CN109916868B - A β-cyclodextrin functionalized carbon dot material - Google Patents

Abstract

The invention discloses a β-cyclodextrin functionalized carbon dot material. The carbon dot material is prepared by bonding β-cyclodextrin to the surface of the carbon dots based on the amide condensation reaction of N, S-doped carbon dots (N,S-CDs). It can be applied to the field of environmental monitoring and the level monitoring of steroid hormones in biological samples, especially for the selective analysis of testosterone.

Description

A β-cyclodextrin functionalized carbon dot material

This application is a division of an invention patent application with the application number "201710206063.6", the application date being March 31, 2017, and the invention title "β-cyclodextrin functionalized carbon dot material and its application in the detection of testosterone" Application.

technical field

The present invention generally relates to the field of environmental monitoring and the level monitoring of steroid hormone substances in biological and food samples, especially to a selective analysis method for testosterone.

Background technique

As a very important steroid androgen, testosterone itself is not only widely involved in the process of biological metabolism and reproductive development as a regulatory factor, but its level in the organism is related to various physiological indicators and even diseases, such as type 2 diabetes, Prostate cancer, coronary artery disease, and kidney stones are associated with testosterone, which is also used in medicine and veterinary medicine due to its important physiological role and enters the environment through medical, domestic and agricultural wastewater discharges. The existing detection and analysis of testosterone mainly rely on high-performance and precise instruments such as HPLC, GC-MS, and HPLC-MS/MS. Although there are ELISA-based rapid detection kits for biological samples that can be applied to the analysis of testosterone content, such analysis requires the pretreatment of the samples and cannot be detected in situ. Therefore, an efficient and simple method that can visualize testosterone levels in situ is not only of great significance for biochemical detection, but also can be extended to the detection of environment and food safety.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to synthesize β-cyclodextrin-functionalized carbon dots, and to provide a convenient, quick, and accurate liquid sample testosterone analysis method based on its selective recognition ability for testosterone.

According to a first aspect of the present invention, a β-cyclodextrin functionalized carbon dot material is provided, the carbon dot material is made of N, S-doped carbon dots (N, S-CDs) based on an amide condensation reaction. It is prepared by fine bonding to the surface of carbon dots.

First of all, cyclodextrins, as functional recognizers, can be divided into α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, δ-cyclodextrin according to the number of glucopyranose molecules that compose them. There are four kinds, of which the first three are the most common. Considering the volume of the target molecule, α-cyclodextrin with only 6 glucopyranose molecules is not suitable for use because its internal cavity volume is too small. Similarly, γ-cyclodextrin, Although the internal space of δ-cyclodextrin is enough to accommodate the target molecule, the corresponding steric hindrance sites are reduced due to its too large space, which reduces the molecular recognition ability, so β-cyclodextrin is selected.

Secondly, in the selection of side chain functional groups, first of all, the end must contain a group that is easy to react with the carboxyl group on the surface of the carbon point, so amino group is selected, and the length of the outer side itself should not be too long, otherwise it is not conducive to photo-excited electron transfer, so choose single (6-amino group) -6-Deoxy) group is a side chain group

In summary, preferably, the β-cyclodextrin is mono(6-amino-6-deoxy)β-cyclodextrin.

Specific case: First dissolve N,S-CDs in deionized water, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCL) and N- Hydroxysuccinimide (NHS), after stirring for a certain period of time, adding mono(6-amino-6-deoxy) β-cyclodextrin, and continuing to stir the reaction, the obtained product was dialyzed using a dialysis bag, and finally freeze-dried to obtain the described β-cyclodextrin functionalized carbon dot material.

Preferably, considering the catalytic mechanism of EDC.HCL, the NHS is slightly excessive so that the molar ratio of EDC.HCL to NHS is between 1:1.5 and 1:2, so N,S-CDs is comparable to EDC.HCL, NHS, monolithic The material ratio of (6-amino-6-deoxy) β-cyclodextrin ranges from the mass ratio (1:3:3.5:10) to (1:3:4:20).

According to a second aspect of the present invention, there is provided a method for detecting testosterone, comprising the following steps:

(1) providing the above-mentioned β-cyclodextrin functionalized carbon dot material;

(2) Take a certain amount of β-cyclodextrin functionalized carbon dot material and (ferrocenylmethyl)trimethylammonium iodide (Fc ⁺ ) to mix and dissolve in water with a certain pH, shake for a certain period of time, and form a fluorescent probe Needle;

(3) configure a series of concentrations of testosterone standard solution, respectively add it to the fluorescent probe system, after oscillating for a certain period of time, use a fluorescence spectrometer to detect the fluorescence intensity at a certain wavelength, and obtain a standard equation after linear fitting;

(4) Add the sample liquid to be detected into the fluorescent probe system, and after oscillating for a certain period of time, use a fluorescence spectrometer to detect the fluorescence intensity at a certain wavelength, and compare with the standard equation to obtain the testosterone content in the sample liquid.

Preferably, the ratio of β-cyclodextrin functionalized carbon dot material to Fc ⁺ is in the range of 1:4 to 1:6 by mass.

Preferably, the water with a certain pH is a phosphate buffer.

Preferably, the oscillation time in steps (2) and (3) is more than 20 minutes.

According to a third aspect of the present invention, there is provided a fluorescent probe system for detecting testosterone, comprising the above-mentioned β-cyclodextrin functionalized carbon dot material, (ferrocenylmethyl)trimethylammonium iodide (Fc ⁺ ) and phosphate buffer.

The present invention adopts a host-guest fluorescent probe system, and then analyzes the testosterone level in the liquid sample through fluorescence spectrum. For the first time, this method uses β-cyclodextrin-functionalized carbon dots as a selective recognition fluorescent probe, which has a good recognition effect on the target analyte testosterone, and greatly reduces the use of organic solvents compared with the traditional method. The pretreatment time and dependence on advanced instruments are shortened; the present invention is a fluorescence colorimetric method based on a host-guest fluorescent probe system, which has good selectivity for testosterone, a steroid androgen, and has a good linear relationship, The detection limit is low, and it can be widely used in the detection of testosterone levels in liquid samples. At the same time, due to the low cytotoxicity and good biocompatibility of the material, it has the potential to be applied to the in situ visual detection of testosterone levels in biological samples.

Description of drawings

FIG. 1 is a graph showing the results of fluorescence spectrum analysis of a series of concentrations of testosterone standard solution using the fluorescent probe system of the present invention.

Detailed ways

1. Instruments and Reagents

Varian CARY Eclipse Fluorescence Spectrometer (Varian, USA)

Testosterone standard (98.0%) was purchased from Shanghai Aladdin Company, and was diluted with ethanol to form a working solution. Androstenedione, progesterone, 17α-hydroxyprogesterone, megestrol acetate, estrone, and estradione were all purchased from Bailingwei; other organic solvents were purchased from Beijing Chemical Factory Reagents.

2. Preparation of β-cyclodextrin-functionalized carbon dots and construction of fluorescent probe system

N,S-CDs were prepared by hydrothermal method using citric acid and L-cysteine according to the literature method. After reacting in a reactor at 200°C for 3 hours, the crude product was purified by dialysis using a dialysis bag with a mass cut-off of 3500 Da. , the obtained N,S-CDs has an obvious absorption peak of 1702cm ^-1 in the infrared spectrum, which proves that its surface is rich in carboxylic acid functional groups, and the energy level peak of 284.5eV obtained by XPS analysis proves that its CC bond is sp ² After hybridization, a graphitized structure is formed.

Dissolve 15mg N,S-CDs in 30mL deionized water, first add 43mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCL) and 51mg N- Hydroxysuccinimide (NHS) was stirred at room temperature for 30min, 200mg of mono(6-amino-6-deoxy)β-cyclodextrin was added, and the reaction was continued for 12h. The resulting product was dialyzed for 48h using a 3500Da dialysis bag. Freeze-drying to obtain the final β-cyclodextrin functionalized carbon dot material.

60 μg of β-cyclodextrin functionalized carbon dot material and 320 μM of Fc ⁺ were dissolved in 3.0 mL of phosphate buffer solution (10 mM, pH=7.4) and shaken for 20 min at room temperature to form a fluorescent probe system.

3. Detection of testosterone level by fluorescence colorimetry

First build a standard equation (as described in detail later), add the sample solution to be detected into the fluorescent probe system, and after oscillating for 20 minutes, use a fluorescence spectrometer to detect the fluorescence intensity at 425 nm at an excitation wavelength of 355 nm, and compare it with the standard equation to obtain The testosterone content in the sample solution.

4. Construction and optimization of fluorescent probe system

The rational construction of the fluorescent probe system is of great significance for the successful detection of the target analyte. A suitable system can improve the detection sensitivity of the target analyte, and the conditional factors affecting the probe system include the pH value of the system and the amount of the quencher. and equilibration time.

1. The effect of pH value of the system

pH is an important factor in fluorescent probe systems, and different pH may significantly affect the stability and intensity changes of fluorescent probes.

In the preliminary experiments, the fluorescence stability of β-cyclodextrin-functionalized carbon dots at different pH values was first verified. 10mM) to make the final concentration 20μg mL ^-1 , and after shaking for 30min, use a fluorescence spectrometer to record the change of the emission light intensity at 425nm under the excitation wavelength of 355nm. The experiment found that in the measured pH range (5.16-9.05), the fluorescence performance of β-cyclodextrin functionalized carbon dots was very stable, and there was no large fluctuation. The detailed experimental results are shown in Table 1.

Table 1

2. Quencher Fc ⁺ dosage

From the experimental results of the previous step, it can be seen that pH has little effect on the fluorescence intensity of the probe, so neutral pH=7.00 was selected as the subsequent test conditions, and a certain amount of β-cyclodextrin functionalized carbon dots and different amounts of Fc ⁺ were dissolved in In pH=7.00 phosphate buffer (10mM), the final concentration of carbon dots was 20μg mL ^-1 , while the concentration of Fc ⁺ was between 0-500μM, and after shaking for 30min, the system was recorded using a fluorescence spectrometer at 425nm at an excitation wavelength of 355nm changes in the emitted light intensity. It can be seen from the results that with the increase of Fc ⁺ concentration, the fluorescence intensity of the system gradually decreased, and the fluorescence intensity of the system remained basically unchanged after more than 300 μM, indicating that the binding of Fc ⁺ to β-cyclodextrin functionalized carbon dots reached saturation at this time. , the specific results are shown in Table 2 below.

Table 2

3. The effect of equilibration time

According to the optimization results in the previous step, the conditions for constructing the probe system were selected: take a certain amount of β-cyclodextrin functionalized Cdots and Fc ⁺ and dissolve them in phosphate buffer (10mM) at pH=7.00 to make the final concentration of Cdots 20μg mL ^{− 1} And the concentration of Fc ⁺ was 320 μM, after shaking for 30 min, a certain amount of testosterone standard solution was added, and a fluorescence spectrometer was used to record the change of the emission light intensity at 425 nm at the excitation wavelength of 355 nm with time. As can be seen from the results, with the increase of time after adding testosterone, the system gradually balances and the fluorescence intensity recovers, and the fluorescence intensity does not change after the balance is completed after 20min. The detailed results are shown in Table 3 below.

table 3

Fifth, the establishment of standard equations

Under the above optimized conditions, a certain amount of β-cyclodextrin functionalized carbon dots and Fc ⁺ were dissolved in phosphate buffer (10 mM) at pH=7.00, so that the final concentration of carbon dots was 20 μg mL ^-1 , while the Fc ⁺ The concentration was 320 μM, and different amounts of testosterone standard solutions (0-480 μM) were added after oscillating for 30 min. After equilibrating at room temperature for 20 min, a fluorescence spectrometer was used to record the change of the emission light intensity at 425 nm at an excitation wavelength of 355 nm. The results are shown in Table 4. It can be seen from the results that the fluorescent probe system has a good response to testosterone that varies in the range of 0-280 μM. After plotting the data using Origin, the results are shown in Figure 1. It can be seen from Figure 1 that the fluorescent probe system has a very good response to testosterone. It has good identification and quantitative analysis ability, and has a good linear relationship between fluorescence intensity and testosterone concentration within a certain range. The linear equation is y(fluorescence intensity, au)=0.2078x(testosterone concentration, μM)+142.73.

Table 4

6. Specificity verification

In order to verify the specificity of the fluorescent probe system for testosterone, a certain amount of β-cyclodextrin-functionalized carbon dots and Fc ⁺ were dissolved in phosphate buffer (10 mM) at pH=7.00 under optimized conditions to make the final The concentration was 20μg mL ^-1 , while the concentration of Fc ⁺ was 320μM. After shaking for 30min, high and low levels (200, 80μM) of testosterone and other steroid hormones (androstenedione, progesterone, 17α-hydroxyprogesterone) were added respectively. , megestrol acetate, estrone, estradione), after equilibrating at room temperature for 20min, use a fluorescence spectrometer to record the change in the emission intensity of the system at 425nm at an excitation wavelength of 355nm, and compare it with the original system before these substances were added. The fluorescence was compared (ΔI/I ₀ ), and the results showed that only testosterone could change the fluorescence intensity of the system at a large level after the addition, so the system had a specific response to testosterone. The specific results are shown in Table 5 below.

table 5

Those skilled in the art should understand that the specific embodiments described above are only for better understanding of the present invention, and are not intended to limit the present invention, and the protection scope of the present invention should be defined by the claims.

Claims (3)

1. A fluorescent probe system for detecting testosterone comprises β -cyclodextrin functionalized carbon dot material, (ferrocenyl methyl) trimethyl ammonium iodide and phosphate buffer;

the β -cyclodextrin functionalized carbon dot material is prepared by bonding β cyclodextrin on the surface of a carbon dot through an amide condensation reaction by virtue of N, S-doped carbon dots.

2. The fluorescent probe system of claim 1, said β cyclodextrin being mono (6-amino-6-deoxy) β cyclodextrin.

3. The fluorescent probe system of claim 1, wherein the β -cyclodextrin functionalized carbon dot material is prepared by dissolving N, S-doped carbon dots in deionized water, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, stirring for a certain period of time, adding mono (6-amino-6-deoxy) β cyclodextrin, continuing stirring for reaction, dialyzing the obtained product with a dialysis bag, and finally freeze-drying to obtain the β -cyclodextrin functionalized carbon dot material.

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