle assembly was 25 nm, and the number of nanoparticles in
the assembly was about 15. Further, a silica glass layer was formed
by the method of step 3 in Example 1, using TEOS by the Stober
method. The final size of the glass beads was about 30 nm. Further,
the PL efficiency was 25%.
* * * * *