CN116144361A - Lecithin functionalized high-brightness fluorescent nano probe and preparation method thereof - Google Patents

Abstract

The invention provides a lecithin functionalized high-brightness fluorescent nanoprobe, wherein the lecithin functionalized high-brightness fluorescent nanoprobe is in the form of quantum dots wrapped in lecithin; the lecithin functionalized high-brightness fluorescent nanoprobe The chemical formula of is [Zn _0.07 ‑Cu _0.03 In _0.1 Se _0.3‑y ](C ₄₂ H ₈₄ O ₉ PN), where 0<y≤0.2. Compared with the prior art, the lecithin-functionalized high-brightness fluorescent nanoprobe is in the form of quantum dots wrapped in lecithin, and has water solubility, stability and biocompatibility. The lecithin functionalized high-brightness fluorescent nanoprobe also has high-brightness fluorescent performance, and can be applied in gene detection, medical diagnosis, biological imaging, targeted drug delivery and the like. In addition, it has the characteristics of non-toxicity, easy operation, strong sensitivity and excellent stability.

Description

Lecithin functionalized high-brightness fluorescent nanoprobe and preparation method thereof

technical field

The invention relates to the field of biomedicine, in particular to a lecithin functionalized high-brightness fluorescent nano-probe and a preparation method thereof.

Background technique

Quantum dots are widely used in the field of cell imaging. Studies have shown that quantum dots can be used to monitor the dynamics of vaccines and amplify immune responses. Artificially synthesized quantum dots can effectively couple antigens and adjuvants to target tissues and cells. , and noninvasively imaged the dynamics of metastases to lymph nodes and intercellular compartments. Therefore, the use of quantum dots as a biological probe has broad prospects in the field of biomedicine.

At present, ternary quantum dots are favored in the biological field because of their low toxicity and good luminous effect. Studies have shown that ternary quantum dots CuInSe ₂ not only have similar properties to II-VI and III-V binary quantum dots, but also are composed of low-toxic elements, with weak light scattering and light attenuation, deeper detection depth, and better spatial Higher resolution. However, in the application process of the biomedical field, the ternary quantum dot CuInSe ₂ does not have water solubility, stability, and biocompatibility, and cannot be directly applied to gene detection, medical diagnosis, bioimaging, targeted drug delivery, etc. .

If the surface ligand replacement of the ternary quantum dot CuInSe ₂ is forced to make it water-soluble, stable, and biocompatible, the quantum dot fluorescence quantum yield of the ternary quantum dot CuInSe ₂ will be reduced, and it will lose nearly Infrared luminescent tissue penetration or fluorescence performance is very weak.

In view of this, it is necessary to provide a lecithin-functionalized high-brightness fluorescent nanoprobe and a preparation method thereof, which not only have water solubility, stability, and biocompatibility, but also have high-brightness fluorescent performance. It provides a new type of probe for gene detection, medical diagnosis, biological imaging, targeted drug delivery and other applications.

Contents of the invention

The main purpose of the present invention is to provide a lecithin functionalized high-brightness fluorescent nanoprobe and its preparation method, aiming to solve the problems in the prior art of the probe's water solubility, stability, and biocompatibility, high-brightness fluorescence performance incompatibility and other issues.

To achieve the above object, the present invention provides a lecithin functionalized high-brightness fluorescent nanoprobe, the lecithin functionalized high-brightness fluorescent nanoprobe is in the form of quantum dots wrapped in lecithin; the lecithin functionalized high-brightness The chemical formula of the fluorescent nanoprobe is [Zn _0.07 -Cu _0.03 In _0.1 Se _0.3-y ](C ₄₂ H ₈₄ O ₉ PN), where 0<y≤0.2.

The present invention also provides a preparation method of lecithin functionalized high-brightness fluorescent nanoprobe, comprising the steps of:

S1, providing selenium precursors and cation precursors.

S2, under the condition of 160-200° C., mixing the selenium precursor and the cation precursor, reacting and cooling to obtain a high-brightness fluorescent nano-probe.

S3, dissolving the high-brightness fluorescent nanoprobe and lecithin in chloroform, removing the solvent to form a film, and dispersing the film in water to obtain the lecithin functionalized high-brightness fluorescent nanoprobe.

Wherein, the lecithin-functionalized high-brightness fluorescent nanoprobe is in the form of quantum dots wrapped in lecithin; the chemical formula of the lecithin-functionalized high-brightness fluorescent nanoprobe is [Zn _0.07 -Cu _0.03 In _0.1 Se _0.3-y ](C ₄₂ H ₈₄ O ₉ PN), wherein, 0<y≤0.2; the cation precursor is a mixed solution containing In ions, Zn ions and Cu ions.

Further, in the step S1, the method of obtaining the selenium precursor includes: adding oleic acid, oleylamine, and dodecanethiol to the selenium powder and mixing to obtain a selenium-containing mixed solution. In an inert gas atmosphere, the selenium precursor is obtained by heating in a water bath at 40-70° C. for 30-90 minutes; wherein, the volume ratio of oleic acid, oleylamine and dodecanethiol is 0.75:0.75:0.5-0.9.

Further, the mass concentration of selenium in the selenium-containing mixed solution is 6.54-23.55 g/L.

Further, the volume ratio of the oleic acid, the oleylamine and the dodecanethiol is 0.75:0.75:0.75-0.9.

Further, the mass concentration of selenium in the selenium-containing mixed solution is 6.54-13.2 g/L.

Further, in the step S1, the method of obtaining the cation precursor includes: preparing a mixed solution containing In ions, Cu ions and Zn ions, and heating to 160 °C in an inert gas atmosphere under vacuum conditions. ~200°C, the cationic precursor is obtained.

Further, in the step S3, the mass concentration ratio of the lecithin and the high brightness fluorescent nanoprobe is 0.03-0.07 g/mL.

Further, in the step S2, the reaction and cooling process specifically include: keeping the injected mixed liquid reacting at the condition of 160-200°C for 20-60 minutes to obtain the reaction liquid, and cooling the reacted liquid to room temperature; after the cooling step, it also includes: carrying out alcohol precipitation and water extraction or differential centrifugation on the cooled product.

Further, the process of extracting the alcohol precipitation and water extraction includes: adding an alcohol precipitant to the cooled reaction solution to obtain a precipitation mixture; discarding the supernatant in the precipitation mixture to obtain a precipitate ; adding a reconstitution agent to the precipitate to redissolve the precipitate to obtain the lecithin functionalized high-brightness fluorescent nanoprobe; wherein, the precipitant includes: ethanol; the reconstitution agent includes: three Chloromethane.

The present invention has the following beneficial effects:

(1) The lecithin-functionalized high-brightness fluorescent nanoprobe provided by the present invention is in the form of quantum dots wrapped in lecithin, and has water solubility, stability and biocompatibility.

(2) The lecithin functionalized high-brightness fluorescent nanoprobe provided by the present invention has high-brightness fluorescence performance, and can be applied in gene detection, medical diagnosis, biological imaging, targeted drug delivery and the like.

(3) The lecithin functionalized high-brightness fluorescent nanoprobe provided by the present invention also has the characteristics of non-toxicity, easy operation, high sensitivity and excellent stability.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. For those skilled in the art, other drawings can also be obtained according to the structures shown in these drawings without creative effort.

Fig. 1 is the quantum dot fluorescence spectrogram of the high-brightness fluorescent nanoprobe obtained in embodiment 1;

Fig. 2 is the physical figure of the high-brightness fluorescent nanoprobe obtained in embodiment 1 under the ultraviolet lamp; Wherein, (a) is the physical figure under the ultraviolet lamp of the high-brightness fluorescent nanoprobe (0.5mL dodecanethiol), (b) is the physical picture of the high-brightness fluorescent nanoprobe (0.6mL dodecanethiol) under the ultraviolet lamp, (c) is the physical picture of the high-brightness fluorescent nanoprobe (0.75mL dodecanethiol) under the ultraviolet lamp; (d) is the physical picture of the high-brightness fluorescent nanoprobe (0.9mL dodecanethiol) under the ultraviolet lamp;

Fig. 3 is the quantum dot fluorescence spectrogram of the high-brightness fluorescent nanoprobe obtained in embodiment 2;

Fig. 4 is the physical figure of the high-brightness fluorescent nanoprobe (70% selenium powder) obtained in embodiment 2 under the ultraviolet lamp;

Fig. 5 is the quantum dot fluorescence spectrogram of the lecithin functionalized high-brightness fluorescent nanoprobe obtained in embodiment 3;

Fig. 6 is the physical picture of the lecithin functionalized high-brightness fluorescent nanoprobe obtained in Example 3 under an ultraviolet lamp; wherein, (a) is the ultraviolet light of the lecithin functionalized high-brightness fluorescent nanoprobe (5mg lecithin) The physical picture under the lamp, (b) is the physical picture of the lecithin functionalized high-brightness fluorescent nanoprobe (7mg lecithin) under the ultraviolet light, (c) is the lecithin functionalized high-brightness fluorescent nanoprobe (9mg lecithin) (d) is the physical picture of lecithin functionalized high-brightness fluorescent nanoprobe (10mg lecithin) under ultraviolet light;

Fig. 7 is the quantum dot fluorescent spectrum comparison diagram of the high-brightness fluorescent nanoprobe obtained in Example 3, the Ca-CuInSe quantum dots prepared in Comparative Example 1, and the Mn-CuInSe quantum dots prepared in Comparative Example 2;

Fig. 8 is the lecithin functionalized high-brightness fluorescent nanoprobe (0.9mL dodecanethiol) obtained in Example 1 and the infrared spectrum comparison chart of pure lecithin; wherein, (a) is the lecithin functionalized high-brightness fluorescent nanoprobe The infrared spectrogram of probe (0.9mL dodecanethiol), (b) is the infrared spectrogram of pure lecithin;

Fig. 9 is the fluorescence lifetime figure of the high brightness fluorescent nanoprobe (0.9mL dodecanethiol) obtained in embodiment 1;

Fig. 10 is the fluorescence lifetime diagram of the lecithin functionalized high-brightness fluorescent nanoprobe (0.9mL dodecanethiol) obtained in Example 1;

Fig. 11 is a physical comparison diagram of the fluorescence intensity of the lecithin functionalized high-brightness fluorescent nanoprobe (0.9mL dodecanethiol) obtained in Example 1 before and after placing it for six months; The physical picture, (b) is the physical picture under the ultraviolet lamp after being placed for six months, (c) is the physical picture under the natural light after being placed for six months;

FIG. 12 is a diagram of the quantum yield of the lecithin functionalized high-brightness fluorescent nanoprobe (0.9 mL dodecanethiol) obtained in Example 1 after being placed for six months.

The realization of the purpose of the present invention, functional characteristics and advantages will be further described with reference to the accompanying drawings in combination with the implementation modes.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the implementation manners in the present invention, all other implementation manners obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

It should be noted that, in the case of no conflict, the following embodiments and the features in the embodiments can be combined with each other. It should also be understood that the terminology used in the embodiments of the present invention is for describing specific implementations, not for limiting the protection scope of the present invention.

Among them, "Wavelength" can be expressed as wavelength; "Intensity" can be expressed as fluorescence intensity; "Wavenumber" can be expressed as wave number; "Transmittance" can be expressed as light transmittance; "Time" can be expressed as time; "Counts" can be expressed is the count; "QY" can be expressed as the quantum yield.

Unless otherwise defined, all technical and scientific terms used in the present invention are consistent with those skilled in the art's grasp of the prior art and the description of the present invention, and can also be used with the methods, equipment, and materials described in the embodiments of the present invention Any methods, apparatus and materials of the prior art similar or equivalent to the practice of the present invention.

When the examples give numerical ranges, it should be understood that, unless otherwise stated in the present invention, the two endpoints of each numerical range and any value between the two endpoints can be selected. The test methods for which specific conditions are not indicated in the following examples are usually in accordance with conventional conditions, or in accordance with the conditions suggested by each manufacturer. The materials or reagents required in the following examples are commercially available unless otherwise specified.

In order to solve the problems in the prior art, such as the water solubility, stability, biocompatibility and incompatibility of high-brightness fluorescent performance of the probe, the present invention provides a lecithin functionalized high-brightness fluorescent nanoprobe, lecithin The functionalized high-brightness fluorescent nanoprobe is in the form of quantum dots wrapped in lecithin; the chemical formula of the lecithin-functionalized high-brightness fluorescent nanoprobe is [Zn _0.07 -Cu _0.03 In _0.1 Se _0.3-y ](C ₄₂ H ₈₄ O ₉ PN), where 0<y≤0.2.

The above lecithin-functionalized high-brightness fluorescent nanoprobe is in the form of quantum dots wrapped in lecithin, and has water solubility, stability and biocompatibility. The lecithin functionalized high-brightness fluorescent nanoprobe also has high-brightness fluorescent performance, and can be applied in gene detection, medical diagnosis, biological imaging, targeted drug delivery and the like. In addition, it has the characteristics of non-toxicity, easy operation, strong sensitivity and excellent stability.

The present invention also provides a preparation method of lecithin functionalized high-brightness fluorescent nanoprobe, comprising the steps of:

S1, providing selenium precursors and cation precursors.

S2, under the condition of 160-200° C., mix the selenium precursor and the cation precursor, react and cool to obtain a high-brightness fluorescent nano-probe.

S3, dissolving the high-brightness fluorescent nanoprobe and lecithin in chloroform, removing the solvent to form a film, and dispersing the film in water to obtain lecithin functionalized high-brightness fluorescent nanoprobe.

Among them, the lecithin-functionalized high-brightness fluorescent nanoprobe is in the form of quantum dots wrapped in lecithin; the chemical formula of the lecithin-functionalized high-brightness fluorescent nanoprobe is [Zn _0.07 -Cu _0.03 In _0.1 Se _0.3-y ](C ₄₂ H ₈₄ O ₉ PN), wherein, 0<y≤0.2; the cationic precursor is a mixed liquid containing In ions, Zn ions and Cu ions.

Specifically, in step S3, the process of removing the solvent to form a film may specifically include: adding high-brightness fluorescent nanoprobes and lecithin into chloroform, and dissolving them on a rotary evaporator with a rotational speed of 100 r/min. Specifically, the dispersion process may be to add ultra-pure water to the liquid after film formation for ultrasonic oscillation.

Further, in step S1, the method of obtaining the selenium precursor includes: adding oleic acid, oleylamine, and dodecanethiol to the selenium powder and mixing to obtain a selenium-containing mixed liquid, and under vacuum conditions, in an inert gas atmosphere In the method, the selenium precursor is obtained by heating in a water bath at 40-70° C. for 30-90 minutes; wherein, the volume ratio of oleic acid, oleylamine and dodecanethiol is 0.75:0.75:0.5-0.9. Specifically, the inert gas can be nitrogen, helium or argon. First, vacuumize the evenly stirred mixed liquid containing selenium, and then introduce the aforementioned inert gas. When the gas is sufficient, start to put in 40~ Stir in a water bath at 70°C and keep warm for 30-90 minutes.

Further, the mass concentration of selenium in the selenium-containing mixed solution is 6.54-23.55 g/L. Experiments have found that when the mass concentration of selenium is <6.5g/L or >23.55g/L, the fluorescence intensity will decrease sharply.

Further, the volume ratio of oleic acid, oleylamine and dodecanethiol is 0.75:0.75:0.75-0.9.

Further, the mass concentration of selenium in the selenium-containing mixed solution is 6.54-13.2 g/L. When the mass concentration of selenium falls within the range of 6.54-13.2 g/L, the finally prepared lecithin-functionalized high-brightness fluorescent nanoprobes all have strong fluorescence brightness, and the effect is better.

Further, in step S1, the method of obtaining the cation precursor includes: preparing a mixed solution containing In ions, Cu ions and Zn ions, and heating to 160-200°C in an inert gas atmosphere under vacuum conditions, Obtain the cation precursor. It should be noted that the inert gas charged here may be the same as the gas used to prepare the selenium precursor. Specifically, vacuumize the mixed solution containing In ions, Cu ions and Zn ions, and then pass in an inert gas. When the gas is sufficient, start to heat up stepwise to 160-200°C.

Further, in step S3, the mass concentration ratio of lecithin and high-brightness fluorescent nanoprobe is 0.03-0.07 g/mL.

Further, in step S2, the reaction and cooling process specifically includes: keeping the injected mixed solution at 160-200°C for 20-60 minutes to react to obtain the reaction solution, and cooling the reacted solution to room temperature; after the cooling step, it also includes: : Carry out alcohol precipitation and water extraction or differential centrifugation on the cooled product.

Further, the process of extracting alcohol precipitation and water extraction includes: adding an alcohol precipitant to the cooled reaction solution to obtain a precipitation mixture; discarding the supernatant in the precipitation mixture to obtain a precipitate; adding The reconstitution solvent redissolves the precipitate to obtain the lecithin functionalized high-brightness fluorescent nanoprobe; wherein, the precipitant includes: ethanol; the reconstitution solvent includes: chloroform.

For further understanding of the present invention, now give an example:

Example 1

Only change the amount of dodecanethiol in the selenium-containing mixture, and carry out 4 groups of experiments

1. Preparation of Selenium Precursor

Weigh 0.0316g of selenium powder, pour it into a clean three-neck flask soaked in aqua regia, add 0.75mL of oleic acid and 0.75mL of oleylamine with a pipette gun, and finally add 0.5mL, 0.6mL, 0.75mL, 0.9 mL of dodecanethiol was stirred evenly to obtain a selenium-containing mixed solution.

Vacuumize in the order of 10min, 5min, and 5min in turn, and then fill with nitrogen. After sufficient nitrogen for the last time, put it in a 50°C water bath for stirring and keep warm for 60min.

2. Preparation of cationic precursor

Weigh 0.0057g cuprous iodide, 0.0292g indium acetate, 0.0128g zinc acetate, pour into a clean three-neck flask soaked in aqua regia, add 4mL octadecene, 0.5mL dodecanethiol (DDT) was placed in a sand bath, vacuumized in the order of 10min, 10min, and 10min, and then filled with nitrogen. After the last sufficient nitrogen, the temperature was raised to 175°C.

3. Synthesis of high-brightness fluorescent nanoprobes

Take 1.2mL of the selenium precursor solution prepared in step 1 and quickly inject it into the cationic precursor solution, stir well, and react at 175°C for 20min. Centrifuge at 8500rpm for 5min, pour off the supernatant and add chloroform as a solvent, put it into a brown bottle and store it at room temperature.

Among them, the obtained high-brightness fluorescent nanoprobe (0.5mL dodecanethiol), high-brightness fluorescent nanoprobe (0.6mL dodecanethiol), high-brightness fluorescent nanoprobe (0.75mL dodecanethiol), high The quantum dot fluorescence spectrum of brightness fluorescent nanoprobe (0.9mL dodecanethiol) is shown in Figure 1; (d) shown.

4. Use a pipette gun to take 0.150mL of the high-brightness fluorescent nanoprobe prepared in step 3. and pour it into a round-bottomed flask, add 0.0070g of lecithin, then add 1mL of chloroform to fully stir to dissolve, and put the round-bottomed The flask was installed on a rotary evaporator with a rotational speed of 100r/min, put into a water bath to keep warm at 37°C, vacuumed with a circulating water-type multi-purpose vacuum pump, and condensed by a low-temperature coolant circulating pump for a total of 30 minutes at the same time, and produced at the bottom of the round bottom flask. Then add 1 mL of ultrapure water for ultrasonic oscillation to obtain lecithin functionalized high-brightness fluorescent nanoprobes.

Example 2

Only changing the amount of selenium powder added, 7 groups of experiments were carried out

1. Preparation of Selenium Precursor

Weigh 0.0157g, 0.0220g, 0.0282g, 0.0316g, 0.0345g, 0.0408g, 0.0471g of selenium powder respectively, pour them into a clean three-necked flask soaked in aqua regia, and add 0.75mL of oleic acid with a pipette gun and 0.75mL oleylamine, and finally add 0.9mL of dodecanethiol and stir evenly to obtain a selenium-containing mixed solution.

Vacuumize in the order of 10min, 5min, and 5min in turn, and then fill with nitrogen. After sufficient nitrogen for the last time, put it in a 50°C water bath for stirring and keep warm for 60min.

2. Preparation of cationic precursor

Weigh 0.0057g cuprous iodide, 0.0292g indium acetate, 0.0128g zinc acetate, pour into a clean three-neck flask soaked in aqua regia, add 4mL octadecene, 0.5mL dodecanethiol (DDT) was placed in a sand bath, vacuumized in the order of 10min, 10min, and 10min, and then filled with nitrogen. After the last sufficient nitrogen, the temperature was raised to 175°C.

3. Synthesis of high-brightness fluorescent nanoprobes

Take 1.2mL of the selenium precursor solution prepared in step 1 and quickly inject it into the cationic precursor solution, stir well, and react at 37°C for 30min. Centrifuge at 8500rpm for 5min, pour off the supernatant and add chloroform as a solvent, put it into a brown bottle and store it at room temperature.

The obtained high-brightness fluorescent nanoprobe (50% selenium powder), high-brightness fluorescent nanoprobe (70% selenium powder), high-brightness fluorescent nanoprobe (90% selenium powder), high-brightness fluorescent nanoprobe (100% Selenium powder), high brightness fluorescent nanoprobe (110% selenium powder), high brightness fluorescent nanoprobe (130% selenium powder), high brightness fluorescent nanoprobe (150% selenium powder) quantum dot fluorescence spectrum 3; wherein, the physical figure of the high-brightness fluorescent nanoprobe (70% selenium powder) under the ultraviolet lamp is as shown in Figure 4.

4. Use a pipette gun to take 0.150mL of the high-brightness fluorescent nanoprobe prepared in step 3. and pour it into a round-bottomed flask, add 0.0070g of lecithin, then add 1mL of chloroform to fully stir to dissolve, and put the round-bottomed The flask was installed on a rotary evaporator with a rotational speed of 100r/min, put into a water bath to keep warm at 37°C, vacuumed with a circulating water-type multi-purpose vacuum pump, and condensed by a low-temperature coolant circulating pump for a total of 30 minutes at the same time, and produced at the bottom of the round bottom flask. Then add 1 mL of ultrapure water for ultrasonic oscillation to obtain lecithin functionalized high-brightness fluorescent nanoprobes.

Example 3

Only change the addition amount of lecithin, carry out 4 groups of experiments

1. Preparation of Selenium Precursor

Weigh 0.0220g of selenium powder, pour it into a clean three-neck flask soaked in aqua regia, add 0.75mL oleic acid and 0.75mL oleylamine with a pipette, and finally add 0.9mL of dodecanethiol and stir evenly. A mixture containing selenium was obtained.

Vacuumize in the order of 10min, 5min, and 5min in turn, and then fill with nitrogen. After sufficient nitrogen for the last time, put it in a 50°C water bath for stirring and keep warm for 60min.

2. Preparation of cationic precursor

Weigh 0.0057g cuprous iodide, 0.0292g indium acetate, 0.0128g zinc acetate, pour into a clean three-neck flask soaked in aqua regia, add 4mL octadecene, 0.5mL dodecanethiol (DDT) was placed in a sand bath, vacuumized in the order of 10min, 10min, and 10min, and then filled with nitrogen. After the last sufficient nitrogen, the temperature was raised to 175°C.

3. Synthesis of high-brightness fluorescent nanoprobes

Take 1.2mL of the selenium precursor solution prepared in step 1 and quickly inject it into the cationic precursor solution, stir well, and react at 175°C for 20min. Centrifuge at 8500rpm for 5min, pour off the supernatant and add chloroform as a solvent, put it into a brown bottle and store it at room temperature.

5. Use a pipette gun to take 0.150 mL of the high-brightness fluorescent nanoprobe prepared in step 3. and pour it into a round bottom flask, add 0.0050 g, 0.0070 g, 0.0090 g, 0.010 g of lecithin, and then add 1 mL of three Thoroughly stir and dissolve the methyl chloride, install the round-bottomed flask on a rotary evaporator with a rotating speed of 100r/min, put it in a water bath to keep warm at 37°C, use a circulating water-type multi-purpose vacuum pump to evacuate, and condense with a low-temperature coolant circulating pump. For a total of 30 minutes, a film was formed at the bottom of the round bottom flask, and then 1 mL of ultrapure water was added for ultrasonic oscillation to obtain lecithin-functionalized high-brightness fluorescent nanoprobes.

The obtained lecithin functionalized high-brightness fluorescent nanoprobe (5mg lecithin), lecithin functionalized high-brightness fluorescent nanoprobe (7mg lecithin), lecithin functionalized high-brightness fluorescent nanoprobe (9mg lecithin), The quantum dot fluorescence spectrogram of lecithin functionalized high-brightness fluorescent nanoprobe (10mg lecithin) is shown in Figure 5; The physical figure under the ultraviolet lamp is (a), (b), (c) in Figure 6 respectively ), (d) shown.

Comparative example 1

1. Preparation of Selenium Precursor

Weigh 0.0220g of selenium powder, pour it into a clean three-neck flask soaked in aqua regia, add 0.75mL oleic acid and 0.75mL oleylamine with a pipette, and finally add 0.9mL of dodecanethiol and stir evenly. A mixture containing selenium was obtained.

Vacuumize in the order of 10min, 5min, and 5min in turn, and then fill with nitrogen. After sufficient nitrogen for the last time, put it in a 50°C water bath for stirring and keep warm for 60min.

2. Preparation of cationic precursor (Ca)

Weigh 0.0057g cuprous iodide, 0.0292g indium acetate, 0.0123g calcium acetate, pour into a clean three-necked flask soaked in aqua regia, add 4mL octadecene, 0.5mL dodecanethiol (DDT) was placed in a sand bath, vacuumized in the order of 10min, 10min, and 10min, and then filled with nitrogen. After the last sufficient nitrogen, the temperature was raised to 175°C.

3. Synthesis of Ca-CuInSe quantum dots

Take 1.2mL of the selenium precursor solution prepared in step 1 and quickly inject it into the cationic precursor solution (Ca), stir well, and react at 175°C for 20min. The precipitating agent was centrifuged at 8500rpm for 5min, the supernatant was poured off and chloroform was added as a solvent, and it was stored in a brown bottle at room temperature.

Comparative example 2

1. Preparation of Selenium Precursor

Weigh 0.0220g of selenium powder, pour it into a clean three-neck flask soaked in aqua regia, add 0.75mL oleic acid and 0.75mL oleylamine with a pipette, and finally add 0.9mL of dodecanethiol and stir evenly. A mixture containing selenium was obtained.

Vacuumize in the order of 10min, 5min, and 5min in turn, and then fill with nitrogen. After sufficient nitrogen for the last time, put it in a 50°C water bath for stirring and keep warm for 60min.

2. Preparation of cationic precursor (Mn)

Weigh 0.0057g cuprous iodide, 0.0292g indium acetate, 0.0172g manganese acetate, pour into a clean three-necked flask soaked in aqua regia, add 4mL octadecene, 0.5mL dodecanethiol (DDT) was placed in a sand bath, vacuumized in the order of 10min, 10min, and 10min, and then filled with nitrogen. After the last sufficient nitrogen, the temperature was raised to 175°C.

3. Synthesis of Mn-CuInSe quantum dots

Take 1.2mL of the selenium precursor solution prepared in step 1 and quickly inject it into the cationic precursor solution (Mn), stir well, and react at 175°C for 20min. The precipitating agent was centrifuged at 8500rpm for 5min, the supernatant was poured off and chloroform was added as a solvent, and it was stored in a brown bottle at room temperature.

Analysis example 1

The high-brightness fluorescent nanoprobe (0.5mL dodecanethiol), the high-brightness fluorescent nanoprobe (0.6mL dodecanethiol), the high-brightness fluorescent nanoprobe (0.75mL dodecanethiol) obtained in step 3 of Example 1 were Alcohol) and high-brightness fluorescent nanoprobe (0.9mL dodecanethiol) for fluorescence intensity comparison, the quantum dot fluorescence spectrum obtained by comparison is shown in Figure 1; the physical picture under the ultraviolet lamp is shown in Figure 2.

It can be seen from Fig. 1 that when the dosage of dodecanethiol (DDT) increases, the fluorescence intensity not only increases obviously, but also the peak blue shifts. From Figure 1, it can be seen that the DDT dosage of 900 μL obviously shows the strongest fluorescence intensity, and the effect of 750 μL is second, but there is a large gap in the fluorescence intensity compared with 600 μL.

According to the observation in Figure 2, it can be seen that under the ultraviolet light, the fluorescence intensity is also obviously enhanced with the increase of the DDT content.

Analysis example 2

The high brightness fluorescent nanoprobe (50% selenium powder), the high brightness fluorescent nanoprobe (70% selenium powder), the high brightness fluorescent nanoprobe (90% selenium powder), the high brightness fluorescent nanoprobe (90% selenium powder) that embodiment 2 step 3 obtains, Nanoprobe (100% selenium powder), high brightness fluorescent nanoprobe (110% selenium powder), high brightness fluorescent nanoprobe (130% selenium powder), high brightness fluorescent nanoprobe (150% selenium powder) for fluorescence Intensity comparison, the quantum dot fluorescence spectrum obtained by comparison is shown in Figure 3; wherein, the physical picture of the high-brightness fluorescent nanoprobe (70% selenium powder) under the ultraviolet lamp is shown in Figure 4 .

It should be noted that, with the addition of 0.0316g (0.4mmol) of selenium powder as the metered value, the high-brightness fluorescent probe obtained under this condition is represented by a high-brightness fluorescent nanoprobe (100% selenium powder). On the basis of measured value, respectively reduce 10%, reduce 30%, reduce 50%, measure value, increase 10%, increase 30%, increase 50%, obtain high-brightness fluorescent nanoprobe (50% selenium powder), high Brightness fluorescent nanoprobe (70% selenium powder), high brightness fluorescent nanoprobe (90% selenium powder), high brightness fluorescent nanoprobe (100% selenium powder), high brightness fluorescent nanoprobe (110% selenium powder) , High brightness fluorescent nanoprobe (130% selenium powder), high brightness fluorescent nanoprobe (150% selenium powder).

According to the observation in Figure 3, it can be seen that when the amount of selenium powder increases, the peak not only decreases obviously, but also the peak blue shifts. However, when the high-brightness fluorescent nanoprobe (50% selenium powder) and the high-brightness fluorescent nanoprobe (90% selenium powder), the peak is not as high as before, and the fluorescence intensity is the strongest at 70%, exceeding the instrument range. Similarly, from the physical picture of the high-brightness fluorescent nanoprobe (70% selenium powder) in Fig. 4 under the ultraviolet lamp, it can also be seen that when the amount of selenium powder is reduced by 30% on the basis of the metered value, the fluorescence brightness is very high.

Analysis example 3

The lecithin functionalized high-brightness fluorescent nanoprobe (5mg lecithin), the lecithin functionalized high-brightness fluorescent nanoprobe (7mg lecithin), the lecithin functionalized high-brightness fluorescent nanoprobe (9mg lecithin) obtained in Example 3 lecithin), lecithin functionalized high-brightness fluorescent nanoprobe (10mg lecithin) carries out fluorescence intensity comparison, and the quantum dot fluorescence spectrum figure obtained by comparison is shown in Figure 5; The physical figure under the ultraviolet lamp is respectively in Figure 6 (a), (b), (c), (d) shown.

It can be seen from Fig. 5 that the wavelength of the emission peak of the obtained lecithin functionalized high-brightness fluorescent nanoprobe is at 627nm, although the content of lecithin has changed, the position of the emission peak basically does not change. It can be seen from Figure 6 that the fluorescence intensity will gradually decrease with the decrease of lecithin content.

Analysis example 4

Example 3, the high-brightness fluorescent nanoprobe obtained in step 3 is Zn-CuInSe in Figure 7; the Ca-CuInSe quantum dot obtained in Comparative Example 1 is Ca-CuInSe in Figure 7; the Mn-CuInSe obtained in Comparative Example 2 Quantum dots, that is, Mn-CuInSe in Figure 7; comparative analysis of fluorescence intensity is performed as shown in Figure 7.

According to the observation in Fig. 7, it can be seen that only the high-brightness fluorescent nanoprobes have strong fluorescence intensity; the Ca-CuInSe quantum dots made in Comparative Example 1 and the Mn-CuInSe quantum dots made in Comparative Example 2 have too weak or almost no fluorescence intensity. It has fluorescent properties, so it cannot have high-brightness fluorescent performance.

Analysis example 5

The lecithin functionalized high-brightness fluorescent nanoprobe and pure lecithin obtained by using 0.9 mL of dodecanethiol in Example 1 were compared and analyzed by infrared spectroscopy. The infrared spectrum of the lecithin functionalized high-brightness fluorescent nanoprobe is shown in Figure 8(a); the infrared spectrum of pure lecithin is shown in Figure 8(b).

According to the observation in Figure 8, it can be seen that the infrared spectrum has a broad absorption band at 2900cm ^- 1-2950cm ^-1 , where there is a stretching vibration peak of _CH2 , and there is P=O stretching vibration at 1200cm ^- 1-1250cm ^-1 , there is a stretching vibration of POC at 1060cm ^-1 -1100cm ^-1 , from which it can be inferred that the lecithin functionalized high-brightness fluorescent nanoprobe has the characteristics of lecithin, and lecithin successfully detects the high-brightness fluorescent nanoprobe The surface of the needle is modified, so that the obtained final product—the lecithin-functionalized high-brightness fluorescent nanoprobe has the characteristic of lecithin hydrophilicity, and the water solubility of the lecithin-functionalized high-brightness fluorescent nanoprobe is increased.

Analysis example 6

The lecithin functionalized high-brightness fluorescent nanoprobe obtained by using 0.9mL dodecanethiol in Example 1, and the high-brightness fluorescent nanoprobe (0.9mL dodecanethiol) obtained in Step 3 of Example 1 were used for quantum dot fluorescence lifetime Compared. The fluorescence lifetime diagram of the high brightness fluorescent nanoprobe (0.9mL dodecanethiol) is shown in Figure 9; the fluorescence lifetime diagram of the lecithin functionalized high brightness fluorescent nanoprobe is shown in Figure 10.

It can be seen from Fig. 9 and Fig. 10 that the photon count decreases gradually as time increases. The data obtained in Fig. 9 and Fig. 10 are fitted by the following second-order function:

I _t ＝B ₁ exp(-t/τ ₁ )+B ₂ exp(-t/τ ₂ )

Among them, τ ₁ is the short lifetime, which is caused by the direct transition of excitons from the conduction band to the valence band, and τ ₂ is the long lifetime, which is related to the recombination of the internal defect states of the quantum dots. It should be noted that the high-brightness fluorescent nanoprobe (0.9mL dodecanethiol) and the lecithin functionalized high-brightness fluorescent nanoprobe are in the water phase.

Before and after the high-brightness fluorescent nanoprobe (0.9mL dodecanethiol) was transferred to water, the short lifetimes of the oil phase and the water phase were 3.49μs (B ₁ : 28.71%) and 3.24μs (B ₁ : 28.41%) respectively. ; The long lifetime values are 9.90 μs (B ₂ : 71.29%) and 10.02 μs (B ₂ : 71.59%), respectively. The results showed that the recombination path of quantum dots did not change significantly before and after water transfer, and the luminescence mechanism was stable, which also indicated that the amphiphile modification did not destroy the structure of quantum dots, and quantum dots still dominated by long-life luminescence (B ₂ accounted for more than 70%).

Analysis Example 7

The lecithin functionalized high-brightness fluorescent nanoprobe obtained in Example 1 using 0.9 mL of dodecanethiol was placed for comparison and analysis after six months. The physical pictures of lecithin-functionalized high-brightness fluorescent nanoprobes were prepared under ultraviolet light on the day of preparation, the physical pictures under ultraviolet light after six months of storage, and the physical pictures of natural light after six months of storage are shown in (a) and (b) of Figure 11 respectively. , (c) shown. The quantum yield after standing for 6 months is shown in Figure 12.

According to the observation in Figure 11, although the fluorescence brightness of the lecithin-functionalized high-brightness fluorescent nanoprobe is not as strong as before, the fluorescence brightness is still not weak. It can be concluded from Figure 12 that the quantum yield of lecithin-functionalized high-brightness fluorescent nanoprobes is still as high as 21.5% after six months. It shows that the lecithin functionalized high-brightness fluorescent nanoprobe has relatively strong stability and is suitable for use as a fluorescent probe for biomedical research.

It can be seen that the lecithin functionalized high-brightness fluorescent nanoprobe solves the problems in the prior art, such as the water solubility, stability, and biocompatibility of the probe, and the incompatibility of high-brightness fluorescent performance, and also has the advantages of non-toxicity. It has the characteristics of high toxicity, easy operation, strong sensitivity and excellent stability.

In summary, in the above-mentioned technical solutions of the present invention, the above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. Equivalent structural transformation, or direct/indirect application in other related technical fields are included in the patent protection scope of the present invention.

Claims (10)

1. The lecithin functional high-brightness fluorescent nano probe is characterized in that the lecithin functional high-brightness fluorescent nano probe is in a lecithin-coated quantum dot form;

the chemical formula of the lecithin functionalized high-brightness fluorescent nano probe is [ Zn ] _0.07 -Cu _0.03 In _0.1 Se _0.3-y ](C ₄₂ H ₈₄ O ₉ PN), wherein 0<y≤0.2。

2. A preparation method of lecithin functionalized high-brightness fluorescent nano-probe is characterized by comprising the following steps:

s1, providing a selenium precursor and a cation precursor;

s2, mixing the selenium precursor and the cation precursor at 160-200 ℃ for reaction and cooling to obtain the high-brightness fluorescent nano probe;

s3, dissolving the high-brightness fluorescent nano probe and lecithin in chloroform, removing a solvent to form a film, and dispersing the film in water to obtain the lecithin functionalized high-brightness fluorescent nano probe;

wherein the lecithin functionalized high-brightness fluorescent nano probe is in a lecithin-coated quantum dot form; the chemical formula of the lecithin functionalized high-brightness fluorescent nano probe is [ Zn ] _0.07 -Cu _0.03 In _0.1 Se _0.3-y ](C ₄₂ H ₈₄ O ₉ PN), wherein 0<y is less than or equal to 0.2; the cation precursor is a mixed solution containing In ions, zn ions and Cu ions.

3. The method for preparing a lecithin-functionalized high-brightness fluorescent nanoprobe according to claim 2, wherein in the step S1, the selenium precursor is obtained by the following steps: adding oleic acid, oleylamine and dodecyl mercaptan into selenium powder, mixing to obtain a selenium-containing mixed solution, and heating in a water bath for 30-90 min at 40-70 ℃ in an inert gas atmosphere under a vacuum condition to obtain a selenium precursor;

wherein, the volume ratio of oleic acid to oleylamine to dodecyl mercaptan is 0.75:0.75:0.5 to 0.9.

4. The method for preparing the lecithin-functionalized high-brightness fluorescent nanoprobe according to claim 3, wherein the mass concentration of selenium in the selenium-containing mixed solution is 6.54-23.55 g/L.

5. The method for preparing a lecithin-functionalized high-brightness fluorescent nanoprobe according to claim 3, wherein the volume ratio of oleic acid to oleylamine to dodecyl mercaptan is 0.75:0.75:0.75 to 0.9.

6. The method for preparing the lecithin-functionalized high-brightness fluorescent nanoprobe according to claim 4, wherein the mass concentration of selenium in the selenium-containing mixed solution is 6.54-13.2 g/L.

7. The method for preparing a lecithin-functionalized high-brightness fluorescent nanoprobe according to claim 2, wherein in the step S1, the method for obtaining the cationic precursor comprises the following steps: preparing a mixed solution containing In ions, cu ions and Zn ions, and heating to 160-200 ℃ In an inert gas atmosphere under a vacuum condition to obtain the cation precursor.

8. The method for preparing a lecithin-functionalized high-brightness fluorescent nanoprobe according to claim 2, wherein in the step S3, the mass concentration ratio of the lecithin to the high-brightness fluorescent nanoprobe is 0.03-0.07 g/mL.

9. The method for preparing the lecithin-functionalized high-brightness fluorescent nanoprobe according to claim 2, wherein in the step S2, the reaction and cooling process specifically comprises: maintaining the injected mixed solution to react for 20-60 min at 160-200 ℃ to obtain a reaction solution, and cooling the reaction solution to room temperature;

the cooling step further comprises the following steps: and carrying out alcohol precipitation water extraction or differential centrifugation on the cooled product.

10. The method for preparing the lecithin-functionalized high-brightness fluorescent nanoprobe according to claim 9, wherein the process for extracting the alcohol precipitation water extraction comprises the following steps: adding an alcohol precipitant into the cooled reaction solution to obtain a precipitation mixed solution; discarding the supernatant in the precipitation mixture to obtain a precipitate; adding a complex solvent into the precipitate to redissolve the precipitate, thereby obtaining the lecithin functionalized high-brightness fluorescent nano probe;

wherein the precipitant comprises: ethanol; the complex solvent comprises: trichloromethane.

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