CN109520979B

CN109520979B - Method for detecting cholesterol in serum

Info

Publication number: CN109520979B
Application number: CN201811304853.9A
Authority: CN
Inventors: 李顺兴; 郑凤英; 李跃海
Original assignee: Minnan Normal University
Current assignee: Minnan Normal University
Priority date: 2018-11-02
Filing date: 2018-11-02
Publication date: 2021-07-20
Anticipated expiration: 2038-11-02
Also published as: CN109520979A

Abstract

The invention discloses a method for detecting cholesterol in serum, which comprises the following steps: (1) mixing beta-CD @ AuNPs, CQDs and a proper amount of cholesterol stock solution prepared by taking absolute ethyl alcohol as a solvent, preparing cholesterol standard solutions with different concentrations, and drawing a working curve under a specific fluorescence condition; (2) obtaining serum to be detected and detecting; (3) and (3) substituting the detection result of the step (2) into the working curve to obtain the concentration of cholesterol in the serum to be detected. The invention not only can overcome the defects of technologies such as chromatography, electrochemistry, colorimetry, plasma absorption spectrometry and the like, but also has the advantages of wide detection linear range, higher sensitivity, good selectivity and the like.

Description

Method for detecting cholesterol in serum

Technical Field

The invention belongs to the technical field of biological small molecule analysis and detection, and particularly relates to a method for detecting cholesterol in serum.

Background

Cholesterol, which was first discovered from gallstones, is widely present in animals, is a basic structural component of all animal cell membranes, plays an important role in maintaining the structural integrity and fluidity of membranes, and requires cholesterol as a raw material for the synthesis of bile acids, vitamin D and hormones. The normal level of total cholesterol in the serum of healthy persons is generally lower than 5.2 mmol.L^-1. Excessive blood cholesterol levels, known as hypercholesterolemia, significantly increase the risk of vascular disease such as coronary heart disease, hypertension and atherosclerosis. On the other hand, the lack of cholesterol is considered to be associated with the occurrence of diseases such as depression, cancer and cerebral hemorrhage. With the improvement of living standard, diseases such as coronary heart disease, diabetes and hypertension caused by abnormal cholesterol are in a growing trend, and cholesterol is classified as a class 3 carcinogen by the international cancer research organization of the world health organization. Therefore, development of a method for detecting serum cholesterolThe method of sterol content is of paramount importance.

Various analytical methods have been developed for the detection of cholesterol content, such as chromatography and electrochemical binding, chromatography, electrochemical methods, colorimetric methods, plasma absorption spectroscopy, and the like. However, most methods require a cumbersome experimental process and are expensive; or rely on the high selectivity catalytic reaction of cholesterol oxidase to carry out cholesterol determination, but this method is unstable and expensive due to the volatile activity of the enzyme; or hazardous chemicals are used in the process. Therefore, it is necessary to develop a method with high selectivity, low cost and environmental protection to further improve the defects of the current method.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for detecting cholesterol in serum.

The technical scheme of the invention is as follows:

a method for detecting cholesterol in serum comprises the following steps:

(1) mixing beta-CD @ AuNPs, CQDs and a proper amount of cholesterol stock solution prepared by taking absolute ethyl alcohol as a solvent, and preparing cholesterol standard solutions with different concentrations; fixing the fluorescence excitation wavelength at 390nm, recording the fluorescence emission spectra of cholesterol standard solutions with different concentrations, and taking the intensity of the fluorescence emission peak Em 528nm as the quantitative basis and the fluorescence recovery efficiency (F-F)₀)/F₀Linear fitting of different cholesterol concentrations, determination of detection limits, F₀F is the intensity of the fluorescence emission peak Em at 528nm corresponding to the concentration of different cholesterol standard solutions; when the concentration of cholesterol is 10-210 mu mol.L^-1Fluorescence recovery efficiency (F-F)₀)/F₀Shows good linear relation with the concentration of cholesterol, and obtains a linear working curve y which is 0.0052x-0.0139, R²＝0.996，y＝(F-F₀)/F₀X is cholesterol concentration; the beta-CD @ AuNPs are gold chloride acid (HAuCl)₄) The solution is taken as a precursor, beta-cyclodextrin (beta-CD) is taken as a reducing agent and a stabilizing agent, the solution is prepared by heating and refluxing in one step,the CQDs are synthesized by taking cetylpyridinium chloride (CPC) solution as a precursor through an ultrasonic one-step method;

(2) preserving fresh blood at room temperature overnight, and centrifuging light yellow supernatant every other day to obtain serum; adding 0-160 mu mol.L into the serum^-1The cholesterol standard solution is subjected to fluorescence measurement according to the parameters in the step (1), and the measurement is repeated at least three times to obtain the intensity of a corresponding fluorescence emission peak Em at 528 nm;

(3) the intensity of the fluorescence emission peak Em 528nm obtained in step (3) was substituted into the above linear working curve to obtain the concentration of cholesterol in serum.

In a preferred embodiment of the present invention, the β -CD @ AuNPs are prepared by the following steps: ultrapure water is added into the reaction vessel in sequence, and 0.1 mol.L^-1And PBS buffer of pH 7.0, 0.01mol. L^-1HAuCl of₄Solution and 0.01mol.L^-1Stirring the beta-CD aqueous solution uniformly, transferring the solution into an oil bath kettle, strongly stirring and refluxing the solution at 100 ℃ for 60min to change the color of the solution from light yellow to light red to wine red, naturally cooling the solution, filtering the solution through a 0.45 mu m filter membrane, and storing the solution at 4 ℃.

More preferably, the ultrapure water, PBS buffer solution and HAuCl are used₄The volume ratio of the solution to the beta-CD aqueous solution is 35: 5: 1: 10.

In a preferred embodiment of the present invention, the CQDs are prepared by a method comprising: to 15 mmol. L^-1Adding 2.0 mol. L to the CPC solution^-1After the ultrasonic treatment is carried out for 30min, the pH value of the solution is adjusted to 7.0 by concentrated hydrochloric acid, the reaction is terminated, and the CQDs solid is obtained by freeze drying after being dialyzed for 24h by a dialysis bag, and is dissolved in water again according to the concentration required by the experiment and stored at 4 ℃; the solution changed color from colorless to light yellow and finally to brown-yellow during sonication.

Further preferably, the volume ratio of the CPC solution to the NaOH solution is 100: 9.

In a preferred embodiment of the present invention, the average particle size of the β -CD @ AuNPs is 24 to 26nm, respectively.

In a preferred embodiment of the present invention, the CQDs have average particle diameters of 2.8 to 3.2nm, respectively.

The invention has the beneficial effects that: the invention not only can overcome the defects of technologies such as chromatography, electrochemistry, colorimetry, plasma absorption spectrometry and the like, but also has the advantages of wide detection linear range, higher sensitivity, good selectivity and the like.

Drawings

FIG. 1 is a Transmission Electron Microscope (TEM) image of (a) β -CD @ AuNPs in example 1 of the present invention; (b) high Resolution Transmission Electron Microscopy (HRTEM) images of single β -CD @ AuNPs; (c) a particle size distribution histogram of beta-CD @ AuNPs; (d) TEM image of CQDs and (d) histogram of particle size distribution.

FIG. 2 is a fluorescence excitation spectrum (Ex. about. 390nm) and an emission spectrum (Em. about. 528nm) of CQDs in example 1 of the present invention, and an ultraviolet-visible absorption spectrum (Abs) of β -CD @ AuNPs, in which 1 to 4 and 5 to 8 are CQDs, β -CD @ AuNPs, CQDs/β -CD @ AuNPs/cholesterol under sunlight and 365nm ultraviolet light, respectively; (b) CQDs, CQDs/beta-CD @ AuNPs and CQDs/beta-CD @ AuNPs are added with 60 mu mol.L^-1Fluorescence spectrum of cholesterol.

FIG. 3 is a graph showing fluorescence emission spectra of CQDs in example 1 of the present invention after adding various concentrations of β -CD @ AuNPs; (b) f₀Curve relating/F to concentration of beta-CD @ AuNPs (F)₀The fluorescence emission peak intensity of CQDs when no beta-CD @ AuNPs are added, F is the fluorescence emission peak intensity after different concentrations of beta-CD @ AuNPs are added, and Ex is 390nm and Em is 528 nm); (c) the curve of the change of fluorescence intensity with time before and after adding beta-CD @ AuNPs (2mL) into CQDs; (d) cholesterol (60. mu. mol. L) is added into CQDs/beta-CD @ AuNPs^-1) Plot of the intensity of the post-fluorescence versus time.

FIG. 4 is a graph showing the fluorescence selective responses of (a) various substances to CQDs/β -CD @ AuNPs in example 1 of the present invention; (b) and (3) fitting a fluorescence standard curve of CQDs/beta-CD @ AuNPs to cholesterol concentration and corresponding colorimetric images.

Detailed Description

The technical solution of the present invention is further illustrated and described by the following detailed description.

Example 1:

1. preparation of beta-CD @ AuNPs and CQDs

The beta-CD @ AuNPs are prepared from chloroauric acid (HAuCl)₄) The solution is used as a precursor, beta-cyclodextrin (beta-CD) is used as a reducing agent and a stabilizing agent, and the gold nanoparticles (beta-CD @ AuNPs) modified by the beta-cyclodextrin are prepared in one step by heating and refluxing. The method comprises the following specific steps: 35mL of ultrapure water and 5mL of PBS buffer (0.1 mol. L) were added to the flask^-1pH 7.0), 1mL of HAuCl₄Solution (0.01 mol. L)^-1) And 10mL of an aqueous solution of beta-CD (0.01 mol. L)^-1) Stirring uniformly, transferring into oil bath, strongly stirring and refluxing at 100 deg.C for 60min to change the color of the solution from light yellow to light red and finally to wine red. After the solution was cooled naturally, it was filtered through a 0.45 μm filter and stored at 4 ℃ (PBS buffer solution (0.1 mol. L) of pH 7.0^-1) Preparation: mixing 0.68g KH₂PO₄29.1mL of 0.1 mol. L was added^-1Then diluted with ultrapure water to 100 mL). The prepared beta-CD @ AuNPs are shown in FIGS. 1(a) - (c).

CQDs are prepared by taking cetylpyridinium chloride (CPC) solution as a precursor and synthesizing the precursor by an ultrasonic one-step method. The method mainly comprises the following steps: to 100mL (15 mmol. L)^-1) To the CPC solution (3) was added 9mL (2.0 mol. L)^-1) After 30min of ultrasound (during which the solution changes from colorless to light yellow and finally to brownish yellow), the pH of the solution was adjusted to 7.0 with concentrated hydrochloric acid to terminate the reaction. Dialyzing with dialysis bag (Mw ≈ 3500) for 24h, freeze drying the solution to obtain Carbon Quantum Dots (CQDs) solid, dissolving the CQDs solid in water according to the concentration required by experiment, and storing at 4 deg.C. The CQDs thus obtained are shown in FIG. 1 (d).

As shown in FIG. 1, the prepared β -CD @ AuNPs and CQDs have red and brown-yellow colors, respectively, are spherical and have average particle sizes of about 25nm and 3nm, respectively.

2. Optical characteristics of the respective substances

As shown in FIG. 2(a), the fluorescence emission peak of CQDs is 528nm, the fluorescence excitation peak symmetrical to the fluorescence emission peak is 390nm, the blue curve is the ultraviolet-visible absorption spectrum of beta-CD @ AuNPs, the maximum absorption is at 528nm, and the maximum absorption is well coincident with the fluorescence emission peak of CQDs. The beta-CD @ AuNPs can efficiently quench the fluorescence of CQDs, and a fluorescence resonance energy transfer system can be constructed, wherein the CQDs are fluorescence energy donors, and the beta-CD @ AuNPs are fluorescence energy acceptors. The CQDs solution and the beta-CD @ AuNPs solution were bright yellow and wine red, respectively, in daylight (1 and 2 of the inset in FIG. 2), and the CQDs fluoresced strongly under 365nm UV light while the beta-CD @ AuNPs did not (5 and 6 of the inset in FIG. 2).

As shown in FIG. 2, the fluorescence emission peak of CQDs is obvious and the intensity is large; when the beta-CD @ AuNPs solution is added, fluorescence quenching occurs, the system solution is light red (3 of an insert in figure 2 (a)), and only weak fluorescence is shown under the irradiation of 365nm ultraviolet light (7 of an insert in figure 2 (a)); after the addition of cholesterol was continued, the fluorescence of CQDs was partially restored (FIG. 2(b)), and the color of the system solution was changed to dark pink (4 in the inset in FIG. 2 (a)), and the fluorescence was restored under 365nm ultraviolet light (8 in the inset in FIG. 2 (a)). Taken together, it is shown that β -CD @ AuNPs efficiently quench the fluorescence of CQDs, while cholesterol restores the fluorescence of CQDs, with a concomitant change in solution color.

3. Optimization of reaction conditions

As shown in FIG. 3(a), the fluorescence intensity of CQDs decreases with the increase in the volume of the added β -CD @ AuNPs, and the fluorescence intensity F of CQDs decreases when the volume of the added β -CD @ AuNPs is 0 to 2000. mu.L (FIG. 3b)₀the/F is well linear with the volume of the added beta-CD @ AuNPs, and when the volume of the beta-CD @ AuNPs is increased continuously, the curve is bent upwards, which shows that the fluorescence quenching efficiency of CQDs is reduced. Therefore, 2mL of β -CD @ AuNPs were selected as the fluorescence quencher and the colorimetric indicator. Because both CQDs and β -CD @ AuNPs are prepared at around pH 7 in solution and are stable at around pH 7. As shown in FIG. 3(c), the fluorescence intensity of CQDs is quenched upon addition of β -CD @ AuNPs, the reaction is rapid, and the reaction time for fluorescence quenching is negligible. But cholesterol (60. mu. mol. L) was added^-1) The fluorescence recovery time is not negligible, as shown in fig. 3(d), after the cholesterol is added, the fluorescence intensity of the CQDs is continuously increased along with the time extension, the fluorescence intensity tends to be stable at 40min, and the fluorescence recovery process is basically completed, so 40min is selected as the reaction time of the fluorescence recovery in the CQDs/β -CD @ AuNPs system.

4. Selective study of CQDs/beta-CD @ AuNPs on serum cholesterol

Performing fluorescence response measurement on main substances in serum, mainly comprising NaCl, KCl and MgCl₂Glucose, uric acid, glycine, aspartic acid, glutamic acid, glutathione (reduced form), ascorbic acid and cholesterol (all at a concentration of 90. mu. mol. L)^-1) As shown in FIG. 4(a), the recovery efficiency of cholesterol on CQDs/β -CD @ AuNPs is the most significant, reaching about 1.2. Overall CQDs/β -CD @ AuNPs are highly selective for cholesterol.

5. The specific detection method of this embodiment is as follows:

(1) 2mL of the prepared beta-CD @ AuNPs was added to 300. mu.L (0.4 mg. multidot.mL)^-1) Adding appropriate amount of cholesterol stock solution (10 mmol. multidot.L with anhydrous ethanol as solvent) into CQDs^-1) The preparation is 0, 10, 30, 50, 70, 90, 110, 130, 150, 170, 190, 210 mu mol & L^-1The volume of the standard cholesterol solution was adjusted to 5mL with ultrapure water. The fluorescence excitation wavelength is fixed at 390nm, the fluorescence emission spectra of cholesterol standard solutions with different concentrations are respectively recorded, and the intensity of a fluorescence emission peak (Em 528nm) is used as a quantitative basis. Recovery of efficiency by fluorescence (F-F)₀)/F₀Linear fitting for different cholesterol concentrations (F)₀F corresponds to the intensity of the fluorescence emission peak with different cholesterol concentrations added, for the intensity of the fluorescence emission peak without cholesterol added), the limit of detection of the method is determined. When the concentration of cholesterol is in the range of 10-210 mu mol.L^-1When the fluorescence is recovered, the efficiency of fluorescence recovery (y ═ F-F)₀)/F₀) Shows a good linear relation (R) with the concentration (x) of cholesterol²＝0.996)，y＝0.0052x-0.0139。

(2) Determination of cholesterol content in animal serum

Fresh, clean pig blood was obtained from the market and stored in blue-capped reagent bottles overnight at room temperature. The light yellow supernatant was centrifuged every other day (4000rpm, 6min) to obtain relatively pure pig serum. The method of (1) was followed by adding 50. mu.L of pig serum and adding 0, 40, 80, 160. mu. mol. L of each^-1The standard cholesterol solution is added to 5mL in constant volume and is added after 40minAnd (4) measuring fluorescence. All experiments were performed in triplicate at room temperature.

(3) And (3) substituting the fluorescence intensity values obtained before and after the step (2) into the linear working curve in the step (1) to obtain the corresponding cholesterol concentration.

The method analyzes and measures the cholesterol in the actual pig serum, and the result is shown in the table 1:

TABLE 1 fluorescence method based on CQDs/beta-CD @ AuNPs for porcine serum cholesterol concentration (. mu. mol. L)^-1) Measurement of (n ═ 3)

In conclusion, the CQDs/beta-CD @ AuNPs fluorescent signal in the method of the embodiment is stable, has the advantages of good water solubility, low biotoxicity and the like compared with other fluorescent probes, utilizes the synergistic advantages of fluorescence resonance energy transfer and subject-object recognition, has wide linear range, high selectivity and low detection limit, and can effectively overcome the defects of methods such as chromatography, electrochemical method, colorimetric method, plasma absorption spectroscopy and the like. Cholesterol gave a linear working curve over different concentration ranges: y is 0.0052x-0.0139, linear range: 10-210. mu. mol. L^-1. The experiment process is accompanied with the change of the solution color, and a method for detecting cholesterol by colorimetric-fluorescent double channels can be further developed.

The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.

Claims

1. A method for detecting cholesterol in serum for non-diagnostic therapeutic purposes, comprising: the method comprises the following steps:

(1) mixing beta-CD @ AuNPs, CQDs and a proper amount of cholesterol stock solution prepared by taking absolute ethyl alcohol as a solvent to obtain mixed solutions containing cholesterol standard solutions with different concentrations; fixing the fluorescence excitation wavelength at 390nm, and recording the fluorescence excitation wavelengthFluorescence emission spectrum of the mixed solution of the cholesterol standard solution, the intensity of the fluorescence emission peak Em =528 nm as the quantitative basis, and the fluorescence recovery efficiency (F-F)₀）/F₀Linear fitting of different cholesterol concentrations, determination of detection limits, F₀The intensity of the fluorescence emission peak Em =528 nm when cholesterol is not added, and the intensity of the fluorescence emission peak Em =528 nm when F corresponds to the concentration of different cholesterol standard solutions; when the concentration of cholesterol is 10-210 mu mol.L^-1Fluorescence recovery efficiency (F-F)₀）/F₀Shows good linear relation with the concentration of cholesterol, and obtains a linear working curve y =0.0052x-0.0139, R²=0.996，y=（F-F₀）/F₀X is cholesterol concentration; the beta-CD @ AuNPs are gold chloride acid (HAuCl)₄) The CQDs are synthesized by a one-step ultrasonic method by taking cetylpyridinium chloride (CPC) solution as a precursor;

(2) preserving fresh blood at room temperature overnight, and centrifuging light yellow supernatant every other day to obtain serum; adding 0-160 mu mol.L into the serum^-1Performing fluorescence measurement according to the parameters of the step (1), and repeating the measurement for at least three times to obtain the intensity of a corresponding fluorescence emission peak Em =528 nm;

(3) substituting the intensity of the fluorescence emission peak Em =528 nm obtained in the step (3) into the linear working curve to obtain the concentration of cholesterol in serum;

the preparation method of the beta-CD @ AuNPs comprises the following steps: 35mL of ultrapure water and 5mL of 0.1 mol. L were added in this order to the reaction vessel^-1And 1mL of 0.01mol. L PBS buffer solution having a pH of 7.0^-1HAuCl of₄Solution and 10mL of 0.01mol. L^-1Stirring the beta-CD aqueous solution uniformly, transferring the solution into an oil bath kettle, stirring and refluxing for 60min at 100 ℃ with strong force, changing the color of the solution from light yellow to light red, finally changing the solution into wine red, naturally cooling the solution, filtering the solution by a 0.45 mu m filter membrane, and storing the solution at 4 ℃;

the preparation method of the CQDs comprises the following steps: to 100mL of 15 mmol. L^-1CPC solution of9mL of 2.0 mol. L was added to the solution^-1After the ultrasonic treatment is carried out for 30min, the pH value of the solution is adjusted to 7.0 by concentrated hydrochloric acid, the reaction is terminated, and the CQDs solid is obtained by freeze drying after being dialyzed for 24h by a dialysis bag, and is dissolved in water again according to the concentration required by the experiment and stored at 4 ℃; the solution changed color from colorless to light yellow and finally to brown-yellow during sonication.

2. The detection method according to claim 1, characterized in that: the average particle size of the beta-CD @ AuNPs is 24-26 nm.

3. The detection method according to claim 1, characterized in that: the CQDs have an average particle size of 2.8-3.2 nm.