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    • 专利摘要:
    Super fluorescent Ag2 S nanoparticles in the near infrared region and method of obtaining. Labeling with fluorescent probes plays an important role in studies at the cellular and tissue level in both biomedical research and in vivo studies. Ag2 S particle probes have the advantage of low or no toxicity, while exhibiting near infrared fluorescence. But the Ag2 S probes have the drawback of stopping a low quantum efficiency (around 0.2%). The present invention describes Ag2 S nanoparticles capable of emitting in the near infrared with a quantum efficiency of 10% (which represents a 50-fold increase with respect to the efficacy of the nanoparticles obtained up to the moment) as well as its synthesis method, which is a simple method that gives rise to colloidal stable particles that can be superficially functionalized. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2773946A1
    申请号:ES201900006
    申请日:2019-01-15
    公开日:2020-07-15
    发明作者:Retama Jorge Rubio；Gonzalez Diego Mendez；Marco Laurenti；Garcia Daniel Jaque；Santos Harrison David Assis
    申请人:Universidad Complutense de Madrid；Universidad Autonoma de Madrid；
    IPC主号:
    专利说明:

    [0001] Super fluorescent Ag 2 S nanoparticles in the near infrared region and method of obtaining
    [0003] TECHNICAL SECTOR
    [0004] The invention belongs to the field of Materials Chemistry for application in Biology and Medicine. More specifically, it refers to obtaining inorganic materials with fluorescent properties for application in advanced systems for image contrast in biological media.
    [0006] BACKGROUND OF THE INVENTION
    [0007] Fluorescent probe labeling plays an important role in studies at the cellular and tissue level both in biomedical research and in vivo studies .
    [0009] In the case of in vivo studies , fluorescent images obtained in the near infrared have a number of advantages that make them unique. On the one hand, the greater penetration capacity of infrared light makes it possible to illuminate and visualize deep tissues that visible light cannot reach. On the other hand, the lower abundance of biological molecules capable of emitting in the near infrared makes it possible to reduce the background signal, allowing to increase the sensitivity of the technique.
    [0011] For this reason, in recent years, great attention has been paid to the development of probes capable of emitting in the near infrared for application in medical imaging, molecular biology, etc. Currently, the most commonly used probes for near infrared emission contain Cd2 +, Hb2 +, Pb2 +, among others. But it would be interesting to develop probes that emit in the near infrared region but with low toxicity.
    [0013] In this sense, Ag 2 S particles constitute an ideal alternative due to their low or no toxicity, while at the same time exhibiting fluorescence in the near infrared. But Ag 2 S probes have the disadvantage of having a low quantum efficiency (around 0.2%), so a chemical modification of their surface is necessary to increase said efficiency. However, its crystalline structure makes such a modification of its surface substantially difficult.
    [0014] All the foregoing makes it of great interest in the current state of the art to develop a method that allows obtaining Ag 2 S probes with high quantum efficiency by means of a simple synthesis method that produces nanoparticles of controlled size, easily dispersible, with high degree of reproducibility and that, in addition, allows a simple surface functionalization for its subsequent application in fluorescent imaging.
    [0016] EXPLANATION OF THE INVENTION
    [0017] The present invention refers to Ag 2 S nanoparticles capable of emitting in the near infrared with a quantum efficiency of 10% (which represents a 50-fold increase with respect to the efficiency of the nanoparticles obtained so far) as well as their synthesis method which is a simple method that gives rise to colloidally stable particles that can be superficially functionalized, which makes them interesting as image contrast agents in biological applications.
    [0019] The Ag 2 S nanoparticle synthesis method comprises the following steps: 1) Preparation of hydrophobic Ag 2 S nanoparticles.
    [0020] a) A source of Ag and a source of S are mixed with a long chain organic molecule containing an amine and / or a thiol and the mixture is reacted at a temperature between 80 ° C and 320 ° C in a closed atmosphere.
    [0021] b) After a passive cooling process, a polar organic solvent is added to the previous mixture, it is centrifuged and the nanoparticles obtained are washed, giving rise to hydrophobic nanoparticles.
    [0022] 2) Ultra-fast laser treatment.
    [0023] 3) Ligand exchange to generate hydrophilic Ag 2 S particles.
    [0025] In step 1a) of preparation of hydrophobic Ag 2 S nanoparticles, the silver source can be any silver nitrate, silver dihydrocarbyl thiophosphate, silver dioctyl sulfosuccinate, silver thiobenzoate, silver acetate, silver dodecanoate, silver tetradecanoate. silver and silver octadecanoate. Among the long chain organic molecules, octylamine, trioctylamine, dodecylamine, octadecylamine, octaenothiol, undecanethiol, dodecanethiol, tridecanethiol, tetradecanethiol, pentadecanethiol, hexadecanethiol, eicosaenothiol, can be used.
    [0027] In step 2) ultrafast laser treatment induces a structural change that eliminates defects and increases the quantum efficiency of nanoparticles. This process is preferably carried out by dispersing the Ag 2 S nanoparticles in chloroform, dichloromethane, dichloroethane, trichloroethane or any organic solvent that contains chlorine. In the sample optimization process, an ultra-fast laser is used for a time interval that depends on specific characteristics of the laser, including: wavelength, pulse length, frequency, applied power density and size of the diameter of the laser. make.
    [0029] Step 3) of ligand exchange is carried out by mixing hydrophobic Ag 2 S nanoparticles obtained in the previous step with an equivalent amount or an excess of some hydrophilic molecule that contains a thiol group in such a way that the surface of the nanoparticles of Ag 2 S is functionalized by the hydrophilic group, thus obtaining hydrophilic particles. The hydrophilic group is any of: mercaptoacetic acid, mercaptopropionic acid, cysteine, cysteinamine, thioctic acid, ammonium mercaptoacetate, or any combination thereof. The organic solvent may be, among others, ethanol, methanol, acetone, chloroform, dichloromethane, toluene, 1-methyl-2-pyrrolidone, or any combination thereof. In this organic solvent, the substitution reaction is carried out at a temperature between 0 and 80 ° C for a period of about 1 hour or more. The reaction time can be modified according to the reactivity and solubility of the stabilizer molecule. The pH of the aqueous solutions used during step 3 is between 7 and 14. After this, the quantum dots are washed with aqueous solutions to remove traces of reagents.
    [0031] The method is simple and provides reproducibility to the system.
    [0033] The particles obtained have a quantum efficiency close to 10% and show high fluorescence, stability and excellent biocompatibility, which is why they can be used as contrast agents both in cell imaging and in vivo imaging .
    [0035] BRIEF DESCRIPTION OF THE DRAWINGS
    [0036] Figure 1. TEM image of Ag 2 S nanoparticles.
    [0038] Figure 2. Quantum efficiency of Ag 2 S nanoparticles before and after laser treatment.
    [0039] Figure 3. Nanoparticle life times before and after laser treatment.
    [0041] Figure 4. Intensity of fluorescence emitted by nanoparticles before and after laser treatment.
    [0043] PREFERRED EMBODIMENT OF THE INVENTION
    [0044] The present invention is further illustrated by the following examples, which are not intended to be limiting of its scope.
    [0046] Example 1:
    [0047] 0.1 mmol of silver diethyldithiocarbamate and 10 g of dodecanethiol are mixed in a reactor and heated to 200 ° C under N 2 for 1 hour. After this, the reaction is allowed to cool naturally and 50 ml of anhydrous ethanol are added. The mixture is centrifuged, washed and dispersed in hexane. The sample thus obtained is identified as monoclinic Ag2S nanoparticles by X-ray diffraction and transmission electron microscopy. The size of the particles is 5 nm as shown in Figure 1. These nanoparticles have an emission spectrum in the infrared as shown in Figure 2.
    [0049] To the above dispersion 0.2 g of thioctic acid and a similar volume of anhydrous ethanol are added and it is redispersed in CHCh. The resulting mixture was sonic and sonic for 4 h; then it is centrifuged and washed with deionized water to obtain Ag 2 S nanoparticles dispersible in water. The fluorescence of the water soluble particles is shown in Figure 3. The quantum yield of the sample is 0.17%.
    [0051] Example 2:
    [0052] 0.1 mmol of silver nitrate, 8 g of dodecanethiol and 5.4 g of oleylamine are mixed in a three-neck flask and the mixture is heated at 180 ° C for 1 h. The solution is then cooled to room temperature and 50 ml of anhydrous ethanol are added. The resulting mixture is centrifuged, washed, and dispersed in chloroform. The sample obtained is characterized by X-ray diffraction and transmission electron microscopy. The results of this characterization show that the synthesized nanoparticles have an Ag 2 S shell, have a monoclinic phase and a small silver core. The size of the nanoparticles is 10 nm. A spectroscopic analysis shows that the nanoparticles have a good fluorescence emission spectrum in the near infrared.
    [0054] 0.2 g of L-cysteine is added to the above cyclohexane dispersion, then an equal volume of anhydrous ethanol is added. The resulting mixture is stirred for 24 h, then centrifuged and washed with deionized water to obtain water-soluble Ag 2 S quantum dots with particle sizes of approximately 8 nm, which still have a very strong fluorescence emission. The quantum yield of the sample is 0.14%
    [0056] Example 3:
    [0057] 3 mmol of AgNO 3 , as a silver source, is dissolved in 10 ml of octadecylamine at 150 ° C (in an oil bath). After 10 minutes the solution takes on a deep metallic blue color, indicating the formation of Ag nanoparticles. Then 1 mmol of L-cystine is added to the premix as a source of sulfur. The reaction is maintained for 20 minutes in an air atmosphere after which hydrophobic Ag 2 S nanoparticles are obtained.
    [0059] The nanoparticles thus obtained have a size of approximately 12 nm and a core-shell structure (Ag core and Ag 2 S shell). These nanoparticles are precipitated by adding anhydrous ethanol and subsequently centrifuged and redispersed in CHCl 3 . The quantum yield of these nanoparticles is 0.2%.
    [0061] Example 4:
    [0062] This example and the following illustrate the treatment of Ag 2 S nanoparticles with ultrafast laser.
    [0064] The hydrophobic particles obtained according to example 3 are dispersed in CHCh or other chlorinated solvents such as CH 2 Cl, CCU, etc. maintaining a concentration of nanoparticles in solution of 1 mg / ml. Subsequently, they are irradiated using an ultra-fast laser at a wavelength of 800 nm and 3W with a beam of 0.8 mm diameter (power density 6W / cm2), with pulses of 50 femto-seconds, at a frequency of 1 kHz for approximately 10 minutes. After that, the sample goes from exhibiting a quantum yield of 0.2% to 10%. This rise in quantum efficiency, as As shown in figure 2, it is linked to an increase in the lifetime of the nanoparticles (figure 3), as well as in the fluorescence of the nanoparticles as shown in figure 4.
    [0066] Example 5:
    [0067] The enhancement reaction can be performed by illuminating the sample with an ultra-fast laser at a wavelength of 400 nm. In this case, the improvement of the nanoparticles is carried out in a shorter time (figure 3a).
    [0069] Example 6:
    [0070] The application of pulses below 500 fs allows to carry out the transformation of the nanoparticles and improve their quantum performance, being, among the preferred values, a pulse of 50 fs as shown in figure 3b. The energy density applied to the sample can also be varied. This affects the speed of transformation of the product and the structure of the nanoparticles. In such a way that power densities greater than 40 W / cm2 damage the nanoparticles and reduce their quantum efficiency. Below these power densities, the quantum efficiency of the nanoparticles improves, being optimal for values of 20 W / cm2 and inefficient for values lower than 3W / cm2.
    权利要求:
    Claims (10)
    [1]
    1. Hydrophilic Ag 2 S nanoparticles characterized in that their surface is functionalized by any hydrophilic group from among: mercaptoacetic acid, mercaptopropionic acid, cysteine, cysteinamine, thioctic acid, ammonium mercaptoacetate, or any combination thereof.
    2. Synthesis method of hydrophilic Ag 2 S nanoparticles comprising the following steps:
    1) Preparation of hydrophobic Ag 2 S nanoparticles.
    a) A source of Ag and a source of S are mixed with a long chain organic molecule containing an amine and / or a thiol and the mixture is reacted at a temperature between 80 ° C and 320 ° C in a closed atmosphere.
    b) After a passive cooling process, a polar organic solvent is added to the previous mixture, it is centrifuged and the nanoparticles obtained are washed, giving rise to hydrophobic nanoparticles.
    [2]
    2) Ultra-fast laser treatment.
    3) Ligand exchange to generate hydrophilic Ag 2 S particles.
    [3]
    3. Ag 2 S nanoparticle synthesis method, according to claim 2, where the silver source can be any silver nitrate, silver dihydrocarbyl-thiophosphate, silver dioctyl sulfosuccinate, silver thiobenzoate, silver acetate, silver dodecanoate , silver tetradecanoate and silver octadecanoate. Among the long chain organic molecules, octylamine, trioctylamine, dodecylamine, octadecylamine, octaenothiol, undecanethiol, dodecanethiol, tridecanethiol, tetradecanethiol, pentadecanethiol, hexadecanethiol, eicosaenothiol can be used.
    [4]
    4. Ag 2 S nanoparticle synthesis method, according to claims 2 and 3, where the laser treatment is carried out by dispersing the Ag 2 S nanoparticles in chloroform, dichloromethane, dichloroethane, trichloroethane or any organic solvent that contains chlorine.
    [5]
    5. Method of synthesis of Ag 2 S nanoparticles, according to claim 4, where an ultra-fast laser is used during a time interval that depends on specific characteristics of the laser among which it is worth highlighting: wavelength, pulse length, frequency, applied power density and size of the beam diameter.
    [6]
    6. Method of synthesis of Ag 2 S nanoparticles, according to claims 2 to 5, where ion exchange is carried out by mixing hydrophobic Ag 2 S nanoparticles with an equivalent amount or an excess of some hydrophilic molecule that contains a thiol group of such that the surface of the Ag 2 S nanoparticles is functionalized by the hydrophilic group, thus obtaining hydrophilic particles.
    [7]
    7. Ag 2 S nanoparticle synthesis method, according to claim 6, where the hydrophilic group is any of: mercaptoacetic acid, mercaptopropionic acid, cysteine, cysteinamine, thioctic acid, ammonium mercaptoacetate, or any combination thereof. The organic solvent may be, among others, ethanol, methanol, acetone, chloroform, dichloromethane, toluene, 1-methyl-2-pyrrolidone, or any combination thereof.
    [8]
    8. Ag 2 S nanoparticle synthesis method, according to claims 6 and 7, wherein the substitution reaction is carried out at a temperature between 0 and 80 ° C for a period of 1 hour or more. The reaction time can be modified according to the reactivity and solubility of the stabilizer molecule. The pH of the aqueous solutions used is between 7 and 14 and the quantum dots are washed with aqueous solutions to remove reagent traces.
    [9]
    9. Synthesis method of Ag 2 S nanoparticles, according to claims 6 and 7. After this, the quantum dots are washed with aqueous solutions to remove reagent remains.
    [10]
    10. Use of the claimed nanoparticles as fluorescent probes.
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    同族专利:
    公开号 | 公开日
    WO2020148347A1|2020-07-23|
    ES2773946B2|2021-03-08|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US20160083647A1|2011-05-30|2016-03-24|Suzhou Institute of Nano-tech and Nano-bionics, Chinese Academy of Sciences|Preparation Method of Near-Infrared Silver Sulfide Quantum Dots|
    CN106753344A|2016-11-25|2017-05-31|清华大学|Silver sulfide quantum dot and preparation method and application|
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    PCT/EP2020/050939| WO2020148347A1|2019-01-15|2020-01-15|Ag2s nanoparticles and methods of production thereof|
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