CN111590087B

CN111590087B - Preparation method of fluorescent gold nanocluster, prepared fluorescent gold nanocluster and application thereof

Info

Publication number: CN111590087B
Application number: CN202010498703.7A
Authority: CN
Inventors: 张群林; 方胡凤; 余欢; 卢麒; 方栒
Original assignee: Anhui Medical University
Current assignee: Anhui Medical University
Priority date: 2020-06-04
Filing date: 2020-06-04
Publication date: 2023-04-18
Anticipated expiration: 2040-06-04
Also published as: CN111590087A

Abstract

The invention discloses a preparation method of a fluorescent gold nanocluster, which relates to the technical field of material preparation and comprises the following steps: the fluorescent gold nanocluster is prepared by reacting chloroauric acid with polyphenol solution by taking polysaccharide as a template. The invention also discloses a preparation method of the fluorescent gold nanocluster and the catechol-borate compound fluorescent gold nanocluster prepared by the preparation method, the prepared catechol-borate compound fluorescent gold nanocluster and application of the catechol-borate compound fluorescent gold nanocluster as a fluorescent probe. The invention has the beneficial effects that: the invention takes the polysaccharide as a template and the polyphenol as a reducing agent to prepare the fluorescent gold nanocluster, which can enhance the solubility and the dispersibility of the obtained fluorescent gold nanocluster; boric acid is used as a protective agent, the prepared catechol-borate compound fluorogold nanocluster has good biocompatibility, physiological requirements are well met, and the catechol-borate compound fluorogold nanocluster has high selectivity in detection of high active oxygen level change in cells.

Description

Preparation method of fluorescent gold nanocluster, prepared fluorescent gold nanocluster and application thereof

Technical Field

The invention relates to the technical field of material preparation, in particular to a preparation method of a fluorescent gold nanocluster, the prepared fluorescent gold nanocluster and application thereof.

Background

With the rapid development of nano technology, the nano fluorescent material as a new fluorescent material draws more and more extensive attention of researchers, and provides a good development opportunity for the design and development of nano biosensors. At present, the widely studied fluorescent nano materials mainly include carbon dots, graphene quantum dots, semiconductor quantum dots, fluorescent metal nanoclusters and the like. The fluorescent metal nanocluster as a new fluorescent material has the advantages of unique optical characteristics, low biological toxicity, good light irradiation stability and biocompatibility, larger Stokes shift, simple and convenient preparation method and the like, and the excellent properties enable the fluorescent metal nanocluster to have wide application prospects in the fields of biological small molecule sensing, biomarker imaging, biomedicine and the like. However, the prior art has the problems of poor dispersibility and solubility of the fluorescent gold nanoclusters and low quantum yield, and the application of the gold nanoclusters is influenced.

Synthesis using template molecules is one of the most effective methods to improve the fluorescence quantum yield of metal nanoclusters. Until now, a large number of templated nanoclusters have been synthesized, for example, patent publication No. CN105441069A discloses a method for synthesizing high fluorescence gold nanoclusters by using small molecules as templates, and silver ions are introduced to synthesize alloy copper-silver nanoclusters.

The zwitterionic compound containing carboxyl and amino is used as a nanocluster ligand, so that the biocompatibility of the nanocluster can be greatly improved, and the fluorescence characteristic of the nanocluster can be remarkably enhanced. Common zwitterionic compounds for synthesizing metal nano cluster particles mainly comprise proteins, polypeptides and some biological small molecules such as amino acids, and the like, in small molecular ligands, the amino acids and amino acid analogues have both protection and reducibility, and simultaneously contain carboxyl and amino electron-rich groups, so that the amino acid and amino acid analogues can be used as most effective surface ligands for promoting fluorescence enhancement, but the applicability of the fluorescent gold nano cluster is poor at physiological pH.

Reactive Oxygen Species (ROS) are produced in metabolic processes and are important signal molecules in biological systems that regulate a variety of physiological functions. However, over-expression of ROS in vivo can lead to oxidative stress, a trigger factor for the induction of a number of diseases.

Active oxygen and active nitrogen (RNS) are in a wide variety. Representative active oxygen is mainly H ₂ O ₂ 、HOCl、·OH、 ¹ O ₂ 、O ₂ ^·- And the like. The representative active nitrogen is mainly ONOO ^- 、HNO、NO·、·NO ₂ And so on. HOCl,. OH, ONOO ^- The three active substances have strong oxidizing power and can be directly usedNucleic acids, proteins, lipids, etc., are oxidized to cause irreparable damage and various serious diseases, and are collectively called high reactive oxygen species (ros). Active molecules in vivo are susceptible to interconversion under certain conditions.

Common detection methods for active oxygen and active nitrogen can be divided into three major categories: electron Spin Resonance (ESR), chromatography (Chromatography), and Spectroscopy (spectrometry). In recent years, the measurement of the hROS tends to be higher in sensitivity and more suitable for a fluorescence analysis method, but the molecular fluorescent probe has the defects of complex synthesis steps, poor photobleaching resistance, high biotoxicity and sensitivity of spontaneous autoxidation, and the presence of other ROS or RNS generates certain interference on the detection of the hROS, so that the analysis selectivity is poor and the like, and further application of the molecular fluorescent probe in the field of bioanalysis and in biological imaging research is limited.

Disclosure of Invention

One of the technical problems to be solved by the invention is that the existing fluorescent gold nanoclusters (AuNCs) are poor in dispersibility and solubility, and the preparation method of the fluorescent gold nanoclusters using the polysaccharide as the template is provided.

The invention solves the technical problems through the following technical means:

a preparation method of a fluorescent gold nanocluster comprises the following steps: and (3) reacting chloroauric acid with a polyphenol solution by taking polysaccharide as a template to prepare the fluorescent gold nanocluster.

Has the advantages that: the method takes the polysaccharide as a template and the polyphenol as a reducing agent to prepare the fluorogold nanocluster, and can enhance the solubility and the dispersibility of the obtained fluorogold nanocluster.

Preferably, the preparation method of the fluorescent gold nanocluster comprises the following steps: dissolving chloroauric acid in water to prepare chloroauric acid aqueous solution, mixing polysaccharide and chloroauric acid aqueous solution, heating to boiling state, adding polyphenol solution, reacting and cooling to obtain the fluorescent gold nanocluster.

Preferably, a buffer solution is added in the preparation process of the fluorescent gold nanocluster to prepare the catechol-borate complex fluorescent gold nanocluster;

the preparation method of the catechol-borate compound fluorescent gold nanocluster comprises the following steps: and (2) taking polysaccharide as a template, adding a buffer solution after the chloroauric acid reacts with the polyphenol solution, and further reacting to obtain the fluorescent gold nanocluster, wherein the buffer solution comprises one or two of boric acid and borate.

Has the beneficial effects that: boric acid is introduced into polysaccharide templated fluorescent AuNCs, and the function of the boric acid is mainly embodied in two aspects: on one hand, the formation of polyphenol borate complex between polyphenol and boric acid enhances the electron donor capability of the surface ligand of the gold nanocluster to a certain extent, and increases the density of delocalized electron cloud, so that the fluorescence of the gold nanocluster is enhanced; on the other hand, the formed polyphenol borate complex blocks or inhibits the oxidation of hydroxyl on polyphenol through strong covalent binding, and the stability of the gold nanocluster at physiological pH is further enhanced.

Preferably, the preparation method of the catechol-borate complex fluorescent gold nanocluster comprises the following steps:

(1) Dissolving chloroauric acid in water to prepare a chloroauric acid aqueous solution, mixing polysaccharide and the chloroauric acid aqueous solution, heating to a boiling state, adding a polyphenol solution, reacting and cooling to prepare the fluorescent gold nanocluster;

(2) And (2) mixing the fluorescent gold nanocluster prepared in the step (1) with a buffer solution for reaction to prepare the catechol-borate compound fluorescent gold nanocluster.

Preferably, the polysaccharide is one or more of soluble starch, glycogen, cellulose, chitosan, cyclodextrin, hyaluronic acid and chondroitin sulfate, and the polyphenol is one or more of catechol or a catechol derivative.

Preferably, the polysaccharide is soluble starch and the polyphenol is levodopa (L-DOPA).

Preferably, the buffer is a borate buffer.

Has the advantages that: the pre-dissolution of the chloroauric acid in the starch enhances the solubility and dispersibility of the fluorescent gold nanoclusters. Boric acid was introduced into starch templated AuNCs, and its role was mainly reflected in two aspects: on one hand, the formation of catechol-borate complex between polyphenol and boric acid enhances the electron donor capability of the surface ligand of the gold nanocluster to a certain extent, and increases the density of delocalized electron cloud, so that the fluorescence of the gold nanocluster is enhanced; on the other hand, the formed catechol-borate complex blocks or inhibits oxidation and adhesion of catechol on L-DOPA through strong covalent bonding, and further enhances the stability of the gold nanoclusters under physiological pH.

Preferably, the soluble starch is subjected to pre-gelatinization treatment to prepare pre-gelatinized starch, and the concentration of the pre-gelatinized starch in chloroauric acid aqueous solution is 0.3-1.5%; dissolving levodopa in water to prepare a levodopa aqueous solution, wherein the concentration of the levodopa aqueous solution is 0.5-4mM, the pH of a borate buffer solution is 7.2-9, the concentration of boric acid in the borate buffer solution is 0.1-1M, and the volume ratio of the fluorogold nanoclusters to the buffer solution is 0.1-4.

Preferably, the concentration of the pregelatinized starch in the chloroauric acid aqueous solution is 1.25%, the concentration of the levodopa solution is 3.45mM, the concentration of boric acid in the borate buffer solution is 0.2M, and the volume ratio of the fluorescent gold nanoclusters to the borate buffer solution is 2.

The second technical problem to be solved by the invention is the fluorescent gold nanocluster prepared by the preparation method.

Has the beneficial effects that: the prepared fluorescent gold nanocluster can enhance the solubility and the dispersibility of the fluorescent gold nanocluster.

The invention aims to solve the third technical problem that the existing fluorogold nanoclusters are poor in stability under the physiological pH condition and cannot meet the physiological requirement, and provides a preparation method of the catechol-borate ester compound fluorogold nanocluster.

A preparation method of catechol-borate compound fluorescent gold nanoclusters comprises the following steps: taking polysaccharide as a template, adding a buffer solution for further reaction after chloroauric acid reacts with a polyphenol solution to prepare the catechol-borate compound fluorescent gold nanocluster; the polyphenol in the polyphenol solution is one or more of catechol or catechol derivatives; the buffer solution comprises one or two of boric acid and borate.

Has the advantages that: boric acid is a common lewis acid that can end-cap L-DOPA via covalent bonds to form a polyphenol-borate complex.

Preferably, the polysaccharide is one or more of soluble starch, glycogen, cellulose, chitosan, cyclodextrin, hyaluronic acid and chondroitin sulfate.

Preferably, the polysaccharide is soluble starch, the polyphenol is levodopa, and the buffer is borate buffer.

Has the advantages that: boric acid is a common lewis acid that can end-cap L-DOPA via covalent bond formation of a catechol-boronate complex. Not only blocks spontaneous oxidation of L-DOPA under physiological pH, but also inhibits the aggregation tendency of nanoclusters caused by the adhesion property of catechol. The synthesis reaction time of the catechol-borate complex fluorogold nanocluster is only 5min.

Preferably, the soluble starch is subjected to pre-gelatinization treatment to prepare pre-gelatinized starch, the concentration of the pre-gelatinized starch in a chloroauric acid aqueous solution is 0.3-1.5%, levodopa is dissolved in water to prepare a levodopa aqueous solution, the concentration of the levodopa aqueous solution is 0.5-4mM, the pH of a borate buffer solution is 7.2-9, the concentration of boric acid in the borate buffer solution is 0.1-1M, and the volume ratio of the fluorescent gold nanoclusters to the borate buffer solution is 0.1-4.

The fourth technical problem to be solved by the invention is to provide the catechol-borate ester compound fluorescent gold nano-cluster prepared by the preparation method.

Has the beneficial effects that: the catechol-borate complex fluorescent gold nanocluster prepared by the invention has strong cyan fluorescence emission at 480nm, the fluorescence quantum yield is 2.8%, the particle size is small, the diameter is about 2.9nm, the size is uniform, and strong fluorescence stability can be still kept after 386nm strong light irradiation for 30 min.

The catechol-borate compound fluorescent gold nanocluster prepared by the method has good monodispersity, the average diameter is 2.9 +/-0.8 nm, the size is uniform, and the dispersion is uniform. The prepared catechol-borate complex fluorogold nanocluster contains 36% of Au ⁺ This plays a very important role in maintaining the stability of the catechol-borate complex fluorogold nanoclusters.

The defect that levodopa is easily oxidized into quinone under physiological conditions is overcome, the pH stability of the gold nanoclusters is further enhanced in a PBS (phosphate buffer solution) with the pH range of 7-10, the fluorescence intensity of the catechol-borate complex fluorogold nanoclusters is kept highest, and the catechol-borate complex fluorogold nanoclusters are proved to have good biocompatibility and better meet physiological required conditions.

The fluorescence intensity of the catechol-boronate complex fluorogold nanoclusters remains stable even at high temperature (60 ℃) and high ionic strength (800 mM NaCl). After half an hour of continuous irradiation, the fluorescence of the catechol-boronate complex fluorogold nanoclusters slightly decreased initially and then stabilized for a long time.

After being stored for 2 months, the fluorescence intensity of the catechol-borate complex fluorescent gold nano-cluster is only reduced by 10%, the anti-aggregation stability is good, and the catechol-borate complex fluorescent gold nano-cluster is well dispersed and dissolved in an aqueous solution.

The fifth technical problem to be solved by the invention is to provide the application of the catechol-borate compound fluorescent gold nanocluster prepared by the preparation method as a fluorescent probe.

Has the advantages that: the existing molecular fluorescent probe has poor photobleaching resistance, high biological toxicity and sensitivity of spontaneous autoxidation, often has the defects of poor selectivity, certain interference of other ROS or RNS and the like, and limits the further application of the molecular fluorescent probe in-vivo research.

The catechol-borate complex fluorescent gold nanoclusters are small in particle size, uniform in size, good in stability under physiological conditions, good in light irradiation resistance and low in cytotoxicity, and the high stability of the catechol-borate complex fluorescent gold nanoclusters greatly promotes the application of the catechol-borate complex fluorescent gold nanoclusters in fluorescent probes under physiological conditions.

Preferably, the catechol-borate complex fluorogold nanocluster is applied to biological imaging as a fluorescent probe.

Preferably, the catechol-borate complex fluorogold nanocluster is applied to specific detection of high-activity oxygen.

Preferably, the catechol-borate complex fluorescent gold nanocluster is combined with o-phenylenediamine for specific detection of high-activity oxygen.

Preferably, the catechol-borate complex fluorescent gold nanocluster is combined with o-phenylenediamine to serve as a ratio fluorescent probe for specifically detecting high-activity oxygen.

Has the beneficial effects that: the catechol-borate complex fluorescent gold nanocluster and the hROS are subjected to indirect reaction, so that unnecessary oxidation reaction of the catechol-borate complex fluorescent gold nanocluster and other ROS is avoided to a certain extent, and the specificity of the catechol-borate complex fluorescent gold nanocluster/o-phenylenediamine sensing hROS is increased. The method for detecting hROS by using the catechol-borate complex fluorescent gold nanocluster/o-phenylenediamine has high selectivity, and greatly meets the requirement of monitoring hROS in a complex biological sample.

The catechol-borate complex fluorescent gold nanocluster/o-phenylenediamine sensing system can detect the change of hROS through the change of two fluorescence signal intensities, and has the advantages of strong selectivity, high response value, good light resistance, high sensitivity of visual imaging monitoring of the hROS level in a living body and good reproducibility.

Preferably, the high active oxygen is OH, clO ^- Or ONOO ^- 。

Preferably, the step of detecting high reactive oxygen species comprises:

(1) Dissolving o-phenylenediamine in water to prepare an o-phenylenediamine aqueous solution, mixing the o-phenylenediamine aqueous solution with a phosphate buffer solution, adding hROS, and carrying out mixed reaction;

(2) Then adding catechol-borate compound fluorescent gold nano-cluster for reaction, and performing fluorescence detection under the excitation of 386nm.

The detection principle is as follows: OPD can be oxidized to 2, 3-Diaminophenazine (DAP), giving a new absorption peak at 430nm and a yellow fluorescence emission at 560nm, the cyan fluorescence of the catechol-boronate complex fluorogold nanoclusters can decrease with increasing concentration of hROS, with a distinguishable change in fluorescence color from cyan (480 nm) to yellow (560 nm), the ratio of fluorescence intensities at 560nm and 480nm (I ₅₆₀ _nm /I ₄₈₀ _nm ) And the concentration of hROS is in a linear relation, so that the catechol-borate compound fluorogold nano-cluster becomes an ideal sensor for quantifying hROS.

Has the advantages that: for OH and ClO ^- And ONOO ^- The detection limits (at SNR of 3) of the ratiometric sensors were calculated to be 0.11. Mu.M, 0.50. Mu.M and 0.69. Mu.M, respectively, and the response concentration ranges covered the normal physiological concentration range of hROS in cells, indicating that SLB-AuNCs have high sensitivity in detecting hROS under physiological conditions. The fluorescence emission bands of SLB-AuNCs and DAP do not interfere with each other, so that the SLB-AuNCs/OPD sensing system can detect the change of hROS through the change of the intensity of two fluorescence signals, and has strong selectivity and high response value.

Preferably, the concentration of the o-phenylenediamine aqueous solution is 0.01-20mM, the reaction time in the step (1) is 1-30min, and the reaction time in the step (2) is 1-25min.

Preferably, the concentration of the o-phenylenediamine aqueous solution is 10mM, the reaction time in the step (1) is 20min, and the reaction time in the step (1) is 20min.

Has the advantages that: concentration of OPD, reaction time required for hROS to oxidize OPD (t) ₁ ) And the duration of action (t) of the OPD oxidation products DAP and SLB-AuNCs ₂ ) The method plays an important role in the signal-to-noise ratio and the sensitivity of the sensor analysis and detection, the larger the fluorescence intensity ratio along with the increase of the OPD concentration in the hROS sensing system, the more gradual the trend of the increase of the fluorescence intensity ratio when the OPD concentration is more than 10mM, and therefore 10mM OPD is selected as the condition of the subsequent experiment. Reaction time required for hROS to oxidize OPD (t) ₁ ) And the reaction time (t) of the OPD oxidation product DAP with SLB-AuNCs ₂ ) The change of the fluorescence intensity ratio is similar to the change of OPD concentration, the ratio increase trend in short time is obvious, and when t is ₁ 、t ₂ When the time reaches 20min, the trend of increasing the fluorescence intensity ratio is not obvious. Therefore, t is selected ₁ ＝20 min，t ₂ =20min as optimum reaction conditions.

Preferably, the application of the catechol-borate complex fluorescent gold nanocluster in combination with o-phenylenediamine in specific detection of high active oxygen comprises organism endogenous high active oxygen monitoring and organism exogenous high active oxygen monitoring.

Preferably, the method for monitoring endogenous hyperreactive oxygen species of an organism comprises the following steps:

(1) After HeLa cell culture, stimulation treatment with Lipopolysaccharide (LPS), then stimulation treatment with phorbol ester (PMA), and after adding OPD culture, adding SLB-AuNCs culture;

(2) And (3) monitoring the fluorescence change in the cells, setting the excitation wavelength to be 405nm, and observing the fluorescence imaging conditions of two channels, namely a cyan channel (450-480 nm) and a yellow channel (560-620 nm).

Has the advantages that: the change in hROS was detected by a change in the intensity of both fluorescent signals, and when the fluorescent signal was detected in the yellow channel, the cyan channel was almost completely quenched, indicating the intracellular production of hROS. The SLB-AuNCs/OPD sensing system is suitable for visually monitoring the endogenous hROS level in living cells.

Preferably, the method for monitoring the in vitro sexual source hROS comprises the following steps:

(1) Adding OPD for culture after HeLa cell culture, and then adding hROS for incubation;

(2) Washing the cells with phosphate buffer, and adding SLB-AuNCs into the cells for culture;

(3) And (3) monitoring the fluorescence change in the cells, setting the excitation wavelength to be 405nm, and observing the fluorescence imaging conditions of two channels, namely a cyan channel (450-480 nm) and a yellow channel (560-620 nm).

Has the advantages that: the pyrocatechol-borate complex fluorescent gold nanoclusters are added with hROS for pretreatment, the cyan channel fluorescent signals almost disappear, bright yellow emission is observed in a yellow channel, the pyrocatechol-borate complex fluorescent gold nanocluster/OPD sensing system regulates specificity to the hROS signals, the fluorescence of the pyrocatechol-borate complex fluorescent gold nanoclusters is rapidly and thoroughly quenched by the OPD oxidized by the hROS, and the endogenous hROS level is successfully detected.

The invention has the advantages that:

(1) The invention takes the polysaccharide as a template and the polyphenol as a reducing agent to prepare the fluorescent gold nanocluster, and can enhance the solubility and the dispersibility of the obtained fluorescent gold nanocluster.

(2) Boric acid is a common lewis acid that can end-cap levodopa via covalent bond formation of a polyphenol-borate complex.

(3) The method improves the defect that levodopa is easily oxidized into quinone under physiological conditions, further enhances the pH stability of the gold nanocluster in a PBS buffer solution with the pH range of 7-10, and ensures that the fluorescence intensity of the catechol-borate complex fluorescent gold nanocluster is kept highest, so that the catechol-borate complex fluorescent gold nanocluster has good biocompatibility and better meets the physiological requirements.

(4) According to the invention, the catechol-borate complex fluorescent gold nanocluster is used for detecting the level change of high active oxygen (hROS) in cells, and the OPD and the hROS are directly interacted, so that unnecessary reaction of the catechol-borate complex fluorescent gold nanocluster and other active oxygen active nitrogen is avoided to a certain extent, and the high selectivity is achieved.

Drawings

FIG. 1 is a fluorescence emission spectrum of L-DOPA synthesized AuNCs (LD-AuNCs), starch templated LD-AuNCs (SLD-AuNCs) and L-DOPA-borate complex functionalized SLD-AuNCs (SLB-AuNCs) in example 8 of the present invention; in the figure, a represents LD-AuNCs; b represents SLD-AuNCs; c represents SLB-AuNCs.

FIG. 2 is a photograph of AuNCs synthesized with and without starch in example 8 of the present invention; wherein FIG. 2A is the addition of starch; fig. 2B shows no starch added.

FIG. 3 is a graph showing the effect of various reaction conditions on the synthesis of SLB-AuNCs in example 10 of the present invention; wherein FIG. 3A represents starch concentration; FIG. 3B shows the L-DOPA concentration, and FIG. 3C shows the boric acid concentration; FIG. 3D shows the volume ratio of boric acid to SLD-AuNCs; FIG. 3E shows the pH of borate buffer; FIG. 3F shows the reaction time.

FIG. 4 is a transmission electron microscope image and size distribution chart of SLB-AuNCs in example 10 of the present invention; wherein FIG. 4A shows transmission electron microscopy imaging of SLB-AuNCs, inset is a close-up of the crystal structure of a single SLB-AuNCs gold cluster; FIG. 4B shows the size distribution of SLB-AuNCs.

FIG. 5 is an X-ray photoelectron spectrum (Au 4 f) of SLB-AuNCs in example 10 of the present invention; wherein a represents the original map, 4f _7/2 And 4f _5/2 Spectral decomposition to correspond to Au ⁰ (vertical line filling) and Au ⁺ (horizontal line filling) two parts.

FIG. 6 is a Fourier transform infrared spectrum of SLB-AuNCs in example 10 of the present invention.

FIG. 7 is a graph showing an ultraviolet absorption spectrum, a fluorescence emission spectrum and a fluorescence lifetime degradation curve of a product in example 10 of the present invention; wherein FIG. 7A shows the ultraviolet absorption spectrum (red line) and fluorescence emission spectrum (blue line) of SLB-AuNCs, and the excitation wavelength is 386nm. Illustration is shown: photographs of SLB-AuNCs under (a) ambient light and (b) a 365nm UV lamp;

FIG. 7B is a UV absorption spectrum of levodopa, boric acid, levodopa-borate complex and SLB-AuNCs; FIG. 7C is the fluorescence lifetime decay curve (excitation at 386 nm) of SLB-AuNCs.

FIG. 8 is a graph showing the results of stability measurement of SLB-AuNCs in example 11 of the present invention; wherein FIG. 8A is a graph showing the effect of pH on the fluorescence intensity of SLB-AuNCs; FIG. 8B is a graph showing the effect of temperature on the fluorescence intensity of SLB-AuNCs; FIG. 8C is a graph showing the effect of ion intensity on the fluorescence intensity of SLB-AuNCs; FIG. 8E is a graph showing the effect of two-month storage time on the fluorescence intensity of SLB-AuNCs; the upper curve in fig. 8E represents 0 days and the lower curve represents 2 months.

FIG. 9 is a graph showing the effect of pH on the fluorescence intensity of SLD-AuNCs and SLB-AuNCs.

FIG. 10 shows a reaction solution of OPD and OH (consisting of H) in example 12 of the present invention ₂ O ₂ And HRP generated).

FIG. 11 is a graph showing the results of measurement of high reactive oxygen species using the fluorescent gold nanoclusters SLD-AuNCs in combination with o-phenylenediamine as a fluorescent probe in example 12 of the present invention, in which FIG. 11A is a picture of the reaction solution when different concentrations of hROS are added under ambient light and UV light; FIG. 11B SLB-AuNCs/OPD vs ClO at different concentrations ^- A fluorescence spectral response map of (a); FIG. 11C is a working curve of SLB-AuNCs/OPD ratio sensor for detecting hROS.

FIG. 12 is a graph showing the results of detection of the hROS sensing mechanism in example 13 of the present invention; wherein FIG. 12A is the fluorescence spectra of SLB-AuNCs, SLB-AuNCs + OPD, SLB-AuNCs + DAP; FIG. 12B shows the UV absorption spectra of OPD, DAP, and the fluorescence spectra of SLB-AuNCs; FIG. 12C is a graph showing the decay of fluorescence lifetime of SLB-AuNCs, LB-AuNCs + DAP; FIG. 12D shows zeta potential measurement values of SLB-AuNCs, OPD, DAP and SLB-AuNCs + DAP.

FIG. 13 is a graph showing the results of various reaction conditions on the measurement of hROS in example 14 of the present invention; wherein FIGS. 13A, 13D, 13G show OPD concentrations; FIGS. 13B,13E,13H show hROS and OPD incubation times; FIG. 13C,13F, 13I shows the reaction time of DAP with SLB-AuNCs.

FIG. 14 is a graph showing the results of selective assays for sensing hROS in example 15 of the present invention.

FIG. 15 is a graph showing the survival rate of HeLa cells after 24 hours of treatment with different concentrations of SLB-AuNCs and OPD in example 16 of the present invention; wherein FIG. 15A shows SLB-AuNCs; fig. 15B shows OPD.

FIG. 16 shows the detection of exogenous hROS (ClO) in HeLa cells in example 16 of the present invention ^- As typical hrs) confocal fluorescence microscopy images; wherein the top image indicates that the cells were incubated with SLB-AuNCs and OPD; intermediate images show cells incubated with OPD and then with ClO ^- Incubation, and finally treatment with SLB-AuNCs; bottom image shows incubation with OPD after pretreatment of cells with NAC, followed by ClO ^- Treatment and final incubation with SLB-AuNCs was continued. The scale bar is equal to 20 μm.

FIG. 17 is a confocal fluorescence microscopy image of endogenous hROS in HeLa cells of example 16 of the present invention; where the top image shows the cells incubated with SLB-AuNCs and OPD. The intermediate image shows the cells pretreated with LPS and PMA and then incubated with OPD and SLB-AuNCs. Bottom panel shows cells pretreated with TEMPO together with LPS, PMA, and then incubated with OPD and SLB-AuNCs.

The scale bar is equal to 20 μm.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Test materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The specific techniques or conditions not specified in the examples can be performed according to the techniques or conditions described in the literature in the field or according to the product specification.

Example 1

Preparing fluorescent gold nanoclusters SLD-AuNCs:

(1) Pre-gelatinization of soluble starch: placing 0.25g of soluble starch in a round-bottom flask, adding 20mL of double distilled water for suspension, stirring and heating on an oil bath pot until the soluble starch is boiled, continuing to heat and boil for 20min after the soluble starch is heated to be transparent and has no obvious particles, and cooling to room temperature to obtain 1.25% of pregelatinized starch for later use;

(2) Thoroughly cleaning the glass instrument by using aqua regia (nitric acid/hydrochloric acid, volume ratio of 1;

(3) Preparing chloroauric acid aqueous solution containing 1.25% pregelatinized starch (final concentration of chloroauric acid aqueous solution is 8.6 × 10) ^-4 M), heating to a vigorous boiling with stirring, 7mL of 8.6X 10 ^-4 M aqueous chloroauric acid solution was added to 1mL of a 3.45X 10 solution ^-3 Keeping the L-3,4-dihydroxyphenylalanine (L-DOPA) in the water solution of M levodopa in a vigorous boiling state, and reacting for 5min. The color of the reaction mixture solution is changed from light yellow to dark brown, the reaction solution is cooled to room temperature, centrifuged at 13500rpm for 10min, reaction by-products with larger particle size are removed, and light brown supernatant is left, thus obtaining the fluorescent gold nanoclusters (Starch-plated L-DOPA synthesized gold nanoclusters) named as SLD-AuNCs.

The preparation method of the chloroauric acid aqueous solution containing 1.25% of pregelatinized starch comprises the following steps: diluting 1mL of chloroauric acid mother liquor with the concentration of 10mg/mL to 35mL by using 1.25% pregelatinized starch prepared in the step (1) to obtain the final concentration of 8.6 × 10 ^- ⁴ Aqueous solution of M in chloroauric acid.

Example 2

This embodiment is different from embodiment 1 in that: the aqueous solution of levodopa was replaced with an aqueous solution of dopamine, the concentration of which in this example was 0.6mM.

Example 3

This embodiment is different from embodiment 1 in that: the levodopa aqueous solution was replaced with a protocatechuic aldehyde aqueous solution, the concentration of which in this example was 1mM.

Example 4

This embodiment is different from embodiment 1 in that: the levodopa aqueous solution was replaced with an aqueous protocatechuic acid solution, the concentration of which in this example was 0.6mM.

Example 5

The present embodiment is different from embodiment 1 in that: the levodopa aqueous solution was replaced with an aqueous catechin solution, the concentration of which in this example was 0.6mM.

Example 6

This embodiment is different from embodiment 1 in that: the aqueous levodopa solution was replaced with an aqueous epicatechin solution having a concentration of 3mM in this example.

Example 7

Preparing fluorescent gold nanoclusters SLB-AuNCs:

the present embodiment is different from embodiment 1 in that: the SLD-AuNCs prepared in example 1 were mixed with borate buffer (boric acid-borax buffer, pH =7.4, boric acid concentration 0.2M) at a volume ratio of 2.

Comparative example 1

This comparative example differs from example 1 in that: no starch was added during the preparation.

Example 8

Effect of starch and boric acid on SLB-AuNCs: the fluorescence emission spectra and fluorescence quantum yields of the products prepared in comparative example 1, example 1 and example 2 were determined.

SLB-AuNCs fluorescence quantum yield determination method: calculated by comparing the integral of the fluorescence emission intensity with the absorbance of a quinine sulfate standard with a known quantum yield of 54%.

Dissolving quinine sulfate in 0.1M H ₂ SO ₄ Preparing a series of low-concentration quinine sulfate solutions. The absorbance of quinine sulfate with different concentrations at the excitation wavelength of the gold nanoclusters is less than or equal to 0.1. The refractive index of the gold nanocluster solution is almost the same as that of the quinine sulfate contrast solution, and is 1.33. The fluorescence quantum yield of SLB-AuNCs can be calculated according to the following equation:

wherein QY _ref Refers to the fluorescence quantum yield, eta and eta of the reference _ref The refractive indexes of the solution of the sample to be measured and the reference substance, I and I _ref Is the integrated integral area value of the fluorescence emission intensity of the sample to be detected and the reference substance, A and A _ref The ultraviolet absorbance values of a sample to be detected and a standard substance are shown.

(1) Template function of starch on SLB-AuNCs synthesis:

as shown in fig. 1, it can be seen that the fluorescence intensity of the produced nanoclusters was significantly increased after adding starch to the chloroauric acid solution before the reaction. In the figure, a represents comparative example 1, b represents example 1, c represents example 2.

The results of fluorescence quantum yield measurement are shown in table 1, using quinine sulfate control (QY =0.54, at 0.1 mh) ₂ SO ₄ Middle) the Quantum Yield (QY) of monodisperse SLB-AuNCs was 2.8%, and the fluorescence enhancement of the gold cluster demonstrated the signal amplification function of starch.

More significantly, as shown in FIG. 2, HAuCl ₄ Pre-dissolution in starch ultimately enhances the solubility and dispersibility of the resulting SLB-AuNCs, which is readily discernible to the naked eye, and the resulting synthetic AuNCs solution without starch has significant insoluble material sticking to the flask wall, which cannot be further dissolved by vigorous shaking. However, in the AuNCs solution synthesized using starch as a template, no insoluble matter was found on the flask wall, and the dispersibility of the fluorescent gold nanoclusters was promoted using starch as a template.

TABLE 1 SLB-AuNCs fluorescence quantum yield

(2) Capping of catechol group with boric acid: the reaction mechanism of L-DOPA with boric acid and the formation of the L-DOPA-borate complex are shown below:

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the formation of catechol borate complex between L-DOPA and boric acid enhances the electron donor capability of the surface ligand of the gold nanocluster to a certain extent, and increases the density of delocalized electron cloud, so that the fluorescence of the gold nanocluster is enhanced; on the other hand, the formed catechol borate compound blocks or inhibits the oxidation and adhesion performance of catechol on L-DOPA through strong covalent bonding, and the stability of the gold nanocluster at physiological pH is further enhanced. The invention takes polysaccharide as a synthesis template, polyphenol as a reducing agent and boric acid as a protective agent to protect o-dihydroxy and high-valence gold ions (Au) in the polyphenol ³ ⁺ ) Is reduced to Au by a reducing agent ⁰ And Au ⁺ Nucleation was performed to generate AuNCs.

As can be seen from b and c in fig. 1, the introduction of boric acid not only further increases the fluorescence intensity of the gold nanoclusters, but also changes the fluorescence color thereof from blue to cyan, and the maximum emission wavelength of the gold nanoclusters is red-shifted from 464nm to 480nm.

Example 9

Optimization of SLB-AuNCs synthesis conditions: the concentration of each reactant, the mixing ratio of SLD-AuNCs and boric acid, and the reaction time during the synthesis process were optimized.

Adjusting the concentration of the pregelatinized starch in the chloroauric acid aqueous solution to 0.3-1.5%, the concentration of the L-DOPA solution to 0.5-4mM, the pH value of the borate buffer solution to 7.2-9, the concentration of boric acid in the borate buffer solution to 0.1-1M, the volume ratio of the boric acid to the SLD-AuNCs to 0.1-4, and the reaction time to 1-20min.

As shown in FIG. 3, the fluorescence intensity of SLB-AuNCs depends on the concentration of starch, L-DOPA and borate buffer. The experimental results show that: when the starch concentration reached 1.25% (FIG. 3A), the L-DOPA concentration reached 3.45mM (FIG. 3B), and the borate buffer reached 0.2M (FIG. 3C), the fluorescence intensity of the SLB-AuNCs prepared by synthesis did not increase significantly any more until the plateau, and therefore, SLB-AuNCs were synthesized using 1.25% starch, 3.45mM L-DOPA, and 0.2M borate buffer. Next, boric acid was covalently complexed with the phthalic diamine group of L-DOPA by mixing SLD-AuNCs with borate buffer. As shown in FIG. 3D, the SLB-AuNCs fluorescence intensity gradually increased with the reaction volume ratio of borate buffer to SLD-AuNCs solution, and when the reaction volume ratio reached 0.5, the fluorescence intensity increased to a maximum value, and the fluorescence intensity started to decrease as the volume ratio continued to increase. Therefore, the optimal volume ratio of SLD-AuNCs to borate buffer is 2:1. as can be seen from the results in fig. 3E, the pH of the borate buffer had a negligible effect on the fluorescence intensity, and a physiological pH of 7.4 was chosen. Finally, the synthesis reaction of AuNCs only requires 5 minutes, as shown in FIG. 3F, and the longer the reaction time, the fluorescence intensity is not further enhanced.

In this example, FIG. 3A shows that when the concentration of pregelatinized starch in an aqueous chloroauric acid solution was 0.3 to 1.5%, the concentration of L-DOPA solution was 3.45mM, the pH of borate buffer was 7.4, the concentration of boric acid in borate buffer was 0.2M, the volume ratio of boric acid to SLD-AuNCs was 0.5, and the reaction time was 5min.

FIG. 3B shows that when the concentration of L-DOPA was 0.5 to 4mM, the concentration of pregelatinized starch in the aqueous chloroauric acid solution was 1.25%, the pH of the borate buffer was 7.4, the concentration of boric acid in the borate buffer was 0.2M, the volume ratio of boric acid to SLD-AuNCs was 0.5, and the reaction time was 5min.

FIG. 3C shows that when the concentration of boric acid in borate buffer was 0.1-1M, the concentration of pregelatinized starch in the aqueous solution of chloroauric acid was 1.25%, the concentration of L-DOPA was 3.45mM, the pH of borate buffer was 7.4, the volume ratio of boric acid to SLD-AuNCs was 0.5, and the reaction time was 5min.

FIG. 3D shows that when the volume ratio of boric acid to SLD-AuNCs is 0.1-4, the concentration of pregelatinized starch in the aqueous chloroauric acid solution is 1.25%, the concentration of L-DOPA solution is 3.45mM, the pH of borate buffer is 7.4, the boric acid concentration in borate buffer is 0.2M, and the reaction time is 5min.

FIG. 3E shows that when the pH of the borate buffer was 7.2-9 and the concentration of pregelatinized starch in the aqueous solution of chloroauric acid was 1.25%, the concentration of L-DOPA solution was 3.45mM, the concentration of boric acid in the borate buffer was 0.2M, the volume ratio of boric acid to SLD-AuNCs was 0.5, and the reaction time was 5min.

FIG. 3F shows that when the reaction time is 1 to 20min, the concentration of pregelatinized starch in the aqueous chloroauric acid solution is 1.25%, the concentration of L-DOPA solution is 3.45mM, the concentration of boric acid in borate buffer is 0.2M, the volume ratio of boric acid to SLD-AuNCs is 0.5, and the pH of borate buffer is 7.4.

Example 10

The SLB-AuNCs prepared in example 2 were characterized:

(1) Performing SLB-AuNCs microscopic morphology by using a Transmission Electron Microscope (TEM) and a high-resolution transmission electron microscope (HRTEM);

(2) Measuring the valence state of gold in the SLB-AuNCs by using an X-ray photoelectron spectroscopy (XPS) method;

(3) Verifying the surface ligand groups of the SLB-AuNCs by Fourier transform infrared spectroscopy (FT-IR) analysis;

(4) The optical properties of SLB-AuNCs were determined by fluorescence spectrophotometry, ultraviolet spectrophotometry and fluorescence lifetime analysis.

And (3) measuring results:

(1) As shown in fig. 4A, the close-up in the inset shows a lattice plane with a lattice spacing of 0.23nm, corresponding to the (111) lattice spacing of face-centered cubic Au. The specific particle size information of the SLB-AuNCs is obtained through image analysis and judgment of 200 single particles, and as shown in FIG. 4B, the prepared SLB-AuNCs have good monodispersity, the average diameter is 2.9 +/-0.8 nm, the size is uniform, and the dispersion is uniform. The relatively small size and good monodispersity of SLB-AuNCs suggest that our SLB-AuNCs have certain advantages for use in bioimaging.

(2) As shown in FIG. 5, the binding energies of 83.8eV and 87.5eV correspond to Au 4f _7/2 And Au 4f _5/2 Electronic track, these results clearly demonstrate Au ⁰ And Au ⁺ In the metal core and surface of SLB-AuNCs, and Au ⁰ And Au ⁺ From catechol group and Au in levodopa ³⁺ Reduction reaction between them. The data show that the AuNCs prepared contain 36% Au ⁺ This plays a very important role in maintaining the stability of SLB-AuNCs.

(3) As shown in fig. 6, the levodopa boronate complexes andthe presence of a starch template. 2933cm ^-1 The peak can be attributed to CH in starch ₂ C-H stretching vibration of 1025cm ^-1 The characteristic peak at (A) is due to C-O-C stretching vibration of the starch. 3378cm appeared in the FT-IR spectrum of SLB-AuNCs ^-1 And 3231cm ^-1 The strong absorption peak is derived from the intermolecular hydrogen bond O-H stretching vibration of starch and the N-H stretching vibration of amino group in L-DOPA attached to the surface. 1643cm in AuNCs infrared spectrum ^-1 And 1436cm ^-1 The characteristic peaks of (A) are derived from asymmetric stretching vibration of carboxyl group and C = C bond in benzene ring of L-DOPA. In addition, the asymmetric B-O stretching vibration is 1366cm ^-1 Characteristic band of (B), boric acid at 928cm ^-1 In-plane bending vibration of O-H and out-of-plane vibration of characteristic peak of borate ester 649cm ^-1 The clear characteristic peaks confirm the existence of levodopa borate complex in SLB-AuNCs.

(4) As shown in fig. 7A, the uv absorption spectrum has no characteristic absorption peak at 520nm indicating the absence of gold nanoparticles (aunps), while a distinct absorption peak at 285nm indicates the formation of gold nanoclusters. From the fluorescence spectrum of SLB-AuNCs, it was found that the maximum emission wavelength was 480nm (excitation at 386 nm). SLB-AuNCs have excellent water solubility and emit intense cyan fluorescence under ultraviolet light (365 nm) irradiation in an aqueous solution.

As shown in FIG. 7B, L-DOPA showed the maximum absorption band at 280 nm. However, the levodopa borate compound has an absorption peak at 285nm, and the accurate matching of the maximum absorption band between the levodopa borate compound and the SLB-AuNCs further proves that the SLB-AuNCs surface ligand is the levodopa borate compound according to the specific ultraviolet spectrum molecular characteristics of the nanocluster.

As shown in FIG. 7C, the mean lifetime of SLB-AuNCs was calculated to be 2.6ns by fitting a bi-exponential curve to the fluorescence lifetime decay curve (386 nm excitation) of SLB-AuNCs.

Example 11

Stability assay of SLB-AuNCs: the stability of SLB-AuNCs prepared in example 2 was measured with respect to pH, high temperature, ionic strength and continuous light.

As shown in FIG. 8A and FIG. 9, in the PBS buffer solution with pH range of 7-10, the fluorescence intensity of SLB-AuNCs is kept highest, and the fluorescence intensity of SLD-AuNCs is higher under acidic condition, which indicates that the physiological applicability of SLB-AuNCs is better than that of SLD-AuNCs, and the SLB-AuNCs has good biocompatibility.

FIGS. 8B and 8C show that the fluorescence intensity of SLB-AuNCs remains stable even at high temperature (60 ℃) and high ionic strength (800 mM NaCl). After half an hour of continuous irradiation, the fluorescence of SLB-AuNCs slightly decreased at the beginning, and then stabilized for a long time (FIG. 8D). As shown in FIG. 8E, after 2 months of storage, the fluorescence intensity of SLB-AuNCs decreased by only 10%, the anti-aggregation stability was good, and the SLB-AuNCs were well dispersed and dissolved in an aqueous solution. The experimental results show that the high stability of SLB-AuNCs will greatly facilitate their application in vitro and in vivo bioimaging under physiological conditions.

Example 12

The application of the fluorescent gold nanocluster SLD-AuNCs combined with o-phenylenediamine as a ratiometric fluorescent probe in detecting high active oxygen is as follows:

construction of SLB-AuNCs/OPD sensing system

(1) 200 μ L of 10mM OPD and 1300 μ L of 30mM phosphate buffer solution (PBS, naH) ₂ PO ₄ -Na ₂ HPO ₄ pH 7.4), then adding 100 mu L of hROS with different concentrations, fully and uniformly mixing, and reacting the mixed solution for 20min at room temperature;

(2) After that, 400. Mu.L of SLB-AuNCs were added to the mixture and incubated at room temperature for 20min, and finally, the fluorescence spectrum was recorded under excitation at 386nm (slit width 5/5 nm).

The experimental results are as follows: the invention introduces o-phenylenediamine (OPD) as a hROS sensitive reagent, the OPD can be oxidized into 2, 3-Diaminophenazine (DAP), and the oxidation product is confirmed to be the DAP by ESI-MS spectrum, as shown in figure 10, M/z =211.09 (M + H) ⁺ ) Consistent with the theoretical values of DAP.

As shown in FIG. 11A, where OPD is oxidized by hROS to produce DAP, the cyan fluorescence of SLB-AuNCs can decrease with increasing concentration of hROS, with a distinguishable change in fluorescence color from cyan (480 nm) to yellow (560 nm). As shown in FIG. 11B, the wavelength difference between the two emission peaks is 80nm, so that the mutual interference is reduced, and the resolution of the hROS ratio detection and the imaging analysis is improved.

As shown in FIG. 11C, the ratio of fluorescence intensities at 560nm and 480nm (I) ₅₆₀ _nm /I ₄₈₀ _nm ) The linear relation with the concentration of hROS shows that SLB-AuNCs is an ideal sensor for quantifying hROS. For OH and ClO ^- And ONOO ^- The detection limits (at SNR of 3) of the ratiometric sensors were calculated to be 0.11. Mu.M, 0.50. Mu.M and 0.69. Mu.M, respectively, and the response concentration ranges covered the normal physiological concentration range of hROS in the cells, indicating that SLB-AuNCs can detect hROS with high sensitivity under physiological conditions.

Example 13

Sensing mechanism for hROS detection

As shown in FIG. 12A, OPD had no effect on SLB-AuNCs fluorescence emission, while SLB-AuNCs decreased in fluorescence intensity and generated a new fluorescence emission peak at 560nm when hROS was present, indicating that hROS was the main cause of fluorescence change. The absorption spectrum of the OPD oxidation product DAP at 430nm overlapped with the fluorescence emission spectrum of SLB-AuNCs at 480nm, resulting in Fluorescence Resonance Energy Transfer (FRET) (FIG. 12B). To further confirm the FRET process, the fluorescence lifetime decay curve of SLB-AuNCs in the presence or absence of DAP was measured by a fluorescence lifetime meter. As shown in FIG. 12C, the fluorescence lifetime of SLB-AuNCs was changed from 2.60ns to 0.66ns in the presence of DAP, which demonstrates the presence of electron or energy transfer process between SLB-AuNCs and DAP. It is well known that a key factor in the occurrence of FRET is that the distance between the fluorescence donor and acceptor should be less than 10nm. The zeta potential measurement in FIG. 11D shows that the SLB-AuNCs surface is negatively charged and the zeta potential value is-10.5 mV. DAP carries an amino group and is electropositive. The zeta potential dropped significantly after SLB-AuNCs and DAP incubation, indicating that there was an electrostatic attraction between SLB-AuNCs and DAP. The electrostatic attraction shortens the distance between SLB-AuNCs and DAP, causing FRET to occur. All the results demonstrate that the mechanism for detecting hROS by SLB-AuNCs/OPD ratiometric fluorescence is fluorescence resonance energy transfer.

Example 14

Optimization of SLB-AuNCs/OPD sensing system: concentration of OPD, reaction time required for hROS to oxidize OPD (t) ₁ ) To be provided withAnd the reaction time (t) of OPD oxidation products DAP and SLB-AuNCs ₂ ) Plays an important role in the signal-to-noise ratio and sensitivity of the sensor analysis detection.

The concentration of the OPD aqueous solution was adjusted to 0.01-20mM, and the reaction time (t) required for oxidizing OPD by hROS was adjusted ₁ ) 1-30min, the reaction time (t) of OPD oxidation product DAP and SLB-AuNCs ₂ ) Is 1-25min.

As shown in FIG. 13, the fluorescence intensity ratio increased with the increase of OPD concentration in the hROS sensing system, and the trend of the increase of fluorescence intensity ratio tended to be flat when the concentration of OPD was more than 10mM, so 10mM OPD was selected as the condition for the subsequent experiment. Reaction time required for hROS to oxidize OPD (t) ₁ ) And the duration of action (t) of the OPD oxidation product DAP with SLB-AuNCs ₂ ) The change of fluorescence intensity ratio is similar to that caused by OPD concentration, the ratio increase trend in short time is obvious, and when t is ₁ 、t ₂ When the time reaches 20min, the trend of increasing the fluorescence intensity ratio is not obvious. Therefore, t is selected ₁ ＝20min，t ₂ And the reaction condition is optimal for 20min.

In this example, when the concentration of the OPD aqueous solution is 0.01-20mM, the reaction time (t) required for hROS to oxidize OPD ₁ ) 20min, the reaction time of the OPD oxidation product DAP and SLB-AuNCs (t) ₂ ) It is 20min.

Reaction time required when hROS oxidize OPD (t) ₁ ) The concentration of OPD aqueous solution is 10mM when the time is 1-30min, and the reaction time of OPD oxidation product DAP and SLB-AuNCs is t ₂ ) It is 20min.

When the OPD oxidation product DAP is reacted with SLB-AuNCs for a certain time (t) ₂ ) 1-25min, the concentration of OPD aqueous solution is 10mM, and the reaction time required for oxidizing OPD by hROS (t) ₁ ) It is 20min.

Example 15

Selective assay for sensing hROS

The selectivity is an important parameter for evaluating the anti-interference capability of the detection method, and the method is used for determining the influence of the potential interference substances on the specificity and the practicability of the SLB-AuNCs/OPD sensing system under the optimal condition. The potential interfering substances mainly comprise other active oxygen, active nitrogen, metal ions, small molecular substances in vivo and the like.

The results are shown in FIG. 14, in which FIG. 14 shows the SLB-AuNCs/OPD sensing system vs. hROS (150. Mu.M ONOO) ^- ；150μM ClO ^- (ii) a 50 μ M. OH), different organisms coexisting (1 mM), ratio of metal ions (1 mM) and other reactive oxygen species fluorescence response (0.5 mM), the ratio of fluorescence intensity reached 1.1 (ONOO) after the experimental group added with hROS ^- )、2.1(ClO ^- ) 2.4 (. OH) and the color of the solution itself and the color of the fluorescence under 365nm UV lamp illumination gradually changed to yellow. The fluorescence intensity ratio of other active oxygen, active nitrogen, metal ions and in vivo small molecular substances is almost consistent with that of a blank group, and the color of the solution and the color of fluorescence are not changed. The SLB-AuNCs/OPD method for detecting the hROS has high selectivity.

Example 16

SLB-AuNCs/OPD cell imaging monitoring of hROS levels: the preparation method of the active oxygen and active nitrogen test solution in the embodiment is the prior art.

(1) SLB-AuNCs and OPD cytotoxicity assays:

HeLa cell culture HeLa cells derived from human cervical cancer tissue were inoculated into a cell culture flask containing 5% of CO at 37 ℃ in a DMEM Medium (Dulbecco's Modified Eagle Medium) containing 10% fetal bovine serum, 100U/L penicillin and 100mg/L streptomycin ₂ The culture is carried out in a humidified incubator. The culture medium was changed every two days and used for the experiment after several days of culture. In this example, heLa cells were purchased from cell resource center of Shanghai academy of sciences of China.

Cytotoxicity assays for SLB-AuNCs with OPD

Cytotoxicity of SLB-AuNCs with o-phenylenediamine was determined by the MTT method. And (3) inoculating the HeLa cells on a 96-well plate, culturing for 24h, adding SLB-AuNCs and o-phenylenediamine with different concentrations respectively after the cells adhere to the wall, and then incubating for 24h. After cell culture, 20. Mu.L of 5mg mL was added to each well ^-1 Thiazole blue tetrazolium bromide (MTT) solution, and incubation in a carbon dioxide incubator was continued for 4h. After the termination of the incubation, the culture supernatant was removed from the wells of the plate and 150. Mu.L of DMSO was added to each well. Light in dark environment at room temperatureAnd rotating for 5min, and dissolving the precipitate formed by the reaction of the living cells and the MTT reagent. And finally, measuring the absorption value of each hole at 490nm by using an ultraviolet microplate reader. Cell viability was calculated according to the following formula:

cell viability (%) = (OD) _Treated /OD _Control )×100％，

Wherein OD _Treated And OD _Control The UV absorbance values measured in the microplate in the presence and absence of SLB-AuNCs and OPD, respectively. Finally, corresponding concentrations of SLB-AuNCs and OPD with cell viability higher than 85% were selected for cell imaging experiments.

(2) Determination of exogenous active oxygen of HeLa cell

The specific steps for monitoring the exogenous active oxygen of the HeLa cell are as follows: heLa was inoculated into a 15mm glass bottom 35mm wide petri dish containing 5% CO at 37% ₂ And culturing in a humid incubator for 24 hours. After the cell confluence reached 60%, 1mM OPD was added and cultured for 2h, then 300. Mu.M ClO was added ^- Incubate at 37 ℃ for 10min. After washing the cells three times with PBS, SLB-AuNCs (200. Mu.g mL) were added to the cells ^-1 ) And culturing for 4h. In the control group, after incubating the cells with OPD (1 mM) for 2h, adding 10mM of N-acetylcysteine (NAC) as a free radical scavenger for 25min, and then, incubating with ClO ^- (300. Mu.M) after 10min of cell treatment, SLB-AuNCs medium (200. Mu.g mL) ^-1 ) The cells were added and the culture was continued for 4h. The cells were washed with PBS wash solution, and the change in fluorescence was observed in each group of cells under a confocal laser microscope.

(3) Determination of endogenous active oxygen of HeLa cells

The specific steps for monitoring the endogenous active oxygen of the HeLa cell are as follows: heLa was inoculated into a 15mm glass bottom 35mm wide petri dish containing 5% CO at 37% ₂ And culturing in a humid incubator for 24 hours. After the cell confluence reached 60%, the experiment was performed. The experimental group was first treated with 1. Mu.g mL of cells ^-1 Lipopolysaccharide (LPS) stimulation treatment for 12h, followed by 10nM phorbol ester (PMA) stimulation of the cells for 1h. Then 1mM OPD was added and cultured for 2h, then 200. Mu.g mL was added to the cells ^-1 SLB-AuNCs were cultured for 4h. In the control group, the active oxygen scavenger was first treated with 1mM TEMPO and 1. Mu.g mL ^-1 LPS Co-productionThe same stimulation treatment was performed for 12h, and the cells were stimulated with 10nM PMA for 1h. Then after incubating the cells with 1mM OPD for 2h, 200. Mu.g mL of the cells were added ^-1 SLB-AuNCs were added to the cells and the culture was continued for 4h. The cells were washed with PBS wash solution, and the change in fluorescence was observed in each group of cells under a confocal laser microscope.

And (3) measuring results:

(1) SLB-AuNCs and OPD cytotoxicity: the biocompatibility of SLB-AuNCs was evaluated by studying the viability of viable cells 24h after exposure to different concentrations of SLB-AuNCs by the MTT method. As observed in FIG. 15A, when the SLB-AuNCs concentration was as high as 300. Mu.g mL ^-1 Cell viability was still higher than 85%. The SLB-AuNCs are proved to have small toxicity and good biocompatibility. The effect of OPD on HeLa cytotoxicity was investigated in the same way, with cell viability higher than 85% after 24h incubation with 1.5mM OPD (fig. 15B). The results indicate that low toxicity OPD should be used for cellular imaging as an indicator.

(2) Imaging and monitoring exogenous hROS of HeLa cells: and (3) observing the change of fluorescence signals caused by different concentrations of hROS in the HeLa cells by adopting a laser confocal fluorescence microscope, setting the excitation wavelength to be 405nm, and observing the fluorescence imaging conditions of two channels, namely a cyan channel (450-480 nm) and a green channel (560-620 nm). As shown in FIG. 15, SLB-AuNCs alone (200. Mu.g mL) was used in the control group ^-1 ) HeLa cells incubated with OPD (1 mM) showed bright cyan fluorescence (FIG. 16, A-D). Indicating that SLB-AuNCs rapidly entered the cells and showed a bluish emission of SLB-AuNCs. The experimental group was pretreated with hypochlorite before addition of SLB-AuNCs, the cyan channel fluorescence signal almost disappeared, while a bright yellow emission was observed in the yellow channel (FIG. 16, E-H). Furthermore, by pre-treating cells with the hROS scavenger N-acetylcysteine (NAC) (10 mM), the cyan fluorescence was recovered, demonstrating the signaling regulation specificity of the SLB-AuNCs/OPD sensing system for hROS (FIG. 16, I-L). The fluorescence of SLB-AuNCs is rapidly and completely quenched by OPD oxidized by hypochlorous acid, and the endogenous hROS can be successfully detected.

(3) Imaging and monitoring endogenous hROS of HeLa cells: cells were stimulated with LPS for 12h, then PMA for 1h, and finally OPD and SLB-AuNCs were added, and a clear increase in fluorescence in the yellow channel and almost complete quenching of the cyan channel was observed by FIG. 17, indicating that LPS/PMA stimulated intracellular hROS production. In contrast, in the control group, when TEMPO cells introduced as a radical scavenger were co-treated with LPS and PMA, fluorescence was restored in the cyan channel and no fluorescence was observed in the yellow channel (FIG. 17, I-L). The results show that TEMPO can effectively eliminate superoxide and inhibit the expression of myeloperoxidase, so that the intracellular hROS level is reduced, and further prove that the SLB-AuNCs/OPD sensing system is suitable for visually monitoring the endogenous hROS level in living cells.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention in its corresponding aspects.

Claims

1. A preparation method of catechol-borate compound fluorescent gold nanoclusters is characterized by comprising the following steps: the method comprises the following steps: taking polysaccharide as a template, reacting chloroauric acid with a polyphenol solution, adding a buffer solution, and further reacting to prepare the catechol-borate compound fluorescent gold nanocluster; the polyphenol in the polyphenol solution is one or more of catechol or catechol derivatives; the buffer solution comprises one or two of boric acid and borate.

2. The method for preparing catechol-borate complex fluorescent gold nanoclusters according to claim 1, wherein: the method comprises the following steps:

(1) Dissolving chloroauric acid in water to prepare chloroauric acid aqueous solution, mixing polysaccharide and the chloroauric acid aqueous solution, heating to a boiling state, adding polyphenol solution, reacting, cooling, and purifying to prepare the fluorogold nanocluster;

3. The method for preparing catechol-boronate complex fluorescent gold nanoclusters according to claim 2, wherein the method comprises the following steps: the polysaccharide is one or more of soluble starch, glycogen, cellulose, chitosan, cyclodextrin, hyaluronic acid and chondroitin sulfate.

4. A catechol-borate complex fluorogold nanocluster produced by the production method according to any one of claims 1 to 3.

5. Use of the catechol-boronate complex fluorogold nanocluster of claim 4 as a fluorescent probe.

6. The application of the catechol-boronate complex fluorescent gold nanocluster as claimed in claim 5 as a fluorescent probe, wherein the fluorescent probe comprises the following components in percentage by weight: the catechol-borate compound fluorogold nano-cluster is applied to biological imaging as a fluorescent probe.

7. The application of the catechol-boronate complex fluorescent gold nanocluster as a fluorescent probe, which is characterized in that: the application of the catechol-borate compound fluorogold nanocluster in specific detection of high-activity oxygen is provided.