A preparation method for chiral silicon quantum dots for detecting peroxynitrite concentration

Abstract

A method for preparing chiral silicon quantum dots for detecting peroxynitrite concentration, which relates to a method for preparing chiral silicon quantum dots. The present invention aims to solve the problem that the fluorescence of melanin is partially quenched by aqueous solution, resulting in a low fluorescence intensity; solve the problem that existing SiQDs cannot detect ONOO ‑ The specific detection is carried out, and the emission range, fluorescence lifetime and quantum yield are low, which makes it impossible to fully utilize the emission light of quantum dots, resulting in poor detection effect and the inability to achieve endogenous detection of cells. Method: 1. Preparation of chiral melanin particles (K / P) by oxidation method ox Solution; 2. Preparation of SO3‑(K / P) by stirring method ox ;3. Preparation of SiQDs-(K / P) by hydrothermal method ox The present invention is used to prepare chiral silicon quantum dots for detecting the concentration of peroxynitrite.

Description

Technical Field

[0001] The invention relates to a method for preparing chiral silicon quantum dots. Background Art

[0002] Peroxynitrite (ONOO - ) is one of the reactive oxygen species and plays a key role in the inflammatory process. - Abnormal concentrations of ONOO may even cause cancer. - It is a highly active isomer of nitrate with strong oxidative potential and can oxidize proteins, lipids, peptides and nucleic acids. - It can lead to fluorescence quenching or fluorescence enhancement. Currently, many excellent materials for the treatment of pneumonia and cancer are being studied. These materials can be combined with ONOO - Fluorescent probes and drugs were used in combination to investigate the effects of different concentrations of ONOO - Perform quantitative detection.

[0003] Based on melanin and ONOO - A direct oxidation relationship between the two can be established by using quantum dots (QDs) to quantitatively analyze ONOO - Detection method. However, despite the potential of melanin, its fluorescence is not suitable for traditional spectroscopy due to its extremely weak signal, which makes fluorescence detection and tracking challenging. Ayala Lampel et al. developed a polymer peptide pigment based on melanin that can spontaneously form different chromophores, called melanin-like particles. Among them, the melanin-like particles synthesized by phenylalanine (K / P) ox Due to its special fluorescence emission intensity, it is very suitable for detecting ONOO - .

[0004] The interaction between melanin-like fluorophores and water molecules in aqueous solution is closely related, which may lead to energy transfer from the excited state of the fluorophore to the surrounding water molecules. In order to alleviate the interaction between molecules, steric hindrance can be introduced. The addition of sulfonic acid groups can lead to (K / P) ox Being in a hydrophobic environment reduces nonradiative energy transfer from the excited state to the surrounding water. For example, Sun et al. developed a novel NIR-II probe, FD-1080J-aggregates, whose luminescence intensity was enhanced by the addition of 1,4-butanesulfonone. Despite this improvement in luminescence intensity, the large particle size of melanin typically leads to severe photobleaching, hindering long-term imaging in vitro and in vivo and making it unsuitable for biomedical applications.

[0005] II-VI semiconductor quantum dots have the advantages of size-tunable emission color, strong fluorescence, and high resistance to photobleaching. However, semiconductor quantum dots with excellent optical properties lack specific detection of ONOO.- Silicon quantum dots (SiQDs) are increasingly being used as fluorescent probes in biological and biomedical research due to their biocompatibility and low toxicity. Although simple methods exist for preparing SiQDs with reasonable monodispersity and quantum yield, their emission range is limited to approximately 150 nm, with low fluorescence lifetime and quantum yield, which cannot fully utilize the emitted light of the quantum dots, resulting in poor detection effects and inability to achieve endogenous detection in cells.

[0006] In summary, the existing melanin-like pigments are partially quenched by aqueous solution, making their fluorescence intensity low; the existing SiQDs cannot - Specific detection is performed, and the emission range, fluorescence lifetime and quantum yield are low, which makes it impossible to fully utilize the emission light of quantum dots, resulting in poor detection effect and inability to detect endogenous cells. Summary of the Invention

[0007] The present invention aims to solve the problem that the fluorescence of melanin is partially quenched by aqueous solution, resulting in a low fluorescence intensity; solve the problem that the existing SiQDs cannot - The emission range, fluorescence lifetime, and quantum yield of the quantum dots are low, making it impossible to fully utilize the emitted light of the quantum dots, resulting in poor detection results and the inability to detect endogenous cells. Furthermore, a method for preparing chiral silicon quantum dots for detecting peroxynitrite concentration is provided.

[0008] A method for preparing chiral silicon quantum dots for detecting peroxynitrite concentration is carried out according to the following steps:

[0009] 1. Preparation of Chiral Melanin-like Particles (K / P) by Oxidation ox Solution:

[0010] ① Dissolving Lys-Tyr-Phe tripeptide in double-distilled water to obtain a tripeptide solution, adjusting the pH of the tripeptide solution to 5-8, and stirring to obtain a translucent hydrogel; dissolving mushroom tyrosinase in phosphate buffer to obtain a mushroom tyrosinase solution; adding the mushroom tyrosinase solution to the translucent hydrogel and stirring, then adding phenylalanine and stirring to obtain a mixed solution, and centrifuging the mixed solution to obtain a supernatant;

[0011] The phenylalanine is D-phenylalanine or L-phenylalanine;

[0012] The volume ratio of the translucent hydrogel to the mushroom tyrosinase solution is 1:(0.1-0.3); the mass ratio of the Lys-Tyr-Phe tripeptide to phenylalanine is 1:(1-3);

[0013] ② Centrifuge the supernatant to obtain aqueous phase I and solid phase I;

[0014] ③. Add double distilled water to solid phase I and sonicate, then centrifuge to obtain aqueous phase II and solid phase II;

[0015] ④. Repeat step 1 ③ for 2 to 3 times on solid phase II, then combine aqueous phase I and aqueous phase II to obtain (K / P) ox solution;

[0016] 2. Preparation of SO3-(K / P) by stirring method ox :

[0017] (K / P) ox The pH of the solution was adjusted to 9-10, and then 1,4-butane sultone was added dropwise, stirred and centrifuged to obtain SO3-(K / P) ox Mixing solution;

[0018] 3. Preparation of SiQDs-(K / P) by hydrothermal method ox :

[0019] Under stirring, SO3-(K / P) ox The solution was mixed with N-[3-(trimethoxysilyl)propyl]ethylenediamine, and then reacted at a temperature of 180°C to 200°C for 2h to 6h, and finally taken out and cooled to room temperature, and SiQDs-(K / P) was obtained after dialysis. ox solution;

[0020] The SiQDs-(K / P) ox SiQDs-(K / P) in solution ox The concentration is 4.05mg / mL~8.1mg / mL.

[0021] The beneficial effects of the present invention are:

[0022] ① Nanofiber gels were synthesized by self-assembling the tripeptide KYF as a supramolecular precursor in phosphate buffer. After the KYF gel was oxidized by mushroom tyrosinase, L-Phe or D-Phe was added to form two fluorescent melanin-like particles, (K / P) ox The solution has an excitation wavelength of 380 nm and an emission wavelength of 550 nm. The innovative addition of sulfonic acid groups to the melanin-like particles increased the fluorescence intensity by 1.5 times. In order to improve the photobleaching of organic macromolecular fluorophores, N-[3-(trimethoxysilyl)propyl]ethylenediamine (DAMO) was added to the melanin-like particles to hydrothermally synthesize highly photostable SiQDs-(K / D- / LP). ox .

[0023] ② For the first time, SiQDs with chiral structure were synthesized, and SiQDs-(K / DP) was studied. ox and SiQDs-(K / LP)ox The prepared chiral silicon quantum dots have higher quantum yield (QY) than existing silicon quantum dots, SiQDs-(K / DP) ox and SiQDs-(K / LP) ox The QY of SiQDs-(K / D- / LP) is 47.66% and 26.07% respectively, and it has a large luminescence range (380-700nm). ox It also retains its melanin-like particles to ONOO - The sensitivity of ONOO can be detected by SiQDs alone. - .

[0024] ③SiQDs-(K / LP) ox The detection range is 2μM~100μM. The first linearity is in the concentration range of 2μM~10μM, R 2 The second linearity is in the concentration range of 20μM to 100μM, where R 2 is 0.994 (based on signal-to-noise ratio S / N=3). The interference experiment results show that SiQDs-(K / LP) ox ONOO - The detection is selective.

[0025] ④SiQDs-(K / LP) ox The SiQDs-(K / LP) ox Detection of ONOO in HepG2 cells - From the Spec3 channel, we can see that after adding LPS for 1h and 2h, SiQDs-(K / LP) ox All of them showed strong fluorescence quenching. This indicates that SiQDs-(K / LP) ox The probe is expected to be used in the detection of inflammatory cells and hepatocellular carcinoma, providing new ideas for future lesion treatment and detection.

[0026] Therefore, the SiQDs-(K / LP) excited by 380 nm ultraviolet light of the present invention has high quantum yield, wide emission range and longer lifespan. ox The preparation method of semiconductor quantum dots is simple, with strong resistance to photobleaching and small toxic side effects. - It integrates multiple functions such as imaging (oxidative stress in cancer cells).

[0027] Figures in the specification

[0028] Figure 1This is a flow chart of the preparation of chiral silicon quantum dots for detecting the concentration of peroxynitrite in Example 1;

[0029] Figure 2 TEM imaging diagram, a is the (K / DP) prepared in step 1④ of Example 1 ox Solution b is (K / LP) prepared in step 1④ of Example 2 ox Solution c is SiQDs-(K / DP) prepared in step 3 of Example 1 ox Solution, d is the SiQDs-(K / LP) prepared in step 3 of Example 2 ox solution;

[0030] Figure 3 is a particle size diagram, a is the (K / DP) prepared in step 1④ of Example 1 ox Solution b is (K / LP) prepared in step 1④ of Example 2 ox Solution c is SiQDs-(K / DP) prepared in step 3 of Example 1 ox Solution, d is the SiQDs-(K / LP) prepared in step 3 of Example 2 ox solution;

[0031] Figure 4 is the UV absorption graph, 1 is the (K / DP) prepared in step 1④ of Example 1 ox Solution 2 is prepared in step 1 (4) of Example 2 (K / LP) ox Solution 3 is SiQDs-(K / LP) prepared in step 3 of Example 1 ox Solution 4 is SiQDs-(K / DP) prepared in step 3 of Example 2 ox solution;

[0032] Figure 5 The excitation light is 380nm and the illumination intensity is 100mW / cm 2 The fluorescence emission intensity of the samples under the condition shown in Figure 1 is SiQDs-(K / DP) prepared in step 3 of Example 1. ox Solution 2 is SiQDs-(K / LP) prepared in step 3 of Example 2 ox Solution 3 is (K / DP) prepared in step 1④ of Example 1 ox Solution 4 is prepared in step 1 (4) of Example 2 (K / LP) ox solution;

[0033] Figure 6 The excitation light is 380nm and the illumination intensity is 100mW / cm 2 The fluorescence lifetime diagram of the sample below, 1 is the SiQDs-(K / LP) prepared in step 3 of Example 2 oxSolution 2 is SiQDs-(K / DP) prepared in step 3 of Example 1 ox Solution 3 is (K / DP) prepared in step 1④ of Example 1 ox Solution 4 is prepared in step 1 (4) of Example 1 (K / LP) ox solution;

[0034] Figure 7 is a quantum yield diagram, a is the (K / DP) prepared in step 1④ of Example 1 ox Solution b is (K / LP) prepared in step 1④ of Example 2 ox Solution c is SiQDs-(K / DP) prepared in step 3 of Example 1 ox Solution, d is the SiQDs-(K / LP) prepared in step 3 of Example 2 ox solution;

[0035] Figure 8 The excitation light is 380nm and the illumination intensity is 100mW / cm 2 The fluorescence intensity diagram below is shown in FIG1 , where 1 is the (K / DP) prepared in step 1④ of Example 1 ox Solution 2 is SO3-(K / DP) prepared in step 2 of Example 1 ox Solution 3 is prepared in step 1 (4) of Example 2 (K / LP) ox Solution 4 is SO3-(K / LP) prepared in step 2 of Example 2 ox solution;

[0036] Figure 9 1 is the circular dichroism spectrum, 2 is the (K / LP) prepared in step 1④ of Example 2 ox Solution 2 is (K / DP) prepared in step 1 (4) of Example 1 ox Solution 3 is SiQDs-(K / LP) prepared in step 3 of Example 2 ox Solution 4 is the SiQDs-(K / DP) prepared in step 3 of Example 1 ox solution;

[0037] Figure 10 SiQDs-(K / LP) prepared in step 3 of Example 2 ox Energy spectrum of the solution;

[0038] Figure 11 Preparation of SiQDs-(K / LP) in step 3 of Example 2 ox X-ray photoelectron spectrum of the solution, a is the full spectrum, b is the C spectrum, c is the N spectrum, d is the O spectrum, e is the S spectrum, and f is the Si spectrum;

[0039] Figure 12The excitation light is 380nm and the illumination intensity is 100mW / cm 2 The fluorescence emission intensity of the samples under the condition shown in Figure 1 is SiQDs-(K / LP) prepared in step 3 of Example 2. ox Solution 2 is prepared in step 1 (4) of Example 2 (K / LP) ox Solution 3 is the SiQDs-Cat solution prepared in the comparative experiment, and 4 is the SiQDs-(K / LP) prepared in step 3 of Example 2. ox ONOO was added to the solution - 5 is the SiQDs-Cat solution prepared for the comparative experiment, in which ONOO - , 6 Example 2 Step 1 ④ prepared (K / LP) ox ONOO was added to the solution - ;

[0040] Figure 13 The excitation light is 380nm and the illumination intensity is 100mW / cm 2 Next, add ONOO - Effects of post-reaction time, temperature, pH value and laser irradiation time on SiQDs-(K / LP) ox 、(K / LP) ox and the effect of SiQDs-Cat, a is the temperature of 30℃, pH 5 and ONOO - The SiQDs-(K / LP) prepared in step 3 of Example 2 was subjected to a solution concentration of 20 μM. ox Effect of different reaction times of the solution, b is the pH 5, reaction 10min and ONOO - The effect of different temperatures under the condition of solution concentration of 20μM, c is the temperature of 30℃, reaction time of 10min and ONOO - The effect of different pH values ​​under the condition of solution concentration of 20 μM, d is the temperature of 30 ° C, pH 5 and ONOO - Effect of laser irradiation time on material stability under the condition of solution concentration of 100 μM. 1 in bd is SiQDs-(K / LP) prepared in step 3 of Example 2. ox Solution 2 is the SiQDs-Cat solution prepared in the comparative experiment, and 3 is the (K / LP) solution prepared in step 1④ of Example 2. ox solution;

[0041] Figure 14 The excitation light is 380nm and the illumination intensity is 100mW / cm 2 Under various concentrations of ONOO - induced SiQDs-(K / LP) oxThe emission light response of a is the SiQDs-(K / LP) prepared in step 3 of Example 2 ox Add 0-300 μM ONOO to the solution - After the fluorescence spectrum, a is 0 μM, 2 is 2 μM, 3 is 4 μM, 4 is 6 μM, 5 is 8 μM, 6 is 10 μM, 7 is 20 μM, 8 is 40 μM, 9 is 60 μM, 10 is 80 μM, 11 is 100 μM, 12 is 200 μM, 13 is 300 μM, b is the SiQDs-(K / LP) prepared in step 3 of Example 2 ox Add 0-300 μM MONOO to the solution - The fluorescence line graph after the experiment is shown in Figure 2. c is the SiQDs-(K / LP) prepared in step 3 of Example 2. ox Add 0-10 μM MONOO to the solution - The fluorescence line graph after d is the SiQDs-(K / LP) prepared in step 3 of Example 2. ox Add 20-100 μM MONOO to the solution - Fluorescence line chart after

[0042] Figure 15 SiQDs-(K / LP) prepared in step 3 of Example 2 ox Solution for imaging endogenous peroxynitrite in HepG2 cells. DETAILED DESCRIPTION

[0043] Specific embodiment 1: This embodiment is a method for preparing chiral silicon quantum dots for detecting the concentration of peroxynitrite, which is as follows:

[0044] 1. Preparation of Chiral Melanin-like Particles (K / P) by Oxidation ox Solution:

[0045] ① Dissolving Lys-Tyr-Phe tripeptide in double-distilled water to obtain a tripeptide solution, adjusting the pH of the tripeptide solution to 5-8, and stirring to obtain a translucent hydrogel; dissolving mushroom tyrosinase in phosphate buffer to obtain a mushroom tyrosinase solution; adding the mushroom tyrosinase solution to the translucent hydrogel and stirring, then adding phenylalanine and stirring to obtain a mixed solution, and centrifuging the mixed solution to obtain a supernatant;

[0046] The phenylalanine is D-phenylalanine or L-phenylalanine;

[0047] The volume ratio of the translucent hydrogel to the mushroom tyrosinase solution is 1:(0.1-0.3); the mass ratio of the Lys-Tyr-Phe tripeptide to phenylalanine is 1:(1-3);

[0048] ② Centrifuge the supernatant to obtain aqueous phase I and solid phase I;

[0049] ③. Add double distilled water to solid phase I and sonicate, then centrifuge to obtain aqueous phase II and solid phase II;

[0050] ④. Repeat step 1 ③ for 2 to 3 times on solid phase II, then combine aqueous phase I and aqueous phase II to obtain (K / P) ox solution;

[0051] 2. Preparation of SO3-(K / P) by stirring method ox :

[0052] (K / P) ox The pH of the solution was adjusted to 9-10, and then 1,4-butane sultone was added dropwise, stirred and centrifuged to obtain SO3-(K / P) ox Mixing solution;

[0053] 3. Preparation of SiQDs-(K / P) by hydrothermal method ox :

[0054] Under stirring, SO3-(K / P) ox The solution was mixed with N-[3-(trimethoxysilyl)propyl]ethylenediamine, and then reacted at a temperature of 180°C to 200°C for 2h to 6h, and finally taken out and cooled to room temperature, and SiQDs-(K / P) was obtained after dialysis. ox solution;

[0055] The SiQDs-(K / P) ox SiQDs-(K / P) in solution ox The concentration is 4.05mg / mL~8.1mg / mL.

[0056] In this embodiment, Lys-Tyr-Phe tripeptide (KYF tripeptide) is used to form a nanofiber gel as a supramolecular precursor. It self-assembles into a translucent hydrogel in a phosphate buffer (aqueous buffer) at physiological pH. The KYF gel is then oxidized using mushroom tyrosinase. D-phenylalanine or L-phenylalanine (D-Phe or L-Phe) is then added to introduce chiral amino acid side chains. 1,4-Butanesultone is then added to increase the sulfonic acid group to increase the steric hindrance between the product and water, thereby increasing the fluorescence intensity. Finally, N-[3-(trimethoxysilyl)propyl]ethylenediamine and (K / P) ox As silicon source, ONOO was synthesized by hydrothermal method. - Sensitive SiQDs-(K / P) ox .

[0057] The beneficial effects of this embodiment are:

[0058] ① Nanofiber gels were synthesized by self-assembling the tripeptide KYF as a supramolecular precursor in phosphate buffer. After the KYF gel was oxidized by mushroom tyrosinase, L-Phe or D-Phe was added to form two fluorescent melanin-like particles, (K / P) ox The solution has an excitation wavelength of 380 nm and an emission wavelength of 550 nm. The innovative addition of sulfonic acid groups to the melanin-like particles increased the fluorescence intensity by 1.5 times. In order to improve the photobleaching of organic macromolecular fluorophores, N-[3-(trimethoxysilyl)propyl]ethylenediamine (DAMO) was added to the melanin-like particles to hydrothermally synthesize highly photostable SiQDs-(K / D- / LP). ox .

[0059] ② For the first time, SiQDs with chiral structure were synthesized, and SiQDs-(K / DP) was studied. ox and SiQDs-(K / LP) ox The prepared chiral silicon quantum dots have higher quantum yield (QY) than existing silicon quantum dots, SiQDs-(K / DP) ox and SiQDs-(K / LP) ox The QY of SiQDs-(K / D- / LP) is 47.66% and 26.07% respectively, and it has a large luminescence range (380-700nm). ox It also retains its melanin-like particles to ONOO - The sensitivity of ONOO can be detected by SiQDs alone. - .

[0060] ③SiQDs-(K / LP) ox The detection range is 2μM~100μM. The first linearity is in the concentration range of 2μM~10μM, R 2 The second linearity is in the concentration range of 20μM to 100μM, where R 2 is 0.994 (based on signal-to-noise ratio S / N=3). The interference experiment results show that SiQDs-(K / LP) ox ONOO - The detection is selective.

[0061] ④SiQDs-(K / LP) ox The SiQDs-(K / LP) ox Detection of ONOO in HepG2 cells - From the Spec3 channel, we can see that after adding LPS for 1h and 2h, SiQDs-(K / LP)ox All of them showed strong fluorescence quenching. This indicates that SiQDs-(K / LP) ox The probe is expected to be used in the detection of inflammatory cells and hepatocellular carcinoma, providing new ideas for future lesion treatment and detection.

[0062] Therefore, the SiQDs-(K / LP) excited by 380 nm ultraviolet light of this embodiment has high quantum yield, wide emission range and longer lifetime. ox The preparation method of semiconductor quantum dots is simple, with strong resistance to photobleaching and small toxic side effects. - It integrates multiple functions such as imaging (oxidative stress in cancer cells).

[0063] Specific embodiment 2: This embodiment differs from specific embodiment 1 in that: the concentration of the Lys-Tyr-Phe tripeptide in the tripeptide solution in step 1 (1) is 10 mM to 30 mM; the concentration of the phosphate buffer in step 1 (1) is 10 mM to 100 mM, with a pH of 5 to 8; and the enzyme activity of the mushroom tyrosinase solution in step 1 (1) is 50 U / mL to 100 U / mL. Other steps are the same as those in specific embodiment 1.

[0064] Specific embodiment 3: This embodiment differs from either specific embodiment 1 or 2 in that: the centrifugation described in steps 1 (1) to 3) and step 2 is performed at a speed of 10,000 to 15,000 rpm for 5 to 10 minutes; the volume ratio of solid phase I to double-distilled water described in step 1 (3) is 1:(2-3); and the ultrasonication described in step 1 (3) is performed at a power of 50 W to 100 W for 5 to 10 minutes. Other aspects are the same as specific embodiments 1 or 2.

[0065] Specific embodiment 4: This embodiment differs from any one of the specific embodiments 1 to 3 in that: the (K / P) in step 1④ ox In solution (K / P) ox The concentration of the compound is 16.2 mg / mL to 32.4 mg / mL. Other aspects are the same as those of the first to third embodiments.

[0066] Specific embodiment 5: This embodiment differs from specific embodiments 1 to 4 in that: in step 2, a NaOH solution with a concentration of 0.01M to 0.1M is used to convert (K / P) ox The pH of the solution is adjusted to 9 to 10. Other steps are the same as those in the first to fourth embodiments.

[0067] Specific embodiment 6: This embodiment differs from specific embodiments 1 to 5 in that in step 2, 1,4-butane sultone is added dropwise at a stirring speed of 5 to 20 r / s and a dropping speed of 0.1 to 0.25 μL / s. Other aspects are the same as specific embodiments 1 to 5.

[0068] Specific embodiment seven: This embodiment differs from any one of the specific embodiments one to six in that: the SO3-(K / P) described in step two ox SO3-(K / P) in solution ox The concentration of the compound is 18.2 mg / mL to 34.4 mg / mL. Other aspects are the same as those of the first to sixth embodiments.

[0069] Specific embodiment eight: This embodiment differs from any one of specific embodiments one to seven in that: (K / P) described in step two ox The volume ratio of the solution to 1,4-butane sultone is 1:(0.03-0.015). Other aspects are the same as those of the first to seventh embodiments.

[0070] Specific embodiment 9: This embodiment differs from any one of specific embodiments 1 to 8 in that: the SO3-(K / P) described in step 3 ox The volume ratio of the solution to N-[3-(trimethoxysilyl)propyl]ethylenediamine is 1:(0.2-0.4). Other aspects are the same as those of the first to eighth embodiments.

[0071] Specific embodiment 10: This embodiment differs from specific embodiments 1 to 9 in that the dialysis in step 3 is specifically performed in a dialysis bag with a molecular weight cut-off of MW=500Da for 6 to 12 hours. Other aspects are the same as specific embodiments 1 to 9.

[0072] The following examples are used to verify the beneficial effects of the present invention:

[0073] Example 1, combined with Figure 1 Specific instructions:

[0074] A method for preparing chiral silicon quantum dots for detecting peroxynitrite concentration is carried out according to the following steps:

[0075] 1. Preparation of Chiral Melanin-like Particles (K / DP) by Oxidation ox Solution:

[0076] ① Dissolve Lys-Tyr-Phe tripeptide in double-distilled water to obtain a tripeptide solution, adjust the pH of the tripeptide solution to 8, and stir at a speed of 15 rpm for 10 minutes to obtain a translucent hydrogel; dissolve mushroom tyrosinase in phosphate buffer to obtain a mushroom tyrosinase solution; add 1 mL of the mushroom tyrosinase solution to 10 mL of the translucent hydrogel, and stir at a speed of 15,000 rpm for 3 hours, then add D-phenylalanine, and stir at a speed of 15,000 rpm for 24 hours to obtain a mixed solution, and centrifuge the mixed solution to obtain a supernatant;

[0077] The mass ratio of the Lys-Tyr-Phe tripeptide to phenylalanine is 1:3;

[0078] The concentration of Lys-Tyr-Phe tripeptide in the tripeptide solution is 30 mM; the concentration of the phosphate buffer is 100 mM, and the pH is 8; the enzyme activity of the mushroom tyrosinase solution is 50 U / mL;

[0079] ② Centrifuge the supernatant to obtain aqueous phase I and solid phase I;

[0080] ③. Add double distilled water to solid phase I and sonicate at 100 W for 10 minutes, then centrifuge to obtain aqueous phase II and solid phase II.

[0081] The volume ratio of the solid phase I to double distilled water is 1:2;

[0082] ④. Repeat step 1③ for 3 times on solid phase II, then combine aqueous phase I and aqueous phase II to obtain (K / DP) ox solution;

[0083] After measuring the (K / DP) in step 1④ ox In solution (K / DP) ox The concentration is 16.2 mg / mL;

[0084] 2. Preparation of SO3-(K / DP) by stirring method ox :

[0085] 10mL (K / DP) was mixed with 0.1M NaOH solution. ox The pH of the solution was adjusted to 10, and then 150 μL of 1,4-butanesulfonate was added dropwise at a stirring speed of 20 r / s and a dropping speed of 0.25 μL / s. The mixture was stirred at a speed of 15 r / s for 24 h and then centrifuged to obtain SO3-(K / DP) ox Mixing solution;

[0086] The SO3-(K / DP) oxSO3-(K / DP) in solution ox The concentration is 18.2 mg / mL;

[0087] 3. Preparation of SiQDs-(K / DP) by hydrothermal method ox :

[0088] Under the condition of stirring speed of 15r / s, 5mL SO3-(K / DP) ox The solution was mixed with 2.0 mL of N-[3-(trimethoxysilyl)propyl]ethylenediamine, and then reacted at 200 ° C for 4 h. Finally, it was taken out and cooled to 20 ° C. After dialysis, SiQDs-(K / DP) was obtained. ox solution;

[0089] The SiQDs-(K / DP) was determined to be ox SiQDs-(K / DP) in solution ox The concentration is 4.05 mg / mL.

[0090] The Lys-Tyr-Phe tripeptide described in step 1① was purchased from Hangzhou Zhuanpeptide Biotechnology Co., Ltd.

[0091] The centrifugation in steps 1 to 3 and step 2 is specifically carried out at a rotation speed of 15,000 rpm for 10 minutes.

[0092] The dialysis described in step 3 is specifically performed in a dialysis bag with a molecular weight cut-off of MW=500Da for 7 hours.

[0093] Example 1 Step 1 (4) prepared (K / DP) ox Solution, SO3-(K / DP) prepared in step 2 ox Solution and SiQDs-(K / DP) prepared in step 3 ox All solutions were stored at 5°C until use.

[0094] Example 2: The difference between this comparative experiment and Example 1 is that: in step 1①, D-phenylalanine is replaced by L-phenylalanine; in step 1④, (K / LP) is obtained. ox Solution; Step 2 to obtain SO3-(K / LP) ox Mixed solution; Step 3 to obtain SiQDs-(K / LP) ox The rest is the same as in Example 1.

[0095] Comparative experiment: The difference between this comparative experiment and Example 1 is that in step 3, 5 mL SO3-(K / DP) ox The solution was replaced with 5 mL of 20 mM catechol aqueous solution. Other steps were the same as those in Example 1.

[0096] The product prepared in step three of the comparative experiment is abbreviated as SiQDs-Cat solution.

[0097] Figure 2 TEM imaging diagram, a is the (K / DP) prepared in step 1④ of Example 1 ox Solution b is (K / LP) prepared in step 1④ of Example 2 ox Solution c is SiQDs-(K / DP) prepared in step 3 of Example 1 ox Solution, d is the SiQDs-(K / LP) prepared in step 3 of Example 2 ox From the transmission electron microscope (TEM) images, we can see that (K / DP) ox and (K / LP) ox All are spherical, with smooth surfaces and good dispersion. SiQDs prepared from chiral melanin-like precursors are well dispersed, have high contrast on ultrathin carbon supports, and show clear edge contours. SiQDs-(K / DP) ox It consists of multiple quantum dots wrapped in an organic material that appears in the form of rice-like particles. On the other hand, SiQDs-(K / LP) ox The surface is not covered with organic matter and is simply dispersed in water.

[0098] Figure 3 is a particle size diagram, a is the (K / DP) prepared in step 1④ of Example 1 ox Solution b is (K / LP) prepared in step 1④ of Example 2 ox Solution c is SiQDs-(K / DP) prepared in step 3 of Example 1 ox Solution, d is the SiQDs-(K / LP) prepared in step 3 of Example 2 ox Solution. (K / DP) ox The size of the particles is relatively uniform, with some larger particles of 223 nm and some particles smaller than 10 nm (TEM). (K / DP) measured by dynamic light scattering (DLS) ox and (K / LP) ox The hydrodynamic average particle sizes of SiQDs-(K / DP) were 96 nm and 160 nm, respectively. ox and SiQDs-(K / LP) ox The average particle sizes of 5.3 nm and 4.2 nm were respectively.

[0099] Figure 4 is the UV absorption graph, 1 is the (K / DP) prepared in step 1④ of Example 1 oxSolution 2 is prepared in step 1 (4) of Example 2 (K / LP) ox Solution 3 is SiQDs-(K / LP) prepared in step 3 of Example 1 ox Solution 4 is SiQDs-(K / DP) prepared in step 3 of Example 2 ox Solution. (K / DP) ox and (K / LP) ox Exhibits broadband UV / Vis absorption. (K / DP) ox and (K / LP) ox They all exhibited an additional unique and intense absorbance at 501 nm, which correlated with the red color of the solution, and a relatively flat peak at 352 nm. ox and SiQDs-(K / LP) ox The UV spectrum shows that the peak at 501 nm disappears, which corresponds to (K / DP) ox and (K / LP) ox The prepared SiQDs solution is light yellow, and the peak at 300nm is attributed to the π-π* transition of the C=C bond. It is worth noting that SiQDs-(K / LP) ox The peak at 300 nm is larger than that of SiQDs-(K / DP) with more C=C bonds. ox Stronger.

[0100] Figure 5 The excitation light is 380nm and the illumination intensity is 100mW / cm 2 The fluorescence emission intensity of the samples under the condition shown in Figure 1 is SiQDs-(K / DP) prepared in step 3 of Example 1. ox Solution 2 is SiQDs-(K / LP) prepared in step 3 of Example 2 ox Solution 3 is (K / DP) prepared in step 1④ of Example 1 ox Solution 4 is prepared in step 1 (4) of Example 2 (K / LP) ox Solution. (K / DP) ox and (K / LP) ox There is an emission peak at 550nm, (K / DP) ox The fluorescence intensity is slightly greater than (K / LP) ox . Prepared SiQDs-(K / LP) ox and SiQDs-(K / DP) ox It has high fluorescence intensity, with an emission peak at 490nm and a wide emission range (380nm to 700nm).

[0101] Figure 6The excitation light is 380nm and the illumination intensity is 100mW / cm 2 The fluorescence lifetime diagram of the sample below, 1 is the SiQDs-(K / LP) prepared in step 3 of Example 2 ox Solution 2 is SiQDs-(K / DP) prepared in step 3 of Example 1 ox Solution 3 is (K / DP) prepared in step 1④ of Example 1 ox Solution 4 is prepared in step 1 (4) of Example 1 (K / LP) ox Solution. SiQDs-(K / LP) ox The lifetime of SiQDs-(K / DP) is the longest, which is 27.219μs. ox The lifetime is 25.169μs. (K / DP) ox and (K / LP) ox The fluorescence lifetimes are 21.394μs and 21.977μs, respectively.

[0102] Figure 7 is a quantum yield diagram, a is the (K / DP) prepared in step 1④ of Example 1 ox Solution b is (K / LP) prepared in step 1④ of Example 2 ox Solution c is SiQDs-(K / DP) prepared in step 3 of Example 1 ox Solution, d is the SiQDs-(K / LP) prepared in step 3 of Example 2 ox Solution. Prepared SiQDs-(K / DP) ox and SiQDs-(K / LP) ox They exhibit high quantum yields of 47.66% and 26.07%, respectively. These values ​​are approximately (K / DP) ox (0.96%) and (K / LP) ox (0.29%) 50 and 90 times.

[0103] Figure 8 The excitation light is 380nm and the illumination intensity is 100mW / cm 2 The fluorescence intensity diagram below is shown in FIG1 , where 1 is the (K / DP) prepared in step 1④ of Example 1 ox Solution 2 is SO3-(K / DP) prepared in step 2 of Example 1 ox Solution 3 is prepared in step 1 (4) of Example 2 (K / LP) ox Solution 4 is SO3-(K / LP) prepared in step 2 of Example 2 oxAs shown in the figure, the innovative addition of sulfonic acid groups to the melanin-like particles increased the fluorescence intensity of the melanin-like particles by 1.5 times. This indicates that the addition of sulfonic acid groups effectively prevents some energy from being transferred from the excited state of the melanin-like fluorophore to the surrounding water.

[0104] Figure 9 1 is the circular dichroism spectrum, 2 is the (K / LP) prepared in step 1④ of Example 2 ox Solution 2 is (K / DP) prepared in step 1 (4) of Example 1 ox Solution 3 is SiQDs-(K / LP) prepared in step 3 of Example 2 ox Solution 4 is the SiQDs-(K / DP) prepared in step 3 of Example 1 ox Solution. SiQDs-(K / DP) ox and SiQDs-(K / LP) ox The CD bands exhibited perpendicular mirror images, with positive (right-handed) and negative (left-handed) curves in the UV region. This indicates that SiQDs with chirality were successfully prepared. ox and (K / LP) ox Compared with the spectral characteristics of SiQDs-(K / DP) ox and SiQDs-(K / LP) ox The CD peaks at 320 nm and 346 nm show a slight blue shift, which indicates that the chirality of SiQDs is mainly attributed to the introduction of chiral ligands and the transfer of chirality from molecular-scale to nanoscale materials.

[0105] Figure 10 SiQDs-(K / LP) prepared in step 3 of Example 2 ox This shows the presence of C, O, and Si elements in the nanoparticles in the TEM image, confirming that the nanoparticles are SiQDs.

[0106] Figure 11 Preparation of SiQDs-(K / LP) in step 3 of Example 2 ox X-ray photoelectron spectrum of the solution, a is the full spectrum, b is the C spectrum, c is the N spectrum, d is the O spectrum, e is the S spectrum, f is the Si spectrum; From the full spectrum, we can see that SiQDs-(K / LP) ox The elements of SiQDs-(K / LP) revealed five main components, including O1s, N 1s, C 1s, S 2p, and Si 2p. oxThe high-resolution C1s XPS spectrum (Figure b) reveals the presence of C=O (288.2 eV), CN (285.5 eV), and CC (284.8 eV). The N1s spectrum (Figure c) displays three peaks corresponding to NH (401.5 eV), N-(C)3 (400.2 eV), and CNC (399.2 eV). The O1s spectrum (Figure d) contains three peaks, including C=O (533.7 eV), CO (532.2 eV), and O-Si (531.0 eV), where the O-Si bond originates from N-[3-(trimethoxysilyl)propyl]ethylenediamine (DAMO). The S 2p spectrum (Figure e) shows peaks at SO2 (168.8 eV) and SO3 (167.6 eV), where the S element originates from 1,4-butanesultone. The Si 2p spectrum (Figure f) shows two peaks at 102.6 and 102.1 eV, which are related to Si-O and Si-C groups, respectively. XPS results further confirm the chiral SiQDs-(K / LP) ox successful synthesis.

[0107] Detection of ONOO - Experiment: 200 μL of SiQDs-(K / LP) prepared in step 3 of Example 2 was added ox Solution (or replaced with the SiQDs-Cat solution prepared in the comparative test, the (K / LP) prepared in step 1④ of Example 2) ox solution), 1.5 mL of pH = 8 Tris hydrochloric acid buffer and 300 μL of different concentrations of ONOO - The solution was pipetted into a 2 mL centrifuge tube, stirred at a rotation speed of 10 r / s for 5 min, and then the fluorescence spectrum was measured at an excitation wavelength of 380 nm.

[0108] Figure 12 The excitation light is 380nm and the illumination intensity is 100mW / cm 2 The fluorescence emission intensity of the samples under the condition shown in Figure 1 is SiQDs-(K / LP) prepared in step 3 of Example 2. ox Solution 2 is prepared in step 1 (4) of Example 2 (K / LP) ox Solution 3 is the SiQDs-Cat solution prepared in the comparative experiment, and 4 is the SiQDs-(K / LP) prepared in step 3 of Example 2. ox ONOO was added to the solution - 5 is the SiQDs-Cat solution prepared for the comparative experiment, in which ONOO - , 6 Example 2 Step 1 ④ prepared (K / LP) ox ONOO was added to the solution -Compared with SiQDs-Cat, SiQDs-(K / LP) ox It has excellent fluorescence intensity and a wider emission range. - After, (K / LP) ox and SiQDs-(K / LP) ox All showed obvious fluorescence quenching, while SiQDs-Cat was not affected. This indicates that SiQDs-(K / LP) synthesized by oxidized tripeptide ox ONOO - The detection is specific.

[0109] Figure 13 The excitation light is 380nm and the illumination intensity is 100mW / cm 2 Next, add ONOO - Effects of post-reaction time, temperature, pH value and laser irradiation time on SiQDs-(K / LP) ox 、(K / LP) ox and the effect of SiQDs-Cat, a is the temperature of 30℃, pH 5 and ONOO - The SiQDs-(K / LP) prepared in step 3 of Example 2 was subjected to a solution concentration of 20 μM. ox Effect of different reaction times of the solution, b is the pH 5, reaction 10min and ONOO - The effect of different temperatures under the condition of solution concentration of 20μM, c is the temperature of 30℃, reaction time of 10min and ONOO - The effect of different pH values ​​under the condition of solution concentration of 20 μM, d is the temperature of 30 ° C, pH 5 and ONOO - Effect of laser irradiation time on material stability under the condition of solution concentration of 100 μM. 1 in bd is SiQDs-(K / LP) prepared in step 3 of Example 2. ox Solution 2 is the SiQDs-Cat solution prepared in the comparative experiment, and 3 is the (K / LP) solution prepared in step 1④ of Example 2. ox Figure a shows that when ONOO - (20 μM) added SiQDs-(K / LP) ox At 1 min, the fluorescence was significantly quenched, and the trend slowed down after 10 min. Therefore, 10 min was selected as the ox and ONOO - The optimal reaction time is . In Figures bd, the effects of temperature, pH and laser irradiation time on the stability of the material are studied. It can be observed that SiQDs-(K / LP) ox The fluorescence intensity of SiQDs-Cat is always much higher than that of (K / LP)ox , pH and laser irradiation time have little effect on it. The highest fluorescence intensity was observed at pH = 5, SiQDs-(K / LP) ox The highest fluorescence intensity is achieved at 30°C. Therefore, 30°C and pH = 5 will be the optimal conditions for detecting ONOO. - The comparative experimental results show that SiQDs-(K / LP) ox Superior optical properties and ONOO compared to SiQDs-Cat - Detection specificity.

[0110] Figure 14 The excitation light is 380nm and the illumination intensity is 100mW / cm 2 Under various concentrations of ONOO - induced SiQDs-(K / LP) ox The emission light response of a is the SiQDs-(K / LP) prepared in step 3 of Example 2 ox Add 0-300 μM ONOO to the solution - After the fluorescence spectrum, a is 0 μM, 2 is 2 μM, 3 is 4 μM, 4 is 6 μM, 5 is 8 μM, 6 is 10 μM, 7 is 20 μM, 8 is 40 μM, 9 is 60 μM, 10 is 80 μM, 11 is 100 μM, 12 is 200 μM, 13 is 300 μM, b is the SiQDs-(K / LP) prepared in step 3 of Example 2 ox Add 0-300 μM MONOO to the solution - The fluorescence line graph after the experiment is shown in Figure 2. c is the SiQDs-(K / LP) prepared in step 3 of Example 2. ox Add 0-10 μM MONOO to the solution - The fluorescence line graph after d is the SiQDs-(K / LP) prepared in step 3 of Example 2. ox Add 20-100 μM MONOO to the solution - Figure a shows the fluorescence line graph after ONOO - With the increase of concentration, the fluorescence intensity at 480nm gradually decreases. The line graph in Figure b shows that the concentration of ONOO between 100 and 300 μM - The concentration range showed a low ΔI, which may lead to inaccurate measurements. Therefore, concentrations greater than 100 μM were not used as standard curves. As shown in Figures c and d, two linear relationships were established for the probe in the concentration range of 2 to 100 μM. The first linear relationship was observed between 2 and 10 μM, and R 2 The value was 0.996, and the limit of detection (LOD) was 99nM. A second linear relationship was observed between 20 and 100μM, R 2The value is 0.994 (based on the signal-to-noise ratio S / N=3).

[0111] SiQDs-(K / LP) ox Solution detection of ONOO in cells - HepG2 cells were seeded in a six-well plate with a sterilized cover glass, culture medium (fetal bovine serum) was added, and they were kept at 37°C and in a 5% CO2 atmosphere for 24 hours to obtain a monolayer of cells. The culture medium was aspirated. 20 μL of SiQDs-(K / LP) prepared in step 3 of Example 2 was added. ox The solution was added to a six-well plate containing 1 mL of PBS to make SiQDs-(K / LP) ox The final concentration of the solution was 100 μg / mL. The cells were incubated at 37°C in a 5% CO₂ atmosphere for 30 minutes, followed by three rinses with PBS. Lipopolysaccharide (LPS) at 10 μg / mL and dimethyl sulfoxide (DMSO) at 10 μg / mL were incubated at 37°C in a 5% CO₂ atmosphere for 1 hour and 2 hours, respectively, followed by three rinses with PBS. Cells were then fixed by adding 1 mL of 2.5% glutaraldehyde solution to each well and incubating at 37°C for 10 minutes. The cells were then washed three times with PBS and the coverslips removed. The prepared samples were then inverted and examined under a confocal microscope.

[0112] Figure 15 SiQDs-(K / LP) prepared in step 3 of Example 2 ox Solution for detecting endogenous peroxynitrite imaging in HepG2 cells. The probe in the figure refers to SiQDs-(K / LP) ox As shown in the figure, HepG2 cells were exposed to SiQDs-(K / LP) ox After incubation for 30 min, strong yellow fluorescence was observed at 514 nm. In addition, when the cells were stimulated to produce ONOO by adding 10 μg / mL LPS, the ONOO - When the yellow fluorescence intensity decreased after 1 h and was almost completely quenched after 2 h, a control experiment was performed by adding DMSO (300 μM) before incubating cells with LPS to prevent the endogenous ONOO - After incubation with DMSO and LPS for 1 h and 2 h, the yellow fluorescence intensity of the 514 nm channel did not change, indicating that the observed fluorescence changes were due to ONOO - The data in the pseudo-color image (Spec3) channel intuitively show the SiQDs-(K / LP) ox The change of fluorescence intensity.

Claims

1. A method for preparing chiral silicon quantum dots for detecting peroxynitrite concentration, characterized in that It is carried out in the following steps:

1. Preparation of Chiral Melanin-like Particles (K / P) by Oxidation ox Solution: ① Dissolve Lys-Tyr-Phe tripeptide in double distilled water to obtain a tripeptide solution, adjust the pH of the tripeptide solution to 5-8, and stir to obtain a translucent hydrogel; dissolve mushroom tyrosinase in phosphate buffer to obtain a mushroom tyrosinase solution; adding a mushroom tyrosinase solution to the translucent hydrogel and stirring, then adding phenylalanine and stirring to obtain a mixed solution, and centrifuging the mixed solution to obtain a supernatant; The phenylalanine is D-phenylalanine or L-phenylalanine; The volume ratio of the translucent hydrogel to the mushroom tyrosinase solution is 1:(0.1-0.3); the mass ratio of the Lys-Tyr-Phe tripeptide to phenylalanine is 1:(1-3); ② Centrifuge the supernatant to obtain aqueous phase I and solid phase I; ③. Add double distilled water to solid phase I and sonicate, then centrifuge to obtain aqueous phase II and solid phase II; ④. Repeat step 1 ③ for 2 to 3 times on solid phase II, then combine aqueous phase I and aqueous phase II to obtain (K / P) ox solution; 2. Preparation of SO3-(K / P) by stirring method ox : (K / P) ox The pH of the solution was adjusted to 9-10, and then 1,4-butane sultone was added dropwise, stirred and centrifuged to obtain SO3-(K / P) ox Mixing solution; 3. Preparation of SiQDs-(K / P) by hydrothermal method ox : Under stirring, SO3-(K / P) ox The solution was mixed with N-[3-(trimethoxysilyl)propyl]ethylenediamine, and then reacted at a temperature of 180°C to 200°C for 2h to 6h, and finally taken out and cooled to room temperature, and SiQDs-(K / P) was obtained after dialysis. ox solution; The SiQDs-(K / P) ox SiQDs-(K / P) in solution ox The concentration is 4.05mg / mL~8.1mg / mL.

2. The method for preparing chiral silicon quantum dots for detecting peroxynitrite concentration according to claim 1, characterized in that The concentration of Lys-Tyr-Phe tripeptide in the tripeptide solution described in step 1① is 10mM to 30mM; the concentration of the phosphate buffer described in step 1① is 10mM to 100mM, and the pH is 5 to 8; the enzyme activity of the mushroom tyrosinase solution described in step 1① is 50U / mL to 100U / mL.

3. The method for preparing chiral silicon quantum dots for detecting peroxynitrite concentration according to claim 1, characterized in that The centrifugation described in steps 1 to 3 and step 2 is specifically performed at a speed of 10,000 rpm to 15,000 rpm for 5 to 10 minutes; the volume ratio of solid phase I to double distilled water described in step 1 3 is 1:(2 to 3); the ultrasound described in step 1 3 is specifically performed at a power of 50 W to 100 W for 5 to 10 minutes.

4. The method for preparing chiral silicon quantum dots for detecting peroxynitrite concentration according to claim 1, characterized in that As described in step 1④ (K / P) ox In solution (K / P) ox The concentration is 16.2mg / mL~32.4mg / mL.

5. The method for preparing chiral silicon quantum dots for detecting peroxynitrite concentration according to claim 1, characterized in that In step 2, a NaOH solution with a concentration of 0.01M to 0.1M is used to convert (K / P) ox The pH of the solution was adjusted to 9-10.

6. The method for preparing chiral silicon quantum dots for detecting peroxynitrite concentration according to claim 1, characterized in that In step 2, 1,4-butane sultone is added dropwise at a stirring speed of 5 r / s to 20 r / s and a dropping speed of 0.1 μL / s to 0.25 μL / s.

7. The method for preparing chiral silicon quantum dots for detecting peroxynitrite concentration according to claim 1, characterized in that SO3-(K / P) described in step 2 ox SO3-(K / P) in solution ox The concentration is 18.2mg / mL~34.4mg / mL.

8. The method for preparing chiral silicon quantum dots for detecting peroxynitrite concentration according to claim 1, characterized in that (K / P) as described in step 2 ox The volume ratio of the solution to 1,4-butanesulfonate is 1:(0.03-0.015).

9. The method for preparing chiral silicon quantum dots for detecting peroxynitrite concentration according to claim 1, characterized in that SO3-(K / P) described in step 3 ox The volume ratio of the solution to N-[3-(trimethoxysilyl)propyl]ethylenediamine is 1:(0.2-0.4).

10. The method for preparing chiral silicon quantum dots for detecting peroxynitrite concentration according to claim 1, characterized in that The dialysis described in step 3 is specifically performed in a dialysis bag with a molecular weight cut-off of MW=500Da for 6 hours to 12 hours.

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