Biosynthesis method of near-infrared silver sulfide quantum dots

Abstract

The invention discloses a biosynthesis method of near-infrared silver sulfide quantum dots. The biosynthesis method comprises the following steps: (1) preparing an escherichia coli-hydrogen sulfide compound: taking a wet thallus of escherichia coli as a catalytic reaction system, putting the escherichia coli into ultrapure water, adding an S source into the ultrapure water, and carrying out oscillatory reaction to obtain the escherichia coli-hydrogen sulfide compound; and (2) preparing the near-infrared silver sulfide quantum dots: adding an Ag source into the escherichia coli-hydrogen sulfide compound obtained in the step (1), and carrying out oscillatory reaction to obtain the near-infrared silver sulfide quantum dots. Escherichia coli is used as a starting strain, biological synthesis of the near-infrared silver sulfide quantum dots is quickly and mildly mediated, and the method has the advantages of being large in synthesis amount, environmentally friendly, green and high in specificity.

Description

Technical Field

[0001] The invention relates to the field of nano-biotechnology, and in particular to a biosynthetic method for near-infrared silver sulfide quantum dots. Background Art

[0002] Quantum Dot (QD) is a semiconductor nanocrystal material widely used in the field of electronic science. Due to its unique optical properties, such as high fluorescence intensity, good stability and size effect, it has gradually been applied to the biomedical field in the past two decades and has become the research focus of in vivo and in vitro fluorescent groups. Since near-infrared II region fluorescence (1000-1700nm, NIR-II) has high tissue penetration depth, low autofluorescence and higher signal-to-noise ratio, it has been widely used in biology.

[0003] Silver sulfide (Ag2S) quantum dots are a new type of quantum dot material that can emit near-infrared II fluorescence. They have attracted much attention due to their ultra-low toxicity, high stability, low cost and good biocompatibility. Like most inorganic semiconductor materials, Ag2S quantum dots are mainly prepared by chemical and physical synthesis methods. However, due to factors such as reaction kinetics and thermodynamics, most inorganic semiconductor materials still need to be synthesized through complex synthetic routes under harsh conditions. It is still challenging to prepare these materials under mild conditions, which has become one of the main factors limiting the biological application of these inorganic semiconductors. In recent years, some researchers have proposed using engineered peptides and phage display libraries from the surface of bacterial cells as templates to synthesize inorganic semiconductor materials, which provides a new strategy for the biosynthesis of inorganic semiconductor materials. Several functional inorganic semiconductor materials such as GaAs nanocrystals, hydroxyapatite, zinc sulfide (ZnS) quantum dots and silver nanoparticles have also been reported to be successfully prepared. However, there are still many problems in the use of biological methods to synthesize Ag2S quantum dots. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a biosynthetic method for near-infrared silver sulfide quantum dots. The method of the present invention not only has a large synthesis amount, but also has the significant advantages of being environmentally friendly, green, and highly specific, and opens up a new way for the synthesis of near-infrared silver sulfide quantum dots.

[0005] The technical solution of the present invention is as follows:

[0006] A biosynthetic method for near-infrared silver sulfide quantum dots, the biosynthetic method comprising the following steps:

[0007] (1) Preparing an E. coli-hydrogen sulfide complex: using wet E. coli cells as a catalytic reaction system, placing the E. coli in ultrapure water, adding a sulfur source thereto, and performing an oscillation reaction to obtain an E. coli-hydrogen sulfide complex;

[0008] (2) Preparation of near-infrared silver sulfide quantum dots: Adding an Ag source to the Escherichia coli-hydrogen sulfide complex obtained in step (1), oscillating the reaction, and obtaining near-infrared silver sulfide quantum dots.

[0009] Furthermore, in step (1), the S source is at least one of cysteine ​​and sodium thiosulfate.

[0010] Furthermore, in step (1), the Escherichia coli includes at least one of Escherichia coli Nissle 1917, Escherichia coli K12, and Escherichia coli BL21.

[0011] Furthermore, in step (1), the concentration of the Escherichia coli in ultrapure water is OD 600 The value is 2-3.

[0012] Furthermore, in step (1), the concentration of the S source in ultrapure water is 2-8 mmol / L.

[0013] Furthermore, in step (1), the rotation speed of the oscillation reaction is 150-250 rpm, the temperature is 32-37° C., and the time is 20-36 h.

[0014] Furthermore, in step (2), the Ag source is silver nitrate.

[0015] Furthermore, the molar ratio of the Ag source to the S source is (0.5-2):1.

[0016] Furthermore, the molar ratio of the Ag source to the S source is preferably 1:1.

[0017] Furthermore, in step (2), the rotation speed of the oscillation reaction is 150-250 rpm, the temperature is 4-50° C., and the time is 3-5 s.

[0018] The beneficial technical effects of the present invention are:

[0019] The present invention provides a method for biosynthesizing near-infrared silver sulfide quantum dots. The method uses wet Escherichia coli cells as a catalytic reaction system and effectively mediates the biosynthesis of near-infrared silver sulfide quantum dots through its rapid and mild characteristics. The method of the present invention not only has a large synthesis amount, but also has the significant advantages of being environmentally friendly, green, and highly specific, and opens up a new way for the synthesis of near-infrared silver sulfide quantum dots.

[0020] The method for biosynthesizing near-infrared silver sulfide quantum dots provided by the present invention successfully prepared near-infrared silver sulfide quantum dots. These quantum dots not only exhibited excellent biocompatibility and high stability, but also had the characteristics of safety and non-toxicity. Therefore, they have broad application prospects in the fields of biological imaging labeling and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The NIR fluorescence emission spectra of the near-infrared silver sulfide quantum dots synthesized in Examples 1-3 of the present invention and the products of Comparative Examples 1-3.

[0022] Figure 2 It is a bar graph of the NIR fluorescence emission intensity at 1050 nm of the near-infrared silver sulfide quantum dots synthesized in Example 1 of the present invention and the products of Comparative Examples 5-9.

[0023] Figure 3 It is a NIR fluorescence emission spectra of the near-infrared silver sulfide quantum dots synthesized in Example 1, Examples 4-6 of the present invention and the product of Comparative Example 4.

[0024] Figure 4 This is a bar graph of the NIR fluorescence emission intensity at 1050 nm of the near-infrared silver sulfide quantum dots synthesized in Example 1 and Examples 7-9 of the present invention.

[0025] Figure 5 This is a bar graph of the NIR fluorescence emission intensity at 1050 nm of the near-infrared silver sulfide quantum dots synthesized in Example 1 and Examples 10-12 of the present invention.

[0026] Figure 6 This is a bar graph of the NIR fluorescence emission intensity of the near-infrared silver sulfide quantum dots synthesized in Example 1 of the present invention in aqueous solutions containing different detergents at 1050 nm.

[0027] Figure 7 This is the HAADF STEM image of the near-infrared silver sulfide quantum dots synthesized in Example 1 of the present invention.

[0028] Figure 8 This is an image after Fourier transformation of the high-resolution transmission electron microscope image of the near-infrared silver sulfide quantum dots prepared in Example 1 of the present invention.

[0029] Fig. 9 This is the EDS element mapping diagram of the silver sulfide quantum dots prepared in Example 1 of the present invention.

[0030] Fig.10 This is an EDX image of the silver sulfide quantum dots prepared in Example 1 of the present invention.

[0031] Fig.11The UV-vis-NIR absorption spectrum and NIR fluorescence emission spectrum of the silver sulfide quantum dots prepared in Example 1-2 of the present invention in an aqueous environment.

[0032] In the figure: A is a UV-vis-NIR absorption spectrum; B is a NIR fluorescence emission spectrum.

[0033] Fig.12 This is a graph showing the fluorescence performance of the silver sulfide quantum dots prepared in Example 1.

[0034] In the figure: A is the fluorescence lifetime diagram; B is the quantum yield diagram.

[0035] Fig.13 This is a simplified structural diagram of the near-infrared silver sulfide quantum dots of the present invention. DETAILED DESCRIPTION

[0036] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0037] Fig.13 The schematic diagram is a structural diagram of the near-infrared silver sulfide quantum dots of the present invention. The wet cells of Escherichia coli are used as the catalytic reaction system, cysteine ​​is added, and after culturing for 24 hours, silver nitrate is added and reacted for 3-5 seconds to obtain near-infrared silver sulfide quantum dots. Escherichia coli is placed in ultrapure water, cysteine ​​is added thereto, and the reaction obtains an Escherichia coli-hydrogen sulfide complex, which is then reacted with silver ions to obtain near-infrared silver sulfide quantum dots.

[0038] The Escherichia coli Nissle 1917, Escherichia coli K12, Escherichia coli BL21, Corynebacterium glutamicum (ATCC 14067), Shewanella (ATCC 700550), and Clostridium butyricum (ATCC 19398) used in the following examples / comparative examples of the present invention can all be purchased from commercial sources.

[0039] Example 1

[0040] A biosynthetic method for near-infrared silver sulfide quantum dots comprises the following steps:

[0041] (1) Escherichia coli Nissle 1917 was inoculated into 100 mL of LB liquid medium and cultured at 37°C for 24 h to obtain an Escherichia coli bacterial liquid;

[0042] (2) After centrifuging 100 mL of E. coli culture at 8000 rpm for 8 min, discard the supernatant and resuspend all the cells in 100 mL of ultrapure water to obtain an E. coli aqueous solution (measured OD 600 2-3), adding cysteine ​​to a final concentration of 4 mmol / L, and shaking the reaction at 37°C and 180 rpm for 24 hours to obtain an Escherichia coli-hydrogen sulfide complex;

[0043] (3) Silver nitrate was added to a final concentration of 4 mmol / L, and the mixture was reacted at 25° C. and 180 rpm for 3-5 seconds to obtain near-infrared silver sulfide quantum dots.

[0044] Example 2-3

[0045] The biosynthesis method of Example 2-3 is basically the same as that of Example 1, except that in Example 2-3, Escherichia coli K12 and Escherichia coli BL21 are used in turn to replace the Escherichia coli Nissle1917 in Example 1.

[0046] Embodiment 4-6

[0047] The biosynthesis methods of Examples 4-6 are basically the same as those of Example 1, except that in Examples 4-6, 4 mmol / L silver nitrate in Example 1 is replaced by silver nitrate having a final concentration of 2 mmol / L, 6 mmol / L, and 8 mmol / L.

[0048] Embodiment 7-9

[0049] The biosynthesis method of Examples 7-9 is basically the same as that of Example 1, except that: in step (2) of Examples 7-9, 2 mL, 50 mL, and 500 mL of E. coli solution are used to replace 100 mL of E. coli solution in step (2) of Example 1, and the solution is subsequently resuspended in 2 mL, 50 mL, and 500 mL of ultrapure water to replace 100 mL of ultrapure water in Example 1.

[0050] Examples 10-12

[0051] The biosynthesis method of Examples 10-12 is basically the same as that of Example 1, except that the reaction temperature of 25°C in step (3) of Example 1 is replaced by the reaction temperatures of 4°C, 37°C, and 50°C in step (3) of Example 1 in sequence.

[0052] Comparative Examples 1-3

[0053] Comparative Examples 1-3 are comparative examples of Example 1. The biosynthesis methods of Comparative Examples 1-3 are basically the same as those of Example 1, except that: Comparative Examples 1-3 sequentially use Corynebacterium glutamicum (ATCC 14067), Shewanella (ATCC700550), and Clostridium butyricum (ATCC 19398) to replace Escherichia coli Nissle 1917 in Example 1.

[0054] Comparative Example 4

[0055] Comparative Example 4 is a comparative example of Example 1. The biosynthesis method of Comparative Example 4 is basically the same as that of Example 1, except that 12 mmol / L silver nitrate is used in Comparative Example 4 to replace 4 mmol / L silver nitrate in Example 1.

[0056] Comparative Examples 5-9

[0057] Comparative Examples 5-9 are comparative examples of Example 1. The biosynthesis method of Comparative Examples 5-9 is basically the same as that of Example 1, with the only difference being that Au(NO3)3, Cd(NO3)2, Zn(NO3)2, Cu(NO3)2, and Pb(NO3)2 are used in Comparative Examples 5-9 to replace the silver nitrate in Example 1.

[0058] Test Case

[0059] (1) Effects of different strains on the synthesis of near-infrared silver sulfide quantum dots

[0060] The near-infrared silver sulfide quantum dots obtained in Examples 1-3 of the present invention and the final products obtained in Comparative Examples 1-3 were taken as test samples, and each test sample was prepared into a sample solution of the same concentration, and tested using an Applied NanoFluorescence (USA)-NS1 near-infrared fluorescence spectrometer. The results are as follows: Figure 1 As shown. Figure 1 The results show that the test samples of Examples 1-3 of the present invention have emission peaks in the near-infrared region, and their emission peaks are concentrated in 1000-1100nm. The test samples of Comparative Examples 1-3 basically coincide with the baseline, indicating that the strains of Comparative Examples 1-3 cannot successfully mediate the biosynthesis of silver sulfide quantum dots, reflecting that Escherichia coli can quickly and highly specifically mediate the biosynthesis of near-infrared silver sulfide quantum dots.

[0061] (2) Effects of different metal ions on Escherichia coli-mediated biosynthesis of quantum dots

[0062] The near-infrared silver sulfide quantum dots obtained in Example 1 of the present invention and the final products obtained in Comparative Examples 5-9 were taken as test samples, and each test sample was prepared into a sample solution of the same concentration, and tested using an Applied NanoFluorescence (USA)-NS1 near-infrared fluorescence spectrometer. The results are as follows: Figure 2 As shown. Figure 2 The results show that only the Ag in Example 1 of the present invention 2+ The silver ion group emits strong fluorescence at 1050 nm, while the fluorescence intensity of other metal ion groups at 1050 nm is basically zero, indicating that Escherichia coli has a high affinity for silver ions and can mediate the instantaneous biosynthesis of silver nanoparticles with high specificity.

[0063] (3) Effect of Ag / S molar ratio on the synthesis of near-infrared silver sulfide quantum dots

[0064] The near-infrared silver sulfide quantum dots obtained in Examples 1 and 4-6 of the present invention and the final product obtained in Comparative Example 4 were taken as test samples, and each test sample was prepared into a sample solution of the same concentration, and tested using an Applied NanoFluorescence (USA)-NS1 near-infrared fluorescence spectrometer. The results are as follows: Figure 3 As shown. Figure 3 The results show that the test sample of Comparative Example 4 has no fluorescence emission in the near-infrared region, while Examples 1 and Examples 4-6 have fluorescence emission of different intensities in the near-infrared region, and the near-infrared imaging brightness of Example 1 is significantly stronger than that of other groups, with high PL brightness characteristics, indicating that the Ag / S molar ratio affects the instantaneous biosynthesis of silver sulfide quantum dots, and the best effect is achieved when the Ag / S molar ratio is 1:1.

[0065] (4) Effect of different reaction volumes on the biosynthesis of near-infrared silver sulfide quantum dots

[0066] The near-infrared silver sulfide quantum dots obtained in Example 1 and Examples 7-9 of the present invention were taken as test samples, and each test sample was prepared into a sample solution of equal concentration, and tested using an Applied NanoFluorescence (USA)-NS1 near-infrared fluorescence spectrometer. The results are as follows: Figure 4 As shown. Figure 4 It can be seen from the results that the test samples of Example 1 and Examples 7-9 have different intensities of fluorescence emission in the near-infrared region, indicating that the instantaneous biosynthesis method of silver sulfide quantum dots mediated by Escherichia coli in the embodiments of the present invention is relatively stable, and the reaction can still proceed stably even if the reaction volume is continuously expanded. The biosynthesis method of the present invention can prepare silver sulfide quantum dots in large quantities.

[0067] (5) Evaluation of the sensitivity of biosynthesized near-infrared silver sulfide quantum dots to reaction temperature

[0068] The near-infrared silver sulfide quantum dots obtained in Example 1 and Examples 10-12 of the present invention were taken as test samples, and each test sample was prepared into a sample solution of equal concentration, and tested using an Applied NanoFluorescence (USA)-NS1 near-infrared fluorescence spectrometer. The results are as follows: Figure 5 As shown. Figure 5 The results show that the test samples of Example 1 and Examples 10-12 have different intensities of fluorescence emission in the near-infrared region, indicating that the Escherichia coli-mediated transient biosynthesis method of silver sulfide quantum dots selected by the present invention is not sensitive to the reaction temperature and can be carried out at room temperature or in a low temperature or heating environment.

[0069] (6) Stability evaluation of near-infrared silver sulfide quantum dots

[0070] The near-infrared silver sulfide quantum dots obtained in Example 1 of the present invention were taken as test samples, and were added to four washing solutions, namely, deionized water, 0.5 wt% Nonidet P-40 aqueous solution, 1 wt% Tween 20 aqueous solution, and 1 wt% Triton X-100 aqueous solution, so that the concentration of the silver sulfide quantum dots in each washing solution was 5 mmol / L. The samples were incubated at 25° C. for 3 h, and then detected using an Applied NanoFluorescence (USA)-NS1 near-infrared fluorescence spectrometer. The results are shown in FIG. Figure 6 As shown. Figure 6 The results show that the presence of high concentration non-ionic detergent has no effect on the silver sulfide quantum dots prepared by the present invention.

[0071] (7) Morphological Characterization of Near-Infrared Silver Sulfide Quantum Dots

[0072] The near-infrared silver sulfide quantum dots obtained in Example 1 of the present invention were observed for their morphology and particle size distribution using a FEI Tecnai G2 Spirit 120KV transmission electron microscope. Figure 7 As shown. Figure 7 The results show that the near-infrared silver sulfide quantum dots synthesized in the present invention are individually dispersed spherical particles in an aqueous environment, and the particle size is 2.65±0.85 nm.

[0073] (8) Crystalline Characterization of Near-Infrared Silver Sulfide Quantum Dots

[0074] The near-infrared silver sulfide quantum dots obtained in Example 1 of the present invention were taken and the sample was transmitted through a FEI Talos F200X high-resolution transmission electron microscope, and an image was obtained by Fourier transform. The results are as follows: Figure 8 As shown in the figure, it can be seen that the near-infrared silver sulfide quantum dots synthesized by the present invention are single crystals, and the interlayer distance of the lattice fringes is 0.28nm, which is equivalent to the (-112) plane of the monoclinic silver sulfide quantum dots.

[0075] (9) Elemental composition of near-infrared silver sulfide quantum dots

[0076] The near-infrared silver sulfide quantum dots obtained in Example 1 of the present invention were analyzed by EDS spectrum analysis. The results are as follows: Fig. 9 As shown. Fig. 9 It can be seen from the results that the near-infrared silver sulfide quantum dots synthesized by the present invention are mainly composed of two elements, Ag and S, and the sulfur element is evenly distributed on the surface of the silver core.

[0077] The near-infrared silver sulfide quantum dots obtained in Example 1 of the present invention were taken and their elemental composition was detected by EDX spectrometer. The results are as follows: Fig.10 As shown. Fig.10 The results show that the near-infrared silver sulfide quantum dots synthesized by the present invention are mainly composed of two elements, Ag and S.

[0078] (10) Luminescence properties of near-infrared silver sulfide quantum dots

[0079] The silver sulfide quantum dots prepared in Example 1-2 of the present invention were placed in a water environment and detected by UV-vis-NIR spectrometer and Applied NanoFluorescence (USA)-NS1 near-infrared fluorescence spectrometer. The photoluminescence was measured using 808nm excitation. The results are as follows: Fig.11 As shown. Fig.11 It can be seen that the near-infrared silver sulfide quantum dots synthesized in Examples 1-2 of the present invention all show NIR-II emission peaks, indicating that the near-infrared silver sulfide quantum dots obtained by the method of the present invention have high photoluminescence properties in the near-infrared region.

[0080] (11) Fluorescence properties of near-infrared silver sulfide quantum dots

[0081] The silver sulfide quantum dots prepared in Example 1 of the present invention were taken and their fluorescence lifetime and quantum yield were measured by Edinburgh FLS-1000 steady-state / transient fluorescence spectrometer. The results are as follows: Fig.12 As shown. Fig.12 It can be seen that the near-infrared silver sulfide quantum dots synthesized by the present invention have excellent fluorescence properties.

[0082] The above is only a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and changes directly derived or associated by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included in the protection scope of the present invention.

Claims

1. A biosynthetic method for near-infrared silver sulfide quantum dots, characterized in that: The biosynthetic method comprises the following steps: (1) Preparing an E. coli-hydrogen sulfide complex: using wet E. coli cells as a catalytic reaction system, placing the E. coli in ultrapure water, adding a sulfur source thereto, and performing an oscillation reaction to obtain an E. coli-hydrogen sulfide complex; (2) Preparation of near-infrared silver sulfide quantum dots: Adding an Ag source to the Escherichia coli-hydrogen sulfide complex obtained in step (1), oscillating the reaction, and obtaining near-infrared silver sulfide quantum dots.

2. The biosynthesis method according to claim 1, characterized in that In step (1), the S source is at least one of cysteine ​​and sodium thiosulfate.

3. The biosynthesis method according to claim 1, characterized in that In step (1), the Escherichia coli includes at least one of Escherichia coli Nissle 1917, Escherichia coli K12, and Escherichia coli BL21.

4. The biosynthesis method according to claim 1, characterized in that In step (1), the concentration of Escherichia coli in ultrapure water is OD 600 The value is 2-3.

5. The biosynthesis method according to claim 1, characterized in that In step (1), the concentration of the S source in ultrapure water is 2-8 mmol / L.

6. The biosynthesis method according to claim 1, characterized in that In step (1), the rotation speed of the oscillation reaction is 150-250 rpm, the temperature is 32-37° C., and the time is 20-36 h.

7. The biosynthesis method according to claim 1, characterized in that: In step (2), the Ag source is silver nitrate.

8. The biosynthesis method according to claim 1, characterized in that The molar ratio of the Ag source to the S source is (0.5-2):

1.

9. The biosynthesis method according to claim 8, characterized in that The molar ratio of the Ag source to the S source is preferably 1:

1.

10. The biosynthesis method according to claim 1, characterized in that: In step (2), the rotation speed of the oscillation reaction is 150-250 rpm, the temperature is 4-50° C., and the time is 3-5 s.

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