CN111100636B

CN111100636B - Styrene cyanine dye functionalized carbon dot sensor and preparation method and application thereof

Info

Publication number: CN111100636B
Application number: CN201911358458.3A
Authority: CN
Inventors: 刘巧玲; 牛秀赟; 赵颖; 张燕; 杨博如; 吕世友; 何青; 赵英英; 樊恩宇
Original assignee: Taiyuan Normal University
Current assignee: Taiyuan Normal University
Priority date: 2019-12-25
Filing date: 2019-12-25
Publication date: 2022-06-24
Anticipated expiration: 2039-12-25
Also published as: CN111100636A

Abstract

The invention relates to a styrene cyanine dye functionalized carbon dot sensor and a preparation method and application thereof, belonging to the technical field of biological fluorescence analysis; aiming at effectively avoiding the interference of incident light background fluorescence; the carbon point sensor is formed by carbonizing a styrene cyanine dye on the surface of an original carbon point; the carbonization is further carried out after the amido on the surface of the carbon point and the aldehyde group of the styrene cyanine dye form a C = N double bond; the carbon dot sensor is used for H in aqueous solution⁺Cys/Hcy shows high sensitivity and selectivity, and has the characteristics of visible light excitation and large Stokes displacement, the nano carbon point sensor based on the styrene cyanine dye can be conveniently loaded in cells and positioned in a lysosome, and H in the organelle lysosome can be well observed at a subcellular level through a laser confocal fluorescence microscope⁺And the amount of Cys/Hcy change.

Description

Styrene cyanine dye functionalized carbon dot sensor and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological fluorescence analysis, relates to a fluorescent dye, and particularly relates to a styrene cyanine dye functionalized carbon dot sensor and a preparation method and application thereof.

Background

Intracellular protons play an important role in cells, and participate in important physiological activities such as cellular metabolism, proliferation and division, signal transduction, and catalysis of enzymes. Intracellular H⁺The abnormal fluctuation of (a) is closely related to the occurrence of some diseases, such as: parkinson's disease, Alzheimer's disease, cancer, etc. The biological thiol cysteine (Cys) and homocysteine (Hcy) in cells are also very important, and they are widely involved in the processes of enzyme activation, protein formation, oxidation-reduction in life systems and the like in life systems. Abnormal Cys and Hcy values are also often associated with the development of such diseases, such as Parkinson's disease, Alzheimer's disease, etc. Thus, intracellular H is detected simultaneously⁺And Cys/Hcy can help us to deeply understand the pathogenesis of the diseases and provide practical help for diagnosis and treatment of related diseases.

Fluorescence analysis technology is one of the most important biological analysis and detection means at present, and is widely and deeply applied to DNA sequencing, drug metabolism analysis, measurement of intracellular tissue components and the like. The fluorescent dye is an important component of a fluorescence analysis system, the property of the fluorescent dye influences and even determines the sensitivity and practical value of the whole fluorescence detection, and even if different fluorescent dyes are applied to the same substance analysis process, the detection results can have larger difference, which mainly depends on the physicochemical property of the fluorescent dye.

Determination of H based on fluorescence analysis⁺Or Cys/Hcy, by methods including treatment with H⁺Proportional-response pH probes (Hou, S.L. et al anal. chem., 2019,91(8), 5455-. These reported probes are directed against only one test substance H⁺Or biological thiol response, and does not measure H simultaneously⁺And biological thiols against major diseases whose pathogenesis is currently unclear, such as: parkinson's disease, Alzheimer's disease, etc., and detecting H simultaneously⁺And abnormal fluctuation of biological thiol, has very important value and significance for research of corresponding pathogenesis and early prevention and diagnosis of related diseases.

Disclosure of Invention

The invention overcomes the defects of the prior art and provides a styrene cyanine dye functionalized carbon dot sensor and a preparation method and application thereof. Can simultaneously detect hydrogen ions (H) in water and cells⁺) Concentration and concentration of cysteine (Cys) or homocysteine (Hcy), the carbon dot sensor for H in aqueous solution⁺And Cys/Hcy showed high sensitivity and selectivity.

In order to achieve the above object, the present invention is achieved by the following technical solutions.

A carbon dot sensor functionalized by a styrene cyanine dye, wherein the carbon dot sensor has the following structural general formula I:

wherein R is:

any ofOne of them; CDs (Carbon Dots, CDs) are Carbon sites, and the Carbon source of the Carbon sites is derived from compounds containing amine groups.

Preferably, the compound containing an amine group is any one of alkylamine, aniline, o-phenylenediamine, m-phenylenediamine, p-phenylenediamine, benzenetriamine and dopamine.

The preparation method of the carbon point sensor functionalized by the styrene cyanine dye comprises the following steps of carbonizing the styrene cyanine dye on the surface of an original carbon point to form the carbon point sensor; the carbonization is to further carbonize after the amino on the surface of the carbon dot and the aldehyde group of the styrene cyanine dye form a C ═ N double bond;

preferably, the structural general formula II of the styrene cyanine dye is as follows:

wherein R is:

any one of them.

Preferably, the original carbon dots are obtained by a hydrothermal synthesis method or a microwave synthesis method; the carbonization process is ethanol reflux.

The application of the carbon dot sensor functionalized by the styrene cyanine dye in detecting the hydrogen ion concentration and the cysteine or homocysteine concentration. The carbon point sensor detects pH of 3.92-7.0 and pKa value of 5.30. Carbon dot sensor in aqueous solution to H⁺And Cys/Hcy shows high sensitivity and selectivity, and shows an on-off-on fluorescence response under the excitation of 450nm, the Stokes shift reaches 105nm, and the interference of incident light background fluorescence can be effectively avoided. A pKa value of 5.30 and a pH of between 5.0 and 5.6 and H⁺A good linear relationship is presented. The nano carbon point sensor based on the styrene cyanine dye can be conveniently loaded in cells and positioned in lysosomes, and H in the organelle lysosome can be well observed at the subcellular level through a laser confocal fluorescence microscope⁺And the amount of Cys/Hcy change.

Compared with the prior art, the invention has the beneficial effects that.

The synthesized styrene cyanine dye functionalized carbon dot sensor serves as H⁺And Cys/Hcy fluorescence sensors belong to the class of photo-induced electron transfer (PET) type sensors. The sensor takes the original carbon spot as a fluorophore, carbonized styrene cyanine dye as an acceptor, and H⁺Binding, resulting in PET, with consequent quenching of fluorescence; with the addition of Cys/Hcy, the photoinduced electron transfer process is stopped, the fluorescence is rapidly recovered, and the fluorescence changes in an 'on-off-on' manner. The sensor based on the carbon dots fully utilizes the good water solubility and excellent biocompatibility of the carbon dots, and effectively avoids toxicity to a life system; in addition, the sensor has the characteristics of excitation in a visible light region and large Stokes displacement, can effectively reduce the damage of ultraviolet excitation light to cells and biological samples and the interference of the fluorescence of the cells or the biological samples, improves the sensitivity of the detection method, and is more favorable for the detection of living bodies and fluorescence imaging.

The fluorescence sensor can be incubated into cells, and H in cell lysosomes is detected by a laser confocal fluorescence microscope⁺And changes in Cys/HCy. The carbon dot sensor based on styrene cyanine dye functionalization has the characteristics of simple synthesis method, high detection sensitivity and good selectivity, and can detect H simultaneously⁺And abnormal fluctuation of biological thiol (Cys/Hcy), has very important value and significance for early prevention and auxiliary diagnosis of related diseases.

Drawings

FIG. 1 is a distribution diagram of the particle size of functionalized carbon dots of a styrenic cyanine dye.

FIG. 2 shows a carbon dot sensor functionalized with a styrenic cyanine dye and H of different concentrations⁺Absorption spectrum in pure water.

FIG. 3 shows a carbon dot sensor functionalized with a styrenic cyanine dye and H at different concentrations⁺Emission spectrum in pure water.

FIG. 4 is a graph of a linear fit of pKa.

FIG. 5 shows a carbon dot sensor functionalized with a styrenic cyanine dye and H⁺After quenching of binding, the emission spectra of Cys were added again at different concentrations.

FIG. 6 shows a carbon dot sensor functionalized with a styrenic cyanine dye and H⁺After the quenching, emission spectra of different concentrations of Hcy were added.

FIG. 7 is an image of cells co-localized with lysosome localization dye Lyso-Tracker Red in lysosomes after incubation of a styrenic cyanine dye functionalized carbon spot sensor into the lysosomes of the cells.

FIG. 8 shows a carbon dot sensor functionalized by a styrene cyanine dye in combination with H⁺The cells were then imaged.

FIG. 9 shows a carbon dot sensor binding H functionalized with a styrenic cyanine dye⁺Thereafter, the cells were imaged after addition of Cys.

FIG. 10 shows a combination of a carbon dot sensor with a functionalized carbon dot of a styrenic cyanine dye with H⁺Thereafter, cells were imaged after addition of Hcy.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail with reference to the embodiments and the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. The technical solution of the present invention is described in detail below with reference to the embodiments and the drawings, but the scope of protection is not limited thereto.

Example 1

A carbon dot sensor functionalized with a styrenic cyanine dye, the carbon dot sensor having the following structural formula:

CDs are original carbon points, and the carbon source of the original carbon points is derived from p-phenylenediamine.

The preparation method of the carbon point sensor functionalized by the styrene cyanine dye comprises the following steps of carbonizing the styrene cyanine dye on the surface of an original carbon point to form the carbon point sensor; the carbonization is further carried out after the amino on the surface of the original carbon point and the aldehyde group of the styrene cyanine dye form a C ═ N double bond;

the structural formula of the reactant styrene cyanine dye is as follows:

the original carbon point adopts a hydrothermal synthesis method, and ethanol reflux is adopted in the carbonization process. The prepared carbon dots have the particle size of about 2.28nm, the crystal lattice is 0.26nm, and the particle size distribution diagram is shown in figure 1.

The structural formula of the styrene cyanine dye functionalized carbon dot sensor is specifically a fluorescence sensor for detecting hydrogen ions and cysteine or homocysteine (Cys/Hcy) in water and cells, for example, the sensor is used for H in water⁺And detection of Cys/Hcy; or for H in subcellular-level lysosomes in living systems⁺And Cys/Hcy detection and fluorescence imaging.

The particle diameter of the functionalized carbon dots based on the styrene cyanine dye is about 2.28nm, the crystal lattice is 0.26nm, and the functionalized carbon dots are aligned to H in aqueous solution⁺And Cys/Hcy shows high sensitivity and selectivity, shows an on-off-on fluorescence response under excitation of 450nm, has a Stokes shift of 105nm, a pKa value of 5.30 and a pH value of between 5.0 and 5.6, and is H⁺A good linear relationship is presented. The nano carbon point sensor based on the styrene cyanine dye can be conveniently loaded in cells and positioned in lysosomes, and H in the organelle lysosome can be well observed at the subcellular level through a laser confocal fluorescence microscope⁺And the amount of Cys/Hcy change.

Test example 1

The carbon dot fluorescence sensor functionalized by the styrenic cyanine dye prepared in example 1 was added to pure water to prepare a 0.02mg/mL solution, and 120. mu. mol/L H was added⁺Adding the solution into a solution containing a styrene cyanine dye functionalized carbon dot fluorescence sensor, wherein the solution is accompanied by H in the solution⁺The concentration is increased, the absorption spectrum is gradually red-shifted, and the absorption intensity is gradually enhancedA well-defined isoabsorption point appears at 425nm, as shown in fig. 2.

Test example 2

The carbon dot fluorescence sensor functionalized by the styrene cyanine dye prepared in example 1 was added to pure water to prepare a 0.02mg/mL solution, and H was added⁺Adding the solution into a solution containing a styrene cyanine dye functionalized carbon dot fluorescence sensor, wherein the solution is accompanied by H in the solution⁺The increasing concentration, the gradual decrease in fluorescence intensity, is indicative of the occurrence of photoinduced electron transfer Process (PET), and the fluorescence exhibits a distinct "on-off" characteristic (FIG. 3) with a pKa of 5.30 (FIG. 4).

Test example 3

Test example 2 incorporating H⁺The aqueous solution of the carbon dots functionalized by the styrene cyanine dye is gradually added with Cys solution with the concentration of 120 mu mol/L, the fluorescence intensity is gradually recovered, which indicates that the photoinduced electron transfer Process (PET) is stopped, and the fluorescence presents obvious 'off-on' characteristics (figure 5).

Test example 4

Test example 1 in which H was bonded⁺The aqueous solution of the carbon dots functionalized by the styrene cyanine dye is gradually added with the Hcy solution with the concentration of 120 mu mol/L, the fluorescence intensity is gradually recovered, which indicates that the photoinduced electron transfer process is stopped, and the fluorescence presents obvious 'off-on' characteristics (figure 6).

Test example 5

The sensor based on the carbon dot functionalized by the styrene cyanine dye prepared in example 1 is used as a fluorescence sensor and is incubated in human laryngeal cancer cell Hep-2 (0.05mg/mL) at 37 ℃ and 5% CO₂After incubation for 1 hour in an incubator, the cells were washed 3 times with PBS buffer, then lysosome-localized Red dye Lyso-Tracker Red (50nmol/L) was added thereto, and the incubation was continued at 37 ℃ with 5% CO₂After incubation in an incubator for 30 minutes, washing with PBS buffer solution for 3 times, placing under a laser confocal fluorescence microscope, fixing the excitation wavelength to 458nm, and the emission wavelengths to 500-580nm and 580-630nm respectively, so that lysosomes in cells based on the functionalized carbon dots of the styrene cyanine dye are imaged as bright yellow fluorescence within the range of 500-580nm, and red fluorescence within the range of 580-630nmThe dye Lyso-Tracker Red shows Red fluorescence in the lysosome, and the dye Lyso-Tracker Red realizes co-localization in the lysosome, which shows that the carbon dot can be localized in the lysosome based on the styrene cyanine dye function to realize H in the lysosome⁺And Cys/Hcy detection. (FIG. 7)

Test example 6

The sensor based on the carbon dot functionalized by the styrene cyanine dye prepared in example 1 is used as a fluorescence sensor and is incubated in human laryngeal cancer cell Hep-2 (0.05mg/mL) at 37 ℃ and 5% CO₂After incubation in an incubator for 30 minutes, washing the cells for 3 times by using PBS buffer solution, placing the cells under a confocal laser scanning microscope, fixing the excitation wavelength to be 458nm, and the emission wavelength to be 500-580nm, and observing lysosome imaging as bright yellow fluorescence; then will contain H⁺The culture solution (120. mu. mol/L) was added to the cells, the fixed emission wavelength was still 500-580nm, and the fluorescence intensity in the cells gradually decreased, showing fluorescence quenching (FIG. 8); then will combine with H⁺When 120. mu. mol/L Cys or 120. mu. mol/L Hcy was added to the cells of (1), the fluorescence intensity in the cells gradually recovered with the lapse of time, and bright yellow fluorescence was exhibited in the lysosomes (FIGS. 9 and 10).

While the invention has been described in further detail with reference to specific preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. The carbon point sensor functionalized by the styrene cyanine dye is characterized by having the following structural formula:

CDs are original carbon points, and carbon sources of the original carbon points are derived from p-phenylenediamine; synthesizing original carbon points by a hydrothermal method;

the carbon dot sensor is a new carbon dot formed by connecting aldehyde group of styrene cyanine dye with amino group on the surface of the original carbon dot through C = N double bond and further carbonizing the surface of the carbon dot.

2. The styrenic cyanine dye-functionalized carbon dot sensor according to claim 1, wherein the carbonizing is refluxing in ethanol.

3. Use of a styrenic cyanine dye functionalized carbon dot sensor according to claim 1 in the detection of hydrogen ion concentration and cysteine or homocysteine concentration.

4. The use of a styrenic cyanine dye-functionalized carbon dot sensor according to claim 3, in which the carbon dot sensor detects a pH =3.92-7.0 and a pKa value of 5.30.