CN114196392A

CN114196392A - Dual-mode ratiometric optical probe for detecting adriamycin based on sulfydryl functionalized carbon dots and preparation method and application thereof

Info

Publication number: CN114196392A
Application number: CN202111288676.1A
Authority: CN
Inventors: 弓晓娟; 张俐; 刘洋; 宋胜梅; 王旭; 董川
Original assignee: Shanxi University
Current assignee: Shanxi University
Priority date: 2021-11-02
Filing date: 2021-11-02
Publication date: 2022-03-18
Anticipated expiration: 2041-11-02
Also published as: CN114196392B

Abstract

The invention belongs to the technical field of fluorescent probes, and provides a dual-mode ratiometric optical probe for detecting adriamycin based on sulfydryl functionalized carbon dots, and a preparation method and application thereof. M-phenylenediamine and p-aminobenzoic acid are used as carbon sources, carbon point CDs are prepared by a one-step hydrothermal method, insoluble substances are removed by centrifugation, unreacted precursor substances and small molecules are removed by dialysis, brown CDs solid powder is obtained by freeze drying, and sulfydryl functionalized carbon point HS-CDs are prepared by performing an amide reaction on carboxyl on the surface of 11-mercaptoundecanoic acid and amino on the surface of CDs. And (3) respectively utilizing fluorescence and ultraviolet detection methods to determine the linear relation between the DOX concentration and the fluorescence intensity ratio and absorbance ratio of HS-CDs. And (3) determining the DOX content and the standard addition recovery rate in the actual sample based on a ratio fluorescence detection method. The probe is simple and convenient to operate, strong in anti-interference performance, low in detection cost and low in technical content, and can be used for quickly and accurately detecting the content of DOX in an actual sample.

Description

Dual-mode ratiometric optical probe for detecting adriamycin based on sulfydryl functionalized carbon dots and preparation method and application thereof

Technical Field

The invention belongs to the technical field of fluorescent probes, and particularly relates to a dual-mode ratiometric optical probe for detecting adriamycin based on sulfydryl functionalized carbon dots, and a preparation method and application thereof.

Background

Doxorubicin, DOX for short, is an anthracycline drug that has been widely used as a chemotherapeutic agent in clinical antitumor therapy. A moderate amount of DOX is effective in inhibiting tumor activity, but produces a series of side effects during treatment: inhibiting bone marrow hematopoiesis, leading to massive hair loss and severe gastrointestinal disorders. Therefore, the method has important significance for accurately monitoring the content of the DOX in the blood and urine of a patient, guiding the dosage of the DOX in the anti-tumor treatment process and furthest weakening the toxic and side effects of the DOX.

The methods for detecting DOX reported so far include: high performance liquid chromatography, electrochemical sensing, raman spectroscopy, and the like. These methods have their own unique advantages, but all suffer from the following drawbacks: expensive instruments and equipment are needed, the technical content is high, the operation is complex, the requirement on experimenters is high, and the like. These defects make the existing detection method unable to be widely applied, so it is urgently needed to develop a DOX detection method which is simple and convenient to operate, high in sensitivity, reliable in detection result and convenient to popularize and use.

The optical probe has excellent sensitivity, selectivity and quick response, and is concerned by researchers. A material can be used as an optical probe to detect a substance when its optical properties respond well to the target. Most of the common optical probes have single signal response, and probes with double signal response are rarely reported. The optical probe with single signal response has some problems, such as susceptibility to interference of experimental environment and low reliability of detection result. The optical probe with double signal responses can well avoid the influence caused by experimental conditions, thereby increasing the reliability of a detection result.

The carbon dot has the advantages of photoluminescence performance, good water solubility, luminescence stability, biocompatibility, low cytotoxicity and the like, so that the carbon dot can be used as an optical probe to be widely applied to detection of metal ions, amino acids, medicines, environmental pollutants and the like. The reported optical probes for detecting DOX based on carbon dots are single-wavelength and single-mode detection, have the defect of poor detection result precision, and are difficult to popularize and use. Therefore, the development of the optical probe for quantitatively detecting DOX based on the ratio of carbon spots and double modes has important significance and wide application prospect.

Disclosure of Invention

The invention aims to provide a dual-mode ratiometric optical probe for detecting adriamycin based on sulfydryl functionalized carbon spots, and a preparation method and application thereof.

The invention is realized by the following technical scheme: a dual-mode ratio optical probe for detecting adriamycin based on sulfydryl functionalized carbon points is characterized in that m-phenylenediamine and p-aminobenzoic acid are used as carbon sources, carbon point CDs are prepared through a one-step hydrothermal method, insoluble substances are removed through centrifugation, unreacted precursor substances and small molecules are removed through dialysis, brown CDs solid powder is obtained through freeze drying, and sulfydryl functionalized carbon point HS-CDs are prepared through an amide reaction between carboxyl on the surface of 11-sulfydryl undecanoic acid and amino on the surface of CDs.

The method for preparing the dual-mode ratiometric optical probe for detecting the adriamycin based on the sulfydryl functionalized carbon dot comprises the following specific steps of:

(1) obtaining precursor substances: accurately weighing 0.05 g of m-phenylenediamine and 0.05 g of p-aminobenzoic acid, adding 10 mL of absolute ethyl alcohol, transferring the solution into a polytetrafluoroethylene high-pressure reaction kettle, and reacting at 180 ℃ for 12 h to obtain a brown solution;

(2) obtainment of a brown solid powder of CDs: after the temperature of the reaction system is cooled to room temperature, performing rotary evaporation on the brown solution to remove ethanol solvent to obtain brown solid, dissolving the brown solid in secondary water, centrifuging the solution at 8000 r/min for 15min, dialyzing the brown supernatant in a dialysis bag of 500-1000 Da for 3 days, and freeze-drying the solution in the dialysis bag to obtain brown solid powder of CDs;

(3) 0.1g of 11-mercaptoundecanoic acid, 0.2 g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC & HCl and 0.05 g N-hydroxysuccinimide NHS are dissolved in 4.5 mL of DMSO together for reaction for 1 h, and carboxyl on the surface of the 11-mercaptoundecanoic acid is activated; then, 0.354 g of CDs was dissolved in 4.5 mL of DMSO and added to the above solution, stirred at room temperature for 48 hours to obtain a brown solution, and after the reaction was completed, lyophilized to obtain a tan HS-CDs powder.

The application of the dual-mode ratiometric optical probe for detecting adriamycin based on sulfydryl functionalized carbon spots in the detection of adriamycin DOX is characterized in that the preparation method of the specific probe stock solution comprises the following steps:

(1) preparation of HS-CDs stock solution: adding 0.01 g of HS-CDs solid powder into 10 mL of secondary water, and stirring to fully dissolve the HS-CDs solid powder to obtain HS-CDs stock solution with the concentration of 1.0 mg/mL;

(2) preparation of DOX stock solution: 13.6 mg DOX powder is accurately weighed, added into 2.5 mL secondary water, stirred and dissolved, and DOX stock solution with the concentration of 10 mM is prepared.

The probe is used as a ratiometric fluorescent probe for detecting DOX, and comprises the following specific steps:

obtaining a linear equation between the DOX content and the fluorescence intensity of HS-CDs: accurately measuring 0.4 mL of the HS-CDs stock solution, and adding 1.6 mL of secondary water into the HS-CDs stock solution, wherein the final concentration of HS-CDs is 0.2 mg/mL; adding DOX stock solutions with different volumes into the solution, and recording fluorescence intensity values F of HS-CDs at 513 nm and 590 nm under the excitation wavelength of 399 nm₅₁₃And F₅₉₀(ii) a Linear fitting of DOX concentration to fluorescence intensity ratio F by Origin software₅₉₀/F₅₁₃Two linear equations are obtained: f₅₉₀/F₅₁₃ = 0.0181 C_DOX + 0.2479，R² = 0.999；F₅₉₀/F₅₁₃ = 0.052 C_DOX - 1.182，R²= 0.999, the corresponding linear range and the lowest detection limit are respectively: 0.25-19.96 [ mu ] M, 0.66 [ mu ] M, 50.33-80.88 [ mu ] M, 0.23 [ mu ] M.

The application of the probe in detecting the DOX content in an actual sample comprises the following specific steps:

(1) centrifuging a sample to be detected for 15min at 3000 r/min, taking supernatant, and diluting the supernatant by 1000 times by using a Du's Phosphate Buffer Solution (DPBS) to obtain a stock solution of the sample to be detected for later use;

(2) adding 400 mu L of HS-CDs stock solution into 1600 mu L of to-be-detected sample stock solution, wherein the final concentration of HS-CDs is 0.2 mg/mL, and measuring the fluorescence intensity F of the solution at 513 nm and 590 nm₅₁₃And F₅₉₀Calculating F₅₉₀/F₅₁₃Will F₅₉₀/F₅₁₃Substitution of linear equation F₅₉₀/F₅₁₃ = 0.0181 C_DOX+ 0.2479 or F₅₉₀/F₅₁₃ = 0.052 C_DOX1.182, calculating to obtain the DOX content in the actual sample.

The sample to be detected is urine, the urine is centrifuged for 15min at 3000 r/min, and the supernatant is filtered by a filter with the aperture of 0.45 mu m; the filtered supernatant was diluted with DPBS.

The probe detects the standard addition recovery rate of DOX in an actual sample by using a ratio fluorescence method, and the method comprises the following specific steps:

(1) taking 1.0 mL DOX stock solution, and diluting to 10 mL to obtain 1.0 mM DOX standard solution;

(2) respectively adding 400 mu L of HS-CDs stock solution into 1600 mu L of to-be-detected sample stock solution to enable the final concentration of HS-CDs in the system to be 0.2 mg/mL; mu.L and 139. mu.L of DOX standard solutions were added to the above system so that the final concentrations of DOX were 9.9. mu.M and 65. mu.M, respectively, and the fluorescence intensities F of the mixed solutions at 513 nm and 590 nm were measured and recorded, respectively₅₁₃And F₅₉₀Calculating F₅₉₀/F₅₁₃(ii) a Substitution of linear equation F₅₉₀/F₅₁₃ = 0.0181 C_DOX+ 0.2479 or F₅₉₀/F₅₁₃ = 0.052 C_DOXAnd (5) 1.182) calculating the content of the DOX in the system, and further calculating the standard recovery rate of the DOX in the actual sample.

The probe is used as a ratiometric photometric probe to detect DOX, and the specific steps are as follows: obtaining a linear equation between the DOX content and the HS-CDs ultraviolet absorption intensity: accurately measuring 0.4 mL HS-CDs stock solution, and adding 1.6 mL secondary waterThe final concentration of HS-CDs is 0.2 mg/mL; adding DOX stock solutions with different volumes into the solution, measuring and recording the absorbance values of the mixed solution at 252 nm and 290 nm, respectively recording as A₂₅₂And A₂₉₀(ii) a Linear fitting of DOX concentration to Absorbance ratio A by Origin software₂₅₂/A₂₉₀To obtain a linear equation: a. the₂₅₂/A₂₉₀ = 0.0156 C_DOX + 1.357（R²= 0.997), corresponding linear ranges and lowest detection limits of 2.50-29.8 μ M and 0.75 μ M, respectively.

The method of the invention has the advantages that: m-phenylenediamine, p-aminobenzoic acid and 11-mercaptoundecanoic acid are common reagents and are easily purchased. The amide reaction between 11-mercaptoundecanoic acid and CDs is the basic reaction, and the reaction difficulty is reduced. The probe is based on fluorescence and luminosity dual-mode ratio detection, and has the advantages of good selectivity, high sensitivity and strong background interference resistance.

Compared with other methods for detecting DOX, the method has the advantages of rapidness, effectiveness, stable performance, strong anti-interference capability, no need of expensive instruments and equipment, simple and convenient operation, low detection cost and the like.

Drawings

FIG. 1 is a graph showing the UV absorption spectrum and the optimal excitation emission fluorescence spectrum of HS-CDs prepared in example 1;

FIG. 2 is a graph showing fluorescence spectra of HS-CDs prepared in example 1 at different excitation wavelengths;

FIG. 3 is an X-ray photoelectron spectrum of HS-CDs prepared in example 1;

FIG. 4 is an infrared spectrum of 11-mercaptoundecanoic acid of example 1 and CDs and HS-CDs prepared;

FIG. 5 shows various ions (Zn) in example 2²⁺, Mn²⁺, Fe²⁺, Co²⁺, Ca²⁺, Hg²⁺, Pb²⁺, Ag⁺, Mg²⁺, Ba²⁺, Cd²⁺, Na⁺, CO₃ ^2-, ClO₄ ^-, ClO₃ ^-, Cl^-, Br^-, F^-, S₂O₃ ^2-, I^-) Detecting an interference experiment result graph of DOX by using a ratio fluorescence method;

FIG. 6 is a graph showing the results of an interference experiment for detecting DOX by the ratiometric fluorescence method for each antibiotic (vancomycin, penicillamine, penicillin G sodium, thiamphenicol, oxytetracycline, gentamicin, azithromycin, tetracycline, kanamycin, lincomycin, amoxicillin, trimethoprim, clarithromycin, sulfadiazine, ursolic acid, oleanolic acid) in example 2;

FIG. 7 is a graph showing the fluorescence spectra of HS-CDs after DOX is added to the HS-CDs solution;

FIG. 8 is a linear fit plot of DOX concentration versus change in fluorescence intensity of HS-CDs; the corresponding linear range and the lowest detection limit are respectively 0.25-19.96 MuM and 0.66 MuM;

FIG. 9 is a linear fit plot of DOX concentration versus change in fluorescence intensity of HS-CDs; the corresponding linear range and the lowest detection limit are respectively 50.33-80.88 mu M and 0.23 mu M;

in fig. 10: (a) is a schematic diagram of fluorescence resonance energy transfer between HS-CDs and DOX; (b) zeta potential diagrams for CDs, 11-mercaptoundecanoic acid, DOX, HS-CDs and HS-CDs + DOX; (c) the emission spectrum of HS-CDs and the excitation spectrum of DOX are shown;

FIG. 11 shows various ions (Zn)²⁺, Mn²⁺, Fe²⁺, Co²⁺, Ca²⁺, Hg²⁺, Pb²⁺, Ag⁺, Mg²⁺, Ba²⁺, Cd²⁺, Na⁺，CO₃ ^2-, ClO₄ ^-, ClO₃ ^-, Cl^-, Br^-, F^-, S₂O₃ ^2-, I^-) Detecting an interference experiment result graph of DOX by using a ratio photometry;

FIG. 12 is a graph showing the results of an interference test for detecting DOX by ratiometric photometry of various antibiotics (vancomycin, penicillamine, penicillin G sodium, thiamphenicol, oxytetracycline, gentamicin, azithromycin, tetracycline, kanamycin, lincomycin, amoxicillin, trimethoprim, clarithromycin, sulfadiazine, ursolic acid, oleanolic acid);

FIG. 13 is a graph showing the change in the UV-VIS absorption spectra of HS-CDs after DOX is added to the HS-CDs solution;

FIG. 14 is a linear fit plot of DOX concentration versus HS-CDs absorbance change.

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, 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.

Example 1: preparation and characterization of HS-CDs

Step one, weighing 0.05 g of m-phenylenediamine and 0.05 g of p-aminobenzoic acid, adding 10 mL of absolute ethyl alcohol, transferring the solution to a polytetrafluoroethylene high-pressure reaction kettle, placing the reaction kettle in an oven, and reacting for 12 hours at 180 ℃ to obtain a brown solution.

And step two, after the temperature of the reaction system is cooled to room temperature, performing rotary evaporation on the brown solution to remove ethanol solvent to obtain brown solid, dissolving the brown solid in secondary water, centrifuging the solution at 8000 r/min for 15 minutes, dialyzing the brown supernatant in a dialysis bag of 500-1000 Da for three days, and freeze-drying the solution in the dialysis bag to obtain brown solid powder of CDs.

Step three, 0.1g of 11-mercaptoundecanoic acid, 0.2 g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 0.05 g N-hydroxysuccinimide (NHS) are dissolved in 4.5 mL of DMSO together to react for 1 h, and the carboxyl on the surface of the 11-mercaptoundecanoic acid is activated; then, 0.354 g of CDs was dissolved in 4.5 mL of DMSO and added to the above solution, stirred at room temperature for 48 hours to obtain a brown solution, and after the reaction was completed, lyophilized to obtain a tan HS-CDs powder.

The properties of the resulting tan HS-CDs powder are characterized in fig. 1, fig. 2, fig. 3 and fig. 4.

In the ultraviolet absorption spectrogram of HS-CDs, three obvious absorption peaks are positioned at 215 nm, 290 nm and 457 nm which are respectively represented by sp²Pi → pi transition of carbon, C = ON → pi of^*Surface defects caused by transition and N, S doping; the optimal excitation and emission wavelengths for HS-CDs in FIG. 1 are 399 nm and 513 nm, respectively, with a Stokes shift of 114 nm.

FIG. 2 is a spectrum of the emission spectra of HS-CDs at different excitation wavelengths, and when the excitation wavelength is changed from 300 nm to 480 nm, the emission wavelength is red-shifted from 498 nm to 519 nm, which shows that the HS-CDs have the optical behavior of excitation wavelength dependence, and different emission wavelength spectra can be collected by adjusting the excitation wavelength. FIG. 3 is an X-ray photoelectron spectrum (XPS) of HS-CDs showing characteristic peaks of O1S (530 eV), N1S (399 eV), C1S (282 eV) and S2p (163 eV), indicating that HS-CDs are mainly composed of C, S, N, O four elements.

FIG. 4 is an infrared spectrum of CDs, 11-mercaptoundecanoic acid, and HS-CDs containing O-N/N-H stretching vibrations of CDs and C-H stretching vibrations of 11-mercaptoundecanoic acid; meanwhile, a new peak (N-H stretching vibration or C-N bending vibration) is generated in the HS-CDs, which shows that 11-mercaptoundecanoic acid is successfully modified on the surfaces of the CDs through an amide reaction, namely the mercapto-functionalized carbon dots HS-CDs are successfully constructed.

Example 2: study of HS-CDs as ratiometric fluorescent probes

Example 1: anti-interference experiment for detecting DOX by ratio fluorescent probe

Step one, preparing HS-CDs stock solution: adding 0.01 g of HS-CDs solid powder into 10 mL of secondary water, and stirring to fully dissolve the HS-CDs solid powder to obtain HS-CDs stock solution with the concentration of 1.0 mg/mL;

step two, preparing DOX stock solution: accurately weighing 13.6 mg DOX powder, adding into 2.5 mL of secondary water, stirring for dissolving, and preparing DOX stock solution with the concentration of 10 mM;

step three, weighing a certain mass of metal ions (Zn)²⁺, Mn²⁺, Fe²⁺, Co²⁺, Ca²⁺, Hg²⁺, Pb²⁺, Ag⁺, Mg²⁺, Ba²⁺, Cd²⁺, Na⁺) And anions (CO)₃ ^2-, ClO₄ ^-, ClO₃ ^-, Cl^-, Br^-, F^-, S₂O₃ ^2-, I^-) 10 mL of secondary water was added to each compound to prepare an ionic stock solution having a concentration of 0.1M.

Weighing a certain mass of antibiotics (vancomycin, penicillamine, penicillin G sodium, thiamphenicol, oxytetracycline, gentamycin, azithromycin, tetracycline, kanamycin, lincomycin, amoxicillin, trimethoprim, clarithromycin, sulfadiazine, ursolic acid, oleanolic acid and adriamycin), adding 5 mL of secondary water, and preparing the antibiotic stock solution with the concentration of 0.1M.

Step five, accurately measuring 0.4 mL of HS-CDs stock solution, adding 1.6 mL of secondary water into the HS-CDs stock solution, wherein the final concentration of the HS-CDs is 0.2 mg/mL, respectively adding 10 mu L of ion stock solution, wherein the final concentration of ions is 0.5 mM, measuring the fluorescence intensity of the mixed solution at 513 nm and 590 nm, and respectively recording the fluorescence intensity as F₅₁₃And F₅₉₀(ii) a 10 μ L DOX solution was added thereto at a final DOX concentration of 0.5 mM, and the fluorescence intensities of the mixed solution at 513 nm and 590 nm were measured and recorded as F₅₁₃And F₅₉₀. The results of the experiment are shown in FIG. 5.

FIG. 5 is an interference study of differential ion contrast ratio fluorimetry to detect DOX, demonstrating that the ions studied do not cause fluorescence of HS-CDs (F)₅₉₀/F₅₁₃) A significant change occurs; also, when the ion of interest is present, the addition of DOX will also cause fluorescence (F) of HS-CDs₅₉₀/F₅₁₃) Obvious changes occur, which shows that HS-CDs have good selectivity for detecting DOX, and meanwhile, the ratio fluorescence detection DOX is not interfered by the researched ions.

And sixthly, accurately measuring 0.4 mL of HS-CDs stock solution, adding 1.6 mL of secondary water into the stock solution, wherein the final concentration of the HS-CDs is 0.2 mg/mL, respectively adding 100 mu L of antibiotic stock solution, wherein the final concentration of the antibiotic is 4.76 mM, measuring the fluorescence intensity of the mixed solution at 513 nm and 590 nm, and respectively recording the fluorescence intensity as F₅₁₃And F₅₉₀(ii) a 10 μ L DOX solution was added thereto at a final DOX concentration of 0.5 mM, and the fluorescence intensities of the mixed solution at 513 nm and 590 nm were measured and recorded as F₅₁₃And F₅₉₀. The results of the experiment are shown in FIG. 6.

FIG. 6 is an interference study of fluorescence detection of DOX with contrast ratio of different antibiotics, demonstrating that the selected antibiotics do not cause fluorescence of HS-CDs (F)₅₉₀/F₅₁₃) A significant change occurs; also, when the selected antibiotic is present, the addition of DOX will also cause the fluorescence of HS-CDs (F)₅₉₀/F₅₁₃) Obvious changes occur, which shows that HS-CDs have good selectivity for detecting DOX, and meanwhile, the ratio fluorescence detection of DOX is not interfered by the selected antibiotics.

Example 2: method for detecting DOX based on HS-CDs ratio fluorescence method

step three, obtaining a linear equation between the DOX content and the fluorescence intensity of HS-CDs: accurately measuring 0.4 mL of HS-CDs stock solution, and adding 1.6 mL of secondary water into the stock solution, wherein the final concentration of HS-CDs is 0.2 mg/mL; adding DOX stock solutions with different volumes into the solution, and recording fluorescence intensity values F of HS-CDs at 513 nm and 590 nm under the excitation wavelength of 399 nm₅₁₃And F₅₉₀. The fluorescence spectrum change is shown in FIG. 7.

Step four, linearly fitting the ratio (F) of DOX concentration to fluorescence intensity by Origin software₅₉₀/F₅₁₃) The results are shown in FIGS. 8 and 9.

FIG. 7 is a graph showing the fluorescence spectra of HS-CD after different concentrations of DOX added with HS-CDs. As can be seen from the figure, with the increase of DOX concentration, the fluorescence intensity of HS-CDs at 513 nm gradually decreases, a new peak is generated at 590 nm, and the fluorescence intensity gradually increases; when the concentration of DOX reaches 97.07. mu.M, the fluorescence intensity of HS-CDs at 513 nm is reduced to the minimum, and the fluorescence intensity at 590 nm reaches the maximum. Indicating that Fluorescence Resonance Energy Transfer (FRET) exists between HS-CDs and DOX, wherein HS-CDs are energy donors and DOX is an energy acceptor. The energy transfer mechanism is shown in FIG. 10 a.

FIG. 8 shows the change in fluorescence intensity of HS-CDs (F)₅₉₀/F₅₁₃) Linear dependence on DOX concentration, linear equation F₅₉₀/F₅₁₃ = 0.0181 C_DOX + 0.2479（R²= 0.999), the corresponding linear range and the lowest detection limit are 0.25-19.96 μ M and 0.66 μ M, respectively. It can be seen that the fluorescence intensity (F) of HS-CDs is at 0.25-19.96. mu.M for DOX concentration₅₉₀/F₅₁₃) There is a good linear relationship with DOX concentration.

FIG. 9 shows the change in fluorescence intensity of HS-CDs (F)₅₉₀/F₅₁₃) Linear dependence on DOX concentration, linear equation F₅₉₀/F₅₁₃ = 0.052 C_DOX - 1.182（R²= 0.999), the corresponding linear range and the lowest detection limit are 50.33-80.88 μ M and 0.23 μ M, respectively. It can be seen that the fluorescence intensity (F) of HS-CDs is at 50.33-80.88 μ M for DOX concentration₅₉₀/F₅₁₃) There is a good linear relationship with DOX concentration.

FIG. 10a is a schematic representation of Fluorescence Resonance Energy Transfer (FRET) between HS-CDs and DOX. Firstly, there is electrostatic interaction between HS-CDs and DOX (FIG. 10 b), which shortens the distance between HS-CDs and DOX, makes the distance between them less than 10 nm, and satisfies the sufficient condition for FRET; secondly, there is a good overlap between the emission spectrum of HS-CDs and the excitation spectrum of DOX (FIG. 10 c), satisfying the requirements for FRET to occur; finally, HS-CDs are used as donors to provide energy, DOX is used as an acceptor to absorb energy, and a ratiometric fluorescent probe is constructed to detect DOX.

Example 3: study of HS-CDs as ratiometric photometric probes

Example 1: anti-interference experiment for detecting DOX by ratio photometric probe

Fifthly, accurately measuring 225 muL HS-CDs stock solution, adding 1275 muL secondary water into the stock solution, wherein the final concentration of the HS-CDs is 0.15 mg/mL, then respectively adding 10 muL ion stock solution, wherein the final concentration of ions is 0.66 mM, and measuring the ultraviolet absorption intensity of the mixed solution at 252 nm and 290 nm, which are respectively marked as A₂₅₂And A₂₉₀(ii) a Then, 10 μ L DOX stock solution was added thereto, the final concentration of DOX at this time was 0.66 mM, and the UV absorption intensities of the mixed solution at 252 nm and 290 nm at this time were measured and recorded as A, respectively₂₅₂And A₂₉₀. The results of the experiment are shown in FIG. 11.

FIG. 11 is an interference study of spectrophotometric detection of DOX at different ion contrast ratios, demonstrating that the ions studied do not contribute to the UV absorption intensity of HS-CDs (A)₂₅₂/A₂₉₀) A significant change occurs; also, the addition of DOX in the presence of the ion of interest likewise results in the UV absorption intensity (A) of HS-CDs₂₅₂/A₂₉₀) Obvious change occurs, which shows that HS-CDs have good selectivity to DOX detection, and the ratio photometry DOX detection is not subject to the researchThe interference of ions is studied.

Step six, accurately measuring 225 muL HS-CDs stock solution, adding 1275 muL secondary water into the stock solution, wherein the final concentration of the HS-CDs is 0.15 mg/mL, respectively adding 100 muL antibiotic stock solution, wherein the final concentration of the antibiotic is 6.25 mM, measuring the ultraviolet absorption intensity of the mixed solution at 252 nm and 290 nm, and respectively recording the ultraviolet absorption intensity as A₂₅₂And A₂₉₀(ii) a Then, 10 μ L DOX stock solution was added thereto, the final concentration of DOX at this time was 0.66 mM, and the UV absorption intensities of the mixed solution at 252 nm and 290 nm at this time were measured and recorded as A, respectively₂₅₂And A₂₉₀. The results of the experiment are shown in FIG. 12.

FIG. 12 is an interference study of spectrophotometry of DOX at contrast ratios for different antibiotics, demonstrating that the selected antibiotics do not cause the UV absorption intensity of HS-CDs (A)₂₅₂/A₂₉₀) A significant change occurs; meanwhile, when the selected ions exist, the addition of DOX can also lead to the ultraviolet absorption intensity (A) of HS-CDs₂₅₂/A₂₉₀) Obvious change occurs, which shows that HS-CDs have good selectivity for detecting DOX, and meanwhile, the ratio photometry method for detecting DOX is not interfered by the selected ions.

Example 2: method for detecting DOX based on HS-CDs ratio photometry

step three, obtaining a linear equation between the DOX content and the HS-CDs ultraviolet absorption intensity: accurately measuring 0.4 mL of HS-CDs stock solution, and adding 1.6 mL of secondary water into the stock solution, wherein the final concentration of HS-CDs is 0.2 mg/mL; adding DOX stock solutions with different volumes into the solution, measuring and recording the absorbance values of the mixed solution at 252 nm and 290 nm, respectively recording as A₂₅₂And A₂₉₀. The change in the UV-visible spectrum is shown in FIG. 13, which shows that HS-CDs are present at 252 nm and 290 nm with the addition of DOX standardsThe ultraviolet absorption intensity is gradually increased, but the increase amplitude is different, so that an optical probe for measuring DOX in a ratiometric manner is constructed.

Step four, linearly fitting the ratio of DOX concentration to absorbance (A) by Origin software₂₅₂/A₂₉₀) The results are shown in FIG. 14. Change in UV absorbance of HS-CDs (A)₂₅₂/A₂₉₀) The linear relation between the concentration and DOX is shown in the figure, and the linear equation is A₂₅₂/A₂₉₀ = 0.0156 C_DOX + 1.357（R²= 0.997), corresponding linear ranges and lowest detection limits of 2.50-29.8 μ M and 0.75 μ M, respectively. It can be seen that the ultraviolet absorption intensity (A) of HS-CDs is when the DOX concentration is between 2.50 to 29.8. mu.M₂₅₂/A₂₉₀) There is a good linear relationship with DOX concentration.

Example 4: detection of DOX content in actual sample

Step one, taking plasma of two different mice, centrifuging for 15min at 3000 r/min, and taking supernatant to obtain serum; serum stock solutions were obtained by 1000-fold dilution with Du's Phosphate Buffer (DPBS), and named serum 1 and serum 2, respectively, for future use.

Step two, taking urine of healthy women, centrifuging for 15min at 3000 r/min, and filtering supernatant by using a filter with the pore size of 0.45 mu m; and taking 100 mu L of filtered supernatant, and diluting 1000 times by using DPBS to obtain urine stock solution for later use.

Adding 400 mu L of HS-CDs stock solution into 1600 mu L of serum 1, serum 2 and urine stock solution, wherein the final concentration of HS-CDs is 0.2 mg/mL, and measuring the fluorescence intensity F of the solution at 513 nm and 590 nm₅₁₃And F₅₉₀Calculating F₅₉₀/F₅₁₃。

Step four, F₅₉₀/F₅₁₃Substitution of linear equation F₅₉₀/F₅₁₃ = 0.0181 C_DOX+ 0.2479 or F₅₉₀/F₅₁₃ = 0.052 C_DOX1.182, calculating the DOX content of the actual sample, and obtaining the result shown in the table 1.

Table 1 shows that the detected serum No. 1, serum No. 2 and urine of healthy women do not contain DOX, and the method can be used for detecting the content of DOX in the serum and urine and is not influenced by other components in the serum and urine. Therefore, the ratio fluorescence method based on HS-CDs is suitable for detecting the DOX content in an actual sample, the detection result is not influenced by other interferents, and the method has potential practical application value.

Table 1: detection results of DOX content in serum No. 1, serum No. 2 and urine

ND: not detected out

Example 5: addition standard recovery experiment of DOX in actual sample

Step one, taking 1.0 mL DOX stock solution, and diluting to 10 mL to obtain 1.0 mM DOX standard solution;

step two, adding 400 mu L of HS-CDs stock solution into 1600 mu L of serum No. 1 solution to enable the final concentration of HS-CDs in the system to be 0.2 mg/mL; mu.L and 139. mu.L of DOX standard solutions were added to the above system so that the final concentrations of DOX were 9.9. mu.M and 65. mu.M, respectively, and the fluorescence intensities F of the mixed solutions at 513 nm and 590 nm were measured and recorded, respectively₅₁₃And F₅₉₀Calculating F₅₉₀/F₅₁₃。

Adding 400 mu L of HS-CDs stock solution into 1600 mu L of serum No. 2 solution to enable the final concentration of HS-CDs in the system to be 0.2 mg/mL; mu.L and 139. mu.L of DOX standard solutions were added to the above system so that the final concentrations of DOX were 9.9. mu.M and 65. mu.M, respectively, and the fluorescence intensities F of the mixed solutions at 513 nm and 590 nm were measured and recorded, respectively₅₁₃And F₅₉₀Calculating F₅₉₀/F₅₁₃。

Step four, adding 400 mu L of HS-CDs stock solution into 1600 mu L of urine stock solution to enable the final concentration of HS-CDs in the system to be 0.2 mg/mL; mu.L and 139. mu.L of DOX standard solutions were added to the above system so that the final concentrations of DOX were 9.9. mu.M and 65. mu.M, respectively, and the fluorescence intensities F of the mixed solutions at 513 nm and 590 nm were measured and recorded, respectively₅₁₃And F₅₉₀Calculating F₅₉₀/F₅₁₃。

Step five, the above F₅₉₀/F₅₁₃Respectively substituting into linear equation F₅₉₀/F₅₁₃ = 0.0181 C_DOX+ 0.2479 or F₅₉₀/F₅₁₃ = 0.052 C_DOXAnd (5) 1.182) calculating the content of the DOX in the system, and further calculating the standard recovery rate of the DOX in the serum No. 1, the serum No. 2 or the urine stock solution.

The results are shown in Table 2. Table 2 shows that the recovery rate of DOX in the three practical samples is between 95.0 and 102.0 percent, and the relative standard deviation is less than 7.12 percent, which indicates that HS-CDs have good reproducibility when used for detecting DOX in the practical samples.

Table 2: results of the standard recovery experiment of DOX in three practical samples in example 7

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A dual-mode ratiometric optical probe for detecting doxorubicin based on thiol-functionalized carbon dots, comprising: m-phenylenediamine and p-aminobenzoic acid are used as carbon sources, carbon point CDs are prepared by a one-step hydrothermal method, insoluble substances are removed by centrifugation, unreacted precursor substances and small molecules are removed by dialysis, brown CDs solid powder is obtained by freeze drying, and sulfydryl functionalized carbon point HS-CDs are prepared by performing an amide reaction on carboxyl on the surface of 11-mercaptoundecanoic acid and amino on the surface of CDs.

2. Method for the preparation of a bimodal ratiometric optical probe for the detection of doxorubicin based on thiol-functionalized carbon dots according to claim 1, characterized in that: the method comprises the following specific steps:

3. The use of the bimodal ratiometric optical probe for the detection of doxorubicin based on thiol-functionalized carbon spots, prepared according to claim 2, for the detection of doxorubicin DOX, characterized in that: the preparation method of the specific probe stock solution comprises the following steps:

4. Use according to claim 3, characterized in that: the probe is used as a ratiometric fluorescent probe for detecting DOX, and comprises the following specific steps: obtaining DOX content and HS-CDs fluorescenceLinear equation between light intensities: accurately measuring 0.4 mL of the HS-CDs stock solution, and adding 1.6 mL of secondary water into the HS-CDs stock solution, wherein the final concentration of HS-CDs is 0.2 mg/mL; adding DOX stock solutions with different volumes into the solution, and recording fluorescence intensity values F of HS-CDs at 513 nm and 590 nm under the excitation wavelength of 399 nm₅₁₃And F₅₉₀(ii) a Linear fitting of DOX concentration to fluorescence intensity ratio F by Origin software₅₉₀/F₅₁₃Two linear equations are obtained: f₅₉₀/F₅₁₃ = 0.0181 C_DOX + 0.2479，R² = 0.999；F₅₉₀/F₅₁₃ = 0.052 C_DOX - 1.182，R²= 0.999, the corresponding linear range and the lowest detection limit are respectively: 0.25-19.96 [ mu ] M, 0.66 [ mu ] M, 50.33-80.88 [ mu ] M, 0.23 [ mu ] M.

5. Use according to claim 4, characterized in that: the application of the probe in detecting the DOX content in an actual sample comprises the following specific steps:

6. Use according to claim 5, characterized in that: when the sample to be detected is urine, centrifuging for 15min at 3000 r/min, and filtering supernatant by using a filter with the pore size of 0.45 mu m; the filtered supernatant was diluted with DPBS.

7. Use according to claim 5, characterized in that: the probe detects the standard addition recovery rate of DOX in an actual sample by using a ratio fluorescence method, and the method comprises the following specific steps:

8. Use according to claim 3, characterized in that: the probe is used as a ratiometric photometric probe to detect DOX, and the specific steps are as follows: obtaining a linear equation between the DOX content and the HS-CDs ultraviolet absorption intensity: accurately measuring 0.4 mL of HS-CDs stock solution, and adding 1.6 mL of secondary water into the stock solution, wherein the final concentration of HS-CDs is 0.2 mg/mL; adding DOX stock solutions with different volumes into the solution, measuring and recording the absorbance values of the mixed solution at 252 nm and 290 nm, respectively recording as A₂₅₂And A₂₉₀(ii) a Linear fitting of DOX concentration to Absorbance ratio A by Origin software₂₅₂/A₂₉₀To obtain a linear equation: a. the₂₅₂/A₂₉₀ = 0.0156 C_DOX + 1.357（R²= 0.997), corresponding linear ranges and lowest detection limits of 2.50-29.8 μ M and 0.75 μ M, respectively.