CN113004889A - Method for synthesizing near-infrared fluorescence/photothermal/photoacoustic silver sulfide quantum dots - Google Patents

Method for synthesizing near-infrared fluorescence/photothermal/photoacoustic silver sulfide quantum dots Download PDF

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CN113004889A
CN113004889A CN202110258263.2A CN202110258263A CN113004889A CN 113004889 A CN113004889 A CN 113004889A CN 202110258263 A CN202110258263 A CN 202110258263A CN 113004889 A CN113004889 A CN 113004889A
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silver sulfide
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张树彪
宣扬
关萌
刘一宁
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Dalian Minzu University
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Abstract

The invention discloses a method for synthesizing near-infrared fluorescent/photothermal/photoacoustic silver sulfide quantum dots, which comprises the following steps of: s1, mixing the components in a molar ratio of 1: adding the R- (+) -lipoic acid and the reducing agent in the (1-3) into ultrapure water, and stirring at normal temperature under the protection of an inert medium to obtain a dihydrolipoic acid solution for later use; s2, adjusting the pH value of the obtained dihydrolipoic acid solution to 7-10; s3, adding a silver source, stirring for 10-30 min, and adjusting the pH value to 7-10. And S4, adding a sulfur-containing compound, and reacting for 1-5 hours at the temperature of 30-90 ℃ to obtain the silver sulfide quantum dots. The silver sulfide quantum dots with the functions of near-infrared imaging, photoacoustic imaging, photothermal therapy and the like are synthesized by a one-step method by using the dihydrolipoic acid as the stabilizer, and the silver sulfide quantum dots are simple to operate, mild in reaction, high in nucleation speed and good in experimental reproduction.

Description

Method for synthesizing near-infrared fluorescence/photothermal/photoacoustic silver sulfide quantum dots
Technical Field
The invention belongs to the field of material chemistry and biomedicine, and particularly relates to a method for synthesizing near-infrared fluorescent/photothermal/photoacoustic silver sulfide quantum dots.
Background
With the development of nanotechnology, nanoprobes with imaging function are widely applied to the biomedical field, and especially play an important role in the aspects of early diagnosis of tumors, active drug screening, even real-time evaluation of treatment effect and the like. The nano probe has the characteristics of high detection sensitivity, capability of providing real-time image data and the like, so that the accuracy of early diagnosis is greatly improved, the misdiagnosis risk is reduced, and a basis is provided for early diagnosis and treatment of tumors. Therefore, the application of the nanoprobe in the early diagnosis and the monitoring of the curative effect of the tumor becomes a necessary trend in development. At present, the nano probe realizes multi-modal imaging and multi-modal treatment, and enough evidence shows that the treatment methods such as photodynamic/photothermal and the like can not only directly kill tumor cells, but also cause the release of tumor antigens, and promote the specific immune response of tumor immunotherapy through efficient antigen uptake and immune adjuvant stimulation.
Quantum Dots (QDs) are semiconductor nanostructures that confine conduction band electrons, valence band holes, and electron-hole pairs in three spatial dimensions, typically 1-10 nm in size, smaller than or close to the exciton bohr radius. Because electrons and holes are limited by quanta, the continuous energy band structure is changed into a discrete energy level structure with molecular characteristics, and after being excited, the fluorescence can be emitted. Based on various physical effects of quantum dots, such as quantum size effect, surface effect, quantum confinement and the like, the quantum dots also have regular atomic arrangement similar to crystals, thereby deriving excellent characteristics of wide excitation spectrum, narrow emission spectrum, negligible photobleaching and the like. The nano probe prepared by the quantum dot can be well applied to the research in the fields of basic science of luminescence and life science, and provides help for high-resolution imaging and accurate diagnosis and treatment of malignant tumors.
Silver sulfide quantum dots are a novel near-infrared emission quantum dot, and are favored due to high biocompatibility, stability and strong light-emitting property. Its extremely low solubility constant (KSP ═ 6.3X 10)-50) Can inhibit the release of silver ions into biological media and ensure high biocompatibility. According to Ag2S quantum dot is preparedThe preparation method of the quantum dots can be roughly divided into two main categories according to different types of solvents adopted in the process: organic phase synthesis and aqueous phase synthesis. The quantum dots prepared in the organic solvent have many excellent properties, such as uniform size, good dispersibility, good stability, high fluorescence yield and the like. In some researches, 1-dodecyl mercaptan (DT) is used as a covalent ligand to prepare oil-soluble Ag with the sizes of 5.4 nm, 7nm and 10nm and the maximum emission peak of 1150nm2The S quantum dots are uniform in size and easy to regulate and control. Although oil-soluble quantum dots have many advantages, the preparation in the organic phase requires severe conditions, such as strict oxygen-free environment, high synthesis temperature, etc., and the synthesis steps are complicated, more toxic reagents are used, the cost is high, and the synthesized quantum dots can only be dissolved in a non-polar solvent. Research reports that alpha-Ag with the maximum emission peak at 1050nm is synthesized in an organic phase at 160-180 DEG C2S quantum dots, the prepared quantum dots are transferred into a water phase for biological imaging by adopting a phase transfer method, and the steps are complicated. Compared with an organic phase synthesis method, the aqueous phase synthesis method solves the problem that the synthesized quantum dots are insoluble in polar solvents. However, the existing aqueous phase synthesis method has long synthesis time, low quantum yield, uneven particle size, complicated steps and harsh synthesis conditions such as high temperature, no oxygen and the like. Therefore, research on a simple aqueous synthesis method for synthesizing Ag2The S quantum dots have important significance for further popularization and application.
Disclosure of Invention
Aiming at the problems, the invention researches and designs a method for synthesizing near-infrared fluorescent/photothermal/photoacoustic silver sulfide quantum dots. The technical means adopted by the invention are as follows:
a method for synthesizing near-infrared fluorescence/photo-thermal/photo-acoustic silver sulfide quantum dots comprises the following steps:
s1, mixing the components in a molar ratio of 1: adding the R- (+) -lipoic acid and the reducing agent in the (1-3) into ultrapure water, and stirring at normal temperature under the protection of an inert medium to obtain a dihydrolipoic acid solution for later use;
s2, adjusting the pH value of the obtained dihydrolipoic acid solution to 7-10;
s3, adding a silver source, stirring for 10-30 min, and adjusting the pH value to 7-10.
S4, adding a sulfur-containing compound, wherein the molar ratio of the dihydrolipoic acid to sulfur atoms in the sulfur-containing compound is more than 38.88, and reacting for 1-5 hours at 30-90 ℃ to obtain the silver sulfide quantum dots.
Preferably, in step S1, the reducing agent is one or more of tris (2-carboxyethyl) phosphine hydrochloride, sodium borohydride and sodium citrate.
Preferably, in step S1, the inert medium is nitrogen or an inert gas.
Preferably, in step S1, the concentration of the dihydroliponic acid solution is 0.0162-0.0486 mol/L.
Preferably, in step S3, the silver source is silver nitrate and/or silver acetate.
Preferably, in step S4, the sulfur-containing compound is one or more of sodium sulfide, thioacetamide, sodium thiosulfate, sodium diethyldithiocarbamate, 3-mercaptopropionic acid, hexamethyldisilazane, and elemental sulfur.
Preferably, in steps S2, S3, NaOH (40%)/CH is used3COOH (99.5%) adjusted the pH.
Fluorescence Imaging (FI) as a molecular Imaging technology has the advantages of high resolution, low acquisition time and the like, and can observe dynamic changes of tumor-related molecules in vivo in real time, so that the Fluorescence Imaging (FI) has great application prospect in the aspect of early diagnosis of tumors. However, some endogenous substances (such as hemoglobin, water, bilirubin and melanin, etc.) in biological tissues have strong absorption and scattering to visible light (400-700nm), so that the light penetration capability of the wavelength band is limited. In contrast, the fluorescence in the near infrared region is absorbed and scattered less by tissues and has a strong penetrating power, so that near infrared fluorescent substances are generally used in vivo imaging. Silver sulfide is a novel low-toxicity quantum dot which appears in recent years, has the optical characteristics of wide excitation, narrow emission, long fluorescence life, strong photobleaching resistance and the like of a conventional quantum dot, has stronger tissue penetration capacity due to the fact that the silver sulfide fluorescence emission is located in a near-infrared two-region, can eliminate the interference of biological autofluorescence, and contributes to the improvement of the signal-to-noise ratio, so that the silver sulfide is a promising quantum dot for living body imaging.
Photoacoustic Imaging (PAI) is another new molecular Imaging technology that has been rapidly developed in recent years. After absorbing the pulse laser energy, the biological tissue generates thermal expansion, and ultrasonic waves are generated along with the thermal expansion; different biological tissues generate different ultrasonic waves, the photoacoustic signals carry light absorption characteristic information of the tissues, and light absorption distribution images in the tissues can be reconstructed by measuring the photoacoustic signals. PAI combines the advantages of high contrast of optical imaging and high tissue penetration depth of ultrasonic imaging, avoids the influence of light scattering in principle, can realize deep tissue imaging similar to ultrasonic imaging, can obtain high contrast imaging similar to optical coherence tomography, breaks through high-resolution optical imaging depth soft limit (about 1mm), can provide high-resolution and high-contrast tissue imaging, and has wide application prospect in the aspects of early diagnosis of tumors, monitoring curative effect and the like. The silver sulfide quantum dots have strong absorption in visible light and near infrared regions, and under the excitation of a certain wavelength, the silver sulfide quantum dots also have good photoacoustic response, so that the silver sulfide quantum dots are an ideal photoacoustic imaging contrast agent.
Since a single imaging mode often cannot provide comprehensive information due to certain defects. By taking advantage of the strong points and making up the weakness, two or more imaging technologies are integrated together, which is a necessary trend for the development of the living body molecular imaging technology, and the integration is also an effective way for early diagnosis and efficacy monitoring of cancer, so that the multi-modal imaging technology comes along. Although FI can give a three-dimensional quantitative result of the distribution of fluorescent molecules in a living body, the FI is insufficient in the capability of acquiring structural information; PAI can provide high resolution and high contrast tissue imaging, therefore, FI and PAI in combination complement each other and are ideal bimodal molecular imaging modalities: FI provides the distribution and location of minute amounts of cancer-associated molecules in tissues, while PAT provides deep tissue and spatial structural information.
Photothermal therapy has gained increasing acceptance and attention as an effective therapeutic means for treating tumors. The photothermal therapy of tumor is to utilize the irradiation of external laser to convert the light energy into heat energy, raise the temperature of tumor tissue to effective treatment temperature (>41 ℃) and maintain for a certain time, and utilize the difference of the temperature tolerance of normal tissue and tumor cell to achieve the treatment purpose of apoptosis of tumor cell and no damage to normal tissue. Photothermal therapy is a new tumor treatment mode, and has the advantages of no damage to normal tissues of a human body, no damage to an autoimmune system and the like, so that the influence is comparable to that of traditional tumor treatment such as surgical treatment, chemical treatment, radiation treatment and the like. In addition, photothermal therapy can also result in the release of tumor antigens, and specific immune response of tumor immunotherapy is promoted through efficient antigen uptake and immune adjuvant stimulation. Similar to photoacoustic imaging, because near-infrared light has good tissue penetration in the human body, laser in the near-infrared region is of interest to researchers as a photothermal light source. Similarly, nanoprobes play an important role in photothermal therapy.
Compared with the prior art, the method for synthesizing the near-infrared fluorescence/photo-thermal/photo-acoustic silver sulfide quantum dots has the beneficial effects that:
1. in the invention, dihydrolipoic acid (DHLA) is used as a stabilizer, the water-soluble silver sulfide quantum dots are synthesized by a one-step method, the operation is simple, anhydrous operation conditions are not required, the reaction is mild, the nucleation speed is high, and the experimental reproduction is good. The synthesized silver sulfide quantum dots have the functions of near-infrared imaging, photoacoustic imaging, photothermal treatment and the like, can be used for diagnosis and treatment of tumors, and realize diagnosis and treatment integration.
2. According to the invention, silver sulfide quantum dots with an emission peak of 1060nm and a particle size of about 7nm can be prepared by changing conditions such as reaction temperature (30-90 ℃), pH (7-10), time (1-5 h) and the like.
3. When the silver sulfide quantum dots are used as the multifunctional nano probe, the influence of the probe on various immune cells in the treatment process of malignant tumors and the function of the probe in immunotherapy are researched by utilizing the excellent contrast capability of the silver sulfide quantum dots, a reliable new method is provided for high-resolution imaging and accurate diagnosis and treatment of malignant tumors, a new thought is provided for preventing tumor migration and recurrence and completely curing the malignant tumors, and the survival rate of patients can be greatly improved when the silver sulfide quantum dots are used for early diagnosis of cancers.
Drawings
Fig. 1 is an XRD spectrum of the silver sulfide quantum dot prepared in example 1 of the present invention.
Fig. 2 is an ultraviolet-visible absorption spectrum of the silver sulfide quantum dot prepared in example 1 of the present invention.
Fig. 3 is an excitation spectrum of the silver sulfide quantum dot prepared in example 1 of the present invention.
Fig. 4 is a Transmission Electron Microscope (TEM) image of the silver sulfide quantum dots prepared in example 1 of the present invention.
Fig. 5 is a particle size distribution of silver sulfide quantum dots prepared in example 1 of the present invention.
Fig. 6 is a zeta potential diagram of the silver sulfide quantum dots prepared in example 1 of the present invention.
Fig. 7 is a graph of photothermal temperature increase and a thermal imaging photograph of the silver sulfide quantum dots prepared in example 1 of the present invention.
Fig. 8 is a photothermal conversion efficiency calculation.
Fig. 9 is a graph of the photo-thermal stability of silver sulfide quantum dots prepared in example 1 of the present invention.
Fig. 10 is a graph showing the results of cytotoxicity experiments performed on the silver sulfide quantum dots prepared in example 1 of the present invention.
Fig. 11 is a graph showing the results of cell photothermal therapy experiments on the silver sulfide quantum dots prepared in example 1 of the present invention.
Fig. 12 is a graph showing the results of cell photothermal therapy experiments on the silver sulfide quantum dots prepared in example 1 of the present invention.
FIG. 13 is a graph of photoacoustic imaging results after 2, 4, 6, and 8 hours of co-incubation of the probe with the cells, respectively.
FIGS. 14 and 15 are graphs showing the results of chemotherapy-photothermal combination therapy of A549 cells and Hela cells by using silver sulfide quantum dots to adsorb antitumor drug adriamycin, respectively.
Detailed Description
Example 1
A method for synthesizing near-infrared fluorescent/photothermal/photoacoustic silver sulfide quantum dots by a one-step method comprises the following steps:
s1, 60mL of ultrapure water is added with R- (+) -lipoic acid (200mg) and reducer tris (2-carboxyethyl) phosphine hydrochloride (278mg) in equal molar ratio, and the mixture is stirred vigorously under the protection of nitrogen at normal temperature overnight to obtain reduced lipoic acid, namely DHLA solution (0.0162mol/L) of dihydrolipoic acid for later use.
And S2, adding alkali to adjust the pH of the DHLA solution to 7.5.
S3, adding 500 mu l AgNO3(0.10mol/L) after stirring for 10min, the pH was adjusted to 7.5.
S4, adding 500 mu lNa2S (0.05mol/L) and reacted at 90 ℃ for 1 h. Thus obtaining the silver sulfide quantum dots with the concentration of about 0.11 mg/mL.
Example 2
S1, 60mL of ultrapure water is added with R- (+) -lipoic acid (400mg) and reducer tris (2-carboxyethyl) phosphine hydrochloride (556mg) with equal molar ratio, and the mixture is stirred vigorously under the protection of nitrogen at normal temperature overnight to obtain reduced lipoic acid, namely DHLA solution (0.0324mol/L) of dihydrolipoic acid for later use.
And S2, adding alkali to adjust the pH of the DHLA solution to 7.5.
S3, adding 500 mu l AgNO3(0.10mol/L) after stirring for 10min, the pH was adjusted to 7.5.
S4, adding 500 mu lNa2S (0.05mol/L) and reacted at 90 ℃ for 1 h. Thus obtaining the silver sulfide quantum dots with the concentration of about 0.11 mg/mL.
Example 3
S1, 60mL of ultrapure water is added with R- (+) -lipoic acid (200mg) and reducer tris (2-carboxyethyl) phosphine hydrochloride (578mg) in equal molar ratio, and the mixture is stirred vigorously under the protection of nitrogen at normal temperature overnight to obtain reduced lipoic acid, namely DHLA solution (0.0162mol/L) of dihydrolipoic acid for later use.
And S2, adding alkali to adjust the pH of the DHLA solution to 7.5.
S3, adding 500 mu l AgNO3(0.10mol/L) after stirring for 10min, the pH was adjusted to 7.5.
S4, adding 1000 mu lNa2S (0.05mol/L) and reacted at 90 ℃ for 1 h. Thus obtaining the silver sulfide quantum dots with the concentration of about 0.11 mg/mL.
Example 4
S1, 60mL of ultrapure water is added with R- (+) -lipoic acid (200mg) and reducer tris (2-carboxyethyl) phosphine hydrochloride (578mg) in equal molar ratio, and the mixture is stirred vigorously under the protection of nitrogen at normal temperature overnight to obtain reduced lipoic acid, namely DHLA solution (0.0162mol/L) of dihydrolipoic acid for later use.
And S2, adding alkali to adjust the pH of the DHLA solution to 7.5.
S3, adding 500 mu l AgNO3(0.10mol/L) after stirring for 10min, the pH was adjusted to 7.5.
S4, adding 1000 mu lNa2S (0.05mol/L) and reacting at 60 ℃ for 1 h. Thus obtaining the silver sulfide quantum dots with the concentration of about 0.11 mg/mL.
Example 5
S1, 60mL of ultrapure water is added with R- (+) -lipoic acid (200mg) and reducer tris (2-carboxyethyl) phosphine hydrochloride (578mg) in equal molar ratio, and the mixture is stirred vigorously under the protection of nitrogen at normal temperature overnight to obtain reduced lipoic acid, namely DHLA solution (0.0162mol/L) of dihydrolipoic acid for later use.
And S2, adding alkali to adjust the pH of the DHLA solution to 7.5.
S3, adding 500 mu l AgNO3(0.10mol/L) after stirring for 10min, the pH was adjusted to 7.5.
S4, adding 1000 mu lNa2S (0.05mol/L) and reacted at 60 ℃ for 5 h. Thus obtaining the silver sulfide quantum dots with the concentration of about 0.11 mg/mL.
Example 6
S1, 60mL of ultrapure water is added with R- (+) -lipoic acid (200mg) and reducer tris (2-carboxyethyl) phosphine hydrochloride (578mg) in equal molar ratio, and the mixture is stirred vigorously under the protection of nitrogen at normal temperature overnight to obtain reduced lipoic acid, namely DHLA solution (0.0162mol/L) of dihydrolipoic acid for later use.
And S2, adding alkali to adjust the pH of the DHLA solution to 7.5.
S3, adding 500 mu l AgNO3(0.10mol/L) after stirring for 10min, the pH was adjusted to 7.5.
S4, adding 1000 mu lNa2S (0.05mol/L) and reacted at 30 ℃ for 5 h. Thus obtaining the silver sulfide quantum dots with the concentration of about 0.11 mg/mL.
Example 7
S1, 60mL of ultrapure water is added with R- (+) -lipoic acid (200mg) and reducer tris (2-carboxyethyl) phosphine hydrochloride (578mg) in equal molar ratio, and the mixture is stirred vigorously under the protection of nitrogen at normal temperature overnight to obtain reduced lipoic acid, namely DHLA solution (0.0162mol/L) of dihydrolipoic acid for later use.
And S2, adding alkali to adjust the pH of the DHLA solution to 7.5.
S3, adding 500 mu l AgNO3(0.10mol/L) after stirring for 10min, the pH was adjusted to 7.5.
S4, adding 500 mu lNa2S (0.05mol/L) and reacted at 30 ℃ for 5 h. Thus obtaining the silver sulfide quantum dots with the concentration of about 0.11 mg/mL.
Example 8
S1, 60mL of ultrapure water is added with R- (+) -lipoic acid (200mg) and reducer tris (2-carboxyethyl) phosphine hydrochloride (578mg) in equal molar ratio, and the mixture is stirred vigorously under the protection of nitrogen at normal temperature overnight to obtain reduced lipoic acid, namely DHLA solution (0.0162mol/L) of dihydrolipoic acid for later use.
And S2, adding alkali to adjust the pH of the DHLA solution to 7.5.
S3, adding 500 mu l AgNO3(0.10mol/L) after stirring for 10min, the pH was adjusted to 7.5.
S4, adding 500 mu lNa2S (0.05mol/L) and reacted at 30 ℃ for 1 h. Thus obtaining the silver sulfide quantum dots with the concentration of about 0.11 mg/mL.
Example 9
S1, 60mL of ultrapure water is added with R- (+) -lipoic acid (200mg) and reducer tris (2-carboxyethyl) phosphine hydrochloride (578mg) in equal molar ratio, and the mixture is stirred vigorously under the protection of nitrogen at normal temperature overnight to obtain reduced lipoic acid, namely DHLA solution (0.0162mol/L) of dihydrolipoic acid for later use.
And S2, adding alkali to adjust the pH of the DHLA solution to 7.5.
S3, adding 500 mu l AgNO3(0.10mol/L) after stirring for 10min, the pH was adjusted to 7.5.
S4, adding 500 mu lNa2S (0.05mol/L) and reacted at 30 ℃ for 2 h. Thus obtaining the silver sulfide quantum dots with the concentration of about 0.11 mg/mL.
Example 10
S1, 60mL of ultrapure water is added with R- (+) -lipoic acid (200mg) and reducer tris (2-carboxyethyl) phosphine hydrochloride (578mg) in equal molar ratio, and the mixture is stirred vigorously under the protection of nitrogen at normal temperature overnight to obtain reduced lipoic acid, namely DHLA solution (0.0162mol/L) of dihydrolipoic acid for later use.
And S2, adding alkali to adjust the pH of the DHLA solution to 7.5.
S3, adding 500 mu l AgNO3(0.10mol/L) after stirring for 10min, the pH was adjusted to 7.5.
S4, adding 500 mu lNa2S (0.05mol/L) and reacted at 90 ℃ for 2 h. Thus obtaining the silver sulfide quantum dots with the concentration of about 0.11 mg/mL.
Example 11
S1, 60mL of ultrapure water is added with R- (+) -lipoic acid (200mg) and reducer tris (2-carboxyethyl) phosphine hydrochloride (578mg) in equal molar ratio, and the mixture is stirred vigorously under the protection of nitrogen at normal temperature overnight to obtain reduced lipoic acid, namely DHLA solution (0.0162mol/L) of dihydrolipoic acid for later use.
And S2, adding alkali to adjust the pH of the DHLA solution to 9.
S3, adding 500 mu l AgNO3(0.10mol/L) stirring for 10min, and then adjusting the pH to 9.
S4, adding 500 mu lNa2S (0.05mol/L) and reacted at 90 ℃ for 1 h. Thus obtaining the silver sulfide quantum dots with the concentration of about 0.11 mg/mL.
Example 12
S1, 60mL of ultrapure water is added with R- (+) -lipoic acid (200mg) and reducer tris (2-carboxyethyl) phosphine hydrochloride (578mg) in equal molar ratio, and the mixture is stirred vigorously under the protection of nitrogen at normal temperature overnight to obtain reduced lipoic acid, namely DHLA solution (0.0162mol/L) of dihydrolipoic acid for later use.
And S2, adding alkali to adjust the pH of the DHLA solution to 8.
S3, adding 500 mu l AgNO3(0.10mol/L) stirring for 10min, and then adjusting the pH to 8.
S4, adding 500 mu lNa2S (0.05mol/L) and reacted at 90 ℃ for 1 h. Thus obtaining the silver sulfide quantum dots with the concentration of about 0.11 mg/mL.
Example 13
S1, 60mL of ultrapure water is added with R- (+) -lipoic acid (200mg) and reducer tris (2-carboxyethyl) phosphine hydrochloride (578mg) in equal molar ratio, and the mixture is stirred vigorously under the protection of nitrogen at normal temperature overnight to obtain reduced lipoic acid, namely DHLA solution (0.0162mol/L) of dihydrolipoic acid for later use.
And S2, adding alkali to adjust the pH value of the DHLA solution to 7.
S3, adding 500 mu l AgNO3(0.10mol/L) after stirring for 20min, the pH was adjusted to 7.
S4, adding 500 mu lNa2S (0.05mol/L) and reacted at 90 ℃ for 1 h. Thus obtaining the silver sulfide quantum dots with the concentration of about 0.11 mg/mL.
Example 14
S1, 60mL of ultrapure water is added with R- (+) -lipoic acid (200mg) and reducer tris (2-carboxyethyl) phosphine hydrochloride (578mg) in equal molar ratio, and the mixture is stirred vigorously under the protection of nitrogen at normal temperature overnight to obtain reduced lipoic acid, namely DHLA solution (0.0162mol/L) of dihydrolipoic acid for later use.
And S2, adding alkali to adjust the pH value of the DHLA solution to 7.
S3, adding 500 mu l AgNO3(0.10mol/L) after stirring for 30min, the pH was adjusted to 7.
S4, adding 500 mu lNa2S (0.05mol/L) and reacted at 90 ℃ for 1 h. Thus obtaining the silver sulfide quantum dots with the concentration of about 0.11 mg/mL.
The silver sulfide quantum dot solution obtained in example 1 was concentrated to 2.5mg/mL using a rotary evaporator, and then prepared into silver sulfide quantum dot solutions of different concentrations using ultrapure water for experiments, and the results were shown in fig. 1 to 15.
As can be seen from FIG. 1, the XRD pattern of the silver sulfide quantum dots prepared by the method is matched with that of a silver sulfide standard card.
As can be seen from fig. 2, the silver sulfide quantum dots have significant absorption values even in the near infrared region, which indicates that the silver sulfide quantum dots have good photo-thermal conversion potential under the irradiation of near infrared light.
As shown in FIG. 3, 1mL of the sample was placed in a glass cuvette, and the luminescence spectrum of the sample was collected by an ihr550 monochromator and a CR131 photomultiplier using a semiconductor laser having a wavelength of 808nm as a light source. The result shows that under the excitation of the wavelength of 808nm, the emission peak is stronger at 1060 nm.
As can be seen from fig. 4, the silver sulfide quantum dots have a uniform size and a diameter of about 7 nm.
Referring to fig. 5, 100 μ L of sample is weighed and dissolved in 1mL of ultrapure water, and the solution system is uniformly dispersed by water bath ultrasonic treatment for 10-20 min. The particle size distribution of the silver sulfide quantum dots was obtained by Dynamic Light Scattering (DLS). The result shows that the hydration radius of the silver sulfide quantum dots is mainly distributed between 6nm and 16nm, and the uniform and stable particle size of the silver sulfide quantum dots can be inferred.
As shown in FIG. 6, 100. mu.L of sample is weighed and dissolved in 1mL of ultrapure water, and then water bath ultrasonic treatment is carried out for 10-20 min to obtain a uniformly dispersed solution system. The Zeta potential of the silver sulfide quantum dots was measured using a Zeta potential analyzer. Three tests were performed on the samples. The result shows that the charge of the silver sulfide quantum dot is-35.5 mV, which is negative charge. The prepared silver sulfide quantum dot solution is good in stability, and the solution is not easy to be damaged and coagulated.
As shown in FIG. 7, 150. mu.L of silver sulfide quantum dots at different concentrations (0.4, 0.8 and 1.2mg/mL) were placed in a detachable microplate well and 1.0W/cm was used2Irradiating the sample with 808nm laser for 5min, and recording the temperature change of the sample along with the laser irradiation by using an infrared thermal imaging instrument. The same method was used to determine the concentration of 1.0mg/mL sample at different laser intensities (1.0, 1.5 and 2W/cm)2) Temperature change of the lower. Each sample was set in 5 replicates. The result shows that when the concentration of the silver sulfide quantum dots is constant, the temperature rise effect is increased along with the increase of the laser power; when the laser power is fixed, the temperature rising effect is increased along with the increase of the concentration of the silver sulfide quantum dots.
As shown in FIG. 8, 1mL of the probe (1mg/mL) was put into a glass cuvette and the power used was 1W/cm2Stopping laser irradiation for 10min at 808nm, recording temperature change by a thermal imager, and calculating the photothermal conversion rate of the probe. According to the formula:
Figure BDA0002968449790000101
in the above formula, QDis＝hs*(Tmax water-Tsurr) Hs-mc/τ, τ is the slope in fig. 8, where θ -T (T ═ mc/τ)t-Tsurr)/(Tmax-Tsurr)，TtThe real time temperature of the probe at T seconds is TmaxThe highest temperature, T, that the probe can reachmax waterThe highest temperature, T, that can be reached under the same conditionssurrThe ambient temperature is adopted, the solution mass is m, the specific heat capacity of water is c, the laser power is I, and the absorption value at 808nm is A808And calculating the photo-thermal conversion efficiency to be 48.94%.
As shown in fig. 9, the drawings are photographs of silver sulfide quantum dot aqueous solutions before and after laser irradiation, respectively.
Irradiating silver sulfide quantum dot aqueous solution under laser with power density of 1.5W/cm2And (5) stopping laser irradiation for 5min, naturally cooling for 5min, and recording the temperature change by using a thermal imaging instrument. And then carrying out twice heating/cooling cycles. The temperature rising and falling curve of the silver sulfide quantum dot aqueous solution after repeated irradiation shows that the silver sulfide quantum dot has good photo-thermal stability.
As shown in FIG. 10, A549 and Hela cells were uniformly seeded in 7 groups per well of about 5X 10 in 96-well plates3One cell, 5 in parallel per group. After the cells are completely attached to the wall, adding silver sulfide quantum dots of 0, 15.6, 31.3, 62.5, 125, 250 and 500 mu g/mL respectively, incubating for 4h, then sucking out the culture medium, washing for 3 times with Phosphate Buffered Saline (PBS), washing off the free silver sulfide quantum dots, and adding a new culture medium. After further incubation for 24h, 10. mu.L of CCK-8 solution was added to each well, and after 1h, the absorbance at 450nm of each well was measured using a microplate reader, and the survival rate of the cells was calculated. The result shows that when the concentration of the silver sulfide reaches 0.5mg/mL, the survival rates of A549 and HeLa are still over 86%, and the toxicity of the silver sulfide quantum dots is low.
As shown in fig. 11, after incubation of the cells with 0.5mg/mL silver sulfide quantum dots for 0, 2, 4, and 8 hours, respectively, live cells were stained with calcein, followed by laser irradiation and non-irradiation. The result shows that the cells which are not irradiated by the laser survive, and indirectly proves that the silver sulfide quantum dots are non-toxic. And the dead cells of the sample irradiated by the laser increase along with the increase of the incubation time of the quantum dots and the cells, which proves that the increase of the incubation time leads to the increase of the quantum dot phagocytosis amount of the cells. And can prove the excellent photo-thermal property of the silver sulfide quantum dots.
As shown in FIG. 12, the cells were incubated with quantum dots containing 0, 125, 250, and 500. mu.g/mL silver sulfide for 4h, using both laser irradiation and non-irradiation treatments. Viable cells were then stained with calcein. The result shows that the cells which are not irradiated by the laser survive, and indirectly proves that the silver sulfide quantum dots are non-toxic. The samples irradiated by the laser have more dead cells with the increase of the concentration of the quantum dots, and the increase of the concentration of the quantum dots proves that the amount of quantum dots phagocytosed by the cells is increased. And can prove the excellent photo-thermal property of the silver sulfide quantum dots.
As shown in fig. 13, a549 cells were seeded in a cell culture flask, after the cells were nearly confluent throughout the flask, media containing 200 μ g/mL silver sulfide quantum dots were added for incubation for 2h, 4h, 8h, and 12h, respectively, after which the cells were washed three times with PBS to remove free probes, the cells were fixed with 2.5% glutaraldehyde for 30min, and carefully scraped off with a cell scraper for photoacoustic imaging. The result shows that the amount of the probes phagocytosed by the cells is obviously increased along with the increase of the incubation time, and the photoacoustic signal is gradually enhanced.
As shown in FIGS. 14 and 15, A549 and Hela cells were uniformly plated in 96-well plates at about 5X 10 cells per well3Culturing for 24 hr, adding adriamycin (DOX) and Ag at different concentrations2S and Ag2S @ DOX probe (DOX concentrations of 0, 2, 4, 6 and 8. mu.g/mL). After further incubation for 6h, the cells were washed 3 times with PBS to remove free material, added 50. mu.L PBS and washed with 1.5W/cm2And (3) continuing culturing for 24h after laser irradiation for 10min, adding 10 mu L of CCK-8 solution into each hole, measuring the absorption value of each hole at 450nm by using a microplate reader after 1h, and calculating the survival rate of the cells. The cell viability was calculated in the same manner, also following the above procedure, using different probes at different concentrations, but without laser irradiation. The results show that the chemotherapy-photothermal combination treatment has the best effect.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.

Claims (6)

1. A method for synthesizing near-infrared fluorescence/photo-thermal/photo-acoustic silver sulfide quantum dots is characterized by comprising the following steps: the method comprises the following steps:
s1, mixing the components in a molar ratio of 1: adding the R- (+) -lipoic acid and the reducing agent in the (1-3) into ultrapure water, and stirring at normal temperature under the protection of an inert medium to obtain a dihydrolipoic acid solution for later use;
s2, adjusting the pH value of the obtained dihydrolipoic acid solution to 7-10;
s3, adding a silver source, stirring for 10-30 min, and adjusting the pH value to 7-10.
S4, adding a sulfur-containing compound, wherein the molar ratio of the dihydrolipoic acid to sulfur atoms in the sulfur-containing compound is more than 38.88, and reacting for 1-5 hours at 30-90 ℃ to obtain the silver sulfide quantum dots.
2. The method for synthesizing the near-infrared fluorescent/photothermal/photoacoustic silver sulfide quantum dot according to claim 1, wherein: in step S1, the reducing agent is one or more of tris (2-carboxyethyl) phosphine hydrochloride, sodium borohydride, and sodium citrate.
3. The method for synthesizing the near-infrared fluorescent/photothermal/photoacoustic silver sulfide quantum dot according to claim 1, wherein: in step S1, the inert medium is nitrogen or an inert gas.
4. The method for synthesizing the near-infrared fluorescent/photothermal/photoacoustic silver sulfide quantum dot according to claim 1, wherein: in step S1, the concentration of the dihydroliponic acid solution is 0.0162-0.0486 mol/L.
5. The method for synthesizing the near-infrared fluorescent/photothermal/photoacoustic silver sulfide quantum dot according to claim 1, wherein: in step S3, the silver source is silver nitrate and/or silver acetate.
6. The method for synthesizing the near-infrared fluorescent/photothermal/photoacoustic silver sulfide quantum dot according to claim 1, wherein: in step S4, the sulfur-containing compound is one or more of sodium sulfide, thioacetamide, sodium thiosulfate, sodium diethyldithiocarbamate, 3-mercaptopropionic acid, hexamethyldisilazane, and elemental sulfur.
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