Nitrogen / sulfur co-doped chiral iron monatomic carbon dot as well as preparation method and application thereof

Abstract

The invention relates to a nitrogen / sulfur co-doped chiral iron monatomic carbon dot and a preparation method and application thereof.The preparation method of the nitrogen / sulfur co-doped chiral iron monatomic carbon dot comprises the steps that dimethyl formamide, iron phthalocyanine and cysteine serve as raw materials, and a nitrogen / sulfur co-doped chiral iron monatomic carbon dot solution is synthesized in one step through a hydrothermal synthesis method; the preparation method comprises the following steps: firstly preparing an iron monatomic carbon dot, then carrying out chiral amino acid modification on the surface of the iron monatomic carbon dot through an amido bond to enhance the chiral structure characteristic, and finally obtaining nitrogen / sulfur co-doped chiral iron monatomic carbon dot powder through centrifugation, dialysis and freeze drying. The nitrogen / sulfur co-doped chiral iron monatomic carbon dot has high chiral dependence catalytic activity, shows catalase-like activity, photodynamic activity and photo-thermal activity at the same time, and can promote accumulation of free iron in bacterial cells and finally induce ferroptosis of bacteria through chiral regulation and control of activity of Fur, FtnA and other bacterial metal transporters.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano-functional materials, and particularly relates to a nitrogen / sulfur co-doped chiral iron single-atom carbon dot and its preparation method and application. Background Art

[0002] Different from the action targets of traditional antibiotics, bacterial ferroptosis is a new antibacterial strategy that induces a programmed death mode similar to ferroptosis by regulating bacterial iron metabolism and oxidative stress responses. However, metal transporters in bacteria play a central role in the uptake, storage, and excretion of iron ions, and have an important regulatory effect on inhibiting the occurrence of bacterial ferroptosis by maintaining iron homeostasis and redox balance in bacteria. Therefore, interfering with the activity of the antioxidant system and regulating bacterial metal transporters may be the key strategy for effectively inducing bacterial ferroptosis.

[0003] Single-atom carbon dots (SACDs) have shown important potential in efficiently generating cytotoxic reactive oxygen species (ROS) and disrupting the bacterial redox homeostasis by virtue of their unique single-atom active sites and excellent catalytic performance, and thus have gradually become the focus of attention in the field of antibacterial research. However, traditional SACDs designs usually lack a chiral-selective catalytic mechanism for bacteria, and the catalytic activity still lags behind that of bioenzymes. At the same time, the effect of SACDs on bacterial metal transporters is not yet clear, which limits the antibacterial and anti-biofilm activities of SACDs and the drug design and development based on the bacterial ferroptosis target.

[0004] Therefore, it is of innovative significance to develop a single-atom carbon dot with high chiral catalytic activity and the ability to regulate bacterial metal transporters. Summary of the Invention

[0005] The purpose of the present invention is to provide a nitrogen / sulfur co-doped chiral iron single-atom carbon dot and its preparation method and application in order to solve the above problems.

[0006] The present invention realizes the above purpose through the following technical solutions:

[0007] The present invention provides a preparation method of a nitrogen / sulfur co-doped chiral iron single-atom carbon dot, and the specific steps are as follows:

[0008] (1) Add iron phthalocyanine and cysteine to dimethylformamide, stir until evenly dispersed, and obtain a reaction mother liquor after ultrasonic treatment; after hydrothermal reaction of the reaction mother liquor, a black solution is obtained;

[0009] (2) Centrifuge, dialyze, and purify the black solution obtained in step (1) to obtain an Fe-NSC solution;

[0010] (3) Add the Fe-NSC solution obtained in step (2) to a mixed solution containing N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and cysteine to obtain a chiral-enhanced Fe-NSC solution;

[0011] (4) Dialyze the chiral-enhanced Fe-NSC solution obtained in step (3) and then freeze-dry it to finally obtain nitrogen / sulfur co-doped chiral iron single-atom carbon dots.

[0012] As a further optimized scheme of the present invention, in step (1), the mass ratio of iron phthalocyanine to cysteine is 1:1 to 3:1.

[0013] As a further optimized scheme of the present invention, in steps (1) and (3), the cysteine includes at least one of D-cysteine, D,L-cysteine, and L-cysteine.

[0014] As a further optimized scheme of the present invention, in step (1), the hydrothermal reaction temperature is 160°C to 200°C, and the reaction time is 6 to 12 h.

[0015] As a further optimized scheme of the present invention, in step (3), the molar ratio of N-hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and cysteine is 1:1:1 to 1:1:3.

[0016] As a further optimized scheme of the present invention, in steps (2) and (4), the molecular weight of the dialysis bag used for dialysis is 2000 to 8000 Da, and the dialysis time is 24 h to 72 h.

[0017] The present invention also provides a nitrogen / sulfur co-doped chiral iron single-atom carbon dot prepared by the above preparation method.

[0018] As a further optimized scheme of the present invention, the nitrogen / sulfur co-doped chiral iron single-atom carbon dot contains four elements of sulfur S, nitrogen N, iron Fe, and carbon C at the same time, and the iron is atomically dispersed. The Fe atoms are coordinated with 4 N atoms on average to form Fe-N4, and there is one S atom connected around each Fe-N4 on average.

[0019] The present invention also provides an application of the above nitrogen / sulfur co-doped chiral iron single-atom carbon dot in the preparation of a nano antibacterial material for inducing bacterial ferroptosis.

[0020] The beneficial effects of the present invention are as follows:

[0021] 1) The present invention synthesizes a nitrogen / sulfur co-doped chiral iron single-atom carbon dot solution in one step by a hydrothermal synthesis method, and then modifies the surface of the iron single-atom carbon dots with chiral amino acids through amide bonds to enhance the chiral structural characteristics. After centrifugation, dialysis, and freeze-drying, a nitrogen / sulfur co-doped chiral iron single-atom carbon dot powder is finally obtained, solving the drawback of partial loss of the chiral structure during the high-temperature process;

[0022] 2) The present invention regulates the electronic structure at the Fe active site by introducing heteroelements S and N, improving the activity of catalyzing hydrogen peroxide (H2O2) to generate hydroxyl radicals (·OH), and disrupting the redox enzyme balance of drug-resistant bacteria;

[0023] 3) The nitrogen / sulfur co-doped chiral iron single-atom carbon dots of the present invention can chirally regulate the activity of drug-resistant bacterial metal transport proteins, promote an increase in intracellular iron ions in bacteria, and trigger lipid peroxidation, ultimately leading to bacterial ferroptosis;

[0024] 4) The nitrogen / sulfur co-doped chiral iron single-atom carbon dots of the present invention also have photodynamic (PDT) and photothermal (PTT) therapeutic activities, synergistically accelerating the process of bacterial ferroptosis.

[0025] 5) The nitrogen / sulfur co-doped chiral iron single-atom carbon dots of the present invention have highly chiral-dependent catalytic activity, and at the same time exhibit catalase-like activity, photodynamic activity, and photothermal activity. They can chirally regulate the activity of bacterial metal transport proteins such as Fur and FtnA, promote the accumulation of free iron in bacteria, and ultimately induce bacterial ferroptosis. Description of the Drawings

[0026] Figure 1 A is a high-resolution transmission electron microscope (HRTEM) image of the product (DFe-NSC) in step 2.1; Figure 1 B is a spherical aberration-corrected high-angle annular dark-field scanning transmission electron microscope (AC-HAADF-STEM) image of the product in step 2.1.

[0027] Figure 2 A is the X-ray absorption near-edge structure spectrum (XANES) of the Fe K-edge of the product in step 2.1; Figure 2 B is the Fourier transform extended X-ray absorption fine structure (FT-EXAFS) fitting curve of the product in step 2.1.

[0028] Figure 3 A is the circular dichroism spectrum (CD spectrum) of cysteine with different chirality (D-cysteine, L-cysteine) and DFe-NSC, LFe-NSC; Figure 3 B is the X-ray photoelectron spectroscopy (XPS) of the product in step 2.1.

[0029] Figure 4A is the catalytic activity of the products (100 μg / mL) of steps 2.1, 2.2, 2.3, and 2.4 (DFe-NSC, DLFe-NSC, LFe-NSC, and Fe-NC) of the TMB probe for H2O2; Figure 4 B is the photothermal temperature change curve of the products (200 μg / mL) of steps 2.1, 2.2, 2.3, and 2.4; Figure 4 C is the electron paramagnetic resonance (EPR) spectrum of the unpaired electrons of the products of steps 2.1, 2.2, 2.3, and 2.4; Figure 4 D is the electron paramagnetic resonance (EPR) spectrum of the O2·- yield after laser irradiation of the products of steps 2.1, 2.2, 2.3, and 2.4.

[0030] Figure 5 It is a graph of the results of the in vitro antibacterial activity of the products (200 μg / mL) of steps 2.1, 2.2, 2.3, and 2.4 determined by the plate counting method.

[0031] Figure 6 A is a statistical graph of the in vitro antibacterial rate of the products (200 μg / mL) of steps 2.1, 2.2, 2.3, and 2.4; Figure 6 B is a statistical graph of the intracellular iron ion levels in bacteria before and after treatment with the products (DFe-NSC, LFe-NSC) of steps 2.1 and 2.3 under different conditions quantified by inductively coupled plasma mass spectrometry (ICP-MS); Figure 6 C is a graph of the morphological changes of bacteria and the distribution map of Fe ions before and after treatment with the products (DFe-NSC, LFe-NSC) of steps 2.1 and 2.3 observed by scanning electron microscopy (SEM).

[0032] Figure 7 It is a graph of the in vitro anti-biofilm activity experiment of the products (200 μg / mL) of steps 2.1, 2.2, 2.3, and 2.4 determined by the crystal violet staining method.

[0033] Figure 8 A and B are two-dimensional (2D) molecular docking modeling and three-dimensional (3D) molecular docking modeling of the products of steps 2.3 and 2.1 with the surrounding residues of the Staphylococcus aureus metal transporter Fur; Figure 8 C and D are two-dimensional (2D) molecular docking modeling and three-dimensional (3D) molecular docking modeling of the products (LFe-NSC, DFe-NSC) of steps 2.3 and 2.1 with the surrounding residues of the Staphylococcus aureus metal transporter FtnA. Detailed implementation method

[0034] The present application will be further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the protection scope of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0035] I. Materials

[0036] 1. The dialysis bag used for dialysis has a molecular weight of 2000 - 8000 Da, and the dialysis time is 24 h - 72 h.

[0037] 2. N-hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), D-cysteine, D,L-cysteine, and L-cysteine are all commercially available products.

[0038] The methods used in the present application are all conventional methods known to those skilled in the art unless otherwise specified. The reagents and other materials used are all commercially purchased products unless otherwise specified.

[0039] II. Methods

[0040] 2.1 Preparation of dextrorotatory nitrogen / sulfur co-doped chiral iron single-atom carbon dots (DFe-NSC)

[0041] The specific preparation method is as follows:

[0042] (1) Add 0.53 g of iron phthalocyanine and 0.24 g of D-cysteine to 30 mL of dimethylformamide, stir until evenly dispersed, and obtain a reaction mother liquor after ultrasonic treatment.

[0043] (2) Place the hydrothermal reaction mother liquor described in step (1) in a hydrothermal reaction device, and carry out a hydrothermal reaction at 180 °C for 12 h to obtain a black solution.

[0044] (3) Centrifuge, dialyze, and purify the black solution obtained in step (2) to obtain a DFe-NSC solution.

[0045] (4) Add the DFe-NSC solution obtained in step (3) to a mixed solution containing 10 mM NHS, 10 mM EDC, and 10 mM D-cysteine to obtain a chiral-enhanced DFe-NSC solution.

[0046] (5) Dialyze the chiral-enhanced DFe-NSC solution obtained in step (4) and then freeze-dry it to finally obtain chiral-enhanced DFe-NSC powder.

[0047] 2.2 Preparation of racemic nitrogen / sulfur co-doped chiral iron single-atom carbon dots (DLFe-NSC)

[0048] The preparation method is as follows:

[0049] (1) Add 0.53 g of iron phthalocyanine and 0.24 g of D,L-cysteine to 30 mL of dimethylformamide, stir until evenly dispersed, and obtain the reaction mother liquor after ultrasonic treatment.

[0050] (2) Place the hydrothermal reaction mother liquor described in step (1) in a hydrothermal reaction device, and carry out hydrothermal reaction at 180 °C for 12 h to obtain a black solution.

[0051] (3) Centrifuge, dialyze and purify the black solution obtained in step (2) to obtain the DLFe-NSC solution.

[0052] (4) Add the DLFe-NSC solution obtained in step (3) to a mixed solution containing 10 mM NHS, 10 mM EDC and 10 mM D,L-cysteine to obtain a chiral-enhanced DLFe-NSC solution.

[0053] (5) Dialyze and then lyophilize the chiral-enhanced DLFe-NSC solution obtained in step (4) to finally obtain the chiral-enhanced DLFe-NSC powder.

[0054] 2.3 Preparation of chiral iron single-atom carbon dots (LFe-NSC) doped with L-nitrogen / sulfur

[0055] The preparation method is as follows:

[0056] (1) Add 0.53 g of iron phthalocyanine and 0.24 g of L-cysteine to 30 mL of dimethylformamide, stir until evenly dispersed, and obtain the reaction mother liquor after ultrasonic treatment.

[0057] (2) Place the hydrothermal reaction mother liquor described in step (1) in a hydrothermal reaction device, and carry out hydrothermal reaction at 180 °C for 12 h to obtain a black solution.

[0058] (3) Centrifuge, dialyze and purify the black solution obtained in step (2) to obtain the LFe-NSC solution.

[0059] (4) Add the LFe-NSC solution obtained in step (3) to a mixed solution containing 10 mM NHS, 10 mM EDC and 10 mM L-cysteine to obtain a chiral-enhanced LFe-NSC solution.

[0060] (5) Dialyze and then lyophilize the chiral-enhanced LFe-NSC solution obtained in step (4) to finally obtain the chiral-enhanced LFe-NSC powder.

[0061] 2.4 Preparation of achiral and sulfur-free doped single-atom iron carbon dots (Fe-NC)

[0062] The preparation method is as follows:

[0063] (1) Add 0.53 g of iron phthalocyanine to 30 mL of dimethylformamide, stir until evenly dispersed, and obtain a reaction mother liquor after ultrasonic treatment.

[0064] (2) Place the hydrothermal reaction mother liquor described in step (1) in a hydrothermal reaction device, and obtain a black solution after hydrothermal reaction at 180 °C for 12 h.

[0065] (3) Centrifuge, dialyze, and purify the black solution obtained in step (2) to obtain an Fe-NC solution, and obtain Fe-NC powder after freeze-drying.

[0066] 2.5. Characterize and verify the activity of the chiral Fe-NSC powders (DFe-NSC, DLFe-NSC, LFe-NSC) prepared in steps 2.1 - 2.3 above and the Fe-NC powder prepared in step 2.4. The results are as follows:

[0067] Figure 1 As shown in A: The HRTEM results show that the DFe-NSC prepared in step 2.1 has a well-dispersed spherical structure, and the particle size is distributed between 5 - 10 nm ( Figure 1 A); The AC-HAADF-STEM results provide clear evidence for the dispersion of iron atoms in DFe-NSC, indicating that DFe-NSC has extremely high atomic utilization efficiency, which is crucial for the efficiency of catalytic reactions ( Figure 1 B).

[0068] Figure 2 As shown: The Fe K-edge XANES spectrum shows that the Fe K-edge of chiral DFe-NSC is between Fe2O3 and FeO, indicating that the mixed valence state of Fe is +2 and +3 ( Figure 2 A); In addition, quantitative least-squares fitting was also performed on the EXAFS spectrum, and the experimental results were in good agreement with the fitting model, confirming that Fe atoms are coordinated with four pyridine-Ns, and sulfur is doped in the second shell of FeN4 in DFe-NSC ( Figure 2 B), and the coordination number of Fe-N was calculated to be 4.17.

[0069] Figure 3 As shown: The CD spectrum shows that LFe-NSC and DFe-NSC have completely opposite chiral characteristics, indicating that the chiral transfer and enhancement strategy successfully synthesized chiral single-atom carbon dots ( Figure 3 A); The XPS analysis results show that DFe-NSC has characteristic peaks of C1s, N1s, O1s, S2p, and Fe2p ( Figure 3 B), further confirming its composition.

[0070] In summary, we successfully synthesized Fe-NSC with chirality and single-atom Fe sites and resolved the coordination environment of its Fe single atoms.

[0071] Figure 4 As shown: The detection results of the TMB probe indicate that the catalase (POD-like) activity of chiral Fe-NSC is affected by chirality. Among them, DFe-NSC exhibits the most significant POD-like activity and catalyzes the generation of ·OH hydroxyl radicals from H2O2 in a concentration-dependent manner ( Figure 4 A). Under 808 nm (1 W / cm 2 ) laser irradiation, Fe-NC, LFe-NSC, DLFe-NSC, and DFe-NSC at 200 μg / mL can all rapidly increase the temperature to above 50 °C ( Figure 4 B). DFe-NSC not only has photothermal therapy (PTT) activity but also exhibits significant photodynamic therapy (PDT) activity under light irradiation. As Figure 4 shown in C, compared with Fe-NC, the g value of DFe-NSC increases, indicating that sulfur doping may increase the number of unpaired 3d electrons in the Fe center, which may promote its photodynamic activity and increase the O2 ·- superoxide anion radical yield ( Figure 4 D). In summary, chiral Fe-NSC simultaneously has POD-like, PTT, and PDT activities, and the POD-like and PDT activities of DFe-NSC are significantly higher than those of Fe-NC, indicating that the chiral structure and sulfur doping synergistically enhance its catalase and photodynamic activities.

[0072] The CFU counting results show that under the conditions of H2O2 and laser irradiation (Larse, abbreviated as "L" in the figure), DFe-NSC exhibits the strongest antibacterial activity, significantly higher than that of Fe-NC and LFe-NSC ( Figure 5 ). The antibacterial activity of chiral Fe-NSC alone is weak, but under the action of physiological concentration H2O2, its antibacterial activity is significantly enhanced, and the synergistic effect of PDT and PTT further improves this effect ( Figure 6 A). In addition, by comparing the antibacterial activity differences between Fe-NC and DLFe-NSC, LFe-NSC, and DFe-NSC, the results show that nitrogen / sulfur co-doping and chirality-dependent catalysis of H2O2 may be the key factors for enhancing antibacterial activity. As Figure 6As shown in C, the bacteria in the control group maintained a smooth and intact morphology, while after treatment with DFe-NSC under H2O2 and laser irradiation conditions, obvious damage appeared on the bacterial surface, accompanied by the outflow of contents. It is worth noting that compared with LFe-NSC, DFe-NSC more significantly caused bacterial damage and intracellular iron ion aggregation( Figure 6 B), which may exacerbate the ferroptosis-like effect of bacteria by promoting iron ion accumulation. The anti-biofilm activities of different chiral Fe-NSCs were quantitatively analyzed by crystal violet staining method. Compared with the control group, DFe-NSC showed the most significantly enhanced anti-biofilm activity under H2O2 and laser irradiation conditions( Figure 7 ). The molecular docking results showed that compared with LFe-NSC, DFe-NSC more significantly inhibited the activities of Fur and FtnA proteins, which may be the main reason for the differences in intracellular iron ion content and ferroptosis in bacteria( Figure 8 A-8D). It is speculated from the molecular docking results that chiral Fe-NSC may regulate the activities of bacterial metal transporters through chirality, especially inhibiting the proteins related to iron efflux, thereby leading to iron ion overload and exacerbating the process of cellular ferroptosis.

[0073] The above-described embodiments only express several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A method for preparing nitrogen / sulfur co-doped chiral iron single atom carbon dots, characterized in that: The specific steps are as follows: (1) adding iron phthalocyanine and cysteine ​​to dimethylformamide, stirring until uniformly dispersed, and obtaining a reaction mother liquor after ultrasonic treatment; and obtaining a black solution after the reaction mother liquor is subjected to a hydrothermal reaction; (2) centrifuging, dialysis and purifying the black solution obtained in step (1) to obtain a Fe-NSC solution; (3) adding the Fe-NSC solution obtained in step (2) to a mixed solution containing N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and cysteine ​​to obtain a Fe-NSC solution with enhanced chirality; (4) The chiral enhanced Fe-NSC solution obtained in step (3) is dialyzed and then freeze-dried to finally obtain nitrogen / sulfur co-doped chiral iron single atom carbon dots.

2. The method for preparing nitrogen / sulfur co-doped chiral iron single atom carbon dots according to claim 1, characterized in that: In step (1), the mass ratio of the iron phthalocyanine to cysteine ​​is 1:1 to 3:

1.

3. The method for preparing nitrogen / sulfur co-doped chiral iron single atom carbon dots according to claim 2, characterized in that: In steps (1) and (3), the cysteine ​​includes at least one of D-cysteine, D,L-cysteine ​​and L-cysteine.

4. The method for preparing nitrogen / sulfur co-doped chiral iron single atom carbon dots according to claim 1, characterized in that: In step (1), the hydrothermal reaction temperature is 160° C. to 200° C., and the reaction time is 6 to 12 hours.

5. The method for preparing nitrogen / sulfur co-doped chiral iron single atom carbon dots according to claim 1, characterized in that: In step (3), the molar ratio of N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and cysteine ​​is 1:1:1 to 1:1:

3.

6. The method for preparing nitrogen / sulfur co-doped chiral iron single atom carbon dots according to claim 1, characterized in that: In steps (2) and (4), the molecular weight of the dialysis bag used for dialysis is 2000 to 8000 Da, and the dialysis time is 24 h to 72 h.

7. A nitrogen / sulfur co-doped chiral iron single atom carbon dot, characterized in that: The method is prepared by any one of claims 1 to 6.

8. The nitrogen / sulfur co-doped chiral iron single atom carbon dots according to claim 7, characterized in that: The nitrogen / sulfur co-doped chiral iron single-atom carbon dots contain four elements: sulfur S, nitrogen N, iron Fe, and carbon C. The iron is dispersed at the atomic level. The Fe atom coordinates with an average of 4 N atoms to form Fe-N4, and each Fe-N4 is connected to an average of one S atom.

9. Use of the nitrogen / sulfur co-doped chiral iron single-atom carbon dots as claimed in claim 7 in the preparation of nano-antibacterial materials that induce bacterial ferroptosis.

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