A laser light-emitting nano recognition probe and a preparation method and application thereof

Abstract

The application discloses a kind of lasing light nano recognition probe and its preparation method and application, belong to biomedical detection and nanophotonics cross field.The lasing light nano recognition probe includes: Au nanosphere forming resonant cavity;Mesoporous silica shell is wrapped in Au nanosphere outer layer, and organic fluorescence gain medium is loaded in mesoporous silica shell;Compact silica encapsulation layer is coated outside mesoporous silica shell, and the surface of compact silica encapsulation layer has amino site;Cortisol targeting recognition molecule is coupled on the amino site by amide bond, and the cortisol targeting recognition molecule is used for specifically combining cortisol molecule.The probe of the application preparation process is simple, and batch synthesis can be carried out, and pump threshold is low.When the probe is used for cortisol detection, sample consumption is less, detection limit is low, and it is suitable for trace cortisol in serum high sensitivity, specific quantitative detection.

Description

Technical Field

[0001] This invention relates to the fields of biomedical detection and nanophotonics technology, specifically to a lasing-luminescent nano-recognition probe, its preparation method, and its application. Background Technology

[0002] Three-level stimulated emission (SPEE) surface plasmon amplification nanoprobes combine nanoscale particle size (<100 nm), ultranarrow emission linewidth (~5 nm), and microsecond-scale luminescence lifetime (~100 μs) (Sci. Adv. 2018;4:eaat0292). Compared with traditional fluorescent probe materials, three-level lasing-luminescent nanoprobes can effectively avoid interference from background fluorescence signals in biological samples. They are a novel nanoprobe material with excellent optical properties and are expected to significantly improve the accuracy and sensitivity of existing spectroscopic detection techniques.

[0003] To fully leverage the superior performance of three-level SPASER nanoprobes in biochemical analytical spectroscopy, the stability of their luminescence and the stability of their preparation methods are crucial. In the development of SPASER probe preparation, the initial in-situ doping method suffered from complex processes and poor uniformity (CN109054803 B). The improved capillary adsorption method utilizes capillary action to achieve physical adsorption of dyes within mesopores, improving reproducibility but introducing new problems such as dye leakage and decreased luminescence stability (J. Phys. Chem. C, 2020, 124, 16553−16560). Existing lasing probes use hyaluronic acid encapsulation to address dye leakage, but hyaluronic acid is prone to enzymatic degradation in biological samples such as serum, resulting in low biostability (e.g., reference document CN118995194A). Furthermore, existing lasing probes lack the ability to specifically recognize and quantitatively detect cortisol. Summary of the Invention

[0004] The purpose of this invention is to overcome three major technical defects in existing lasing-luminescent nanoprobes: 1) poor luminescence stability due to dye leakage; 2) poor biostability due to the easy enzymatic degradation of encapsulation materials (such as hyaluronic acid) in biological samples; and 3) lack of specific recognition and quantitative detection capability for cortisol. Therefore, this invention provides a lasing-luminescent nanoprobe with high luminescence stability, high biostability, and specific cortisol recognition capability, as well as its preparation method.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a lasing-luminescent nano-recognition probe, comprising: Au nanospheres forming the resonant cavity; The Au nanospheres are encased in a mesoporous silica shell, within which an organic fluorescent gain medium is loaded. A dense silica encapsulation layer covering the mesoporous silica shell, wherein the surface of the dense silica encapsulation layer has amino sites; A cortisol-targeting recognition molecule is coupled to the amino site via an amide bond, the cortisol-targeting recognition molecule being used to specifically bind to cortisol molecules.

[0006] The Au nanospheres have a particle size of 15 nm to 20 nm; the mesoporous silica shell has a thickness of 20 nm to 25 nm; and the dense silica encapsulation layer has a thickness of 1 nm to 2 nm.

[0007] The spontaneous emission spectrum of the organic fluorescent gain medium overlaps with the resonant absorption curve of the Au nanosphere, thus satisfying the energy level matching principle.

[0008] The organic fluorescence gain medium is 2',7'-difluorofluorescein (OG488); the cortisol targeting recognition molecule is a carboxyl-modified cortisol aptamer.

[0009] The base sequence of the cortisol aptamer is: 5'-COOH-ATG GGC AAT GCG GGG TGG AGA ATGGTT GCC GCA CTT CGG C-3'.

[0010] The lasing wavelength is 547 nm, the pump threshold is ≤1 mJ / cm², and the full width at half maximum (FWHM) of the lasing spectrum is ≤5 nm.

[0011] Furthermore, this invention also provides a method for preparing the aforementioned lasing-luminescent nano-recognition probe, comprising the following steps: Step 1: Synthesize Au nanospheres as a resonant cavity using a modified Frens reduction method; Step 2: Using Au nanospheres as the core, a mesoporous silica shell is grown on their surface by the sol-gel method, and then the shell is centrifuged, washed, and resuspended in ultrapure water; Step 3: Add organic fluorescent gain medium to ultrapure water and load the organic fluorescent gain medium into the mesoporous silica shell by capillary adsorption. Step 4: Add 3-aminopropyltrimethoxysilane (APTMS) to the material solution for in-situ growth to form a dense silica encapsulation layer on the outer layer of the mesoporous silica shell and introduce surface amino sites. After vigorous stirring for 3 h, centrifuge and wash, and resuspend in ultrapure water. Step 5: After activating the cortisol-targeting recognition molecule in the dark, it is coupled to the probe surface through an amide bonding reaction. After incubation, it is centrifuged and washed, and then resuspended in PBS buffer to obtain the lasing-luminescent nano-recognition probe.

[0012] In step 5, the cortisol targeting recognition molecule used is a cortisol aptamer with carboxyl modification; its activation method is: activated by EDC / NHS at 30°C in the dark for 30 min; the centrifugation and washing method is: centrifuged at 8000 r / min for 10 min and washed 3 times; after centrifugation and washing, it is resuspended in PBS buffer at pH=7.4.

[0013] Furthermore, this invention also provides the application of the aforementioned lasing-luminescent nano-recognition probe in the detection of cortisol in serum, comprising the following steps: The lasing-luminescent nanoprobe solution was mixed and incubated with a serum sample to be tested. A magnetic microsphere solution was then added to initiate a competitive reaction. The magnetic microspheres were coupled with cortisol antigen. After magnetic separation, the lasing spectral intensity of the supernatant was measured. The magnetic microspheres were used to compete with cortisol molecules in the serum sample to bind to the cortisol aptamer on the lasing-luminescent nanoprobe. Serum cortisol concentration was determined based on lasing spectral intensity.

[0014] The lasing-luminescent nano-recognition probe was incubated with the serum sample for 0.5 h. The concentration of the added magnetic microsphere solution was 1 mg / mL; the volume ratio of the magnetic microsphere solution to the lasing luminescent nano-recognition probe solution was 1:10-1:20; and the competitive reaction time was 0.5 h.

[0015] This invention provides a lasing-luminescent nanoprobe, its fabrication method, and its application. Au nanospheres are used as a plasmon resonant cavity, a mesoporous silica layer loaded with an organic fluorescent gain medium is used as the gain medium, and a dense silica layer is used as an encapsulation layer. This constructs a lasing-luminescent nanoprobe with both high luminescence stability and high biostability, featuring a narrow bandwidth and a low pump threshold. Compared with existing technologies, it has the following advantages: 1. A pioneering "simultaneous encapsulation / amylation" process solves two major challenges: dye leakage and biocompatibility. This invention utilizes APTMS to grow a dense silica layer in situ on the outer layer of a mesoporous silica layer, achieving the dual functions of "sealing to prevent leakage" and "surface amination" in one step. Compared with the existing technology using hyaluronic acid encapsulation, dense silica exhibits superior biocompatibility, does not undergo enzymatic degradation in complex biological samples such as serum, and significantly improves the probe's practicality in biological detection.

[0016] 2. Unexpectedly enhanced luminescence was achieved. Experiments showed that after APTMS amination modification, the lasing intensity of the probe was approximately three times that before modification, while the emission peak position and full width at half maximum (FWHM) remained unchanged.

[0017] 3. Excellent performance in detecting trace amounts of cortisol. When using the probe of this invention for cortisol detection, only 5 μL of sample is required, with a wide linear detection range (0.1 nM~20 nM) and a detection limit as low as 0.064 nM. Simultaneously, this method exhibits good specificity and anti-interference ability against common interfering substances such as glucose, ascorbic acid, β-estradiol, progesterone, and testosterone, making it suitable for highly sensitive and specific quantitative detection of real serum samples.

[0018] 4. The preparation process is simple and can be mass-produced, solving the problem of poor uniformity in existing technologies. This invention uses capillary adsorption to load dyes, combined with simultaneous encapsulation / amination with APTMS. The process involves fewer steps, milder conditions, and better repeatability, avoiding the problems of complex processes and poor product uniformity in in-situ doping methods (such as CN109054803 B), and has good prospects for industrialization.

[0019] 5. This invention fills a functional gap in the use of lasing probes for the trace detection of cortisol. Existing lasing-luminescent nanoprobes lack specific recognition capabilities for cortisol. This invention, through coupling with a cortisol aptamer, is the first to apply lasing probe technology to the quantitative detection of cortisol, expanding the application scope of lasing probes in the biomedical detection field and providing a widely applicable technical platform for the highly sensitive detection of other small biological molecules. Attached Figure Description

[0020] Figure 1 This is a schematic cross-sectional view of a lasing-luminescent nano-recognition probe provided in Embodiment 1 of the present invention; Figure 2 This is a flowchart illustrating the fabrication process of a lasing-luminescent nano-recognition probe according to Embodiment 2 of the present invention; Figure 3 The images are transmission electron microscope (TEM) images of the lasing probe before and after amination treatment with APTMS in Example 2 of the present invention; wherein, (a) is a TEM image of spaser before amination, with a mesoporous silica shell thickness of about 23.8 nm; (b) is a TEM image of spaser-NH2 after amination, with an overall shell thickness of about 25.7 nm and a dense silica encapsulation layer thickness of about 2 nm. Figure 4 The image shows the luminescence performance of the lasing-luminescent nano-identification probe prepared in Example 2 of the present invention; wherein, (a) is the lasing-luminescence spectrum at different pump energies, with a characteristic emission wavelength of 547 nm; and (b) is the curve of lasing intensity and full width at half maximum (FWHM) as a function of pump energy. Figure 5 The ultraviolet-visible absorption spectrum (a) and lasing emission spectrum (b) of the probe under different modification states in Embodiment 2 of the present invention are shown. Figure 6 This is a schematic diagram illustrating the detection principle of a lasing-luminescent nano-recognition probe for detecting cortisol in serum, provided in Embodiment 3 of the present invention. Figure 7 The diagram shows the working curve of cortisol detection in Embodiment 3 of the present invention; wherein, (a) is the lasing emission spectrum of cortisol at different concentrations; (b) is the linear relationship between the logarithm of cortisol concentration and relative lasing intensity, with a detection range of 0.1 nM to 20 nM, a detection limit of 0.8074 nM, a linear regression equation of Y=0.00175lg(X)+0.02298, and a correlation coefficient R²=0.9825; Figure 8 The diagram shows the specificity and anti-interference performance of the probe; (a) is the probe specificity verification result, with the ordinate being the effective lasing intensity at 547 nm after subtracting the blank background; (b) is the anti-interference verification result of co-incubation with interfering substances, with the ordinate being the lasing intensity at 547 nm, and the error bar being the standard deviation of three parallel experiments (n=3). Detailed Implementation

[0021] To make the objectives, 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. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Example 1 like Figure 1 The present invention provides a lasing-luminescent nano-recognition probe, comprising, from the inside out: Au nanospheres 1, a mesoporous silica shell 2, a dense silica encapsulation layer 4, and a cortisol-targeting recognition molecule 5; wherein, the Au nanospheres form a resonant cavity; the mesoporous silica shell 2 wraps around the Au nanospheres, and an organic fluorescent gain medium 3 is loaded inside the mesoporous silica shell; the dense silica encapsulation layer 4 covers the mesoporous silica shell 2, and the surface of the dense silica encapsulation layer 4 has amino sites; the cortisol-targeting recognition molecule is coupled to the amino sites by amide bonds; the cortisol-targeting recognition molecule is used to specifically bind to cortisol molecules.

[0023] Specifically, in this embodiment, the Au nanospheres have a particle size of 15 nm to 20 nm; the mesoporous silica shell has a thickness of 20 nm to 25 nm; and the dense silica encapsulation layer has a thickness of 1 nm to 2 nm.

[0024] Furthermore, in this embodiment, the spontaneous emission spectrum of the resonance absorption curve of the organic fluorescent gain medium 3 overlaps with that of the Au nanosphere 1, thereby satisfying the energy level matching principle.

[0025] Specifically, in this embodiment, the organic fluorescence gain medium is 2',7'-difluorofluorescein (OG488); the cortisol targeting recognition molecule is a carboxyl-modified cortisol aptamer.

[0026] Further, in this embodiment, the base sequence of the cortisol aptamer is: 5'-COOH-ATG GGC AATGCG GGG TGG AGA ATG GTT GCC GCA CTT CGG C-3'.

[0027] Furthermore, in this embodiment, the lasing-luminescent nanoprobe has a lasing wavelength of 547 nm, a pump threshold ≤ 1 mJ / cm², and a full width at half maximum (FWHM) of the lasing spectrum ≤ 5 nm. The pump wavelength is 490 nm.

[0028] Example 2 like Figure 2 As shown, Embodiment 2 of the present invention provides a method for preparing a lasing-luminescent nano-recognition probe as described in Embodiment 1, comprising the following steps: Step 1: Au nanospheres were synthesized as resonant cavities using a modified Frens reduction method (trisodium citrate reduction method).

[0029] Specifically, the preparation method is as follows: Take 1 mL of 1% (w / v) tetrachloroauric acid solution, dilute it to 100 mL with ultrapure water, and then transfer it to a flask. Heat and stir the system at 110–130 °C. After the solution boils, quickly add 2.5–3.5 mL of 1% (w / v) trisodium citrate solution, and continue the reaction for 15–20 min, then stop heating. Allow the reaction solution to cool naturally to room temperature to obtain a gold nanosphere dispersion with an average particle size of 15 nm–20 nm.

[0030] Step 2: Using Au nanospheres as the core, a mesoporous silica shell is grown on their surface by the sol-gel method to obtain Au@mSiO2 nanoparticles coated with mesoporous silica. After centrifugation and washing, the Au@mSiO2 nanoparticles are resuspended in ultrapure water to obtain a dispersion of Au@mSiO2 nanoparticles.

[0031] Specifically, the preparation method is as follows: Take 1 mL of the gold nanosphere dispersion prepared in step 1, add 60-100 μL of 0.1 M hexadecyltrimethylammonium bromide solution, and incubate for 2 h. Then adjust the pH of the system to about 10 with NaOH solution, and add 40-60 μL of 4% tetraethoxysilane ethanol solution. Stir the reaction at room temperature for 12 h to obtain Au@mSiO2 nanoparticles coated with mesoporous silica. After the reaction, centrifuge at 8000-10000 r / min for 10 min, wash 3 times, and the final precipitate is Au@mSiO2 nanoparticles coated with mesoporous silica. Resuspend the Au@mSiO2 nanoparticles in 1 mL of ultrapure water to obtain the Au@mSiO2 nanoparticle dispersion.

[0032] Step 3: Add an organic fluorescent gain medium to the Au@mSiO2 nanoparticle dispersion. Utilize capillary adsorption to load the organic fluorescent gain medium onto the mesoporous silica shell, thus obtaining a dispersion of Au@mSiO2 nanoparticles doped with the organic fluorescent gain medium, denoted as spaser.

[0033] In this embodiment, the preparation method of spaser is as follows: 40 μL of 2 mM dimethyl sulfoxide solution of 2',7'-difluorofluorescein is added to the Au@mSiO2 nanoparticle dispersion obtained in step 2, and the mixture is left to stand in the dark for 2 days to obtain spaser dispersion.

[0034] Step 4: Add 3-aminopropyltrimethoxysilane (APTMS) to the spaser dispersion for in-situ growth, forming a dense silica encapsulation layer on the outer layer of the mesoporous silica shell and introducing surface amino sites. After vigorous stirring for 3 h, centrifuge and wash, and resuspend in ultrapure water to obtain a dispersion containing spaser-NH2 nanoparticles.

[0035] Specifically, in this embodiment, 12 μL of 1 mM APTMS ethanol solution was slowly added to the spaser dispersion, and the mixture was vigorously stirred for 3 h under near-neutral conditions (pH ≈ 7). Subsequently, it was centrifuged at 8000 r / min for 10 min, washed three times, and resuspended in ultrapure water. The obtained product was characterized by transmission electron microscopy, and the results are as follows: Figure 3 As shown in (b), this step achieves APTMS amination modification of the lasing-luminescent nano-recognition probe on one hand, and forms a dense silica layer on its surface on the other hand, sealing the mesopores on the probe surface, thereby effectively preventing the leakage of dye molecules.

[0036] Step 5: After activating the cortisol-targeting recognition molecule in the dark, it is coupled to the probe surface through an amide bonding reaction. After incubation, it is centrifuged and washed, and then resuspended in PBS buffer to obtain the lasing-luminescent nano-recognition probe.

[0037] Specifically, the coupled cortisol-targeting molecule is a carboxyl-modified cortisol aptamer, and the coupling steps are as follows: (1) Take 100 μL of carboxylated cortisol aptamer solution, add 50 μL of EDC / NHS activator mixture with a concentration of 0.96 μg / mL, and activate at 30℃ in the dark for 30 min to activate the carboxyl activity on the aptamer surface.

[0038] (2) Add 500 μL of spaser-NH2 dispersion prepared in step (4) of Example 1 to the activated cortisol aptamer solution and incubate at 37°C for 2.5 h; then add 175 μL of 5% (w / v) bovine serum albumin solution and continue incubation for 0.5 h. After the reaction is complete, centrifuge at 8000 r / min for 10 min, remove the supernatant, wash 3 times, and finally resuspend in PBS buffer at pH=7.4 to obtain the dispersion containing the lasing luminescent nano-recognition probe, denoted as spaser-NH2-aptamer nanoparticle dispersion.

[0039] In this embodiment, the luminescence performance of the lasing-luminescent nano-recognition probe is tested as follows.

[0040] A nanosecond pulsed laser (Quanta-Ray INDI, Spectrophysic, Inc., USA) with a repetition rate of 20 Hz was selected as the pump source, with a wavelength of 490 nm. Spectral signals were acquired using a high-sensitivity CCD fiber optic spectrometer (Thorlabs, CCS200). Pump energy density gradients of 0.1, 0.2, 0.5, 1, 2, 3, 4, and 5 mJ / cm² were set, and the emission spectra of the lasing-luminescent nanoprobe were acquired. The results are as follows: Figure 4 As shown in (a), the central wavelength of the probe's emission is approximately 547 nm. According to... Figure 4 In section (a), the input-output curves are plotted, as shown below. Figure 4 As shown in (b), the lowest pump threshold of the probe was measured to be approximately 1 mJ / cm², and the full width at half maximum (FWHM) of the lasing spectrum was approximately 5 nm.

[0041] Subsequently, the absorption and lasing emission spectra of the centrifuged spaser nanoparticles, spaser-NH2 nanoparticles, and spaser-NH2-aptamer nanoparticles were compared and analyzed. Figure 5As shown in (a), the characteristic absorption peak of the UV-Vis absorption spectrum exhibits a red shift (525 nm before amination modification, 526 nm after amination modification, and 531 nm after aptamer coupling), confirming that the aptamer has been successfully bound to the surface of the lasing-luminescent nanoprobe. Figure 5 As shown in Figure (b), the above treatment and modification processes did not change the luminescence characteristics of the probe; the emission peak remained stable at 547 nm, and the full width at half maximum (FWHM) was approximately 5 nm. Furthermore, the comparison between spaser nanoparticles and spaser-NH2 nanoparticles indicates that the luminescence intensity of the probe was significantly enhanced after amination modification, approximately three times that before modification. This result confirms that in-situ growth of a dense silica layer on the outer layer of mesoporous silica using APTMS can effectively prevent fluorescent dye leakage. In addition, the luminescence intensity of spaser-NH2 nanoparticles was comparable to that of spaser-NH2-aptamer nanoparticles, indicating that modifying the surface of spaser-NH2 nanoparticles with cortisol aptamer molecules had no effect on their luminescence performance.

[0042] Example 3 Embodiment 3 of the present invention provides an application of a lasing-luminescent nano-recognition probe in the detection of cortisol in serum, such as... Figure 6 As shown, the process includes the following steps: mixing and incubating the lasing-luminescent nanoprobe solution with a serum sample to be tested, adding a magnetic microsphere solution to conduct a competitive reaction, wherein the magnetic microspheres are coupled with cortisol antigen; after the competitive reaction, using magnetic separation technology to separate the supernatant from the substrate, and measuring the lasing spectral intensity of the supernatant after magnetic separation; the magnetic microspheres are used to compete with cortisol molecules in the serum sample to bind to the cortisol aptamer on the lasing-luminescent nanoprobe; and determining the cortisol concentration in the serum based on the lasing spectral intensity.

[0043] The specific operating steps are as follows: (1) Prepare cortisol solutions of different concentrations (0 nM, 10 nM, 20 nM, 40 nM, 60 nM, 100 nM, 200 nM, 500 nM, 2000 nM), and add 5 μL of each solution to 500 μL of lasing-luminescent nano-recognition probe solution, and incubate at 37℃ for 0.5 h; wherein, the concentration of the lasing-luminescent nano-recognition probe solution is 1.3 nM; (2) Add 30 μL of magnetic nanoparticle mixture modified with cortisol antigen (1 mg / mL) to the above reaction system, and perform competitive reaction for 0.5 h, followed by magnetic separation using a magnetic rack; (3) The spectral signals of the above samples were collected according to the method described in Example 3, and the results are as follows: Figure 7As shown. In the concentration range of 0–20 nM, the linear regression equation between relative lasing intensity (Y) and cortisol concentration (X, nM) is Y = 0.00175 lg(X) + 0.02298, with a correlation coefficient R² = 0.9825.

[0044] To verify the specificity of the lasing-luminescent nanoprobe for cortisol detection and its detection stability in complex biological samples, specificity tests and interference resistance tests were conducted, as follows: (1) Specificity test: Solutions of glucose (5 mM), ascorbic acid (23 mM), β-estradiol (0.08 pM), progesterone (50 nM), testosterone (10 nM), and cortisol (500 nM) were prepared respectively. 5 μL of each of these solutions was added to 500 μL of lasing luminescent nanoparticle recognition probe solution, and incubated at 37 ℃ for 0.5 h. Then, 30 μL of 1 mg / mL magnetic beads were added to each solution, and the reaction was allowed to proceed competitively for 0.5 h. Magnetic separation was then performed using a magnetic rack, and the lasing intensity of the supernatant was finally measured. The results are as follows: Figure 8 As shown in (a). With the effective intensity of the cortisol group as 100%, the relative response intensity is: The results showed that the probe's responses to glucose, ascorbic acid, β-estradiol, progesterone, and testosterone were 29.5%, 28.4%, 30.7%, 18.2%, and 23.9%, respectively, all significantly lower than those in the cortisol group, indicating good specificity.

[0045] (2) Anti-interference test: Cortisol solution was mixed with glucose, ascorbic acid, β-estradiol, progesterone, and testosterone and incubated. A blank control group (probe solution only) and a pure cortisol control group (probe + cortisol solution only) were also set up. Signals in each group were detected. Results are as follows: Figure 8 As shown in (b), with the pure cortisol group as 100%, the signal retention rate was... The results showed that the signal retention rates for each group were 129.4%, 105.9%, 100.0%, 82.4%, and 111.8%, respectively. The signal change rates for β-estradiol, ascorbic acid, and testosterone were less than 12%, with no significant interference. Glucose and progesterone showed slight fluctuations, which could be eliminated through pretreatment. The probes are suitable for detection in serum samples.

[0046] In summary, this invention provides a lasing-luminescent nanoprobe, its preparation method, and its application. Using Au nanospheres as a plasmonic resonant cavity, a mesoporous silica layer loaded with an organic fluorescent gain medium as the gain medium, and a dense silica layer as the encapsulation layer, a narrow-bandwidth, low-pump-threshold lasing-luminescent nanoprobe with both high luminescence stability and high biological stability is constructed. This probe can achieve luminescence enhancement and exhibits excellent detection performance for trace amounts of cortisol, with a low detection limit. Furthermore, it possesses good specificity and anti-interference capabilities, making it suitable for highly sensitive and specific quantitative detection of real serum samples.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A lasing-luminescent nano-recognition probe, characterized in that, include: Au nanospheres forming the resonant cavity; The Au nanospheres are encased in a mesoporous silica shell, within which an organic fluorescent gain medium is loaded. A dense silica encapsulation layer covering the mesoporous silica shell, wherein the surface of the dense silica encapsulation layer has amino sites; A cortisol-targeting recognition molecule is coupled to the amino site via an amide bond, the cortisol-targeting recognition molecule being used to specifically bind to cortisol molecules.

2. The lasing-luminescent nano-recognition probe according to claim 1, characterized in that, The Au nanospheres have a particle size of 15 nm to 20 nm; the mesoporous silica shell has a thickness of 20 nm to 25 nm; and the dense silica encapsulation layer has a thickness of 1 nm to 2 nm.

3. The lasing-luminescent nano-recognition probe according to claim 1, characterized in that, The spontaneous emission spectrum of the organic fluorescent gain medium overlaps with the resonant absorption curve of the Au nanosphere, thus satisfying the energy level matching principle.

4. The lasing-luminescent nano-recognition probe according to claim 1, characterized in that, The organic fluorescence gain medium is 2',7'-difluorofluorescein (OG488); the cortisol targeting recognition molecule is a carboxyl-modified cortisol aptamer.

5. The lasing-luminescent nano-recognition probe according to claim 4, characterized in that, The base sequence of the cortisol aptamer is: 5'-COOH-ATG GGC AAT GCG GGG TGG AGA ATG GTT GCC GCA CTT CGG C-3'.

6. The lasing-luminescent nano-recognition probe according to claim 1, characterized in that, The lasing wavelength is 547 nm, the pump threshold is ≤1 mJ / cm², and the full width at half maximum (FWHM) of the lasing spectrum is ≤5 nm.

7. The method for preparing a lasing-luminescent nano-recognition probe according to claim 1, characterized in that, Includes the following steps: Step 1: Synthesize Au nanospheres as a resonant cavity using a modified Frens reduction method; Step 2: Using Au nanospheres as the core, a mesoporous silica shell is grown on their surface by the sol-gel method, and then the shell is centrifuged, washed, and resuspended in ultrapure water; Step 3: Add organic fluorescent gain medium to ultrapure water and load the organic fluorescent gain medium into the mesoporous silica shell by capillary adsorption. Step 4: Add 3-aminopropyltrimethoxysilane (APTMS) to the material solution for in-situ growth to form a dense silica encapsulation layer on the outer layer of the mesoporous silica shell and introduce surface amino sites. After vigorous stirring for 3 h, centrifuge and wash, and resuspend in ultrapure water. Step 5: After activating the cortisol-targeting recognition molecule in the dark, it is coupled to the probe surface through an amide bonding reaction. After incubation, it is centrifuged and washed, and then resuspended in PBS buffer to obtain the lasing-luminescent nano-recognition probe.

8. The method for preparing a lasing-luminescent nano-recognition probe according to claim 7, characterized in that, In step 5, the cortisol targeting recognition molecule used is a cortisol aptamer with carboxyl modification; its activation method is: activated by EDC / NHS at 30°C in the dark for 30 min; the centrifugation and washing method is: centrifuged at 8000 r / min for 10 min and washed 3 times; after centrifugation and washing, it is resuspended in PBS buffer at pH=7.

4.

9. The application of the lasing-luminescent nanoprobe according to claim 1 in the detection of cortisol in serum, characterized in that, Includes the following steps: The lasing-luminescent nanoprobe solution was mixed and incubated with a serum sample to be tested. A magnetic microsphere solution was then added to initiate a competitive reaction. The magnetic microspheres were coupled with cortisol antigen. After magnetic separation, the lasing spectral intensity of the supernatant was measured. The magnetic microspheres were used to compete with cortisol molecules in the serum sample to bind to the cortisol aptamer on the lasing-luminescent nanoprobe. Serum cortisol concentration was determined based on lasing spectral intensity.

10. The application of the lasing-luminescent nano-recognition probe according to claim 9 in the detection of cortisol in serum, characterized in that: The lasing-luminescent nano-recognition probe was incubated with the serum sample for 0.5 h. The concentration of the added magnetic microsphere solution was 1 mg / mL; the volume ratio of the magnetic microsphere solution to the lasing luminescent nano-recognition probe solution was 1:10-1:20; and the competitive reaction time was 0.5 h.

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