A high-sensitivity and high-selectivity fluorescent molecular imprinting probe, a preparation method and application thereof

Abstract

The application discloses a high-sensitivity and high-selectivity fluorescent molecular imprinting probe as well as a preparation method and application thereof. Glucose and trisodium citrate are used as raw materials, C-dots are generated by using a hydrothermal synthesis method, then AgNO3 and HAuCl4 are reacted to obtain Ag@Au NPs@C-dots, then a molecular imprinting technology is used on the surface of the Ag@Au NPs@C-dots, and serotonin is used as a template molecule to synthesize MIP. After eluting the template molecule, the Ag NPs@N / GODs@MIP fluorescent probe which retains the recognition site capable of specifically recognizing serotonin is obtained. The recognition site on the probe can be used as a switch to adsorb serotonin with different concentrations, and the serotonin can effectively quench the fluorescence intensity of the probe, so that a new method for detecting serotonin is established.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of fluorescent probe preparation, in particular to a high-sensitivity and high-selectivity fluorescent molecular imprinting probe and a preparation method and application thereof. BACKGROUND

[0002] Bananas contain 5-hydroxytryptamine which can make people feel happy and peaceful, and can be used as high-quality food for treating depression. The lack of 5-hydroxytryptamine makes people prone to depression. It has been reported that banana pulp can be used to prevent and even alleviate depression, anemia and hypertension. The detection and analysis of trace nutrients such as 5-hydroxytryptamine in bananas can provide data support for the analysis of nutritional functions, component extraction and genetic breeding of bananas. At present, the detection of 5-hydroxytryptamine in bananas mainly includes electrochemical method and liquid chromatography method. However, these methods still have some problems in practical application, such as poor reproducibility and stability of electrochemiluminescence, and time-consuming and high cost of high-performance liquid chromatography. Therefore, it is of great significance to study a new method for detecting 5-hydroxytryptamine with high sensitivity, high selectivity and good stability, which is fast and simple.

[0003] Quantum dots have a characteristic size of less than 10 nm and many unique structures, physical and chemical properties and photochemical properties, and have become a rising star in the field of the intersection of carbon materials and traditional QDs (TQD). Compared with traditional semiconductor quantum dots (TQD) and organic dyes, C-dots have the advantages of simple synthesis, excellent fluorescence properties, easy surface modification, abundant carbon sources in nature, low price, low toxicity and high anti-photobleaching property. In addition, doping of different metal ions not only makes C-dots exhibit more excellent optical properties, but also endows C-dots with new intrinsic properties, improves the quantum yield and application ability of C-dots. The linear response range of the method is 0.005-100 muM, and the detection limit is 0.35 nM (S / N=3). Especially compared with single-metal nanoparticles, double-metal nanoparticles enhance the magnetic, optical and catalytic properties. This may be due to the unique interparticle interaction in the double-metal composition, which leads to good synergy between the two metals, promotes molecular adsorption and activation. Although carbon quantum dots have many advantages, their selectivity and recognition ability for target molecules are weak, and their anti-interference ability for target substances in complex matrix is not strong, and their recognition ability needs to be further improved.

[0004] In order to realize the rapid detection of target molecules, the introduction of specific recognition elements is an effective way to improve the selective recognition ability of C-dots and other probes. MIP is a technology for creating artificial recognition sites in a polymer matrix, which is complementary to the template in terms of size, shape and spatial arrangement of functional groups. Purnendu et al. used citric acid and urea as raw materials to prepare nitrogen-doped graphene quantum dots, and then fixed the bimetallic Au / Ag core-shell on the surface thereof to detect anticancer hydroxyurea by molecular imprinting. The results show that there is no matrix effect, cross-reactivity and false positive. Therefore, MIP is expected to become an ideal recognition element for fluorescent probes. SUMMARY

[0005] Therefore, the present application provides a high-sensitivity and high-selectivity fluorescent molecular imprinting probe, a preparation method and application thereof. Glucose and trisodium citrate are used as raw materials, C-dots are generated by a hydrothermal synthesis method, AgNO3 and HAuCl4 are reacted to obtain Ag@Au NPs@C-dots, and then a MIP is synthesized on the surface of the Ag@Au NPs@C-dots by using a molecular imprinting technology with serotonin as a template molecule. After eluting the template molecule, the Ag NPs@N / GODs@MIP fluorescent probe which retains the recognition site capable of specifically recognizing serotonin is obtained. The recognition site on the probe can be used as a switch to adsorb serotonin of different concentrations, and the serotonin can effectively quench the fluorescence intensity of the probe, so that a new method for detecting serotonin is established.

[0006] To achieve the above object, the present application adopts the following technical solutions:

[0007] Firstly, the present application provides a preparation method of a high-sensitivity and high-selectivity fluorescent molecular imprinting probe, comprising the following steps:

[0008] Step one: synthesis of Ag@AuNPs@C-dots

[0009] (1.1) Dissolve glucose and trisodium citrate in deionized water, ultrasonically disperse uniformly, and then transfer to a high-temperature reaction kettle with a polytetrafluoroethylene liner at 250 DEG C for 3h. After cooling, remove the reaction liquid and centrifuge at 12000rpm / min for 10min, take the supernatant, dialyze in deionized water for 12h, and then vacuum dry at 60 DEG C to obtain carbon quantum dot powder;

[0010] (1.2) The carbon quantum dots powder prepared in step (1.1) is dispersed in deionized water, and AgNO3, trisodium citrate, and NaOH are added under stirring, and then HAuCl4 and NH2OH are added, and the mixture is stirred at 50°C for 30 min, and then the reaction solution is filtered through a 0.22 μm microporous filter membrane, and vacuum dried at 60°C for 5 h to obtain Ag@AuNPs@C-dots;

[0011] Step two: synthesis of Ag@AuNPs@C-dots / MIP

[0012] (2.1) 1×10 -4 mol / L of a serotonin solution, Ag@AuNPs@C-dots, monomer methacrylic acid, toluene, crosslinking agent, and initiator are added to a flask and ultrasonically dissolved for 10 min, and then polymerized at 45°C under N2for 5 h, and then dispersed in deionized water by ultrasonication, and centrifuged at 10,000 rpm / min to obtain a reaction product;

[0013] (2.2) The reaction product is washed with deionized water, and then eluted with a formic acid:acetic acid solution to elute the serotonin, to obtain Ag@AuNPs@C-dots / MIP, which is a high-sensitivity and high-selectivity fluorescent molecular imprinting probe.

[0014] Preferably, the weight ratio of glucose to trisodium citrate in step (1.1) is 3:1.

[0015] Preferably, the concentration of AgNO3 in step (1.2) is 0.1 mol / L, the concentration of trisodium citrate is 0.1 mol / L, the concentration of NaOH is 25 mmol / L, the concentration of HAuCl4 is 0.5 mmol / L, and the concentration of NH2OH is 6.25 mmol / L.

[0016] Further, in step (1.2), the weight ratio of carbon quantum dots powder: AgNO3: trisodium citrate: NaOH: HAuCl4: NH2OH is 0.2 g: 50 μL: 250 μL: 3 mL: 5 mL: 5 mL.

[0017] Preferably, in step (2.1), the weight ratio of serotonin solution: Ag@AuNPs@C-dots: monomer methacrylic acid: toluene: crosslinking agent: initiator is 10 mL: 20 mg: 0.68 mL: 100 μL: 3.15 mL: 30 mg.

[0018] Preferably, the crosslinking agent is ethylene glycol dimethacrylate, and the initiator is AIBN.

[0019] Preferably, the deionized water washing in step (2.2) is repeated for 3 times, and the volume ratio of formic acid to acetic acid in the formic acid: acetic acid solution is 5:1.

[0020] The application further provides a high-sensitivity and high-selectivity fluorescent molecular imprinting probe prepared by the method.

[0021] The application further provides the high-sensitivity and high-selectivity fluorescent molecular imprinting probe prepared by the method or the application of the high-sensitivity and high-selectivity fluorescent molecular imprinting probe, namely the application in trace serotonin detection.

[0022] Further, specifically, the Ag@AuNPs@C-dots / MIP is dissolved in a 0.1 mol / L PBS buffer solution with pH=7.4, the to-be-detected substance is added and fully reacted for 7 min, and the fluorescence intensity before and after the addition is detected.

[0023] According to the technical scheme, compared with the prior art, the application provides a high-sensitivity and high-selectivity fluorescent molecular imprinting probe, a preparation method and application thereof, and has the following beneficial effects.

[0024] Glucose and trisodium citrate are used as raw materials, C-dots are generated by a hydrothermal method, then AgNO3 and HAuCl4 are reacted to obtain Ag@AuNPs@C-dots, then the molecular imprinting technology is used on the surface of the Ag@AuNPs@C-dots, serotonin is used as a template molecule, and MIP is synthesized. After eluting the template molecule, the AgNPs@N / GODs@MIP fluorescent probe which retains the recognition site capable of specifically recognizing serotonin is obtained. The recognition site on the probe can adsorb serotonin with different concentrations, and the serotonin can effectively quench the fluorescence intensity of the probe, so that a new method for detecting serotonin is established. The introduction of the gold-silver bimetallic material improves the fluorescence performance of the C-dots, and the fluorescence intensity and sensitivity of the probe are very high. The introduction of the MIP effectively improves the ability of the sensor to specifically recognize serotonin. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only embodiments of the application, and those skilled in the art can obtain other drawings according to the provided drawings without any creative effort.

[0026] Figure 1Characterization of Ag@Au NPs@C-dots, where: (A) TEM image of Ag@Au NPs@C-dots, (B) Statistical size distribution of Ag@Au NPs@C-dots, (C) XRD spectrum of Ag@Au NPs@C-dots, (D) XPS spectrum of Ag@Au NPs@C-dots;

[0027] Figure 2 Characterization of MIP, where: (A) FTIR spectrum of 5-HT and MIP, (B) Probe EIS spectrum;

[0028] Figure 3 Fluorescence performance detection chart of Ag@Au NPs@C-dots / MIP probe, where: (A) UV absorption spectrum of Ag@Au NPs@C-dots probe, (B) Fluorescence spectrum of Ag@Au NPs@C-dots probe: a. Excitation spectrum; b. Emission spectrum;

[0029] Figure 4 Effect of different concentrations of serotonin on the fluorescence intensity of Ag@Au NPs@C-dots: a-e (0, 50, 500, 2500, 5000 × 10 -11 mol / L) serotonin;

[0030] Figure 5 Effect of pH value of buffer and quenching reaction time on detection of serotonin by Ag@Au NPs@C-dots / MIP probe, where: (A) Effect of pH value on fluorescence intensity of probe, (B) Effect of reaction time on fluorescence intensity of probe;

[0031] Figure 6 Fluorescence response of Ag@Au NPs@C-dots / MIP probe to serotonin, where: (A) Fluorescence response of Ag@Au NPs@C-dots / MIP probe to different concentrations of serotonin: a-p: (0, 5, 25, 50, 100, 200, 300, 400, 500, 750, 1000, 2000, 2500, 4000, 5000, 6000, 7000) × 10 -11 mol / L serotonin, (B) Calibration curve;

[0032] Figure 7 Fluorescence response of Ag@Au NPs@C-dots / MIP probe to serotonin and interfering substances, where: a. 2 × 10 -9 mol / L of serotonin, b. 2 × 10 -9 mol / L of serotonin and 1 × 10 -7mol / L of norepinephrine, dopamine, vitamin C, vitamin B6, glucose, fructose, imidacloprid, thiamethoxam, beta-carotene mixture; n = 5;

[0033] Figure 8 For the reproducibility of Ag@AuNPs@C-dots / MIP probe, n = 5;

[0034] Figure 9 For the stability of Ag@AuNPs@C-dots / MIP probe, n = 5. DETAILED DESCRIPTION

[0035] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0036] Example 1

[0037] (1.1) Accurately weigh 3.0 g of glucose and 1.0 g of trisodium citrate, dissolve them in 30 mL of deionized water, ultrasonic for 5 min, then transfer the uniformly dispersed liquid to a polytetrafluoroethylene lined high temperature reaction kettle, set the oven temperature to 250℃, and set the heating reaction time to 3 h, after the reaction kettle is fully cooled, remove the reaction liquid, at this time the reaction liquid is dark yellow. Centrifuge the reaction liquid at a speed of 12000 rpm / min for 10 min, take the supernatant and dialyze in deionized water for 12 h to obtain a light yellow carbon dot solution, and vacuum dry at 60℃ to obtain carbon quantum dot powder;

[0038] (1.2) Take 0.2 g of carbon quantum dot powder and disperse it in 50 mL of deionized water, under stirring, add 50 μL of 0.1 mol / L AgNO3 and 250 μL of 0.1 mol / L trisodium citrate, then add 3 mL of 25 mmol / L NaOH, and stir under a 50℃ water bath for 30 min. Next, add 5 mL of 0.5 mmol / L HAuCl4 and 5 mL of 6.25 mM NH2OH, and stir the mixed solution for another 30 min. Finally, filter the reaction solution through a 0.22 μm microporous filter membrane, and vacuum dry at 60℃ for 5 h to obtain Ag@AuNPs@C-dots.

[0039] Step two: synthesis of Ag@AuNPs@C-dots / MIP

[0040] (2.1) Add 10 mL of 1×10 -4The reaction product was obtained by ultrasonic dispersion of the reactants in 50 mL deionized water and centrifugation at 10,000 rpm / min.

[0041] (2.2) The oligomers and unreacted monomers produced during the reaction were washed away with deionized water, and the process was repeated three times. Then, 20 mL of a formic acid:acetic acid solution (5:1, V:V) was used to elute the template serotonin from the molecularly imprinted polymer. Finally, the reaction product was redissolved in 10 mL of a PBS buffer (0.1 mol / L, pH = 7.4) and stored at 4°C for later use.

[0042] Experimental Example

[0043] Fluorescence detection method:

[0044] 600 μL of the Ag@AuNPs@C-dots probe solution was taken, different concentrations of serotonin were added, and the volume was made up to 2 mL with 0.1 mol / L PBS at pH = 6.8. After 7 min of reaction, the fluorescence intensity before and after the addition of serotonin was detected.

[0045] The excitation wavelength was Ex = 400 nm, the emission spectrum range was 400-700 nm, the excitation wavelength slit was 5 nm, the emission spectrum slit was 5 nm, and the photomultiplier tube was 700 V.

[0046] Sample pretreatment:

[0047] 5.0 g of chopped banana sample was weighed, 10 mL of acetone was added, and the mixture was shaken in a vortex shaker for 5 min. Then, the mixture was reacted under magnetic stirring for 2 h, followed by centrifugation at 5000 rpm / min at 4°C for 1 h. The supernatant was removed and evaporated to dryness in a 35°C water bath. The residue was dissolved in 1 mL of a PBS buffer (0.1 mol / L, pH = 7.4) and stored in a 4°C refrigerator for later use.

[0048] I. Characterization of Ag@AuNPs@C-dots

[0049] As shown in FIG. 1, the Ag@AuNPs@C-dots have a diameter of about 2-3 nm, and the surface of the Ag@AuNPs@C-dots is covered with a layer of carbon dots. Figure 1As shown, by SEM it can be seen that the synthesized Ag@AuNPs@C-dots are core-shell spherical nanoparticles with C-dots as the core, the particle distribution is relatively uniform, and the size is relatively uniform, which shows that the synthesized Ag@AuNPs@C-dots have good dispersity; the size of the synthesized Ag@AuNPs@C-dots is in the range of 15-75 nm, and the average size is about 42 nm; the XRD pattern of the synthesized Ag@AuNPs@C-dots shows that the Ag@AuNPs@C-dots have been successfully synthesized.

[0050] II. MIP characterization

[0051] Fourier infrared spectroscopy (FI-IR) and electrochemical impedance spectroscopy (EIS) were used to characterize the Ag@AuNPs@C-dots / MIP probe and the adsorption and elution of 5-HT.

[0052] As shown in FIG. 2A, the FT-IR spectrum of the Ag@AuNPs@C-dots / MIP probe shows that the characteristic peaks of 5-HT are all retained during the preparation of the MIP, with only slight changes in position. However, when the 5-HT is removed from the MIP, the peaks disappear. In addition, EIS proves the generation of the Ag@AuNPs@C-dots / MIP. The above results prove that serotonin has been successfully imprinted on the probe. Figure 2 Figure 2 As shown in FIG. 2A, the FT-IR spectrum of the Ag@AuNPs@C-dots / MIP probe shows that the characteristic peaks of 5-HT are all retained during the preparation of the MIP, with only slight changes in position. However, when the 5-HT is removed from the MIP, the peaks disappear. In addition, EIS proves the generation of the Ag@AuNPs@C-dots / MIP. The above results prove that serotonin has been successfully imprinted on the probe.

[0053] III. Fluorescent properties of the Ag@AuNPs@C-dots / MIP probe

[0054] The optical properties of the fluorescent probe were investigated using ultraviolet-visible absorption spectroscopy and fluorescence spectroscopy, as shown in FIG. 3A and FIG. 3B. Figure 3 As shown in FIG. 3A, the Ag@AuNPs@C-dots / MIP has a strong absorption at 493 nm. As shown in FIG. 3B, the maximum excitation wavelength of the material is near 398 nm (curve a), and the maximum emission wavelength of the probe is obtained at about 530 nm (curve b) when the probe is excited at 398 nm. The large Stokes shift of the probe effectively avoids the overlap between the excitation and emission spectra. Moreover, the emission spectrum of the probe has high intensity, a symmetrical and narrow peak shape, which shows that the probe has good fluorescent properties. The quantum yield of a fluorescent substance is an important parameter affecting the fluorescent probe. The quantum yield of the fluorescent substance can be obtained by comparing the integral fluorescence intensity (i.e., the area included in the corrected fluorescence spectrum) of the fluorescent sample to be measured and the reference fluorescent standard substance with a known quantum yield, as well as the absorbance of the incident light (ultraviolet-visible light) at the same excitation wavelength, and then calculating the values according to a specific formula: Figure 3

[0055] ​​

[0056] where Φ u , Φ s is the fluorescence quantum yield of the test substance and the reference standard substance; F u , F s is the integrated fluorescence intensity of the test substance and the reference substance; A u , A s is the absorbance of the test substance and the reference substance at the incident light of the excitation wavelength (A = εbc). In the experiment, Rhodamine B (RhB) solution was used as the reference fluorescence standard substance. The quantum yield of RhB was 0.89, and the related parameters are shown in Table 1:

[0057] Table 1 Comparison of the related parameters of the quantum yield of RhB and Ag@AuNPs@C-dots

[0058]

[0059] Four, five-hydroxytryptamine quenches Ag@AuNPs@C-dots / MIP probe

[0060] As Figure 4 shown, with the addition of different concentrations of five-hydroxytryptamine, the probe captures more and more five-hydroxytryptamine through MIP, and the fluorescence intensity of Ag@AuNPs@C-dots / MIP is continuously reduced, indicating that five-hydroxytryptamine can effectively quench the fluorescence intensity of the probe. First, the fluorescence lifetime of the probe in the presence of different concentrations of five-hydroxytryptamine was determined, and the detection results showed that the intrinsic lifetime τ0 of the material was 20.35 ns, and when 50, 500, 2500, and 5000 x 10 -11 mol / L of five-hydroxytryptamine was added to the system, the fluorescence lifetime of Ag@AuNPs@C-dots / MIP was 20.01, 19.28, 18.74, and 17.28 ns, respectively. When the concentration of five-hydroxytryptamine increased from 0 mol / L to 5000 x 10 -11 mol / L, the fluorescence lifetime of the probe decreased by 307 ns, and the lifetime of the probe changed greatly. Therefore, the fluorescence quenching of five-hydroxytryptamine on Ag@AuNPs@C-dots probe is mainly a dynamic process, and electron transfer occurs to cause fluorescence quenching. The electron-donating group (such as -NH2, -OH) in five-hydroxytryptamine can transfer electrons to the excited-state empty electron orbital, so that the photo-excited single cannot directly jump to the original ground state orbital to emit fluorescence, thereby quenching the fluorescence of Ag@AuNPs@C-dots / MIP.

[0061] Five, optimal conditions of the experiment

[0062] The effects of the pH value of the buffer and the quenching reaction time on the detection of five-hydroxytryptamine by Ag@AuNPs@C-dots / MIP probe were studied. As Figure 5As shown, when the pH value of the buffer solution was in the range of 6.0-7.6, the detection rate of 1×10 -9 The ratio of the quenching value of the probe fluorescence before and after the addition of 5-hydroxytryptamine (ΔIF =

[0063] F0-F1, F0 is the fluorescence intensity before the addition of serotonin, and F1 is the fluorescence intensity before serotonin.) As the pH value increases from 6.0, △IF shows different changes, but at pH = 6.8, △IF reaches its maximum value. Therefore, PBS with pH = 6.8 was selected as the optimal system buffer. In addition, the effect of the reaction time between serotonin and the fluorescent probe on the quenching effect was studied. As the MIP on the probe captured more and more serotonin, the fluorescence of the probe was continuously quenched. When the reaction time reached 7 minutes, the fluorescence intensity quenching value △IF reached its maximum and remained unchanged. Therefore, the reaction time between serotonin and the probe was completed within 7 minutes, and the reaction time was selected to be 7 minutes.

[0064] 6. Fluorescence response of Ag@AuNPs@C-dots / MIP probe to serotonin

[0065] Under the optimal experimental conditions, different concentrations of serotonin were added to react with the probe, and the fluorescence intensity of the Ag@AuNPs@C-dots / MIP probe was detected before and after the addition of serotonin. The fluorescence intensity quenching value △IF=F0-F1 was calculated and a calibration curve was drawn. Figure 6 As shown in Figure 2, as the concentration of 5-HT increases, the fluorescence intensity of the probe is continuously quenched. The logarithm of the 5-HT concentration, ln(c), is 5×10 -11 -7000×10 -11 mol / L has a good linear relationship with the logarithm of the fluorescence quenching intensity ln(△IF) of the probe, and its linear regression equation is: ln(△IF)=0.59ln(c)(10 -11 mol / L)+3.34, r=0.9981, the detection limit is 1.08×10 -11 mol / L(DL=KS b / a, K=3). Compared with the existing reported methods, the present method has higher sensitivity in detecting serotonin.

[0066] VII. Selectivity of Ag@AuNPs@C-dots / MIP Probe

[0067] The ability of Ag@AuNPs@C-dots / MIP probe to selectively recognize the target molecule serotonin from interferents was studied. Figure 7 As shown, according to the experimental method, 1×10 -9 mol / L 5-HT fluorescence intensity (F0) and 1×10 -9serotonin 1 x 10 -7 mol / L norepinephrine, dopamine, vitamin C, vitamin B6, glucose, fructose, imidacloprid, thiamethoxam, β-carotene mixed fluorescence intensity (F1). The relative deviation RD = [(F0-F1) / F0] x 100% of two detection results was calculated, and the result was 1.95%, which indicated that the fluorescence intensity of the probe had little change after adding the interferent. The above results showed that the probe had good selective recognition ability for serotonin.

[0068] Eight, reproducibility and stability of Ag@AuNPs@C-dots / MIP probe

[0069] The reproducibility and stability of Ag@AuNPs@C-dots / MIP probe for detecting serotonin were studied, as shown in Figures 8-9 Under the same conditions, 1 x 10 -9 mol / L serotonin was added to five Ag@Au NPs@C-dots / MIP probes, and the fluorescence intensity before and after adding serotonin was detected, and △IF was calculated. The results showed that the relative standard deviation (RSD) of the △IF values of five experiments was 2.61%. This indicated that the method had good reproducibility. In addition, the prepared Ag@AuNPs@C-dots / MIP fluorescence probe was reacted with 1 x 10 -9 mol / L serotonin, and the fluorescence intensity was detected every 5 min, Figure 9 The results showed that the fluorescence intensity of the probe after 30 min was 99.8% of that after complete reaction, which indicated that the fluorescence probe had good stability.

[0070] Nine, actual sample detection

[0071] The fluorescence probe was applied to the detection of banana samples, and the standard addition recovery test was carried out, and the results are shown in Table 2.

[0072] Table 2 sample determination and recovery rate analysis results

[0073]

[0074] Table 2 shows that the detection results of serotonin in the sample are consistent with the HPLC method, the recovery rate of the method is 86.7-103.5%, and the RSD is less than 2.0%. The above results show that the fluorescence probe method can be used for actual sample detection.

[0075] The application synthesizes an Ag@AuNPs@C-dots fluorescent composite nanoprobe, and realizes the ultra-sensitive detection of serotonin in bananas by using the probe. The addition of Ag / Au bimetal in the composite nanoprobe effectively improves the fluorescence performance, so that the probe has stable and strong fluorescence emission spectrum and high quantum yield; and the introduction of MIP makes the probe show excellent selective recognition ability to the target molecule serotonin, and can effectively exclude interference. Therefore, the probe has the characteristics of high sensitivity, high selectivity and simple operation in the application of serotonin detection in bananas.

[0076] The various embodiments are described in the present specification in a progressive manner, and each embodiment focuses on the difference from other embodiments, and the same or similar parts between the various embodiments can be mutually referred to. For the device disclosed by the embodiments, since it corresponds to the method disclosed by the embodiments, the description is relatively simple, and the relevant parts can be referred to the method part.

[0077] The above description of the disclosed embodiments enables a person skilled in the art to implement or use the application. Various modifications to the embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the application. Therefore, the application will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a high-sensitivity and high-selectivity fluorescent molecular imprinting probe, characterized in that, The method comprises the following steps: Step one: synthesis of Ag@Au NPs@C-dots (1.1) Dissolve glucose and trisodium citrate in deionized water, and then transfer the uniformly dispersed solution into a high-temperature reaction kettle with a polytetrafluoroethylene liner, and react at 250°C for 3 hours. After the reaction is completed, the reaction solution is cooled and centrifuged at 12000 rpm / min for 10 minutes. The supernatant is dialyzed in deionized water for 12 hours, and then vacuum dried at 60°C to obtain carbon quantum dot powder; (1.2) The carbon quantum dot powder prepared in step (1.1) is dispersed in deionized water, and then AgNO3, trisodium citrate and NaOH are added under stirring. The mixture is stirred in a water bath at 50°C for 30 minutes, and then HAuCl4 and NH2OH are added. The mixture is stirred for another 30 minutes, and then filtered through a 0.22 μm microporous filter. The filtrate is vacuum dried at 60°C for 5 hours to obtain Ag@Au NPs@C-dots; Step two: synthesis of Ag@Au NPs@C-dots / MIP (2.1) 1 x 10 -4 mol / L of serotonin solution, Ag@AuNPs@C-dots, monomer methacrylic acid, toluene, crosslinking agent, initiator into a flask ultrasonic 10 min to dissolve thoroughly, polymerization under N2 at 45℃ for 5h, then ultrasonic dispersion in deionized water, 10000 rpm / min centrifugal to get the reaction product; (2.2) The reaction product is washed with deionized water, and then eluted with a formic acid:acetic acid solution to obtain Ag@Au NPs@C-dots / MIP, which is a high-sensitivity and high-selectivity fluorescent molecular imprinting probe.

2. The method according to claim 1, wherein the method is characterized by, In step (1.1), the weight ratio of glucose to trisodium citrate is 3:

1.

3. The method according to claim 1, wherein the method is characterized by, In step (1.2), the concentrations of AgNO3, trisodium citrate, NaOH, HAuCl4 and NH2OH are 0.1 mol / L, 0.1 mol / L, 25 mmol / L, 0.5 mmol / L and 6.25 mmol / L, respectively.

4. The method according to claim 3, wherein the method is characterized by, In step (1.2), the amounts of carbon quantum dot powder, AgNO3, trisodium citrate, NaOH, HAuCl4 and NH2OH are 0.2 g, 50 μL, 250 μL, 3 mL, 5 mL and 5 mL, respectively.

5. The method according to claim 1, wherein the method is characterized by, In step (2.1), the amounts of serotonin solution, Ag@Au NPs@C-dots, monomer methacrylic acid, toluene, crosslinking agent and initiator are 10 mL, 20 mg, 0.68 mL, 100 μL, 3.15 mL and 30 mg, respectively.

6. The method for preparing a high-sensitivity and high-selectivity fluorescent molecular imprinting probe according to claim 1 or 5, characterized in that, The crosslinking agent is ethylene glycol dimethacrylate, and the initiator is AIBN.

7. The method according to claim 1, wherein the method is characterized by, In step (2.2), the deionized water is washed for 3 times, and the volume ratio of formic acid to acetic acid in the formic acid:acetic acid solution is 5:

1.

8. A high-sensitivity and high-selectivity fluorescent molecular imprinting probe prepared by the method of any one of claims 1-7.

9. Use of the high-sensitivity and high-selectivity fluorescent molecular imprinting probe prepared by the method of any one of claims 1-7 or the high-sensitivity and high-selectivity fluorescent molecular imprinting probe of claim 8 in detection of trace serotonin.

10. Use according to claim 9, characterized in that, The Ag@Au NPs@C-dots / MIP is dissolved in 0.1 mol / L PBS buffer solution with pH=7.4, and then the to-be-detected substance is added and reacted for 7 minutes. The fluorescence intensity before and after the addition is detected.

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