TWI438005B - Fluorescent gold nanocluster matrix - Google Patents

Description

Fluorescent gold nano cluster aggregate

The present invention relates to a nanoclusters, and more particularly to a fluorescent gold nanon cluster aggregate.

When the size of the metal or semiconductor particles is small enough, a quantum confinement effect will be produced, that is, the charge and energy of the particles are quantized, and such tiny clusters are called quantum dots. Quantum dot electrons are arranged very tightly. Because quantum quantification effects can excite different colors of fluorescence, quantum dots absorb energy waves with higher energy and produce energy step jumps. When electrons fall from high-energy state to low-energy state, Light with a longer wavelength (reddish light) is emitted. Quantum dots of different particle sizes emit fluorescence of different wavelengths. For example, cadmium selenide (CdSe) emits blue fluorescence when the particle diameter is 2.1 nm, and green fluorescence when the particle diameter is 5 nm. When the particle size is close to 10 nm, The fluorescence it emits is close to red.

Quantum dots have fluorescent brightness compared to traditional organic dye molecules Strong, stable light, and the ability to emit a variety of different wavelengths of the wave with a single wavelength of laser. The transmitted wave is a narrow and symmetrical waveform that can be repeatedly excited, so the fluorescence aging can last. These characteristics attract the attention of scientists. The application of nano-quantum dots is becoming more and more diverse, and it has the potential to replace traditional dyes. Therefore, it is more promising in the application of biomedical engineering.

In recent years, quantum dots, with their excellent optical properties, have successfully overcome the bottlenecks faced by biological and medical optical probes in the past, and have become an important nanomaterial for the design of a new generation of fluorescent probes. From the three-dimensional image of cells, long-term live cell monitoring, single-molecule dynamic intracellular tracking, development of long-acting optical sensors, cancer diagnosis and treatment, breakthroughs have been made, coupled with the rapid industrialization of quantum dots and its billion business opportunities. It has become a model for the extremely successful application of nanotechnology. However, traditionally, water-soluble quantum dots based on toxic heavy metal materials such as cadmium or lead are commercially available. The impact of the extension on the environment and human health is gradually being paid attention to, and it is the dilemma facing the full development of its biomedical applications.

Metal gold is one of the earliest researched nanomaterials. It is called colloidal gold in biological research and its particle size is between 1-100nm. Gold quantum dots have a high electron density, good contrast under an electron microscope, and a relatively high biocompatibility, which has been shown to emit different colors of fluorescence by changing the size of their clusters. It can be applied to the production of multi-dimensional biomedical calibration or optical components, but because the process is quite difficult, it is necessary to use expensive dendrimer as gold quantum. The coating material of the point is time-consuming and difficult to mass-produce, thus limiting the development of its vast biomedical applications. Therefore, the development of a simple and mass-produced gold quantum dot formation technology is the focus of the industry's development.

In view of the above background of the invention, in order to comply with industrial requirements, the present invention provides a cluster of fluorescent gold nanoclusters.

The present invention is characterized in that a fluorescent gold nanocluster is provided, and the surface of the fluorescent gold nano-cluster has a dihydrolipoic acid (DHLA) ligand. Wherein the fluorescent gold nano-cluster is produced by the interaction between the dihydrolipoic acid ligand and the above-mentioned gold nano-cluster, and the particle size range of the fluorescent gold nano-clustered cluster is 0.5 nm to 3 nm, in addition, the above-mentioned fluorescent gold nano clusters have a wavelength of 400 to 1000 nm.

Another feature of the present invention is to provide a Fluorescent gold nanocluster matrix, wherein the fluorescent gold nano cluster assembly system is formed by stacking a plurality of gold nanoclusters, the gold The nano-cluster particle size ranges from 0.5 nm to 3 nm, and the surface of the above-mentioned gold nano-clusters has an alkanethiol ligand, wherein each of the above-mentioned gold nano-clusters passes through the surface of the alkane. a force between the thiol ligands, mutually attracting the stack to form the above-mentioned Fluorescent gold nanocluster matrix, and the above-mentioned fluorescent gold nano cluster assembly system by the above-mentioned gold nano-cluster The clustering of the clusters produces a fluorescent property, and in addition, the above-mentioned fluorescent gold nanoparticle aggregates have a photoreceptor wavelength in the range of 400 to 1000 nm.

Another feature of the present invention is to provide a method for forming a metal nanocluster, which first provides a mixed solution comprising a first metal precursor and a surfactant. And a reducing agent and a solvent are subjected to a reduction reaction in the mixed solution to form a metal nanoparticle, and further, a second metal precursor is added after forming the nano metal particles. So that the number of particles of the second metal precursor is greater than the total number of the nano metal particles, because the concentration of the nano metal particles and the second metal precursor are very different, resulting in an unbalanced coexisting system, the nano metal The particles thus collapse into metal nanoclusters of smaller particle size to form an equilibrium system wherein the metal nanoclusters have a particle size ranging from 1 nm to 4 nm.

In accordance with the above objects, the present invention discloses a fluorescent gold nanon cluster aggregate and a method of forming the same, which can be applied to various metals to form various metal nanoclusters. Wherein, the above-mentioned fluorescent gold nano cluster aggregates can be used as bioprobes and have Applications: fluorescent biological labels, clinical medical imaging agents, and clinical medical testing, tracking and treatment.

The orientation of the invention discussed herein is a fluorescent gold nanoparticle cluster. In order to fully understand the present invention, a detailed description will be presented. Obviously, the practice of the invention is not limited to the specific details that are apparent to those skilled in the art. On the other hand, well-known components or steps are not described in detail to avoid unnecessarily limiting the invention. The preferred embodiments of the present invention are described in detail below, but the present invention may be widely practiced in other embodiments, and the scope of the present invention is not limited by the scope of the following patents. .

One embodiment of the present invention discloses a Fluorescent gold nanocluster having a dihydrolipoic acid (DHLA) ligand on the surface of the fluorescent gold nano-cluster. The fluorescent gold nano cluster is produced by the interaction between the dihydrolipoic acid ligand and the nano cluster, and the particle size range of the fluorescent gold nano cluster is 0.5 nm. Up to 3 nm, in addition, the above-mentioned fluorescent gold nano-clusters have a wavelength of light-emitting fluorescence ranging from 400 to 1000 nm.

In a preferred embodiment of the present embodiment, the fluorescent gold nano-clusters further comprise a spacer, and one of the spacers is bonded to the dihydrolipoic acid (DHLA) ligand, and The other end of the spacer bond has a specific group, wherein the spacer comprises an oligomer or a polymer, and the specific group comprises one of the following groups: a chemical functional group, a crosslinking molecule, a sugar , fluorescent molecules, paramagnetic molecules, biomolecules and drugs.

Wherein the above oligomer or polymer comprises one of the following groups or any combination thereof: polyols, polyether polyols, polyester polyols, polycarbonate Polycarbonate polyols, polycaprolactone polyols, polyacrylate polyols, polyethylene glycol (PEG), dextran, and copolymers thereof.

In another preferred embodiment of the present embodiment, the fluorescent gold nano cluster further comprises a spacer, the spacer is bonded to the dihydrolipoic acid (DHLA) ligand, and the spacer itself There is a specific group, wherein the spacer comprises one of the following groups: a chemical functional group, a crosslinking molecule, a saccharide, a fluorescent molecule, a paramagnetic molecule, a biomolecule, and a drug.

According to still another embodiment of the present invention, a fluorescent gold nanocluster matrix is disclosed, wherein the fluorescent gold nano cluster assembly system is formed by stacking a plurality of gold nano clusters. The Jinnai cluster has a particle size ranging from 0.5 nm to 3 nm, and the above-mentioned Jinnai cluster has an alkanethiol ligand, wherein each of the above-mentioned gold nanoclusters passes through the surface thereof. The interaction between the alkanethiol ligands attracts the stacks to form the above-mentioned Fluorescent gold nanocluster matrix, and the above-mentioned fluorescent gold nano cluster assembly system is made up of the above-mentioned gold nanoparticles. The aggregation of the clusters produces a fluorescent property, and in addition, the above-mentioned fluorescent gold nanoparticle aggregates have a wavelength of light-emitting fluorescence ranging from 400 to 1000 nm.

In a preferred embodiment of the present embodiment, the surface of the fluorescent gold nano-clustered aggregate is coated with a spacer, one end of the spacer is bonded to the alkanethiol ligand, and the other end of the spacer is bonded. A specific group is formed, wherein the spacer comprises an amphoteric polymer or oligomer, and the specific group comprises one of the following groups: a chemical functional group, a crosslinking molecule, a saccharide, a fluorescent molecule, a paramagnetic molecule , biomolecules and drugs.

The above amphoteric polymer or oligomer comprises one of the following groups or any combination thereof: poly (maleic anhydride); PMA], polymer of 1-octadecene maleic anhydride [Poly ( Maleic anhydride-alt-1-octadecene); PMAO] with polyacrylic acid (PAA) and its derivatives.

In another preferred embodiment of the present embodiment, the surface of the fluorescent gold nanoparticle aggregate is coated with a spacer, the spacer is bonded to the alkanethiol ligand, and the spacer itself has a spacer A specific group, wherein the spacer comprises one of the following groups: a chemical functional group, a crosslinking molecule, a saccharide, a fluorescent molecule, a paramagnetic molecule, a biomolecule, and a drug.

Another embodiment of the present invention discloses a method for forming a metal nanocluster. First, a mixed solution is provided. The mixed solution comprises a first metal precursor, a surfactant (surfactant). And a reducing agent and a solvent are subjected to a reduction reaction in the mixed solution to form a metal nanoparticle, wherein the nano metal particles have a surface plasma absorption property.

Furthermore, after forming the nano metal particles, a second metal precursor is added, so that the number of particles of the second metal precursor is greater than the total number of the nano metal particles, because the nano metal particles and the second metal precursor are The concentration of the substance is very different, resulting in an unbalanced coexisting system. The above-mentioned nano metal particles are thus broken into metal nanoclusters having a smaller particle size to form a balance system, wherein the above metal nanoclusters The particle size ranges from 1 nm to 4 nm.

Furthermore, the first metal precursor and the second metal precursor may be the same or different, wherein the first metal precursor and the second metal precursor are selected from one of the following groups: chlorination Gold (AuCl ₃ ), tetrachloroauric acid (HAuCl ₄ ), gold bromide (AuBr ₃ ), tetrabromoic acid (HAuBr ₄ ).

The above surfactant is selected from one of the following groups or any combination thereof: didodecyldimethylammonium bromide (DDAB), tetraoctyl ammonium bromide (Tetraoctylammonium bromide; TOAB), Tetrabutylammonium bromide (TBAB). The reductant is selected from one of the following groups or any combination thereof: tetrabutylammonium borohydride (TBAB), sodium borohydride (NaBH ₄ ), and vitamin C (Ascorbic Acid). The solvent is toluene or chloroform.

On the other hand, after forming the above-mentioned metal nanoclusters, a Ligand-binding reaction is carried out, and the above-mentioned reaction is to bond a ligand to the surface of the above-mentioned metal nanoclusters to form a ligand. Lagged-capped metal nanocluster.

The above ligand is selected from one of the following groups: dihydrolipoic acid (DHLA), dodecanethiol (dodecanethiol; DDT), bis(p-sulfonatophenyl)phenylphosphine; BSPP], triphenylphosphine.

Furthermore, the above Ligand-binding reaction is a thiol-related ligand binding reaction, and the above reaction is to bond the above thiol-related ligand to the above-mentioned metal naphthalene. The surface of the rice clusters forms a thiol-capped metal nanocluster.

The above thiol-related ligand is selected from one of the following groups: dihydrolipoic acid (DHLA), dodecanethiol (DDT), dithiol succinic acid (meso-) 2,3-dimercaptosuccinic acid; DMSA), glutathione (GSH), 1,6-hexanedithiol (1,6-hexanedithiol).

Wherein the thiol-capped metal nanocluster is a fluorescent metal nanocluster, and the particle size range of the fluorescent metal nano cluster is 0.5 nm to 3 nm.

And, after forming the above-mentioned Fluorescent metal nanocluster, performing a functional group coating (functional The reaction is such that the above-mentioned fluorescent metal nanoclusters have at least one functional group property, wherein the functional group coating reaction is a bioconjugation reaction.

The functional group of the above functional coating reaction is selected from one of the following groups: a chemical functional group, a crosslinking molecule, a saccharide, a fluorescent molecule, a paramagnetic molecule, a biomolecule, and a drug.

According to the above embodiments, the fluorescent metal nano clusters such as the fluorescent gold nano clusters and the fluorescent gold nanocluster matrix can be used as bioprobes. It has the following applications: fluorescent biological label, clinical medical imaging developer, and clinical medical testing, tracking and treatment.

Example 1 Fluorescent Golden Nano Cluster (1) Preparation of fluorescent gold nano cluster AuNC-DHLA

(I) First, the dodecyldialkylammonium bromide salt is dissolved in toluene for use as a pre-preparation solution (100 mM), and secondly, gold chloride or tetrabromogold acid is dissolved in the dodecyl group. In the alkylammonium bromide salt (25 mM) to form a metal gold precursor solution, then mix 0.625 mL of citric acid with 1 mL of the pre-prepared solution and stir, then re-inject 0.8 mL of metal gold before stirring. Liquid to obtain a dark red solution in which an excess of methanol is added until the dark red solution becomes an opaque blue-violet solution, and the gold nanoparticles are induced to accumulate by methanol, and further, the unreacted residual reaction in the solution is measured using a centrifuge. Reagents and too small gold nanoparticles are removed.

The purified gold nanoparticle is redissolved in the dodecyldialkylammonium bromide salt solution to form a dark red solution, and secondly, the metal gold precursor solution is added and stirring is continued until the dark red solution is turned into Light yellow transparent liquid. At this time, the above-mentioned gold nanoparticles have been broken into clusters of gold nanoparticles having a small particle size. Wherein, the above-mentioned gold nanoparticles have surface plasma properties (520-530 nm), while the gold nano-clusters have no such properties, and the absorption spectrum thereof is as shown in the first figure, wherein gold chloride is used as the first metal precursor. And (A) is a gold nanoparticle; (B) is a gold nanoparticle using gold chloride as a second metal precursor; (C) is a second metal precursor using tetrabromoic acid Jinnai clusters.

(II) 0.0322 g of tetrabutylammonium bromide powder was dissolved in 2.5 mL of the dodecyldialkylammonium bromide salt solution until the powder was completely dissolved. Then add 0.052g of lipoic acid to the above solution until no bubbles are generated in the solution, so that the lipoic acid is reduced to dihydrolipoic acid. In order to avoid incomplete reduction of lipoic acid, an excess of reducing agent is added. The ammonium bromide powder was determined to have no bubble generation. Furthermore, 2.5 mL of the gold nano-cluster solution is mixed into the dihydrolipoic acid solution and continuously stirred. At this time, the mixed solution turns opaque and appears yellow-brown, thereby forming a fluorescent Chennai. Rice clusters.

Among them, the spectrum of absorption, photoluminescence (PL), and photoluminescence excitation (PLE) of the fluorescent gold nano cluster AuNC-DHLA is shown in the second figure.

(2) Fluorescent gold nano-cluster biomolecule grafting

First, 10 μl of fluorescent gold nano cluster solution was uniformly mixed with 10 μl of X-PEG-amine (3 mM in ddH 2 O) to form a mixed solution, followed by addition of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution (EDC, 8mM in ddH2O), shaking reaction for two hours, according to which a biomolecule grafting reaction is completed, wherein the above PEG system can be vitamin (biotin) or avidin (avidin), and the above-mentioned biomolecule grafting reaction diagram is as shown in the third figure. Shown. In addition, the modified fluorescent gold nanocapsules were purified by colloidal electrophoresis (2% agarose, 75 V) and centrifuged in SBB (sodium borate buffer, pH=9) with a 100 kDa molecular sieve.

Example 2 Fluorescent gold nano cluster aggregates (1) Preparation of fluorescent gold nano cluster AuNC-DDT

A gold nanocluster solution is provided, which is formed as described in Example I(I). The above-mentioned gold nano-cluster solution was continuously stirred for 10 minutes, and slowly dropped into a solution of a thiol group-containing carbon chain molecule dodecanethiol (1:1 by volume), and stirred for 1 hour to carry out particle surface. The ligand is modified and replaced, and at this time, the solution produces a significant turbidity, whereby luminescent nano-nano cluster aggregates are formed.

(2) Fluorescent gold nano-cluster biomolecule grafting

200 μl of fluorescent gold nano cluster solution was mixed with 200 μl of galactose solution (80 mM in ddH ₂ O), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 30 mM in ddH ₂ O) crosslinking agent was added. The solution was shaken for two hours, and the galactose molecule was grafted onto the surface of the fluorescent gold nanoparticle cluster by EDC and the indoleamine bond with the amine galactose, and centrifuged at 100 kDa molecular sieve to remove excess galactose.

Among them, 20 μl of the grafted galactose-galactin cluster was mixed with 20 μl of lectin RCA120 (1 mg/ml), and reacted for 20 minutes to observe whether agglutination occurred or not to determine whether the galactose molecule was successfully grafted. To the surface of the fluorescent gold cluster. In addition, the modified fluorescent gold nano-clusters were purified by colloidal electrophoresis (2% agarose, 75V), recovered by dialysis membrane, and centrifuged in SBB (sodium borate buffer, pH=9) with 100kDa molecular sieve. in. The sixth picture shows the high scores Schematic representation of a sub-modified agglomerate of fluorescent gold nanoclusters with an alkanethiol ligand.

Through statistical analysis, it is found that the upward trend of the absorption spectrum is consistent with the fluorescence spectrum, indicating that the fluorescence characteristics of the Au-DDT cluster are related to the degree of self-assemble aggregation of the cluster. With the generation of Au-S bonds, As the cluster assembly structure becomes more and more obvious, the fluorescence is generated and the intensity is enhanced. Among them, the Au-DDT fluorescent clusters are excited by the excitation light at 325 nm, 345 nm and 365 nm wavelengths respectively, and the red emission fluorescence is generated at 600 nm, and the peaks are not present. A displacement is generated, indicating that the red light of the Au-DDT cluster is a fluorescent characteristic rather than a general scattered light; in addition, the emitted light is fixed at 580 nm and 600 nm, respectively, and the wavelength of the most suitable fluorescent excitation light is measured, and both are found. A peak is generated at the 325 nm position, indicating that the Au-DDT fluorescent gold cluster is excited at a wavelength of 325 nm, and the maximum intensity of red fluorescent radiation can be obtained at 600 nm, as shown in the fourth figure.

Referring to the fifth figure, the HAuCl ₄ precursor has a characteristic absorption peak at 370 nm, and 6 nm nano gold particles are generated by the TBAB reducing agent, and a surface plasma resonance absorption peak appears at 520 nm; the HAuCl ₄ solution is continuously added to make the nano The gold particles disintegrated into clusters, and the absorption peak at 520 nm disappeared, indicating that the cluster was less than 5 nm. Except that the original HAuCl ₄ appeared at the peak of 370 nm, a new absorption peak was generated at 310 nm, and finally DDT molecules were added to produce a fluorescent gold cluster Au-DDT. Due to the hydrophobic interaction between the carbon chain molecules, the clusters form a self-assembled structure, and the absorption spectrum is significantly increased in the infrared range, which indicates that the aggregation occurs. Among them, the absorption spectra of the products of the various stages of the synthesis of the gold nanoparticles and the gold nanoparticles: (A) HAuCl ₄ precursor (0.625 mM) (B) nano gold particles (C) gold nano clusters (D) AuNC -DDT fluorescent nano gold clusters.

Obviously, many modifications and differences may be made to the invention in light of the above description. It is therefore to be understood that within the scope of the appended claims, the invention may be The above are only the preferred embodiments of the present invention, and are not intended to limit the scope of the claims of the present invention; all other equivalent changes or modifications which are not departing from the spirit of the present invention should be included in the following claims. Within the scope.

The first figure is the absorption spectrum of the gold nanoparticles and the gold nano clusters in the first example of the present invention; the second figure is the absorption of the fluorescent gold nano cluster AuNC-DHLA in the first example of the present invention (absorption) ), photoluminescence (PL), photoluminescence excitation (PLE) spectrum; third diagram is the first example of the invention, fluorescent gold nano cluster AuNC-DHLA biomolecule Schematic diagram of the branch reaction; the fourth figure is the photoexcited fluorescence spectrum (PLE) and photoexcitation spectrum (PL) of the fluorescent gold cluster AuNC-DDT in the second example of the present invention; the fifth figure is in the second example of the present invention. , absorption spectra of gold nanoparticles and fluorescent gold nanoclusters AuNC-DDT; Fig. 6 is a schematic view showing the aggregate of fluorescent gold nanoclusters having an alkanethiol ligand modified by an amphoteric polymer in the second embodiment of the present invention.

Claims (6)

A fluorescent gold nano-aggregate aggregate formed by a regular stack of a plurality of gold nanoclusters having a particle size ranging from 0.5 nm to 3 nm, and The surface of the gold nanoclusters has an alkanethiol ligand, wherein each of the golden nanoclusters is attracted to each other through a force between the alkanethiol ligands on the surface thereof to form the fluorescent light. a gold nano-aggregate aggregate, and the fluorescent gold nano-aggregate aggregation system generates fluorescence by the aggregate of the golden nano-clusters, wherein the fluorescent gold nano-aggregate aggregate is light-excited The fluorescence wavelength is 600 nm. The fluorescent gold nano-clustered aggregate according to claim 1, wherein a surface of the fluorescent gold nano-aggregate aggregate is coated with a spacer, and one end of the spacer is bonded to the alkyl sulfide. An alcohol ligand, and the other end of the spacer is bonded to a specific group. The fluorescent gold nanoparticle aggregate according to claim 2, wherein the spacer comprises an amphoteric polymer or an oligomer. a fluorescent gold nano-aggregate aggregate as described in claim 3, The amphoteric polymer or oligomer comprises one of the following groups or any combination thereof: poly (maleic anhydride); PMA], a polymer of 1-octadecene maleic anhydride [ Poly(maleic anhydride-alt-1-octadecene); PMAO] and polyacrylic acid (PAA). The fluorescent gold nano-clustered aggregate according to claim 2, wherein the specific group comprises one of the following groups: a chemical functional group, a crosslinking molecule, a saccharide, a fluorescent molecule, Paramagnetic molecules, biomolecules and drugs. The fluorescent gold nano cluster aggregate according to claim 1, wherein the fluorescent gold nano cluster aggregation system can be used as a bioprobe and has the following applications: biological fluorescence Fluorescent biological label, clinical medical imaging developer, and clinical medical testing, tracking and treatment.