Preparation method and application of novel lactic acid detection fluorescent probe

Abstract

The invention relates to a preparation method and application of a novel lactic acid detection fluorescent probe, and belongs to the technical field of fluorescent probes. The preparation method of the fluorescent probe comprises the following steps: synthesizing a core-shell type up-conversion nano material CS-UCNPs; carrying out surface modification on the core-shell type up-conversion nano material CS-UCNPs; coupling of the core-shell type up-conversion nano material CS-UCNPs and an L-lactic acid aptamer is carried out; and synthesizing the magnetic molybdenum disulfide nanosheet MoS2. The L-lactic acid aptamer, the core-shell UCNPs and the magnetic MoS2 are combined for high-sensitivity detection of the lactic acid content, and compared with a traditional blood lactic acid detection method, the method has the advantages of being non-invasive, non-invasive and the like; and the technology has the advantages of simplicity in operation, high specificity, quick response and the like, has a wider detection range and a lower detection limit, and is expected to be applied to actual sample detection work.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and in particular to a preparation method and application of a novel fluorescent probe for detecting lactic acid. Background Art

[0002] Lactate concentration data has important reference value. The measurement of venous blood lactate is an important diagnostic indicator for assessing shock and hypoxia. The time course of lactate levels and their changes is a prognostic criterion for predicting the development of sepsis, trauma, shock and burn damage. When the blood perfusion in the tissue is insufficient, the oxygen supply is insufficient and cannot meet the needs of metabolism. This imbalance leads to tissue hypoxia and lactic acidosis. If it is not corrected immediately, it will lead to progressive cell damage, multiple organ failure, and even death. Lactate is also of great significance in sports training. Nowadays, in the field of sports training, blood lactate indicators are mainly used to evaluate exercise intensity, provide a theoretical basis for coaches to formulate training plans, use blood lactate for sports selection, and assess the recovery of body fatigue. As people's understanding of lactate continues to deepen, while studying lactate, people pay more attention to the correlation between exercise and pathological conditions and changes in blood lactate concentration, as well as the differences and laws of blood lactate reactions under different conditions, which is the direction that future researchers will continue to explore.

[0003] Existing lactate detection methods mainly include instrumental analysis, chemical analysis and biosensor methods. Instrumental analysis mainly includes laboratory equipment such as high performance liquid chromatography and liquid chromatography-mass spectrometry for offline determination of lactate, but the disadvantages of complex sample pretreatment, expensive equipment and instruments, long analysis time and high operation requirements make it difficult to apply to rapid on-site detection. In chemical analysis, titration analysis is often used for lactate detection, but the sample processing process is complicated, the operation is cumbersome, time-consuming and labor-intensive, and a suitable means of indicating the end point, such as indicators, potentiometers, etc., must be available. Biosensors are analytical tools for detecting specific analytes. Their key elements are transducers and data analysis and visualization tools. They are often based on electrochemical, optical, piezoelectric and other transduction methods to quantitatively detect target targets through signal changes. Based on the above technical defects, how to establish a lactate detection method with good specificity, fast response and sensitive detection has become a technical problem that needs to be solved in the prior art. Summary of the invention

[0004] Based on the above technical problems to be solved by the present invention, the present invention adopts a probe prepared by coupling core-shell upconversion fluorescent nanomaterials (CS-UCNPs), magnetic molybdenum disulfide materials (MoS2) and L-lactic acid aptamers, which has simple steps, easy operation and high sensitivity.

[0005] One of the purposes of the present invention is to provide a method for preparing a novel fluorescent probe for lactic acid detection, characterized in that the method comprises:

[0006] S11, synthesis of core-shell upconversion nanomaterials CS-UCNPs;

[0007] S12, surface modification of the core-shell upconversion nanomaterial CS-UCNPs;

[0008] S13, coupling of the core-shell upconversion nanomaterial CS-UCNPs with L-lactic acid aptamer;

[0009] S14. Synthesis of magnetic molybdenum disulfide nanosheets MoS2.

[0010] Furthermore, the method comprises:

[0011] S21, synthesizing single-core UCNPs by solvothermal method; the single-core UCNPs are NaYF4:Yb,Er;

[0012] S22, coating the NaYF4 shell on the single-core UCNPs by a layer-by-layer assembly method to obtain the core-shell upconversion nanomaterial CS-UCNPs;

[0013] S23, modifying streptavidin (SA) on the surface of the CS-UCNPs by surface amination and classical glutaraldehyde coupling method;

[0014] S24, incubating with a biotin-modified lactic acid aptamer at 37° C. with shaking to prepare a fluorescent probe;

[0015] S25. The magnetic molybdenum disulfide nanosheets MoS2 are synthesized by functionalizing the surface of MoS2 nanosheets with dopamine hydrochloride and fixing iron ions.

[0016] Furthermore, the synthesis method of the UCNPs comprises:

[0017] S31, mixing yttrium chloride hexahydrate, ytterbium chloride hexahydrate, erbium chloride hexahydrate, oleic acid, and 1-octadecene, dispersing them, dissolving all the solids, and obtaining a compound solution;

[0018] S32, heating and stirring the compound solution, and placing the reaction system in a vacuum mode to obtain a solution from which oxygen is removed from the system;

[0019] S33, when no bubbles emerge from the solution, setting the solution to a nitrogen mode, adjusting the temperature to 100° C. to 140° C., switching to a vacuum mode, and obtaining a compound from which bound water molecules are removed;

[0020] S34, changing the system temperature to room temperature, adding a methanol solution containing ammonium fluoride and sodium hydroxide, adjusting the temperature to 15°C to 25°C, and stirring for 0.1 to 2.0 hours; preferably, adjusting the temperature to 20°C, and stirring for 0.5 hours; obtaining a primary product of NaYF4:Yb,Er coated with oleic acid (OA) on the surface;

[0021] S35, setting the mode to pass nitrogen, heating the solution, and switching to a vacuum mode to obtain a compound solution from which methanol has been removed;

[0022] S36, setting the nitrogen gas flow mode, heating the compound solution from which methanol has been removed to 300° C. to 350° C. for 0.5 h to 2.5 h; preferably, heating to 320° C. for 1.5 h; obtaining the preliminarily purified surface OA-coated NaYF4:Yb,Er;

[0023] S37, cool to room temperature, add ethanol to precipitate the product, wash the precipitate with cyclohexane, and dry it to obtain solid OA-UCNPs with surface OA-coated UCNPs; disperse the OA-UCNPs with cyclohexane to obtain mononuclear UCNPs dispersed with cyclohexane.

[0024] Further, the ratio of the amount of yttrium chloride hexahydrate: ytterbium chloride hexahydrate: erbium chloride hexahydrate is n 六水合氯化钇:六水合氯化镱:六水合氯化铒 , the n 六水合氯化钇:六水合氯化镱:六水合氯化铒 =(30-50):(5-7):(0.5-5); preferably, the n 六水合氯化钇:六水合氯化镱:六水合氯化铒 =40:9:1.

[0025] Furthermore, in the S33, the compound from which bound water molecules are removed is orange-yellow and oily.

[0026] Furthermore, the preparation of the core-shell upconversion nanomaterial CS-UCNPs comprises:

[0027] The NaYF4 shell was coated on the mononuclear UCNPs by a layer-by-layer assembly method, YCl3·6H2O was dissolved in OA and ODE, and the mixture was heated to 100°C to 140°C in a nitrogen atmosphere for degassing; vacuum was switched to remove water, and after the mixture was cooled to room temperature, the mononuclear UCNPs dispersed in cyclohexane were added dropwise, and then methanol containing NH4F and NaOH was added dropwise, and the mixture was stirred at room temperature; the solution was heated to 100°C to 140°C for condensation and reflux to remove methanol and cyclohexane, and the mixture was heated to 250°C to 390°C in a nitrogen atmosphere for 0.5h to 2.0h. After the reaction was completed, the mixture was washed and stored with ethanol and cyclohexane, respectively.

[0028] Furthermore, the surface modification method of CS-UCNPs is to modify the surface of CS-UCNPs by ligand exchange method, comprising:

[0029] Add polyethyleneimine into pure water and fully dissolve it; then add CS-UCNPs solution with cyclohexane as solvent and stir at room temperature for 12h to 36h; after the reaction, centrifuge the mixture, collect the precipitate, and wash it with ethanol several times to remove the unreacted PEI; finally, after freeze-drying for 2h to 10h, surface amino-modified core-shell UCNPs, PEI-CS-UCNPs, are obtained; then streptavidin SA is connected to the surface of PEI-CS-UCNPs by the classic glutaraldehyde coupling method; Tris-HCl, PEI-CS-UCNPs and glutaraldehyde solution are mixed evenly at room temperature and sonicated; continue to slowly shake at room temperature for 0.5h to 5h, resuspend in Tris-HCl solution after centrifugal washing, add SA, and shake and incubate at room temperature to obtain SA-coupled core-shell UCNPs, SA-CS-UCNPs.

[0030] Furthermore, the method for preparing the magnetic molybdenum disulfide nanosheets MoS2 comprises:

[0031] Sodium molybdate, thiourea and PEG-20000 are dissolved in pure water. After vigorous stirring at room temperature, the solution is transferred to a polytetrafluoroethylene-lined autoclave. The autoclave is then placed in a muffle furnace and heated to 180°C to 260°C for 12 to 36 hours. After the reaction is completed, the reaction is washed with pure water and solid MoS2 is obtained after vacuum freeze-drying. The solid MoS2 is dispersed in a Tris-HCl solution and subjected to ultrasound to obtain MoS2 nanosheets. Dopamine hydrochloride is then added and the mixture is slowly shaken at room temperature for 2 to 6 hours. The obtained material is washed with ultrapure water. The prepared material is dissolved in water, subjected to ultrasonic treatment to completely dissolve the ferric chloride and ferrous sulfate heptahydrate, and shaken vigorously for 0.5 h to 2 h to ensure that the iron ions are fully fixed on the functionalized MoS2 nanosheets; ammonia water and citric acid are quickly added, and the mixture is shaken vigorously for 0.5 h to 2 h to obtain a black precipitate; the black precipitate is subjected to magnetic separation, washed with acetone, and dried at 40 ° C to 90 ° C for 0.5 h to 2 h to obtain the magnetic molybdenum disulfide nanosheet MoS2.

[0032] The second purpose of the present invention is to provide an application of a new fluorescent probe for lactic acid detection, wherein the application of the new fluorescent probe for lactic acid detection is the application of the fluorescent probe in lactic acid standard solutions of different concentrations; the lactic acid standard solutions of different concentrations include 0, 1mM, 3mM, 10mM, 15mM or 30mM.

[0033] Furthermore, the application method comprises:

[0034] Add lactic acid standards of different concentration gradients to Apt-CS-UCNPs dispersed in Tris-HCl buffer, and incubate at 37°C for 5 to 45 minutes; add the magnetic molybdenum disulfide nanosheets MoS2, mix thoroughly, and continue to react at 37°C for 5 to 45 minutes; after the reaction is completed, perform magnetic separation, place the supernatant in a fluorescent cuvette, and measure the fluorescence intensity of the supernatant at 545 nm using a fluorescence spectrophotometer under 980 nm excitation light.

[0035] Compared with the prior art, the present invention proposes a preparation method and application of a novel fluorescent probe for lactic acid detection, which adopts upconversion fluorescence sensing technology, has good specificity, fast response, and sensitive detection, and has the following beneficial effects:

[0036] 1. The present invention combines L-lactic acid aptamer, core-shell UCNPs, and magnetic MoS2 for highly sensitive detection of lactic acid content. Compared with the traditional blood lactate detection method, it has the characteristics of being non-invasive and non-intrusive. This technology provides a new technical approach for non-invasive sweat lactate detection.

[0037] 2. The present invention establishes a low-cost, easy-to-synthesize, and highly sensitive upconversion fluorescent aptamer sensing method, and successfully synthesizes core-shell UCNPs, which have an obvious emission peak at 545nm under the excitation of 980nm laser. At the same time, MoS2 nanosheets are used as the substrate, and Fe3O4 particles are grown on their surface to make them magnetic. During the detection process, magnetic separation can be used to reduce background interference, thereby improving the detection sensitivity.

[0038] 3. In the application of lactic acid detection, the present invention shows that in the range of 0-30mM, there is a good linear relationship between the logarithmic value of the concentration of the lactic acid standard and the fluorescence intensity value, and the detection limit of lactic acid is 0.38mM. This activity range is similar to the level of lactic acid physiologically expressed in human sweat. This technology has the advantages of simple operation, high specificity, rapid response, etc., with a wide detection range and a low detection limit, and is expected to be applied in actual sample detection work. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 A schematic diagram of a fluorescent probe detection principle of an embodiment of the present invention is shown;

[0040] Figure 2 A transmission electron microscope image of a fluorescent probe according to an embodiment of the present invention is shown;

[0041] Figure 3 A Zeta potential diagram of a fluorescent probe according to an embodiment of the present invention is shown;

[0042] Figure 4 A transmission electron microscope image of a magnetic MoS2 according to an embodiment of the present invention is shown;

[0043] Figure 5 A circular dichroism spectrum of a lactic acid aptamer binding to lactic acid according to an embodiment of the present invention is shown;

[0044] Figure 6 A graph showing the change in fluorescence intensity of a fluorescent probe detecting lactic acid of different concentrations according to an embodiment of the present invention is shown;

[0045] Figure 7 A standard curve fitting diagram of a fluorescent probe for detecting lactic acid of different concentrations according to an embodiment of the present invention is shown. DETAILED DESCRIPTION

[0046] The exemplary embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present application are shown in the accompanying drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0047] In the following examples, unless otherwise specified, the technical means adopted are conventional means well known to those skilled in the art, and the reagents and materials in the present invention are obtained from the market or other public channels.

[0048] The experimental materials and equipment involved in the present invention mainly include but are not limited to:

[0049] Reagents:

[0050] Polyethyleneimine (PEI, 98%), glutaraldehyde (C5H8O2, 50%), ferrous sulfate heptahydrate (FeSO4·7H2O, 99.95%), sodium acetate (CH3COONa, 99%), ammonium fluoride (NH4F, AR), dopamine hydrochloride (C8H 11 NO2·HCl, AR), sodium hydroxide (NaOH, 98%), methanol (CH3OH, AR), ammonia water (NH3·H2O, 25%), sodium molybdate (Na2MoO4·2H2O, 99%), yttrium chloride hexahydrate (YCl3·6H2O, 99.99%), thulium chloride hexahydrate (TmCl3·6H2O, 99.99%), oleic acid (OA, AR), 1-octadecene (1-ODE, AR), erbium chloride hexahydrate (ErCl3·6H2O, 99.99%) were purchased from Sigma-Aldrich, USA. Ferric chloride (FeCl3·6H2O, 99%), thiourea ((NH2)2CS, 99%), citric acid (C6H8O7·H2O, 99%), and PEG-20000 (HO(C2H4O)nH, AR) were purchased from Shanghai Yien Chemical Technology Co., Ltd.

[0051] instrument:

[0052] UV-visible spectrophotometer (TU-1901, Beijing Pujinjie General Instrument Co., Ltd.), XRD diffractometer (BrukerD8, Bruker Daltonics), 980nm semiconductor laser emitter (LOS-BLD-0980-2W, Hitech Optoelectronics Co., Ltd.), TEM (JEM-2100, JEOL Ltd.), X-ray photoelectron spectrometer (ESCALAB-250Xi, Thermo Fisher Scientific China Co., Ltd.), Fourier transform infrared spectrophotometer (NicoletiN10, Thermo Fisher Scientific China Co., Ltd.), fluorescence spectrophotometer (F97pro, Shanghai Lingguang Technology Co., Ltd.), Zeta potential analyzer (Mastersizer-3000, Malvern Panalytical, UK).

[0053] The preparation method and application of a novel fluorescent probe for lactic acid detection described in the present invention mainly include the following technical principles: the probe constructed by the present invention is prepared by coupling core-shell upconversion fluorescent nanomaterials (CS-UCNPs), magnetic molybdenum disulfide materials (MoS2) and L-lactic acid aptamers, with simple steps, convenient operation and high sensitivity; the L-lactic acid aptamer is used as the recognition element, and it shows a specific response to L-lactic acid; the core-shell upconversion fluorescent nanomaterial has high sensitivity and good optical stability, and realizes the quantitative detection of L-lactic acid through the principle of fluorescence resonance energy transfer with magnetic molybdenum disulfide materials (MoS2). Figure 1 As shown, the fluorescence intensity change of the supernatant at 545 nm was measured by a fluorescence spectrophotometer under 980 nm laser excitation, and a linear relationship was established with the lactic acid content in the solution.

[0054] Based on the above principle, the present invention proposes a preparation method and application of a novel fluorescent probe for lactic acid detection, including:

[0055] The single-core UCNPs are synthesized by a solvothermal method; the single-core UCNPs are NaYF4:Yb,Er;

[0056] The NaYF4 shell is coated on the single-core UCNPs by a layer-by-layer assembly method to obtain the core-shell upconversion nanomaterial CS-UCNPs;

[0057] Streptavidin SA was modified on the surface of the CS-UCNPs by surface amination and classical glutaraldehyde coupling method;

[0058] The fluorescent probe was prepared by incubating with the biotin-modified lactic acid aptamer at 37°C with shaking;

[0059] The magnetic molybdenum disulfide nanosheet MoS2 is synthesized by functionalizing the surface of MoS2 nanosheet with dopamine hydrochloride and fixing iron ions.

[0060] Example 1

[0061] The invention provides a method for synthesizing UCNPs.

[0062] Mainly include:

[0063] (1) 0.8 mmol of yttrium chloride hexahydrate (YCl3·6H2O), 0.18 mmol of ytterbium chloride hexahydrate (YbCl3·6H2O), 0.02 mmol of erbium chloride hexahydrate (ErCl3·6H2O), 9 mL of oleic acid (OA), and 15 mL of 1-octadecene (1-ODE) were added to a 100 mL glass three-necked flask, and then dispersed in an ultrasonic cleaner for 10 min to dissolve all the solids to form a solution.

[0064] (2) A high temperature resistant magnet was placed in the three-necked flask, and the three-necked flask was then placed on a magnetic stirring electric heater for magnetic stirring. The reaction system was placed in a vacuum mode by switching the double-row tube for 0.5 h to remove the oxygen dissolved in the system.

[0065] (3) When no bubbles emerge in the solution, switch the double-row tube to nitrogen flow mode, adjust the temperature to 120°C, and then quickly switch the double-row tube to vacuum mode to remove the bound water molecules in the compound at this temperature. At this time, the solution observed in the system is slightly orange-yellow and oily.

[0066] (4) Change the system temperature to room temperature, then slowly add 10 mL of a methanol solution containing 4 mmol of ammonium fluoride (NH4F) and 2.5 mmol of sodium hydroxide (NaOH) dropwise, adjust the temperature to 20°C, and stir for 0.5 h.

[0067] (5) Switch the double-row tube to nitrogen flow mode, heat the solution to 120° C., and then switch the double-row tube to vacuum mode. Under this condition, methanol is removed due to condensation reflux.

[0068] (6) Switch the double-row tube to nitrogen flow mode and heat the system to 320 °C for 1.5 h under nitrogen environment.

[0069] (7) After the reaction is completed, wait until it cools down to room temperature, add ethanol to the system for precipitation, centrifuge at 13000r / min for 15min, wash the precipitate with cyclohexane three times, place it in a drying oven, and vacuum dry it at 60℃ overnight to obtain OA-coated UCNPs solid (OA-UCNPs), which are dispersed with cyclohexane. The NaYF4 shell is coated on the mononuclear UCNPs by layer-by-layer assembly method. Take 1mmol YCl3·6H2O and dissolve it in 9mL OA and 15mL ODE, place it in a 100mL three-necked flask and heat it to 120℃ in a nitrogen atmosphere for degassing, then switch to vacuum to remove water, wait until it cools down to room temperature, drop the mononuclear UCNPs dispersed in cyclohexane into the three-necked flask, and then drop 10mL methanol (CH3OH) containing 4mmoL NH4F and 2.5mmoL NaOH, and stir at room temperature for 0.5h. The solution was heated to 120°C for condensation reflux to remove CH3OH and cyclohexane, and then heated to 320°C under a nitrogen atmosphere for 1.5 h. After the reaction was completed, it was washed three times with ethanol and cyclohexane respectively and then stored.

[0070] result:

[0071] See also Figure 2 ,The results of transmission electron microscopy showed that the particle size of the nanoparticles of the UCNPs sample was distributed between 28-55nm, with an elliptical or polygonal crystal structure, clear particle morphology, and uniform dispersion in the solvent without obvious agglomeration, indicating that the synthesized samples were of high quality and could be used for further surface modification.

[0072] Example 2

[0073] The present invention provides a surface modification method of CS-UCNPs.

[0074] Mainly include:

[0075] The surface of CS-UCNPs was modified by ligand exchange method. The specific operation was as follows: prepare 5 mL of pure water in a 50 mL beaker, add 300 mg of polyethyleneimine (PEI) to fully dissolve it. Then add 10 mL of CS-UCNPs solution (2 mg / mL, solvent is cyclohexane), place a magnetic bar, and perform intense magnetic stirring at room temperature for 24 hours. After the reaction, the mixture was centrifuged at 12000 r / min for 15 minutes, the precipitate was collected, and washed with ethanol several times to remove the unreacted PEI. Finally, after freeze drying for 6 hours, the surface amino core-shell UCNPs (PEI-CS-UCNPs) were obtained. Streptavidin (SA) was then connected to the surface of PEI-CS-UCNPs by the classic glutaraldehyde coupling method. 1 mL Tris-HCl (0.01 M, pH 7.0), 2 mg PEI-CS-UCNPs and 250 μL glutaraldehyde (25%, diluted with pure water) solution were mixed evenly at room temperature and ultrasonicated for 10 min. The mixture was slowly shaken at room temperature for 2 h, and then resuspended in Tris-HCl solution after centrifugal washing. 100 μL 2 mg / mL SA was added, and the mixture was shaken and incubated at room temperature overnight to obtain SA-coupled core-shell UCNPs (SA-CS-UCNPs).

[0076] result:

[0077] See also Figure 3 SA was coupled on the surface of PEI-CS-UCNPs by the glutaraldehyde method, and the Zeta potential change before and after coupling was measured. The surface potential of PEI-modified CS-UCNPs was 7.17 mV, which was due to the positive charge of the amino group on PEI. After modification with SA, the Zeta potential dropped to 4.71 mV, indicating that SA combined with some amino groups on the surface of CS-UCNPs, resulting in a decrease in the surface positive charge, indicating that SA was successfully modified on PEI-CS-UCNPs, and the next step of fluorescent probe preparation could be carried out.

[0078] Example 3

[0079] The invention provides a method for preparing a fluorescent probe.

[0080] Mainly include:

[0081] The prepared SA-CS-UCNPs (1 mL, 2 mg / mL) and the biotin-modified lactic acid aptamer (50 μL, 10 μM) were incubated with slow shaking overnight at 37° C. The incubated mixture was centrifuged, washed three times, and resuspended in Tris-HCl for storage to prepare the UCNPs probe (Apt-CS-UCNPs).

[0082] result:

[0083] See also Figure 3 On the basis of SA-CS-UCNPs, the aptamer modified with biotin was connected by surface SA, and the Zeta potential of the product was further reduced to -0.67 mV. This result was attributed to the isoelectric point of the aptamer sequence being between 4 and 4.5, which caused the surface of the aptamer-connected complex to have a negative charge, thereby affecting the overall potential. This shows that the aptamer was successfully modified on SA-CS-UCNPs, and the fluorescent probe for detecting lactic acid was successfully prepared.

[0084] Example 4

[0085] The invention provides a method for preparing magnetic MoS2 nanosheets.

[0086] Mainly include:

[0087] 1.21g sodium molybdate (Na2MoO4·2H2O), 1.52g thiourea ((NH2)2CS), 30mg PEG-20000 were dissolved in 30mL pure water, and after vigorous stirring at room temperature for 0.5h, the solution was transferred to a polytetrafluoroethylene-lined autoclave, and then the autoclave was placed in a muffle furnace and heated to 220°C for 24h. After the reaction was completed, it was washed with 30mL pure water 4 times, and solid MoS2 was obtained after vacuum freeze drying. 100mg solid MoS2 was dispersed in 75mL Tris-HCl solution (0.01M, pH8.5), and ultrasonicated for 2h to obtain MoS2 nanosheets. 150mg dopamine hydrochloride (C8H 11 NO2·HCl), slowly shake at room temperature for 4h, wash the obtained material three times with ultrapure water and then freeze-dry in vacuum to obtain MoS2 nanosheets functionalized with polydopamine. Take 100mg of the obtained material and dissolve it in 200mL of water. After ultrasonic treatment for 5min, 6.0g of ferric chloride (FeCl3·6H2O) and 3.1g of ferrous sulfate heptahydrate (FeSO4·7H2O) are completely dissolved in the above solution and shaken vigorously for 1h to ensure that the iron ions are fully fixed on the functionalized MoS2 nanosheets. Then quickly add 20mL of ammonia water (25%) and 7.0g of citric acid and shake vigorously for 1h. After magnetic separation, the obtained black precipitate is washed six times with acetone and dried at 65℃ for 1h to obtain the final product.

[0088] result:

[0089] See also Figure 4Transmission electron microscopy shows that the prepared magnetic MoS2 nanocomposite has specific morphological characteristics, in which the size of MoS2 flakes is roughly distributed between 200-300nm, and the surface is evenly loaded with Fe3O4 nanoparticles with a particle size of about 5-10nm, which makes the MoS2 nanocomposite have certain magnetism.

[0090] Example 5

[0091] The invention proposes an application experiment of using a fluorescent probe to detect lactic acid of different concentrations.

[0092] Mainly include:

[0093] 100 μL of Apt-CS-UCNPs (2 mg / mL) dispersed in Tris-HCl buffer (pH 7.0) was added to a centrifuge tube, and then 100 μL of lactic acid standard with different concentration gradients (0-30 mM) was added, and incubated at 37 ° C for 25 min. 100 μL of magnetic MoS2 solution (0.6 mg / mL, solvent is Tris-HCl buffer) was added to the centrifuge tube, mixed thoroughly, and continued to react at 37 ° C for 20 min. After the reaction, the centrifuge tube was placed on a magnetic stand for magnetic separation for 1 min, and the supernatant was placed in a fluorescent cuvette. Under 980 nm excitation light, the fluorescence intensity of the supernatant at 545 nm was measured by a fluorescence spectrophotometer. Three parallel controls were set for each test sample.

[0094] result:

[0095] See also Figure 5 The circular dichroism results of lactic acid and aptamer showed that the spatial configuration of the aptamer changed significantly after binding to the lactic acid molecule, indicating that there was a specific interaction between the aptamer and lactic acid, which means that the aptamer can recognize and bind to the lactic acid molecule.

[0096] For further information, see Figure 6 and Figure 7, within the range of 0-30mM, there is a good linear relationship between the logarithmic value of the concentration of the lactic acid standard and the fluorescence intensity value. The results show that the detection limit of the fluorescent probe for lactic acid is 0.38mM, and lactic acid (concentration range 5-20mM) in human sweat can be accurately monitored. Through non-invasive sweat lactic acid monitoring, athletes and coaches can understand the physical condition of athletes in real time, thereby optimizing training plans, preventing overtraining, and ultimately improving athletic performance. Therefore, the fluorescent probe has great application potential in athlete training and coaching guidance. In addition, the fluorescent probe may also be applied to clinical environments. By continuously monitoring the lactic acid concentration, doctors can promptly detect changes in the patient's condition and take corresponding treatment measures. The present invention provides a new idea for further improving the sensitivity of biosensors, realizing sensor miniaturization and wearable device integration. By continuously optimizing the design of sensors, it is expected to provide athletes, patients and ordinary people with more convenient and accurate lactic acid monitoring services.

[0097] It should be noted that the term "comprises" or any other variation thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only these elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprises..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0098] The above are only embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included within the scope of the claims of the present application.

Claims

1. A method for preparing a novel fluorescent probe for lactic acid detection, characterized in that: The method comprises: S1. Synthesis of core-shell upconversion nanomaterials CS-UCNPs; S2, surface modification of the core-shell upconversion nanomaterial CS-UCNPs; S3, coupling of the core-shell upconversion nanomaterial CS-UCNPs with L-lactic acid aptamer; S4. Synthesis of magnetic molybdenum disulfide nanosheets MoS2.

2. The method for preparing the novel fluorescent probe for lactic acid detection according to claim 1, characterized in that: The method comprises: S21, synthesizing single-core UCNPs by solvothermal method; the single-core UCNPs are NaYF4:Yb,Er; S22, coating the NaYF4 shell on the single-core UCNPs by a layer-by-layer assembly method to obtain the core-shell upconversion nanomaterial CS-UCNPs; S23, modifying SA on the surface of the CS-UCNPs by surface amination and classical glutaraldehyde coupling method; S24, incubating with a biotin-modified lactic acid aptamer at 37° C. with shaking to prepare a fluorescent probe; S25. The magnetic molybdenum disulfide nanosheets MoS2 are synthesized by functionalizing the surface of MoS2 nanosheets with dopamine hydrochloride and fixing iron ions.

3. The method for preparing the novel fluorescent probe for lactic acid detection according to claim 1, characterized in that: The synthesis method of the UCNPs comprises: S31, mixing yttrium chloride hexahydrate, ytterbium chloride hexahydrate, erbium chloride hexahydrate, oleic acid, and 1-octadecene, dispersing them, dissolving all the solids, and obtaining a compound solution; S32, heating and stirring the compound solution, and placing the reaction system in a vacuum mode to obtain a solution from which oxygen is removed from the system; S33, when no bubbles emerge from the solution, setting the solution to a nitrogen mode, adjusting the temperature to 100° C. to 140° C., switching to a vacuum mode, and obtaining a compound from which bound water molecules are removed; S34, changing the system temperature to room temperature, adding a methanol solution containing ammonium fluoride and sodium hydroxide, adjusting the temperature to 15°C to 25°C, and stirring for 0.1 to 2.0 hours; preferably, adjusting the temperature to 20°C, and stirring for 0.5 hours; obtaining a surface OA-coated NaYF4:Yb,Er primary product; S35, setting the mode to pass nitrogen, heating the solution, and switching to a vacuum mode to obtain a compound solution from which methanol has been removed; S36, setting the nitrogen gas flow mode, heating the compound solution from which methanol has been removed to 300° C. to 350° C. for 0.5 h to 2.5 h; preferably, heating to 320° C. for 1.5 h; obtaining the preliminarily purified surface OA-coated NaYF4:Yb,Er; S37, cool to room temperature, add ethanol to precipitate the product, wash the precipitate with cyclohexane, and dry it to obtain solid OA-UCNPs with surface OA-coated UCNPs; disperse the OA-UCNPs with cyclohexane to obtain mononuclear UCNPs dispersed with cyclohexane.

4. The method for preparing the novel fluorescent probe for lactic acid detection according to claim 3, characterized in that: The molar ratio of the yttrium chloride hexahydrate: the ytterbium chloride hexahydrate: the erbium chloride hexahydrate is n 六水合氯化钇:六水合氯化镱:六水合氯化铒 , the n 六水合氯化钇:六水合氯化镱:六水合氯化铒 =(30-50):(5-7):(0.5-5); preferably, the n 六水合氯化钇:六水合氯化镱:六水合氯化铒 =40:9:

1.

5. The method for preparing the novel fluorescent probe for lactic acid detection according to claim 3, characterized in that: In the above S33, the compound from which the bound water molecules are removed is orange-yellow and oily.

6. The method for preparing the novel fluorescent probe for lactic acid detection according to claim 1, characterized in that: The preparation of the core-shell upconversion nanomaterial CS-UCNPs comprises: The NaYF4 shell was coated on the mononuclear UCNPs by a layer-by-layer assembly method, YCl3·6H2O was dissolved in OA and ODE, and the mixture was heated to 100°C to 140°C in a nitrogen atmosphere for degassing; vacuum was switched to remove water, and after the mixture was cooled to room temperature, the mononuclear UCNPs dispersed in cyclohexane were added dropwise, and then methanol containing NH4F and NaOH was added dropwise, and the mixture was stirred at room temperature; the solution was heated to 100°C to 140°C for condensation and reflux to remove methanol and cyclohexane, and the mixture was heated to 250°C to 390°C in a nitrogen atmosphere for 0.5h to 2.0h. After the reaction was completed, the mixture was washed and stored with ethanol and cyclohexane, respectively.

7. The method for preparing the novel fluorescent probe for lactic acid detection according to claim 1, characterized in that: The surface modification method of CS-UCNPs is to modify the surface of CS-UCNPs by ligand exchange method, including: Add polyethyleneimine into pure water and fully dissolve it; then add CS-UCNPs solution with cyclohexane as solvent and stir at room temperature for 12h to 36h; after the reaction, centrifuge the mixture, collect the precipitate, and wash it with ethanol several times to remove the unreacted PEI; finally, after freeze-drying for 2h to 10h, surface amino-modified core-shell UCNPs, PEI-CS-UCNPs, are obtained; then streptavidin is connected to the surface of PEI-CS-UCNPs by the classic glutaraldehyde coupling method; Tris-HCl, PEI-CS-UCNPs and glutaraldehyde solution are mixed evenly at room temperature and sonicated; continue to slowly shake at room temperature for 0.5h to 5h, resuspend in Tris-HCl solution after centrifugal washing, add SA, and shake and incubate at room temperature to obtain SA-coupled core-shell UCNPs, SA-CS-UCNPs.

8. The method for preparing the novel fluorescent probe for lactic acid detection according to claim 1, characterized in that: The preparation method of the magnetic molybdenum disulfide nanosheet MoS2 comprises: Sodium molybdate, thiourea and PEG-20000 are dissolved in pure water. After vigorous stirring at room temperature, the solution is transferred to a polytetrafluoroethylene-lined autoclave. The autoclave is then placed in a muffle furnace and heated to 180°C to 260°C for 12 to 36 hours. After the reaction is completed, the reaction is washed with pure water and solid MoS2 is obtained after vacuum freeze-drying. The solid MoS2 is dispersed in a Tris-HCl solution and subjected to ultrasound to obtain MoS2 nanosheets. Dopamine hydrochloride is then added and the mixture is slowly shaken at room temperature for 2 to 6 hours. The obtained material is washed with ultrapure water. The prepared material is dissolved in water, subjected to ultrasonic treatment to completely dissolve the ferric chloride and ferrous sulfate heptahydrate, and shaken vigorously for 0.5 h to 2 h to ensure that the iron ions are fully fixed on the functionalized MoS2 nanosheets; ammonia water and citric acid are quickly added, and the mixture is shaken vigorously for 0.5 h to 2 h to obtain a black precipitate; the black precipitate is subjected to magnetic separation, washed with acetone, and dried at 40 ° C to 90 ° C for 0.5 h to 2 h to obtain the magnetic molybdenum disulfide nanosheet MoS2.

9. Application of a novel fluorescent probe for lactic acid detection, characterized in that: The fluorescent probe is prepared according to the method for preparing a novel fluorescent probe for lactic acid detection according to any one of claims 1 to 8; The application of the novel fluorescent probe for lactic acid detection is the application of the fluorescent probe in lactic acid standard solutions of different concentrations; the lactic acid standard solutions of different concentrations include 0, 1mM, 3mM, 10mM, 15mM or 30mM.

10. The use of the novel fluorescent probe for lactic acid detection according to claim 9, characterized in that: The applications include: Add lactic acid standards of different concentration gradients to Apt-CS-UCNPs dispersed in Tris-HCl buffer, and incubate at 37°C for 5 to 45 minutes; add the magnetic molybdenum disulfide nanosheets MoS2, mix thoroughly, and continue to react at 37°C for 5 to 45 minutes; after the reaction is completed, perform magnetic separation, place the supernatant in a fluorescent cuvette, and measure the fluorescence intensity of the supernatant at 545 nm using a fluorescence spectrophotometer under 980 nm excitation light.

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