Biosensor and kit for detecting NADH (Nicotinamide Adenine Dinucleotide) as well as preparation method and application of biosensor and kit and detection method of NADH

Abstract

The invention discloses a biosensor and a kit for detecting NADH (Nicotinamide Adenine Dinucleotide), a preparation method and application of the biosensor and the kit, and a detection method of the NADH, and belongs to the technical field of analysis and detection. The invention provides a biosensor for detecting NADH (nicotinamide adenine dinucleotide). The biosensor consists of an up-conversion nano material and a probe A, the invention provides a kit for detecting NADH, and the kit is composed of the biosensor and an NADH standard substance. The invention also provides preparation methods and applications of the biosensor and the kit. A detection method of the NADH is provided, and the concentration of the NADH in the sample to be detected is calculated according to the standard curve; the method further comprises the steps that the surface of the up-conversion nano material is modified with mesoporous silica, the mesoporous silica loads a probe A, the surface of the mesoporous silica is modified with beta-cyclodextrin to enhance biocompatibility, and the probe A quenches fluorescence of UCNPs. The invention provides an up-conversion fluorescence ratio probe which is efficient, sensitive and high in specificity and is used for detecting trace NADH (Nicotinamide Adenine Dinucleotide Hormone) in cells.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a biosensor for detecting NADH, a reagent kit, a preparation method and application thereof, and a method for detecting NADH. Background Art

[0002] Reduced nicotinamide adenine dinucleotide (NADH) and its oxidized form (NAD+) are important biomolecules widely present in living cells. As a pair of enzyme cofactors, NADH / NAD+ play a key role in more than 300 complex enzymatic reactions. The ratio of NADH to its oxidized form NAD+ is a key indicator reflecting the overall redox state of many physiological or pathological processes, including energy metabolism, mitochondrial function, calcium homeostasis, gene expression, cell death, embryonic development, blood flow, aging, cancer and diabetes. In addition, NADH has been widely used to detect enzyme activity in diagnostic diseases, including tumor formation and ischemia. Some experts believe that NADH is beneficial for dehydrogenase-based biosensor and biofuel cell research and can be used to treat Parkinson's disease and depression. The detection of the redox state of NADH / NAD+ is crucial for determining the metabolic state of cells. Therefore, the development of high-precision NADH detection methods has attracted widespread attention.

[0003] At present, conventional laboratory NADH detection methods include chemiluminescence, photoelectrochemical method, electrochemical detection method and high performance liquid chromatography. Although the method can meet the needs of NADH detection, the practical application is often limited by the complexity of the operating instrument and the specialization of the operating steps, and in situ detection cannot be performed. At the same time, NADH rapid colorimetric analysis tools based on different functionalized gold nanoparticles, metal complexes, etc. have the advantages of simple operation, practicality and high efficiency, and show good prospects in actual NADH detection. However, these methods often require high-cost sensing materials, such as gold-based nanomaterials. Based on the defects of the above-mentioned detection methods, it is of great practical significance to develop a sensitive, low-cost and low-toxic NADH detection technology. Summary of the invention

[0004] Based on the above technical problems to be solved by the present invention, the present invention is based on the spironolactone ring-opening mechanism, and the specific interaction between probe A and NADH causes the ultraviolet absorbance value of probe A within a certain wavelength range to be significantly reduced, and its ultraviolet absorption peak area has a large overlap with the emission peak area of ​​UCNPs, thereby causing fluorescence resonance energy transfer (FRET) between UCNPs and probe A. A biosensor for detecting NADH, a kit, a preparation method and application thereof, and a method for detecting NADH are proposed.

[0005] One of the purposes of the present invention is to provide a biosensor for detecting NADH, the biosensor comprising a probe A and an upconversion nanomaterial UCNPs; further, the preparation of the probe A comprises preparing a near-infrared rhodamine dye by reacting 9-(2-carboxyphenyl)-6-(diethylamino)-1,2,3,4-tetrahydroxyammonium perchlorate with quinoline-3-carboxaldehyde; the near-infrared rhodamine dye and quinoline-3-carboxaldehyde are added to CH3COOH to obtain a mixture, extracted with dichloromethane, washed with water and salt to obtain a compound; the compound and MeOTf are added to dichloromethane, extracted, the organic layer is collected, dried and filtered to obtain the probe A; the probe A is a fluorescence quencher; further, the preparation of the upconversion nanomaterial UCNPs comprises the synthesis of UCNPs and the surface modification of UCNPs; the upconversion nanomaterial UCNPs is a nanoparticle; the particle size of the upconversion nanomaterial is 25nm to 30nm.

[0006] Furthermore, the UCNPs are fluorescent probe components; further, the UCNPs are NADH-sensitive fluorescent ratio probes; further, the UCNPs are surface-modified with a layer of mesoporous silica, loaded with the NADH-specific recognition molecule probe A, and the outer layer is modified with a surface ligand; the surface ligand is β-cyclodextrin.

[0007] Furthermore, the synthesis of the UCNPs includes the solvothermal synthesis of NaYF4:Yb,Er,Tm UCNPs, the method comprising:

[0008] S31, RECl3·6H2O, OA, and 1-ODE are mixed and dispersed to obtain a compound in which all solids are dissolved;

[0009] S32, stirring and evacuating the compound to obtain a compound from which oxygen has been removed;

[0010] S33, removing bound water molecules in the compound to obtain an orange-yellow oily solution;

[0011] S34, adding NH4F and a methanol solution containing NaOH, adjusting the temperature, stirring, and obtaining a methanol solution;

[0012] S35, removing methanol from the methanol-containing solution to obtain a methanol-removed solution;

[0013] S36, heating the methanol removal solution and continuing;

[0014] S37, adding ethanol for precipitation, centrifuging to obtain the precipitate, washing with cyclohexane, and vacuum drying to obtain OA-UCNPs solid OA-UCNPs with surface coated with OA.

[0015] Furthermore, the surface modification of the UCNPs includes forming a mesoporous silica shell layer on the surface of the OA-UCNPs, and the method includes:

[0016] S41, taking hexadecyltrimethylammonium bromide, dissolving it, adding chloroform, stirring until the solution is clear, and obtaining a reaction solution;

[0017] S42, adding water, ethanol and NaOH to the reaction solution, stirring, adding TEOS, stirring, adding APTES to obtain a product;

[0018] S43, centrifuging the product, washing with ethanol, transferring it into ethanol containing ammonium nitrate, and stirring it to obtain an ethanol dispersion of the nano fluorescent probe;

[0019] S44, placing monosubstituted β-CD p-toluenesulfonate in the ethanol dispersion of the nano fluorescent probe, stirring and reacting in a nitrogen atmosphere to obtain UCNPs@mSiO2@β-CD.

[0020] A second object of the present invention is to provide a kit for detecting NADH; further, the kit includes a biosensor and a NADH standard.

[0021] Furthermore, the NADH standard series comprises standard solutions with a concentration gradient; further, the concentration of the standard solution is 0-100nM; preferably, the concentration of the standard is 0, 10nM, 20nM, 30nM, 40nM, 50nM, 60nM, 70nM, 80nM, 90nM and 100nM.

[0022] The third object of the present invention is to provide a method for preparing the biosensor or the kit, comprising:

[0023] S71, preparing the probe A;

[0024] S72, preparation of the upconversion nanomaterial UCNPs and combination with the probe A;

[0025] S73, performing subpackaging, packaging, and / or storage.

[0026] The fourth object of the present invention is to provide an application of the biosensor or the kit in detecting NADH in cells; preferably, the cells include PC12 cells or p 0 cell.

[0027] A fifth object of the present invention is to provide a method for detecting NADH, wherein the method uses the biosensor or the kit, and the method comprises:

[0028] S91, establish a standard curve;

[0029] S92, detecting the sample to be tested;

[0030] S93. Calculate the concentration of NADH in the sample to be tested according to the standard curve.

[0031] Further, the detection method includes a fluorescent probe element, and the fluorescent probe element is an upconversion nanoparticle UCNPs having a core-shell structure;

[0032] The detection method determines the NADH content by calculating the ratio of the emission peak to the reference emission peak.

[0033] Compared with the prior art, the present invention proposes a biosensor for detecting NADH, a kit, a preparation method and application thereof, and a method for detecting NADH, which have the following beneficial effects:

[0034] 1. The present invention provides an efficient, sensitive and highly specific upconversion fluorescence ratio probe to detect trace amounts of NADH in cells. Compared with some similar NADH sensors, the fluorescence ratio probe designed in the present invention has excellent sensitivity (LOD = 0.053nM) and high selectivity, which can effectively reduce the impact of most potential interferences in biological systems.

[0035] 2. The present invention uses UCNPs with a core-shell structure as a fluorescent probe component to develop a NADH-sensitive fluorescent ratio probe. A layer of mesoporous silica is modified on its surface to load NADH-specific recognition molecules (probe A), and the outermost layer is modified with β-cyclodextrin as a surface ligand to enhance biocompatibility.

[0036] 3. The fluorescent ratio probe prepared by the present invention has been successfully applied to monitor the fluctuation of NADH level in mitochondria of living cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 A synthetic route of probe A according to an embodiment of the present invention is shown;

[0038] Figure 2 A transmission electron microscope image of an up-conversion fluorescence ratio probe according to an embodiment of the present invention is shown;

[0039] Figure 3 The figure shows the fluorescence intensity change of different concentrations of NADH detected by an up-conversion fluorescence ratio probe according to an embodiment of the present invention and the standard curve fitting diagram;

[0040] Figure 4 An upconversion fluorescence ratio probe for detecting PC12 cells and p 0 Diagram of the application of NADH in cells;

[0041] Figure 5A specificity study diagram of a biosensor according to an embodiment of the present invention is shown;

[0042] Figure 6 A schematic diagram of the detection principle of an up-conversion fluorescence ratio probe according to an embodiment of the present invention is shown;

[0043] Figure 7 A comparison chart of the detection results of an upconversion fluorescence ratio probe according to an embodiment of the present invention and a commercial NADH kit is shown. DETAILED DESCRIPTION

[0044] 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.

[0045] 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.

[0046] The experimental materials and equipment involved in the present invention mainly include but are not limited to:

[0047] Reagents:

[0048] All water used in the experiments was ultrapure water (Milli-Q, 18 MΩcm, Millipore); YCl·6H2O, YbCl·6H2O, ErCl3·6H2O, FeCl3·6H2O, CH3COONa, Na3C6H5O7, oleic acid (OA), 1-octadecene (ODE), tetraethyl orthosilicate (TEOS), (3-aminopropyl)triethoxysilane (APTES), CTAB, and NH4NO3 were purchased from Shanghai Yien Chemical Technology Co., Ltd. (all of analytical grade); PBS buffer was purchased from Beijing Solebold Technology Co., Ltd.; ammonium fluoride, ethylene glycol, sodium hydroxide, methanol, and cyclohexane were purchased from MacLean Biochemical Technology Co., Ltd. (all of analytical grade); glutaraldehyde (50%), NADH, and β-CD were purchased from Aladdin Biochemical Technology Co., Ltd.; all other reagents were purchased from commercial suppliers and used without further purification.

[0049] instrument:

[0050] The morphology of the materials was analyzed by JEM 2100 transmission electron microscope (TEM, Japan Electron Optical Laboratory, Japan) and SU3500 scanning electron microscope (SEM, Hitachi High-Tech Corporation); Fourier transform infrared spectrum (FTIR) was measured by Nicoleti S50 spectrophotometer (Thermo Fisher Scientific, USA); fluorescence intensity was recorded by F97 pro fluorescence spectrophotometer (Shanghai Lingguang Technology Co., Ltd., Shanghai, China) and 980 nm semiconductor laser (Hi-Tech Optoelectronics Co., Ltd., Beijing, China); UV absorption spectrum was recorded by TU-1901 UV-visible spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., Beijing, China); Zeta potential was measured by Mastersizer 3000 110 instrument (Malvern Panalytical, UK).

[0051] The technical principle of the biosensor, kit, preparation method and application thereof and NADH detection method described in the present invention mainly includes: based on the ring-opening mechanism of spironolactone, probe A specifically interacts with NADH, resulting in changes in the ultraviolet absorbance value of probe A within a certain wavelength range, and the ultraviolet absorption peak area overlaps with the emission peak area of ​​UCNPs to a large extent, resulting in FRET between UCNPs and probe A. The NADH content can be determined by calculating the ratio of the emission peak to the reference emission peak.

[0052] Based on the above principle, the present invention proposes a detection method for detecting NADH, comprising:

[0053] Upconversion nanoparticles (UCNPs) with a core-shell structure were selected as fluorescent probe elements. A layer of mesoporous silica was modified on the surface to load NADH-specific recognition molecule probe A (also serving as a fluorescence quencher); the outermost layer was connected by a surface ligand β-cyclodextrin. Based on the ring-opening mechanism of spironolactone, the specific interaction between probe A and NADH resulted in a significant decrease in the ultraviolet absorbance value of probe A within a certain wavelength range, and its ultraviolet absorption peak area had a large overlap with the emission peak area of ​​UCNPs, resulting in fluorescence resonance energy transfer (FRET) between UCNPs and probe A. The NADH content can be determined by calculating the ratio of the emission peak to the reference emission peak.

[0054] The nanosensor consists of a UCNPs core with two strong luminescent emission peaks at 546 and 662 nm, which serve as upconversion fluorescent probes. The core is surrounded by a mesoporous SiO2 (mSiO2) shell with probe A loaded in the pores. NADH sensing is achieved through a fluorescence resonance energy transfer (FRET) process between probe A and UCNPs. The shell is further modified with a layer of β-cyclodextrin (β-CD) surface ligands to improve the stability and biocompatibility of the nanoconjugate in physiological environments and to prevent macromolecules such as enzymes from entering the mesoporous channels while still allowing NADH to permeate freely. The reaction of NADH with probe A induces the opening of the spiro ring, which is accompanied by a weakening of the strong absorption of probe A at 500-600 nm, overlapping with the green emission (546 nm) spectrum of UCNPs. Therefore, the presence of NADH increases the I 546 / I 662 ratio, thereby allowing for quantitative measurement of NADH.

[0055] Example 1

[0056] The invention provides a biosensor for detecting NADH.

[0057] Mainly include:

[0058] Probe A and upconversion nanomaterials. In this embodiment, the upconversion nanomaterial is NaYF4:Y b ,Er.

[0059] (1) Synthesis of probe A: Figure 1 As shown, 9-(2-carboxyphenyl)-6-(diethylamino)-1,2,3,4-tetrahydroxyammonium perchlorate and quinoline-3-carboxaldehyde are refluxed and condensed in acetic acid solution to obtain near-infrared rhodamine dye. NADH recognition molecular probe A was prepared by methylating rhodamine dye with methyl trifluoromethanesulfonate in dichloromethane solution at room temperature. Near-infrared rhodamine dye (475 mg, 1 mmol) and quinoline-3-carboxaldehyde (157 mg, 1 mmol) were added to 10 mL CH3COOH and stirred at 80 ° C for 5 hours. After the reaction mixture was cooled to room temperature, the solution was extracted with 50 mL dichloromethane and washed with water and brine respectively. The obtained compound (256 mg, 1 mmol) and MeOTf (163 mg, 1 mmol) were added to 10 mL dichloromethane solution and stirred at room temperature for 5 hours. 50 mL H2O was added to the above solution and extracted with 40 mL dichloromethane. The organic layer was collected, dried over anhydrous MgSO4 and filtered to obtain probe A.

[0060] (2) Synthesis of UCNPs

[0061] Including the solvothermal synthesis of NaYF4:Yb,Er,Tm UCNPs:

[0062] a) 1mmol RECl3·6H2O (0.8mmolYCl3·6H2O, 0.18mmolYbCl3·6H2O, 0.02mmolErCl3·6H2O, 0.002mmolTmCl3·6H2O, and the doping ratio of lanthanide elements was adjusted and optimized on this basis), 9mL OA, and 15mL 1-ODE were placed in a 100mL glass three-necked flask, and then placed in an ultrasonic cleaner for dispersion for 10 minutes to dissolve all the solids to form a solution.

[0063] b) 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 oxygen dissolved in the system.

[0064] c) 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.

[0065] d) Change the system temperature to room temperature, then slowly add 10 mL of a methanol solution containing 4 mmol NH4F and 2.5 mmol NaOH dropwise, adjust the temperature to 20°C, and stir for 0.5 h.

[0066] e) 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.

[0067] f) Switch the double-row tube to nitrogen flow mode and heat the system to 320° C. for 1.5 h under nitrogen environment.

[0068] g) After the reaction is completed, the temperature is cooled to room temperature, ethanol is added to the system for precipitation, and the mixture is centrifuged at 13,000 r / min for 15 min. The precipitate is washed three times with cyclohexane, placed in a drying oven, and vacuum dried overnight at 60° C. to obtain UCNPs solids coated with OA on the surface (OA-UCNPs).

[0069] (3) Surface modification of UCNPs

[0070] In order to form a mesoporous silica shell on the surface of OA-UCNPs, 0.1 g of hexadecyltrimethylammonium bromide was first added to 20 mL of water in a round-bottom flask and dissolved by ultrasonication. Then, 5 mg mL -1UCNPs were dissolved in 2 mL of chloroform solution and stirred at room temperature overnight until the solution became clear. Then, 10 mL of the above reaction solution was added to a mixture containing 20 mL of water, 3 mL of ethanol and 75 μL of 0.4 mol·L -1 In a round-bottom flask containing NaOH, 150 μL TEOS was added dropwise at 30°C and stirred for 48 h; then, 32 μL APTES was added dropwise to the flask and stirred for 24 h. The product was centrifuged and washed with ethanol three times, then transferred to 50 mL ethanol containing 0.3 g ammonium nitrate and stirred at 45°C for 24 h. 100 mg of monosubstituted β-CD p-toluenesulfonate was placed in an ethanol dispersion of the nanofluorescent probe (2 mg mL -1 ) was stirred in a nitrogen atmosphere at 40°C for 24 h to prepare UCNPs@mSiO2@β-CD. Figure 2 As shown, the surface modification of the fluorescent ratio probe is completed.

[0071] In this embodiment, the above-mentioned probe A, upconversion nanomaterials and NADH standard together constitute a kit, wherein the concentration of the NADH standard is 0-100 nM (specifically 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nM).

[0072] result:

[0073] See also Figure 1 The synthetic near-infrared rhodamine dye was prepared by methylation with methyl trifluoromethanesulfonate in a dichloromethane solution at room temperature to obtain NADH recognition molecular probe A. The nuclear magnetic resonance and mass spectrometry analysis of probe A showed that 1 HNMR (400MHz, methanol-d4): δ9.63 (s, 1H), 9.32 (s, 1H), 8.49 (dd, J = 18.5, 8.6Hz, 2H) ,8.30–8.27(m,1H),8.06–8.05(m,2H),7.78(d,J=7.5Hz,1H),7.73(t,J=7.4Hz ,1H),7.31–7.25(m,1H),7.22–7.11(m,3H),4.74(d,J=1.4Hz,3H),3.71(q,J=7 .2Hz, 4H), 3.10 (d, J = 8.5Hz, 2H), 2.42 (s, 2H), 1.88 (s, 2H), 1.34–1.31 (m, 6H). High-resolution mass spectrum of probe A: m / z [MC 35 H 33 N2O3] + =529.2488, calculated value: 529.2486. This indicates that the preparation of probe A is successful.

[0074] Example 2

[0075] The invention provides a method for detecting NADH by using an up-conversion fluorescence ratio probe biosensor.

[0076] Mainly include:

[0077] (1) Establishment of standard curve

[0078] To establish a standard curve, the fluorescence signal response values ​​of NADH at different concentrations (0, 10, 20, 30, 40, 50, 60, 70 nM) were detected respectively.

[0079] (2) Application of NADH detection in cells

[0080] To verify the ability of the fluorescent ratiometric probe to detect changes in NADH levels in the mitochondrial respiratory chain, cells lacking mitochondrial genes (ρ 0 PC12 cells). 0 Cells, also known as mitochondrial DNA-deficient cells, are a cell model for studying mitochondrial diseases and determining mitochondrial targeting. The specific experimental operation is to add a certain amount of ethidium bromide (5 μg mL -1 ), uridine (50 μg mL -1 ) and pyruvic acid (50 μg mL -1 ), and formulated into a special culture medium that can induce mitochondrial damage. PC12 cells using this culture medium were continuously cultured in a constant temperature incubator at 5% CO2 and 37°C. When the cells covered 80% to 90% of the bottom of the culture flask, 1:2 cell passage was performed, and the culture time was 30 days. 0 Differences in the ability to monitor fluorescence imaging in cells.

[0081] (3) Specificity study

[0082] The luminescence intensity ratio (I 546 / I 662 ) in a 2mL EP tube, the probe mother solution (2mg / mL, 200μL), PBS buffer (10mM, pH=7.0, 600μL) and NADH and interfering substances (including some active oxygen and nitrogen substances, H2O2, glutathione, homocysteine, cysteine, glycine, serine, tyrosine, K + Mg 2+ , Ca 2+ , Cu 2+ , Fe 3+ , HCO3- , S 2- , ClO - , CN - 、ONOO - , 1μM, 200μL), mix evenly, react at 25℃ in the dark for 0.5h, test the fluorescence intensity values ​​at wavelengths of 549nm and 662nm respectively, and calculate the ratio of the two (each group of experiments was repeated 3 times, and the average value of the measurements was taken to ensure the accuracy of the experimental results).

[0083] result:

[0084] See also Figure 3 , in the range of 0-100 nM, the NADH concentration and the I 546 / I 662 The ratio shows a good linear relationship. Figure 3 Display the linear equation fitting result as I 546 / I 662 =0.15585C+0.08492(R 2 =0.9928), and the detection limit is 0.053nM (S / N=3). This range is suitable for measuring the NADH level in living cells (~0.3mM). The present invention has a lower minimum detection limit and higher sensitivity than the prior art.

[0085] See also Figure 4 , wild-type PC12 cells and ρ 0 When cells were incubated with glucose (20 mM) for 30 min, endogenous NADH levels increased, but ρ 0 The change of endogenous NADH level in cells was smaller than that in PC12 cells. The results showed that the action site of the probe in cells can reach the level of mitochondrial organelles, thereby realizing the detection of NADH level in mitochondria, further confirming the application value of fluorescence ratio probes in biological systems.

[0086] See also Figure 5 After the addition of NADH, the upconversion luminescence (UCL) emission at 546 nm was restored, which may be due to the interruption of energy transfer from UCNPs to probe A. In contrast, other oxidative chemicals (H2O2, glutathione, homocysteine, cysteine, glycine, serine, tyrosine, K + Mg 2+ , Ca 2+ , Cu 2+ , Fe 3+ , HCO 3- , S 2- , ClO - , CN - 、ONOO - ) No significant changes were detected.

[0087] Example 4

[0088] The invention provides a test kit preparation method experiment.

[0089] Mainly include:

[0090] (1) Synthesis of probe A: 9-(2-carboxyphenyl)-6-(diethylamino)-1,2,3,4-tetrahydroxyammonium perchlorate and quinoline-3-carboxaldehyde were refluxed and condensed in acetic acid solution to obtain near-infrared rhodamine dye. NADH recognition molecular probe A was prepared by methylating rhodamine dye with methyl trifluoromethanesulfonate in dichloromethane solution at room temperature. Near-infrared rhodamine dye (475 mg, 1 mmol) and quinoline-3-carboxaldehyde (157 mg, 1 mmol) were added to 10 mL CH3COOH and stirred at 80°C for 5 hours. After the reaction mixture was cooled to room temperature, the solution was extracted with 50 mL dichloromethane and washed with water and brine respectively. The obtained compound (256 mg, 1 mmol) and MeOTf (163 mg, 1 mmol) were added to 10 mL dichloromethane solution and stirred at room temperature for 5 hours. 50 mL H2O was added to the above solution and extracted with 40 mL dichloromethane. The organic layer was collected, dried with anhydrous MgSO4 and filtered to obtain probe A. (2) Preparation of upconversion nanofluorescent probe: NaYF4:Yb,Er,Tm UCNPs were synthesized by solvothermal method, and then TEOS, APTES and monosubstituted β-CD p-toluenesulfonate were added to modify the surface of UCNPs to obtain upconversion nanofluorescent probe. (3) Preparation of kit: The above-mentioned probe A, upconversion nanofluorescent probe and NADH standard together constitute a kit. Among them, the concentration of NADH standard is 0-100nM (specifically 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100nM). When using the kit, first take 1 mg of probe A and dissolve it in the up-conversion nano fluorescent probe (concentration of 2 mg / mL) to prepare the probe mother solution, then add different concentrations of NADH standard, mix evenly, react at 25°C in the dark for 0.5 h, test the fluorescence intensity values ​​at wavelengths of 549 nm and 662 nm respectively, calculate the ratio of the two (each group of experiments was repeated 3 times, and the average value of the measurements was taken to ensure the accuracy of the experimental results), draw a standard curve, and finally perform the above detection steps on the analyte, and calculate the NADH concentration in the analyte through the standard curve.

[0091] result:

[0092] See also Figure 6 , a set of fluorescence sensing technology kits for detecting NADH was prepared, which can accurately and quickly detect NADH in the concentration range of 0-100nM.

[0093] Comparative Example

[0094] The present invention proposes a comparative evaluation experiment between the fluorescent ratio probe of the present invention and a commercially available NADH detection kit.

[0095] Mainly include:

[0096] Detection method of commercially available NADH kit:

[0097] 1. Sample preparation:

[0098] Take about 1×10 6 PC12 cells were centrifuged at 600×g for 5 minutes, the culture medium was aspirated, and 200 μL of ice-bath pre-cooled NAD+ / NADH extract was added with a pipette at a ratio of 200 μL of extract per 1 million cells, and the cells were gently pipetted to promote cell lysis; the lysis process can be performed at room temperature or on ice; then centrifuged at 12000×g, 4°C for 5-10 minutes, and the supernatant was taken as the sample to be tested.

[0099] 2. Preparation of the kit:

[0100] a. Preparation of NADH standard: Pipette 655 μL NADH preparation solution and fully dissolve 5 mg NADH provided in this kit to obtain 10 mM NADH standard; please appropriately divide the 10 mM NADH standard and store it at -80℃ in the dark.

[0101] b. Setting up the NADH standard curve: Dilute the 10mM NADH standard with NAD+ / NADH extract to an appropriate concentration gradient. For example, for the initial test, you can set the concentrations to 0, 0.25, 0.5, 1, 2, 4, 6, 8, and 10μM. When testing, add 20μL of the standard to each well of the 96-well plate, which is equivalent to 0, 5, 10, 20, 40, 80, 120, 160, and 200pmol of NADH per well. If necessary, in subsequent experiments, the concentration range of the standard can be appropriately adjusted according to the NADH content in the sample; the point with a concentration of 0μM is the blank control point, which only contains NAD+ / NADH extract. Note: Since NADH is very unstable, it needs to be used as soon as possible after preparation.

[0102] 3. Sample determination:

[0103] Pipette 20 μL of the sample diluted with NAD+ / NADH extract into a 96-well plate. Set up duplicate wells for the sample to reduce experimental errors. Incubate at 37°C in the dark for 10 minutes; then add 10 μL of color development solution to each well, mix well, and incubate at 37°C in the dark for 10-20 minutes. At this time, orange-yellow formazan will be formed. Measure the absorbance at 450nm. If the color is lighter, the incubation time can be appropriately extended to 30-60 minutes.

[0104] 4. Calculation of NAD+ / NADH in the sample:

[0105] Calculate the average absorbance of each point in the standard group and subtract the absorbance of the blank control group to obtain the absorbance of each standard; draw a standard curve with NADH concentration as the horizontal axis and absorbance as the vertical axis.

[0106] Comparison method: NADH with a known concentration of 0.05 μM was detected using the commercially available NADH kit and the fluorescent probe designed by the present invention, and the detection capabilities of the two were evaluated.

[0107] result:

[0108] See also Figure 7 , the detection value of the commercially available NADH kit was calculated to be 0.04884 μM, and the detection value of the fluorescent probe designed by the present invention was 0.06687 μM. The detection values ​​of the two were statistically analyzed, and the results showed that there was no significant difference between the detection results of the probe and the kit, indicating that the fluorescent probe designed by the present invention has the ability to accurately detect NADH, and has the advantages of simple operation and short detection time.

[0109] 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.

[0110] 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 biosensor for detecting NADH, characterized in that: The biosensor comprises a probe A and upconversion nanomaterials UCNPs; The preparation of the probe A includes preparing a near-infrared rhodamine dye by reacting 9-(2-carboxyphenyl)-6-(diethylamino)-1,2,3,4-tetrahydroxyammonium perchlorate with quinoline-3-carboxaldehyde; adding the near-infrared rhodamine dye and quinoline-3-carboxaldehyde to CH3COOH to obtain a mixture, extracting with dichloromethane, washing with water and salt to obtain a compound; adding the compound and MeOTf to dichloromethane, extracting, collecting the organic layer, drying and filtering to obtain the probe A; the probe A is a fluorescence quencher; The preparation of the upconversion nanomaterial UCNPs includes the synthesis of UCNPs and the surface modification of UCNPs; the upconversion nanomaterial UCNPs is nanoparticles; the particle size of the upconversion nanomaterial is 25nm-30nm.

2. The biosensor for detecting NADH according to claim 1, characterized in that: The UCNPs are fluorescent probe components; The UCNPs are NADH-sensitive fluorescence ratio probes; The surface of the UCNPs is modified with a layer of mesoporous silica, which is loaded with the NADH-specific recognition molecule probe A, and the outer layer is modified with a surface ligand; the surface ligand is β-cyclodextrin.

3. The biosensor for detecting NADH according to claim 1, characterized in that: The synthesis of the UCNPs includes the solvothermal synthesis of NaYF4:Yb,Er,Tm UCNPs, the method comprising: S31, RECl3·6H2O, OA, and 1-ODE are mixed and dispersed to obtain a compound in which all solids are dissolved; S32, stirring and evacuating the compound to obtain a compound from which oxygen has been removed; S33, removing bound water molecules in the compound to obtain an orange-yellow oily solution; S34, adding NH4F and a methanol solution containing NaOH, adjusting the temperature, stirring, and obtaining a methanol solution; S35, removing methanol from the methanol-containing solution to obtain a methanol-removed solution; S36, heating the methanol removal solution and continuing; S37, adding ethanol for precipitation, centrifuging to obtain the precipitate, washing with cyclohexane, and vacuum drying to obtain OA-UCNPs solid OA-UCNPs with surface coated with OA.

4. The biosensor for detecting NADH according to claim 1, characterized in that: The surface modification of the UCNPs comprises forming a mesoporous silica shell layer on the surface of the OA-UCNPs, and the method comprises: S41, taking hexadecyltrimethylammonium bromide, dissolving it, adding chloroform, stirring until the solution is clear, and obtaining a reaction solution; S42, adding water, ethanol and NaOH to the reaction solution, stirring, adding TEOS, stirring, adding APTES to obtain a product; S43, centrifuging the product, washing with ethanol, transferring it into ethanol containing ammonium nitrate, and stirring it to obtain an ethanol dispersion of the nano fluorescent probe; S44, placing monosubstituted β-CD p-toluenesulfonate in the ethanol dispersion of the nano fluorescent probe, stirring and reacting in a nitrogen atmosphere to obtain UCNPs@mSiO2@β-CD.

5. A kit for detecting NADH, characterized in that: The kit is prepared by the biosensor for detecting NADH according to any one of claims 1 to 4; The kit includes a biosensor and a NADH standard.

6. The kit for detecting NADH according to claim 5, characterized in that: The NADH standard is a standard solution with a series of concentration gradients; The concentration of the standard solution is 0-100 nM; Preferably, the concentration of the standard is 0, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM and 100 nM.

7. A method for preparing the biosensor according to any one of claims 1 to 4 or the kit according to claim 6, comprising: S71, preparing the probe A; S72, preparation of the upconversion nanomaterial UCNPs and combination with the probe A; S73, performing subpackaging, packaging, and / or storage.

8. Use of the biosensor according to any one of claims 1 to 4 or the kit according to claim 6 in detecting NADH in cells; Preferably, the cells include PC12 cells or p 0 cell.

9. A method for detecting NADH, characterized in that: The detection method uses the biosensor according to any one of claims 1 to 4 or the kit according to claim 6, and the detection method comprises: S91, establish a standard curve; S92, detecting the sample to be tested; S93. Calculate the concentration of NADH in the sample to be tested according to the standard curve.

10. The detection method according to claim 9, characterized in that: The detection method includes a fluorescent probe element, wherein the fluorescent probe element is an upconversion nanoparticle UCNPs having a core-shell structure; The detection method determines the NADH content by calculating the ratio of the emission peak to the reference emission peak.

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