阅读说明：本技术 一种利用量子点敏化上转换纳米材料检测凝血酶的方法 (Method for detecting thrombin by using quantum dot sensitized up-conversion nano material ) 是由 刘志洪 余甜雨 于 2019-09-23 设计创作，主要内容包括：本发明公开一种利用量子点敏化上转换纳米材料检测凝血酶的方法,包括以下步骤：(1)制备适配体TBA1修饰的上转换纳米粒子(UCNPs-TBA1)；(2)制备适配体TBA2修饰的Ag<Sub>2</Sub>Se量子点(Ag<Sub>2</Sub>Se QDs-TBA2)；(3)绘制凝血酶检测的标准曲线；(4)检测待测样品中的凝血酶浓度。本发明解决了利用FRET传感器检测的信背比低的问题,实现了待测样品中凝血酶的高灵敏检测,检出限可达0.091 nM。(The invention discloses a method for detecting thrombin by using quantum dot sensitized up-conversion nano materials, which comprises the following steps: (1) preparing aptamer TBA1 modified up-conversion nanoparticles (UCNPs-TBA 1); (2) preparation of aptamer TBA2 modified Ag 2 Se quantum dot (Ag) 2 Se QDs-TBA 2); (3) drawing a standard curve of thrombin detection; (4) and detecting the concentration of thrombin in the sample to be detected. The invention solves the problem of low signal-to-back ratio of detection by using the FRET sensor, realizes high-sensitivity detection of thrombin in a sample to be detected, and has the detection limit of 0.091 nM.)

1. A method for detecting thrombin by using quantum dot sensitized up-conversion nanoparticles is characterized by comprising the following steps:

(1) preparing aptamer TBA1 modified up-conversion nanoparticles;

(2) preparation of aptamer TBA2 modified Ag₂Se quantum dots: mixing Ag with water₂Se quantum dots react with aptamer TBA2 with one end modified with amino to obtain TBA2 modified Ag₂Se quantum dots;

(3) standard curve for thrombin detection was plotted: the up-conversion nano particles obtained in the step (1) and the Ag obtained in the step (2)₂Se quantum dots are added into a buffer solution, thrombin with different amounts is added into the buffer solution for incubation, the incubated solution is excited by a laser light source to obtain fluorescence intensity, and when the concentration of the thrombin is set to be 0, the obtained blank sample intensity is marked as F₀At a fluorescence ratio F/F₀Taking the concentration of thrombin in the buffer solution as an abscissa to draw a standard curve;

(4) and (3) diluting the sample to be detected with a buffer solution, measuring the fluorescence intensity under the same condition as the step (3), and further obtaining the thrombin concentration in the sample to be detected according to the standard curve obtained in the step (3).

2. The method for detecting thrombin according to claim 1, wherein the absorption spectrum and Ag of the upconversion nanoparticles are₂The emission spectra of the Se quantum dots overlap.

3. The method for detecting thrombin according to claim 1, wherein the upconversion fluorescent nanoparticle is NaYF₄Yb and Er with spherical shape and particle size of 20-28 nm; the Ag is₂The Se quantum dots have the particle size of 3-4nm, are spherical in shape and have carboxyl on the surface.

4. The method for detecting thrombin according to claim 1, wherein the 5' -end of each of the single-stranded nucleic acid TBA1 and the single-stranded nucleic acid TBA2 is modified with amino groups, and the sequences are as follows: 5' -NH₂-TTTTTAGTCCGTGGTAGGGCAGGTTGGGGTGACT-3 'and 5' -NH₂-TTTTTGGTTGGTGTGGTTGG-3’。

5. The method of claim 1, wherein the upconversion is sensitized by quantum dotsA method for detecting thrombin by using nanoparticles, which is characterized in that in the step (2), Ag₂The proportion of Se quantum dots to TBA2 in the reaction is 5 mu mol: (0.5-3) nmol.

6. The method for detecting thrombin according to claim 1, wherein in the step (3), the buffer solution is HEPES buffer solution and has a pH of 7.0-7.5.

7. The method for detecting thrombin according to claim 1, wherein in the step (3), thrombin is added to the buffer solution in an amount ranging from 0 to 125nM, based on the concentration of thrombin in the buffer solution.

8. The method for detecting thrombin by using quantum dot sensitized up-conversion nanoparticles as claimed in claim 1, wherein in step (3), Ag obtained in step (2)₂The adding amount of the Se quantum dots is 0.08-0.4 mu M in terms of the concentration of the Se quantum dots in the buffer solution.

9. The method for detecting thrombin by using quantum dot sensitized up-conversion nanoparticles as claimed in claim 1, wherein in step (3), said incubation time is 2-5h and incubation temperature is 37 ℃.

10. The method according to claim 1, wherein the sample to be tested is a plasma sample, and the prothrombin in the plasma sample needs to be pre-treated to convert the prothrombin into prothrombin.

Technical Field

The invention belongs to the field of biosensing and analysis, and particularly relates to a fluorescence detection method for thrombin by utilizing quantum dot sensitized up-conversion nano materials.

Background

The rare earth ion doped up-conversion nanoparticles can continuously absorb two or more low-energy photons and emit one high-energy photon. The properties of long-wave excitation and short-wave emission can effectively avoid the interference of autofluorescence and scattered light from a biological sample, so the upconversion nanoparticles are widely applied to the field of biological analysis and detection. In addition, due to the characteristic of avoiding simultaneous excitation with other substances, the upconversion nanoparticles are often used in combination with Fluorescence Resonance Energy Transfer (FRET) technology as an energy donor of the FRET system, while nano-materials with large molar absorptivity, such as nano-gold, manganese dioxide, and carbon nano-materials, are often used as energy acceptors of the FRET system. In the detection process, firstly, energy donor-acceptor is assembled to construct a FRET system, and at the moment, the fluorescence of the up-conversion nano particles is quenched by the acceptor; and after the target object appears, the fluorescence of the up-conversion nano particles is recovered by changing the absorption spectrum of the receptor or the distance between the donor and the receptor, so that the target object is detected. It can be seen that the detection sensitivity of this "quenching-boosting" based response mode is limited by the quenching efficiency and the boosting efficiency.

The particle size of the up-conversion nanoparticles is usually tens of nanometers, and luminescent ions are doped in host crystal lattices, so that the luminescent ions serving as energy donors are far away from an external energy acceptor due to the structural characteristics and exceed the effective distance range for energy transfer, so that the energy transfer efficiency is low, and the fluorescence quenching degree is limited. The carbon nano material generally has higher quenching efficiency, but the non-specific adsorption effect on the up-conversion nano particles is stronger, so that the fluorescence recovery process is limited. Either low quenching efficiency or low boosting efficiency will severely limit the signal-to-noise ratio and sensitivity of the assay. In view of the above, there is still a need in the art to find new detection methods to improve the signal-to-back ratio and achieve highly sensitive detection of tumor markers.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects of the prior art, and provide a method for detecting thrombin by using quantum dot sensitized up-conversion nanoparticles, quantitatively detect thrombin in diluted serum, solve the problem of low signal-to-back ratio of detection by using a FRET sensor, realize high-sensitivity detection of thrombin in serum, and the detection limit can reach 0.091 nM.

The technical scheme provided by the invention for solving the technical problems is as follows:

a method for detecting thrombin by using quantum dot sensitized up-conversion nanoparticles comprises the following steps:

(1) preparation of aptamer TBA1 modified upconversion nanoparticles: preparing surface oleic acid coated up-conversion nanoparticles by taking rare earth oleate as a precursor through a coprecipitation method, removing the ligand to obtain ligand-free modified up-conversion nanoparticles with bare surfaces, performing surface functionalization by using polyacrylic acid to ensure that the surfaces of the up-conversion nanoparticles are rich in carboxyl, and reacting the surface functionalized up-conversion nanoparticles with an aptamer TBA1 with amino modified at one end to obtain TBA1 modified up-conversion nanoparticles;

(2) preparation of aptamer TBA2 modified Ag₂Se quantum dots: ag modified with carboxyl group₂Carrying out coupling reaction on the Se quantum dots and an aptamer TBA2 with one end modified with amino to obtain TBA2 modified Ag₂Se quantum dots;

(3) standard curve for thrombin detection was plotted: the up-conversion nano particles obtained in the step (1) and the Ag obtained in the step (2)₂Se quantum dots are added into the buffer solution, thrombin with different amounts is added into the buffer solution for incubation, the incubated solution is placed into a cuvette and is excited by a 980nm laser light source to obtain fluorescence intensity, and when the concentration of the thrombin is set to be 0, the obtained blank sample intensity is recorded as F₀At a fluorescence ratio F/F₀Taking the concentration of thrombin in the buffer solution as an abscissa to draw a standard curve;

(4) and (3) diluting the sample to be detected with a buffer solution, measuring the fluorescence intensity under the same condition as the step (3), and further obtaining the thrombin concentration in the sample to be detected according to the standard curve obtained in the step (3).

According to the scheme, the absorption spectrum and Ag of the up-conversion nano particles₂Emission spectra of Se quantum dots overlap, and Ag₂Se quantum dots absorb at 980 nm.

According to the scheme, the upconversion fluorescent nanoparticle is composed of NaYF₄Yb and Er are spherical, the particle size is 20-28nm, and the crystal phase is a hexagonal phase; the Ag is₂The particle size of the Se quantum dots is about 3-4nm, and the appearance of the Se quantum dots is spherical.

According to the scheme, the 5' ends of the single-stranded nucleic acid TBA1 and the single-stranded nucleic acid TBA2 are modified with amino groups, and the sequences are respectively as follows: 5' -NH₂-TTTTTAGTCCGTGGTAGGGCAGGTTGGGGTGACT-3 'and 5' -NH₂-TTTTTGGTTGGTGTGGTTGG-3’。

According to the scheme, the Ag₂The proportion of Se quantum dots and TBA2 in the coupling reaction is 5 mu mol: (0.5-3) nmol.

According to the scheme, the buffer solution is HEPES buffer solution with the concentration of 10mM and the pH value of 7.2. The buffer solution can also be replaced by serum diluted by the buffer solution, and the dilution multiple of the serum is 20-100 times.

According to the above scheme, in the step (3), the thrombin is added to the buffer solution in an amount of 0 to 125nM, based on the concentration in the buffer solution.

According to the scheme, in the step (3), the concentration of the upconversion nanoparticles obtained in the step (1) in the buffer solution is 0.04-0.06 mg/mL; ag obtained in step (2)₂The concentration of Se QDs-TBA2 in the buffer solution is 0.08-0.4. mu.M.

According to the scheme, the incubation time is 2-5h, and the incubation temperature is 37 ℃.

The principle of the invention is as follows: the sandwich type aptamer sensor for detecting the thrombin is constructed by utilizing the fact that the thrombin comprises two different DNA aptamer binding sites and through the specific recognition effect of aptamers. Two aptamers (TBA1 and TBA2) of thrombin are respectively modified on the surfaces of the up-conversion nanoparticles and the quantum dots, when the thrombin exists in the system, the TBA1 and the TBA2 specifically recognize the thrombin to form an aptamer secondary structure, the distance between the quantum dots and the up-conversion nanoparticles is shortened, the quantum dots transfer the excited state energy of the quantum dots to the up-conversion nanoparticles, the luminescence of the up-conversion nanoparticles is enhanced, and the enhanced degree of the up-conversion luminescence is positively correlated with the concentration of the thrombin, so that the quantitative detection of the thrombin is realized.

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

1. the method utilizes the characteristics of near-infrared excitation and visible emission of the upconversion fluorescent nano material, can directly detect in a serum sample, does not need a preliminary treatment step, is simple to operate, and is more suitable for practical application.

2. According to the invention, the fluorescence enhancement times of the system can be effectively improved by optimizing the relative concentrations of the quantum dots and the aptamers;

3. the invention utilizes the advantage of large light absorption coefficient of quantum dots, can effectively enhance the luminescence of the up-conversion nano material, improves the detection signal-to-back ratio, solves the problem of low signal-to-back ratio of detection by using a FRET sensor, realizes high-sensitivity detection of thrombin in serum, and has the detection limit of 0.091 nM.

Drawings

FIG. 1 is a diagram of the detection principle of thrombin by quantum dot sensitized up-conversion nano material.

FIG. 2 is a graph of the fold increase in fluorescence intensity as a function of thrombin concentration for different incubation times.

FIG. 3 is a graph of thrombin concentration and fluorescence increase factor after different amounts of TBA2 labeled quantum dots.

FIG. 4 is a graph of thrombin concentration versus fluorescence increase after reaction of varying amounts of TBA 2-QDs.

FIG. 5 is a graph of thrombin concentration versus fluorescence increase at various dilutions in serum.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments. In the following examples, the up-conversion nanomaterials and their modifications were made as follows:

1. oil phase NaYF₄The synthesis of Yb and Er: 6.4mL of oleic acid, 10.4mL of 1-octadecene and 0.8mmol of Ln (oleate)₃(Ln ═ Y: Yb: Er ═ 78:20:2) was placed in a three-neck flask, the temperature was raised to 50 ℃ and 3.2mmol of NH was added₄F and 10mL of 2mmol of NaOH in methanol are reacted for 30min at 50 ℃; and then raising the temperature to 100 ℃ under the protection of argon, pumping out methanol gas in the three-neck flask by using a vacuum pump, finally raising the temperature to 290 ℃, reacting at the temperature for 90min, naturally cooling to room temperature, adding a proper amount of ethanol, centrifugally separating, collecting precipitate, washing twice by using a mixed solution of cyclohexane and ethanol in a volume ratio of 1:2, and finally dispersing the precipitate in the cyclohexane. The shape of the upconversion fluorescent nanoparticle is spherical, and the particle size is about 24nm, namely the upconversion fluorescent nanoparticle coated with oleic acid on the surface.

And (3) ligand removal: taking 50mg of the synthesized upconversion nanoparticles coated with the surface oleic acid, adding excessive ethanol, centrifuging, collecting precipitate, adding the precipitate and 30mL of ethanol into a single-neck flask together, adjusting the pH value to 1, performing ultrasonic treatment for 3 hours, centrifuging after the ultrasonic treatment to obtain precipitate, centrifuging and cleaning once by using the ethanol with the pH value of 4, centrifuging and cleaning twice by using ethanol and ultrapure water respectively, and finally dispersing the precipitate in 20mL of ultrapure water. The process is a de-ligand process, and the up-conversion nano particles (UCNPs) without ligand modification and with bare surfaces are obtained.

2. Modification of upconversion nanoparticles with polyacrylic acid:

adding 200mg of polyacrylic acid, 233mg of sodium bicarbonate and 4mL of UCNPs with the concentration of 12.5mg/mL and exposed surfaces into a single-neck flask, violently stirring for 12 hours at room temperature, centrifugally collecting precipitates, and centrifugally cleaning for three times by using ultrapure water to obtain polyacrylic acid modified up-conversion nanoparticles, which are marked as PAA-UCNPs and modified with carboxyl on the surfaces.

3. Carboxyl modified Ag₂Preparing Se quantum dots: first, 2.5. mu.L (0.01mmol) (TMS) was added under an inert gas atmosphere₂Se and 80mg LiN (SiMe)₃)₂Selenium precursor was prepared by dissolving in a mixture of 0.5mL TOP and 1mL ODE. Stirring 16.7mg (0.1mmol) AgAc, 130 μ L (0.75mmol) 1-octanol and 5mL ODE under argon protection for one hour, slowly heating to 160 ℃, quickly injecting selenium precursor, then reacting at 130 ℃ for 30min, cooling to room temperature, washing with ethanol, dispersing in a nonpolar solvent to obtain a solution containing oily Ag₂Solution of Se quantum dots.

Adding dropwise octylamine modified polyacrylate dissolved in chloroform into the solution containing oily Ag₂Fully mixing the Se quantum dots in a chloroform solution, and drying the solvent at room temperature through rotary evaporation; the rotary evaporated solid was dispersed in borate buffer (pH 12.0, 50X 10)^-3M), filtering and purifying by using a polypropylene column filled with Superdex 200prep grade to obtain Ag modified by carboxyl₂And (4) Se quantum dots.

The resulting carboxyl-modified Ag₂After carboxyl of the Se quantum dots is activated by EDC and NHS, coupling reaction can be carried out on the Se quantum dots and an aptamer TBA2 modified with amino at one end.

4. Coupling of TBA1 and PAA-UCNPs: 1mg of PAA-UCNPs was added to 1mL of 2- (N-morpholine) ethanesulfonic acid MES (10mM, pH 5.5) buffer solution, sonicated for 5min, then 0.5mg of EDC. HCl and 1mg of Sulfo-NHS were added, and after shaking at room temperature for 40min, the precipitate was centrifuged, washed twice with ultrapure water, the precipitate was dispersed in 1mL of HEPES (10mM, pH 7.2) buffer solution, and 1nmol of TBA1 was added and shaken overnight to obtain a coupled product. The coupled product was collected by centrifugation, washed twice with ultrapure water and finally dispersed in 1mL of Tris buffer (10mM, pH 7.4) as TBA1-UCNPs and stored at 4 ℃ until use.